BIOCHEMICAL CHARACTERIZATION OF IN VITRO SALT TOLERANT CELL LINES AND REGENERATED PLANTS OF SUGARCANE (SACCHARUM SPP.

HYBRID)

NEELMA MUNIR DEPARTMENT OF BOTANY UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN

BIOCHEMICAL CHARACTERIZATION OF IN VITRO SALT TOLERANT CELL LINES AND REGENERATED PLANTS OF SUGARCANE (SACCHARUM SPP. HYBRID)

A THESIS SUBMITTED TO THE UNIVERSITY OF THE PUNJAB IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANY

BY NEELMA MUNIR

DEPARTMENT OF BOTANY UNIVERSITY OF THE PUNJAB LAHORE, PAKISTAN

IN THE NAME OF GOD THE MOST BENEFICIENT THE MOST MERCIFUL

DEDICATED TO ALL MY FAMILY MEMBERS Especially To My Husband AHMED SHERAZ My Daughter SHIZA And My Son AHMED ALI

CONTENTS
Title CERTIFICATE ACKNOWLEDGEMENTS ABBREVIATIONS/UNIT ABBREVIATIONS ABSTRACT LIST OF ANNEXURES LIST OF FIGURES LIST OF TABLES Chapter 1: INTRODUCTION Chapter 2: LITERATURE REVIEW 2.1 Tissue Culture Studies in Sugarcane 2.1.1 Callus Induction and Proliferation 2.1.2 Plant Regeneration from Callus Cultures 2.2 Biochemical Aspects of Salinity Tolerance in Plants 2.2.1 Proteins as Biochemical Markers of Salinity Tolerance 2.2.2 Antioxidant Enzymes as Biochemical Markers of Salinity Tolerance 2.2.2.1 Studies on Antioxidant Enzymes in Callus Cultures under Salt Stress 2.2.2.2 Studies on Antioxidant Enzymes in Seedlings under Salt Stress 2.2.2.3 Studies on Antioxidant Enzymes in Plants under Salt Stress 2.2.3 Non-Enzymatic Antioxidants as Markers of Salinity Tolerance 2.3 Effect of Salt Stress on Graminaceous Crops 2.3.1 Selection of Salt Tolerant Callus Cultures 2.3.2 Regeneration of Salt Tolerant Cell Lines 2.4 Salt vs. Water Stress 2.5 Effect of Salt Stress in Sugarcane 2.5.1 Effect of Salt Stress on Sugarcane Plants Page Number i ii iii v x xi xxiii 1 9 10 10 13 17 18 23 23 26 28 32 35 36 39 44 47 48

2.5.2 Effect of Salt Stress on Sugarcane Callus Cultures Chapter 3: MATERIALS AND METHODS 3.1 Media Preparation 3.1.1 Preparation of Stock Solutions 3.1.2 Growth Regulators 3.1.3 Preparation of Medium from the Stocks 3.2 Sterilization 3.2.1 Sterilization of Glassware 3.2.2 Sterilization of the media 3.2.3 Sterilization of Working Area of Laminar Airflow Cabinet 3.2.4 Sterilization of Surgical Tools 3.3 Plant Material 3.3.1 Source of plant material 3.3.2 Explant preparation and disinfection

52 57 57 57 57 57 58 58 58 58 59 59 59 59

3.4 Culture Conditions

3.5 Biochemical Studies 3.5.1 Quantitative estimation of Protein 3.5.1.1 Extraction of Protein 3.5.1.2 Estimation of Soluble Protein Contents 3.5.2 Quantitative Estimation of Peroxidase, Catalase and Superoxide Dismutase 3.5.2.1 Enzyme extraction 3.5.2.2 Estimation of Enzymes 3.5.2.2.1 Estimation of Peroxidase 3.5.2.2.2 Estimation of Catalase 3.5.2.2.3 Estimation of Superoxide Dismutase 3.6 Experimental Plan 3.6.1 Callus Formation and Proliferation 3.6.2 Plant Regeneration from the Callus Cultures

60
60 60 60 60 61 61 62 62 63 64 65 65 66

3.6.3 Effect of Various Salt Treatments on Callus Cultures 3.6.4 Polyethylene Glycol (PEG) pretreatment Experiment 3.6.5 Ascorbic acid Pretreatment Experiment 3.7 Statistical Analysis ANNEXURES Chapter 4: CALLUS INDUCTION, MAINTENANCE AND REGENERATION OF SUGARCANE (cv. SPF 234 and cv. HSF 240) RESULTS 4.1 Callus Induction and Proliferation of Sugarcane (cv. SPF 234 and cv. HSF 240) 4.2 Plant Regeneration From Callus Cultures of Sugarcane (cv. SPF 234 and cv. HSF 240) 4.3 Rooting of the Regenerated Shoots of Sugarcane (cv. SPF 234 and cv. HSF 240) 4.4 Hardening and Acclimatization of in vitro-grown Plants of Sugarcane (cv. SPF 234 and cv. HSF 240) DISCUSSION Chapter 5: EFFECT OF SALT STRESS ON CALLUS CULTURES OF SUGARCANE (cvs. SPF 234 and HSF 240) RESULTS 5.1 Effect of Salt Stress on Morphological Characteristics of Sugarcane Callus Cultures 5.2 Effect of Salt Stress on Fresh Weights, Callus Browning and Necrosis in Sugarcane Callus Cultures 5.3 Effect of Salt Stress on Regeneration Potential of Sugarcane Callus Cultures 5.4 Rooting of the Regenerated Plantlets

67 68 68 69 70

80 80 80 85 89 92 93

98 98 98 111

115 120

DISCUSSION Chapter 6: EFFECT OF SALT STRESS ON SOLUBLE PROTEIN CONTENTS AND ANTIOXIDANT ENZYME AVTIVITIES IN SUGARCANE CALLUS CULTURES AND REGENERATED PLANTS

124

129

RESULTS 6.1 Soluble Protein Contents (mg/g tissue) of Sugarcane (Saccharum spp. Hybrid, cv. SPF 234 and cv. HSF 240) Callus Cultures Maintained on 9 Different Salt Stress Treatments (0-160 mM NaCl) Supplemented to MS Medium at Day 90, 120 or 150 under Dark Conditions (27 2 C) 6.2 Peroxidase Activity (mg/g tissue) of Sugarcane Callus Cultures Maintained on 9 Different Salt Stress Treatments Supplemented to MS Medium at Day 90, 120 or 150 Under Dark Conditions (27 2 C) 6.3 Catalase Activity (units/ml enzyme) of Sugarcane Callus Cultures Maintained on 9 Different Salt Stress Treatments Supplemented to MS Medium at Day 90, 120 or 150 Under Dark Conditions (27 2 C) 6.4 Superoxide Dismutase Activity (units/mg protein) of Sugarcane Callus Cultures Maintained on 9 Different Salt Stress Treatments Supplemented to MS Medium at Day 90, 120 or 150 Under Dark Conditions (27 2 C) 6.5 Soluble Protein Contents (mg/g tissue) of the Regenerated Plants of Sugarcane (cvs. SPF 234 and HSF 240) 6.6 Peroxidase, Catalase and Superoxide Dismutase Activities of the Regenerated Plants of Sugarcane (cvs. SPF 234 and HSF 240) DISCUSSION

129

129

133

136

139

143 145 148

Chapter 7: ROLE OF POLYETHYLENE GLYCOL (PEG) IN IMPROVING SALT (NaCl) TOLERANCE OF SUGARCANE RESULTS 7.1 Effect of Polyethylene Glycol (PEG) Pretreatment on Fresh Weights, Browning and Necrosis in Sugarcane Callus Cultures 7.2 Effect of Polyethylene Glycol (PEG) Pretreatment on Soluble Protein Contents of Sugarcane Callus Cultures 7.3 Effect of Polyethylene glycol (PEG) Pretreatment on Peroxidase Activity of Sugarcane Callus Cultures 7.4 Effect of Polyethylene glycol (PEG) Pretreatment on Catalase Activity of Sugarcane Callus Cultures 7.5 Effect of Polyethylene Glycol (PEG) Pretreatment on Superoxide Dismutase Activity of Sugarcane Callus Cultures 7.6 Effect of Polyethylene Glycol (PEG) Pretreatment on Regeneration Potential of Sugarcane (cv. SPF 234 and cv. HSF 240) Callus Cultures DISCUSSION Chapter 8: ROLE OF ASCORBIC ACID IN IMPROVING SALT (NaCl) TOLERANCE OF SUGARCANE RESULTS 8.1 Effect of Ascorbic Acid Pretreatment on Fresh weight, Browning and Necrosis in Sugarcane Callus Cultures 8.2 Effect of Ascorbic Acid Pretreatment on Soluble Protein Contents and Antioxidant Enzyme Activities in Callus Cultures of Sugarcane (cv. SPF 234) 8.2.1 Soluble Protein Contents 8.2.2 Peroxidase Activity 8.2.3 Catalase Activity 8.2.4 Superoxide Dismutase Activity

157 157 157 166 171 176 181 186 191

196 196 196

203 203 203 204 204

8.3 Effect of Ascorbic Acid Pretreatment on Soluble Protein Contents and Antioxidant Enzyme Activities in Callus Cultures of Sugarcane (cv. HSF 240) 8.3.1 Soluble Protein Contents 8.3.2 Peroxidase Activity 8.3.3 Catalase Activity 8.3.4 Superoxide Dismutase Activity 8.4 Effect of Ascorbic Acid Pretreatment on Fresh Weight, Browning and Necrosis in in vitro-grown Plants of Sugarcane (cvs. SPF 234 and HSF 240) 8.5 Effect of Ascorbic Acid Pretreatment on Soluble Protein Contents and Antioxidant Enzyme Activities in in vitro-grown Sugarcane Plants (cvs. SPF 234 and HSF 240) 8.5.1 Soluble Protein Contents 8.5.2 Peroxidase Activity 8.5.3 Catalase activity 8.5.4 Superoxide dismutase activity DISCUSSION LITERATURE CITED

208 208 208 209 209

212

219 219 220 220 221 226 230

Certificate
This is to certify that the research work entitled Biochemical characterization of in vitro salt tolerant cell lines and regenerated plants of sugarcane (Saccharum spp. Hybrid described in this thesis by Ms. Neelma Munir is an original work of the author and has been carried out under my direct supervision. I have personally gone through all the data, results, materials reported in the manuscript and certify their correctness and authenticity. I further certify that the material included in this thesis has not been used in part or full in a manuscript already submitted or in the process of submission in partial or complete fulfillment of the award of any other degree from any institution. I also certify that the thesis has been prepared under my supervision according to the prescribed format and I endorse its evaluation for the award of Ph.D. degree through the official procedures of the University of the Punjab, Lahore.

Supervisor: Dr. Faheem Aftab

Assistant Professor Department of Botany University of the Punjab, Lahore Date: ____________

ii

ACKNOWLEDGEMENTS
All praises belongs to Almighty Allah Who had blessed me the potential to complete this research work and compile this thesis. I would like to record my sentiments of indebtedness to my respected supervisor Dr. Faheem Aftab, Assistant professor, Department of Botany, University of the Punjab, Lahore, for his scholarly guidance, illustration, constructive criticism, keen interest, cooperation and encouragement which was the real source of inspiration for me during my research work. I wish to say thanks to Prof. Dr. Shahida Hasnain, Dean Faculty of Life Sciences, Focal Person Higher Education commission, Islamabad, Pakistan, for his corporation and for smooth running of my HEC merit scholarship program. I wish to express my gratitude to Prof. Dr. Javed Iqbal, Former Chairman, Department of Botany, Currently, Director School of Biological Sciences, University of the Punjab, Lahore for his very kind behavior and for developing my interest in the field of plant tissue culture. I am thankful to Dr. Rass Masood Khan, Chairman, Botany Department, University of the Punjab, Lahore for providing best research facilities. I owe gratitude and sincere thanks to Dr. Humera Afrasiab (Assistant Professor, Botany Department) for her suggestions and guidance during my research work. I am thankful to Higher Education Commision, Islamabad, Pakistan, for the providing me financial support under Merit Scholarship Scheme for PhD studies in Science and Technology which facilitated my research work greatly. My sincere thanks are to my lab fellows Zahoor Ahmed Sajid, M. Akram, Adeela Haroon and Sadia Basharat who willingly helped me to achieve this goal. I offer my heartiest gratitude to my Parents, In Laws, brother (Nauman) and sister (Afsheen) for their encouragement, love and countless prayers at every stage for my success in life. Last but not least I wish to say thanks to my husband Ahmed Sheraz, my daughter Shiza, and my son Ahmed Ali for their love that has strengthened me in my weakness. NEELMA MUNIR

iii

ABBREVIATIONS/UNIT ABBREVIATIONS
2,4-D BA BAP CH Conc. cv. cvs. EDTA Fig. h IAA IBA L. MS NAA NBT PEG pH PVP ROS rpm SDS SOD spp. TDZ UV v/v W w/v 2,4-Dichlorophenoxy acetic acid 6-Benzyladenine 6-Benzylaminopurine Casein Hydrolysate Concentration Cultivar Cultivars Ethylenediaminetetraacetic acid Figure Hour Indole-3-acetic acid Indole-3-butyric acid Linnaeus Murashige and Skoog (1962) basal medium Naphthalene acetic acid Nitroblue tetrazolium Polyethylene glycol Hydrogen ion concentration Polyvinylpolypyrrolidone Reactive oxygen species Revolutions per minute Sodium dodecyl sulphate Superoxide dismutase Species Thidiazuron Ultraviolet Volume/Volume Watt Weight/Volume

iv dS m-1 g g l-1 l M mg l-1 ml ml l-1 M mM mM l-1 nm ppm Decisiemens per meter Gram Gram per liter Liter Molar Milligram per liter Milliliter Milliliter per liter Micromolar Millimolar Millimoles per liter Nanometer Parts per million

ABSTRACT
Soil salinity is one of the major abiotic stresses affecting growth and productivity of important crops worldwide. Sugarcane is a glycophyte crop of major economical value in tropical and subtropical countries of the world where salinity is an ever-increasing problem. Sugarcane plants grown under stressed conditions show not only arrested growth of various parts but also a decrease in sucrose content. During the recent years, tissue culture technique has gained importance in producing plants with improved salt tolerance through selection of salt-tolerant cells lines and their subsequent regeneration. During the present work, protocols were established for callus induction, maintenance and regeneration of the two sugarcane cultivars (cv. SPF 234 and cv. HSF 240). It was observed that the best medium for callus induction and maintenance was MS medium supplemented with 13.5 M 2,4-D. Callus cultures at day 120 were shifted to the various regeneration media. It was observed that among the different media used, MS medium supplemented with 8.87 M BAP and 0.5 M TDZ was the best in terms of all the growth parameters studied for both the cultivars. However, the maximum regeneration frequency in cv. SPF 234 was greater (85 %) as compared to cv. HSF 240 (76 %). Best rooting (95 %) of the regenerated shoots in cv. SPF 234 was obtained on MS medium supplemented with 2 M IBA at day 30. Almost similar results were recorded for in vitro-grown plants of cv. HSF 240 for percentage root formation on MS medium supplemented with 1 or 2 M IBA with 94 and 93 % rooting respectively. Hardening of sugarcane plants was also successfully accomplished for both the sugarcane cultivars.

vi During the study, 60-days-old calluses were shifted to MS media containing various NaCl levels (0-160 mM). The effect of salt stress on sugarcane callus browning, necrosis and fresh weights was observed at day 90, 120 and 150. It was found that with an increase in salt concentration, callus necrosis also increased. Callus cultures at day 120 were transferred to the regeneration medium (MS + 8.87 M BAP + 0.5 M TDZ). The data for regeneration frequency, number of shoots per culture vessel, shoot length, number of roots per culture vessel and root length were recorded at day 30 upon transfer of callus cultures to the regeneration medium. The regeneration frequency was less in the plants regenerated from NaCl-treated callus cultures as compared to control (calluses grown on 0 mM NaCl concentration). It was found that callus cultures of cv. SPF 234 retained 82 % regeneration potential after treatment with 100 mM salt concentration but this frequency decreased sharply at 120 mM salt concentration and only 10 % callus cultures retained regeneration potential. It was also observed that 75 % of the callus cultures of cv. HSF 240 were capable of plant regeneration after 100 mM NaCl treatment. It was also observed during the present study that the number of regenerated shoots from callus cultures treated with various NaCl concentrations was generally greater as compared to the control calluses maintained at 0 mM NaCl. The present investigation also highlights the changes in soluble protein contents and antioxidant enzyme (peroxidase, catalase and superoxide dismutase) activities in response to salt stress at day 90, 120 and 150. Quantitative analysis of the soluble protein contents during the present work indicated that soluble protein contents in the callus cultures of both the sugarcane cultivars (cv. SPF 234 and cv. HSF 240) significantly decreased in response to various NaCl treatments. Results of the present study also indicated that when salt was

vii supplied to the growth media, generally there was a significant increase in peroxidase, catalase and superoxide dismutase activity of the callus cultures as compared to the control in both the cultivars. During the present work, significant difference was observed in the soluble protein contents of the regenerated plants from callus cultures treated with different NaCl concentrations. An interesting observation during the present work was that the plants regenerated from salt-treated callus cultures had generally more antioxidant enzyme activities as compared to the control plants (regenerated from non-treated callus cultures). Salt and water stress show high degree of similarity. An experiment was performed during the present work to study the possible effect of PEG in improving salt tolerance. Sugarcane calluses were treated with four different salt concentrations after giving 1 % PEG pretreatment. The salt concentrations consisted of a control (0 mM NaCl) and three other salt concentrations including one sublethal NaCl level, one above and one below the sublethal NaCl concentration for each cultivar. Data for fresh weights, callus necrosis, soluble protein contents and activities of peroxidase, catalase and superoxide dismutase were recorded at day 90 and 120. No significant change in fresh weights was recorded as a result of PEG pretreatment both at day 90 as well as 120. Less necrosis was observed in the callus cultures of both sugarcane cultivars after PEG pretreatment when subcultured on salt medium as compared to the non-pretreated cultures maintained at the same salt level. PEG pretreatment enhanced the biosynthesis of soluble protein contents in the callus cultures of both the sugarcane cultivars as compared to the non-pretreated controls maintained at the same salt level except in callus cultures of cv. HSF 240 at day 120. A comparison of the PEGpretreated and non-pretreated callus cultures of cv. SPF 234 at day 90 indicated that

viii generally at each salt concentration, PEG-pretreated callus cultures had relatively greater peroxidase, catalase and superoxide dismutase activity as compared to non-pretreated callus cultures maintained at the same salt level. At day 120, only superoxide dismutase activity was increased as a result of PEG pretreatment. In callus cultures of cv. HSF 240, PEG pretreatment affected the peroxidase activity only at day 120 while a significant increase in catalase activity was recorded for this cultivar both at day 90 and 120. PEG-pretreated callus cultures of cv. HSF 240 at day 90 had greater superoxide dismutase activities as compared to their non-pretreated controls maintained at the same NaCl level but at day 120 the effect of PEG pretreatment on SOD activity was non-significant. PEG pretreatment had no effect on regeneration potential of both the cultivars. Ascorbic acid pretreatment to callus cultures was not proven to improve salt tolerance of callus cultures rather it caused more browning and necrosis of the callus tissues. It was also observed that ascorbic acid pretreatment caused decrease in fresh weights of sugarcane callus cultures. A significant effect of ascorbic acid pretreatment was recorded on soluble protein contents, peroxidase and catalase activities in callus cultures of cv. SPF 234 at day 90 but ascorbic acid pretreatment had no significant effect on SOD activity of callus cultures at day 90. Interestingly, the exogenous supply of ascorbic acid to in vitro-grown sugarcane plants improved their salt tolerance as indicated by the studied morphological as well as biochemical parameters. It was also observed that non-pretreated in vitro-grown plants of both sugarcane cultivars could tolerate up to 100 mM NaCl level but the pretreated plants of cv. SPF 234 and cv. HSF 240 could survive up to 160 and 140 mM NaCl concentration respectively. Furthermore, much less yellowing of leaves in ascorbic acid-pretreated plants was observed as compared to non-pretreated plants at the same salt concentration. A

ix significant increase in the fresh weights, number of shoots per culture vessel and shoot length was observed after ascorbic acid pretreatment in both the sugarcane cultivars. A general decrease in root length was recorded after ascorbic acid pretreatment in the plants of cv. SPF 234. Ascorbic acid pretreatment had a significant effect on soluble protein contents as well as activities of peroxidase, catalase and superoxide dismutase in cv. SPF 234. The same trends were observed in in vitro-grown plants of cv. HSF 240 for peroxidase and catalase activity but no significant effect of ascorbic acid pretreatment was recorded on soluble protein contents or superoxide dismutase activity.

LIST OF ANNEXURES
Annexure Number 1: 2: 3: 4: 5: 6: 7: 8: 9: 10: 11: 12: 13A: 13B: Title Formulation of MS Medium (Murashige and Skoog, 1962) for the preparation of Stock Solutions Preparation of Stock Solutions for MS (Murashige and Skoog, 1962) Medium Preparation of Stock Solutions of Growth Regulators Preparation of 1 liter MS Medium Composition of Different Media Used for Callus induction/ Maintenance Composition of different media used for plant regeneration from callus cultures Composition of Different Rooting Media Composition of Different MS Media Used in Salt Stress Experiments 0.1 M Phosphate Buffer (pH 7.2) for Extraction of Proteins and Enzymes Composition of Biuret Reagent for Protein Estimation Reagents for Peroxidase Estimation Reagents for Catalase Estimation Reagents for Superoxide Dismutase Estimation Preparation of Reaction Mixture Page Number 70 71 72 73 73 75 75 76 76 77 77 78 78 79

xi

LIST OF FIGURES
Figure Number 4.1: 4.2: 4.3: 4.4: 4.5: 4.6: 4.7: 4.8: 4.9: 4.10: 4.11: 4.12: 4.13: 4.14: 4.15: 4.16: 4.17: 4.18: Title A 10-days-old callus of cv. SPF 234 (1x) A 10-days-old callus of cv. HSF 240 (1x) A callus culture of cv. SPF 234 at day 30 (1x) A 30-days-old callus of cv. HSF 240 (1x) A 60-days-old callus of cv. SPF 234 (2x) A 60-days-old callus of cv. HSF 240 (2x) Soot initiation (arrows) in callus culture of cv. SPF 234 (1.5x ) Soot initiation (arrows) in callus culture of cv. HSF 240 (2x ) Stereomicrograph showing shoot initiation in callus culture of cv. SPF 234 (20x) Stereomicrograph showing shoot initiation in callus culture of cv. HSF 240 (20x ) Regenerated shoots of cv. SPF 234 at day 30 after subculture on MS basal medium (1.4x) Regenerated shoots of cv. HSF 240 at day 30 after subculture on MS basal medium (1.6x) Root development of regenerated shoots of cv. SPF 234 on MS basal medium supplemented with 2 M IBA (1.8x) Root development of regenerated shoots of cv. HSF 240 on MS basal medium supplemented with 1 M IBA (2x) Well-developed plants of cv. SPF 234 on MS basal medium (1x) Page Number 81 81 81 81 81 81 86 86 86 86 86 86 90 90 90

Well-developed plants of cv. HSF 240 on MS basal medium (1x) 90 Plants of cv. SPF 234 grown under ex vitro condition (1.5x) Plants of cv. HSF 240 grown under ex vitro condition (1.5x) 92 92

xii

5.1:

Callus morphology of sugarcane (Saccharum spp. hybrid cv. SPF 234) on MS basal medium supplemented with 13.5 M 2, 4-D at day 60 under dark conditions at 27 2 C (2x) A well proliferating callus culture at day 90 (1.2x) Callus morphology at day 120 (1.5x) A callus culture at day150 (1.2x) First subculture at 20 mM NaCl concentration at day 90 (1.2x) Greenish callus with somewhat whitish portions undergoing browning (farthest from the viewer; indicated by an arrow) at day 120 (1.2x) Brownish-yellow callus at day 150 (1x) A callus culture (day 90) transferred to 40 mM NaCl concentration (1.2x) Greenish-yellow callus at day120 (1.2x) A decreased callus growth potential at day150 (1x) A 90-days-old greenish-yellow callus with signs of necrosis at 60 mM NaCl concentration (1.4x) A callus culture with some brown and greenish-yellow portions at day 120 (1.2x) Brownish-yellow callus at day 150 (1.2x) Callus (day 90) at 80mM NaCl concentration showing signs of necrosis (1.2x) A 120-day-old off-white callus with patches of reddishbrown color (1.2x) A callus culture showing blackish-brown portion (on the lower side) at day150 (1.4x) Callus necrosis at day 90 (1.2x). A 120-day-old callus showing category A necrosis (1.3x).

98 99 99 99 100

5.2: 5.3: 5.4: 5.5: 5.6:

100 100 100 100 100 101 101 101 101 101 101 102 102

5.7: 5.8: 5.9: 5.10: 5.11: 5.12: 5.13: 5.14: 5.15: 5.16: 5.17: 5.18:

xiii

5.19: 5.20: 5.21: 5.22: 5.23: 5.24: 5.25: 5.26:

Yellowish-brown callus with somewhat translucent appearance at day 150 (1.2x). Callus at 120mM concentration at day 90 on MS basal medium (1x) Blackish-brown callus at 120 mM NaCl concentration showing category A necrosis at day 120 (1.2x) Callus with retarded growth (day 120) at 120 mM NaCl concentration (1.2x) Callus at day 90 showing browning (arrows) at 140 mM NaCl (1x) Callus at day 150 with lower blackish portion (1x) A necrotic callus culture showing category A necrosis at 140 mM NaCl (1x) Callus morphology of sugarcane (cv. SPF 234) at 160 mM NaCl level supplemented to MS medium at day 90 under dark at 27 2 C (1.2x) Callus morphology of sugarcane (Saccharum spp. hybrid cv. HF 240) on MS basal medium supplemented with 13.5 M 2, 4-D at day 60 under dark at 27 2 C (1.4x) A greenish-yellow 90-days-old callus culture at 0 mM NaCl (1.1x) Proliferating callus (day 120) at 20 mM NaCl concentration (1.3x) Callus with translucent appearance at day150 at 0 mM NaCl concentration (0.8x) Callus morphology at 20 mM NaCl concentration at day 90 (1x) Off-white callus culture with a little brown portion at day 120 (1.1x) Callus at day150 at 20 mM NaCl concentration showing some browning (1.2x).

102 102 102 102 103 103 103 103

5.27:

105 105 105 105 106 106 106

5.28: 5.29: 5.30: 5.31: 5.32: 5.33:

xiv 5.34: 5.35: 5.36: 5.37: 5.38: 5.39: 5.40: 5.41: 5.42: 5.43: 5.44: 5.45: 5.46: 5.47: 5.48: 5.49: Initiation of callus necrosis (category E) in 90-days-old callus at 40 mM NaCl concentration (1x). Callus browning (day 120) at 40 mM NaCl concentration (1.1x) Callus culture with smooth appearance having brown patches at day150 (1.2x) A 90-day-old callus upon transfer to 60 mM NaCl concentration (1.1x) Callus culture (day 120) with reddish-brown patches (1.22x) Brownish-yellow callus with reduced proliferation at day 150 (1.1x) A 90-days-old callus culture transferred to 80 mM NaCl concentration (1.1x). Brownish-yellow callus with some off-white portion at day 120 (1.2x) A 150-days-old yellowish-brown necrotic (category A) callus (1.1x) Translucent callus showing signs of necrosis at 100 mM NaCl concentration (1x). A callus culture with some black portion due to necrosis at day 120 (1.2x) Category A necrosis observed in a callus culture at day 150 (0.8x) Brownish off-white callus at day 90 (1.2x). A 120-days-old category A necrotic callus with a small whitish portion (1.4 x) A callus culture at 120 mM NaCl (day 150) that has already turned dark brown to black in color (1x) Callus necrosis at 140 mM NaCl level at day 90 (1.1x)

107 107 107 107 107 107 108 108 108 108 108 108 109 109 109 109

xv 5.50: 5.51: A 90-days-old dry and necrotic callus culture at 160 mM NaCl (1x) Initiation of shoot regeneration from a callus treated with 20 mM NaCl at day 25 after shifting to regeneration medium (1.2x) Shoot regeneration at day 30 from a callus treated with 60 mM NaCl (1 x) Transfer of shoots to MS basal medium (1x) Plants regenerated from callus treated with 100 mM NaCl medium shifted to MS basal medium at day 30 (1x) Shoot initiation from a 40 mM NaCl treated callus culture at day 30 (1.2x) Somewhat elongated shoots at day 15 after transfer to MS basal medium (1x) Regenerated shoots from a callus culture treated with 80 mM NaCl (1.2x) Shoots regenerated from a callus treated with 100 mM NaCl at day 30 (0.8x) Root initiation in in vitro-grown shoots of cv. SPF 234 at day 20 on MS medium supplemented with 2 M IBA (1.1x) Well-developed roots of the sugarcane plants (cv. HSF 240) regenerated from a callus culture treated with 100 mM NaCl on MS medium supplemented with 1 M IBA at day 30 (1.2) In vitro plants of sugarcane (cv. SPF 234) regenerated from callus culture treated with 120 mM NaCl (0.8x) In vitro-grown plants of cv. HSF 240 regenerated from callus culture treated100mM NaCl level (1x) Soluble protein contents in callus cultures of cv. SPF 234 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C

109

117 117 117 117 117 117 117 117

5.52: 5.53: 5.54: 5.55: 5.56: 5.57: 5.58: 5.59:

120

5.60:

120

5.61:

121 121

5.62: 6.1:

131

xvi

6.2:

Soluble protein contents in callus cultures of cv. HSF 240 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C Peroxidase activity in callus cultures of cv. SPF 234 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C Peroxidase activity in callus cultures of cv. HSF 240 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C Catalase activity in callus cultures of cv. SPF 234 maintained on different NaCl levels at day 90, 120, or 150 under dark conditions at 27 2 C Catalase activity in callus cultures of cv. HSF 240 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C Superoxide dismutase activity in callus cultures of cv. SPF 234 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2C Superoxide dismutase activity in callus cultures of cv. HSF 240 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C Off-White non-pretreated callus culture showing category E necrosis at 0 mM NaCl level (1.2x) Callus culture maintained at 0 mM salt level after PEG pretreatment at day 120 (1.4x) A callus culture without PEG pretreatment at day 120 (1x) Category D necrosis in callus maintained at 100 mM NaCl level at day 120 after PEG pretreatment (1.4x) Brownish-yellow, non-pretreated callus culture maintained at 120 mM NaCl level (1.2x) A callus culture maintained at 120 mM NaCl level after PEG pretreatment (1.1x)

131

6.3:

134

6.4:

134

6.5:

137

6.6:

137

6.7:

141

6.8:

141 160

7.1:

7.2: 7.3: 7.4: 7.5: 7.6:

160 161 161 161 161

xvii

7.7:

A reddish-brown (category A necrosis) non-pretreated necrotic callus culture maintained at 140 mM NaCl level (1.1x) PEG-pretreated callus with some off-white portion (category B necrosis) maintained at 140 mM NaCl at day 90 (1x) A non-pretreated callus culture transferred to salt-free medium (1.2x) A PEG-pretreated greenish-yellow callus (category E necrosis) on 0 mM NaCl level at day 120 (1x) A callus culture maintained at 80 mM NaCl level without PEG pretreatment (1.2x) PEG-pretreated callus at 80 mM salt concentration at day 120 (2x) A 120-days-old non-pretreated callus culture transferred to 100 mM NaCl (1.2x) Brownish off-white callus culture maintained at 100 mM NaCl level after PEG pretreatment (1.4x) Yellowish-brown non-pretreated callus culture maintained at 120 mM NaCl level (1.1x) PEG-pretreated 90-days-old off-white callus culture showing category B necrosis with browning in the lower portion at 120 mM NaCl concentration (1.2x) Effect of Polyethylene glycol pretreatment on soluble protein contents in callus cultures of sugarcane (cv. SPF 234) at day 90 and 120 Effect of Polyethylene glycol pretreatment on soluble protein contents in callus cultures of sugarcane (cv. HSF 240) at day 90 and 120 170 Effect of Polyethylene glycol on peroxidase activity in callus cultures of sugarcane (cv. SPF 234) at day 90 and 120

162

7.8:

162 162 162 163 163 164 164 164

7.9: 7.10: 7.11: 7.12: 7.13: 7.14: 7.15: 7.16:

164

7.17:

170

7.18:

7.19:

175

xviii

7.20:

Effect of Polyethylene glycol on peroxidase activity in callus cultures of sugarcane (cv. HSF 240) at day 90 and 120 Effect of Polyethylene glycol on catalase activity in callus cultures of sugarcane (cv. SPF 234) at day 90 and 120 Effect of Polyethylene glycol on catalase activity in callus cultures of sugarcane (cv. HSF 240) at day 90 and 120 Effect of Polyethylene glycol on superoxide dismutase activity in callus cultures of sugarcane (cv. SPF 234) at day 90 and 120 Effect of Polyethylene glycol on superoxide dismutase activity in callus cultures of sugarcane (cv. HSF 240) at day 90 and 120 Pretreated callus culture with a high number of regenerating shoots at day 30 upon transfer to regeneration medium after treatment with 100 mM NaCl level (1.5x) Well-developed shoots on MS medium regenerated from PEG-pretreated callus culture maintained at 100 mM NaCl (1.2x) Shoot initiation from callus culture of cv. SPF 234 maintained at 120 mM NaCl level after PEG pretreatment (1.4x) Rooting of the regenerated shoots treated with 120 mM NaCl after PEG pretreatment (1.2x) Shoot initiation from callus culture of cv. SPF 234 maintained at 80 mM NaCl level after PEG pretreatment (1x) Bunch of shoots developed from callus culture maintained at 100 mM NaCl level after PEG pretreatment (1.5x)

175

7.21:

180

7.22:

180

7.23:

185

7.24:

185

7.25:

187

7.26:

187

7.27:

187 187

7.28: 7.29:

189

7.30:

189

xix 7.31: Well-developed in vitro-grown plants from callus cultures treated with 100 mM NaCl concentration after PEG pretreatment (1.2x) A control callus culture maintained at 0 mM NaCl level (1.2x) Non-pretreated callus culture maintained at 100 mM NaCl level (1.4x) A callus culture maintained at 120 mM NaCl level (1x) Callus morphology at 140 mM NaCl level (1.4x) Some browning initiating (arrow) at 0 mM NaCl (1.2x) Ascorbic acid pretreated callus culture at 100 mM NaCl concentration showing necrosis (1.2 x) A 90-days-old callus maintained at 120 mM NaCl level after ascorbic acid pretreatment (0.9x) Necrotic callus culture after Ascorbic acid pretreatment at day 90 (1x) Callus morphology at 0 mM NaCl level (1 x) A 90-days-old callus at 80 mM NaCl level without ascorbic acid pretreatment (1.2 x) A callus culture at 100 mM NaCl level showing category B necrosis (1x) Non-pretreated callus culture maintained at 120 mM NaCl level (1.1 x) Off-white callus with some black portion at 0 mM NaCl showing necrosis after ascorbic acid pretreatment (1.2x) Callus browning after ascorbic acid pretreatment at 80 mM NaCl concentration (0.9x) Callus maintained at 100 mM NaCl level after ascorbic acid pretreatment (1x)

189 198 198 198 198 198 198 198 198 199 199 199 199

8.1: 8.2: 8.3: 8.4: 8.5: 8.6: 8.7: 8.8: 8.9: 8.10: 8.11: 8.12: 8.13:

200 200 200

8.14: 8.15:

xx 8.16: Ascorbic acid-pretreated callus at 120 mM NaCl level (1.2x) Effect of Ascorbic acid pretreatment on soluble protein contents of callus cultures of sugarcane (cv. SPF 234) at day 90 Effect of Ascorbic acid pretreatment on peroxidase activity of callus cultures of sugarcane (cv. SPF 234) at day 90 Effect of Ascorbic acid pretreatment on catalase activity of callus cultures of sugarcane (cv. SPF 234) at day 90 Effect of Ascorbic acid pretreatment on superoxide dismutase activity of callus cultures of sugarcane (cv. SPF 234) at day 90 Effect of Ascorbic acid pretreatment on soluble protein contents of callus cultures of sugarcane (cv. HSF 240) at day 90 Effect of Ascorbic acid pretreatment on peroxidase activity of callus cultures of sugarcane (cv. HSF 240) at day 90 Effect of Ascorbic acid pretreatment on catalase activity of callus cultures of sugarcane (cv. HSF 240) at day 90 Effect of Ascorbic acid pretreatment on superoxide dismutase activity of callus cultures of sugarcane (cv. HSF 240) at day 90 Non-pretreated control in vitro plants of cv. SPF 234 at 0 mM NaCl level (1.8 x) Ascorbic acid-pretreated plants at day 30 maintained at 0 mM NaCl level (0.5 x) Ascorbic acid-pretreated in vitro plants maintained at 40 mM NaCl level at day 30 (0.6x)

200

8.17:

207

8.18:

207

8.19:

207

8.20:

207

8.21:

211

8.22:

211

8.23:

211

8.24:

211 212 212 215

8.25: 8.26: 8.27:

xxi 8.28: Initiation of browning in plants maintained at 100 mM NaCl concentration after ascorbic acid pretreatment (0.8x) Effect of 100 mM NaCl on non-pretreated plants of cv. SPF 234 (1x) Ascorbic acid-pretreated plants of cv. SPF 234 at 160 mM NaCl level (0.7x) Necrosis of non-pretreated sugarcane plants (cv. SPF 234) at 160 mM NaCl level as a result of NaCl stress (1x) Ascorbic acid-pretreated plant of cv. HSF 240 maintained at 0 mM NaCl level at day 30 (0.5x) Plant of cv. HSF 240 after ascorbic acid pretreatment at 40 mM NaCl level (0.5x) Non-pretreated plant of cv. HSF 240 maintained at 60 mM NaCl level showing yellowing of some leaves due to NaCl stress (0.7 x) Necrotic plant of cv. HSF 240 at 100 mM NaCl level without ascorbic acid pretreatment (1x) Ascorbic acid-pretreated plant at 100 mM NaCl level as compared to non-treated control at the same NaCl level (1.5x) Browning of non-pretreated plant at day 15 upon transfer to MS medium supplemented with 140 mM NaCl level (1.5x) Ascorbic acid-pretreated plant upon transfer to MS medium supplemented with 140 mM NaCl at day 15 (1x) 218 Effect of Ascorbic acid pretreatment on soluble protein contents of in vitro-grown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30

215 215 216

8.29: 8.30: 8.31:

216 217 217

8.32: 8.33: 8.34:

217 218

8.35: 8.36:

218

8.37:

218

8.38:

8.39:

223

xxii 8.40: Effect of Ascorbic acid pretreatment on peroxidase activity of in vitro-grown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Effect of Ascorbic acid pretreatment on catalase activity of in vitro-grown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Effect of Ascorbic acid pretreatment on superoxide dismutase activity of in vitro-grown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Effect of Ascorbic acid pretreatment on soluble protein contents of in vitro-grown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Effect of Ascorbic acid pretreatment on peroxidase activity of in vitro-grown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Effect of Ascorbic acid pretreatment on catalase activity of in vitro-grown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Effect of Ascorbic acid pretreatment on superoxide dismutase activity of in vitro-grown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30

223

8.41:

223

8.42:

223

8.43:

225

8.44:

225

8.45:

225

8.46:

225

xxiii LIST OF TABLES Table Number 4.1: Title Page Number

Effect of MS medium supplemented with various levels of growth regulators on callus induction, fresh weight and morphological characteristics in sugarcane (cv. SPF 234) under dark conditions at 27 2 Effect of MS medium supplemented with various levels of growth regulators on callus induction, fresh weight and morphological characteristics in sugarcane (cv. HSF 240) under dark conditions at 27 2 C Effect of BAP, TDZ and NAA supplemented to MS medium on regeneration frequency of 2-months-old callus cultures of sugarcane (cv. SPF 234) under 16 h photoperiod at 27 2 C Effect of BAP, TDZ and NAA supplemented to MS medium on regeneration frequency of 2-months-old callus cultures of sugarcane (cv. HSF 240) under 16 h photoperiod at 27 2 C Effect of IBA and BAP on the rooting potential of regenerated shoots of sugarcane (cv. SPF 234) under 16 h photoperiod at 27 2 C Effect of IBA and BAP on the rooting potential of regenerated shoots of sugarcane (cv. HSF 240) under 16 h photoperiod at 27 2 C Morphology of sugarcane callus cultures (cv. SPF 234) at different NaCl levels (0-160 mM) supplemented to MS medium under dark conditions at day 90, 120 and 150 Morphology of sugarcane callus cultures (cv. HSF 240) at different NaCl levels (0-160 mM) supplemented to MS medium under dark conditions at day 90, 120 and 150

82

4.2:

83

4.3:

87

4.4:

88

4.5:

91

4.6:

91

5.1:

104

5.2:

110

xxiv 5.3: Effect of different NaCl levels (0-160 mM) supplemented to MS medium on fresh weights of sugarcane (cvs. SPF 234 and HSF 240) callus cultures under dark conditions at day 90, 120 and 150 Effect of different concentrations of NaCl (0-160 mM) supplemented to MS medium on callus browning and necrosis in sugarcane under dark conditions at day 90, 120 and 150 Regeneration potential of 120-days-old NaCl-treated callus cultures of sugarcane (cv. SPF 234) Regeneration potential of 120-days-old NaCl-treated callus cultures of sugarcane (cv. HSF 240) Rooting potential of regenerated shoots of sugarcane (cv. SPF 234) under 16 h photoperiod Rooting potential of regenerated shoots of sugarcane (cv. HSF 240) under 16 h photoperiod Soluble protein contents of sugarcane callus cultures treated with different NaCl concentrations (0-160 mM) at day 90, 120 and 150 Peroxidase activities of sugarcane callus cultures treated with different NaCl concentrations (0-160 mM ) at day 90, 120 and 150 Catalase activity of sugarcane callus cultures treated with different NaCl concentrations (0-160 mM) at day 90, 120 and 150 Superoxide dismutase activity of sugarcane callus cultures treated with different NaCl concentrations (0-160 mM) at day 90, 120 and 150 Soluble protein contents of regenerated plants from 0-160 mM NaCl-treated sugarcane callus cultures Peroxidase, catalase and superoxide dismutase activities of regenerated plants from 0-160 mM NaCl-treated callus cultures of sugarcane (cv. SPF 234)

112

5.4:

114 118 119 122 123

5.5: 5.6: 5.7: 5.8: 6.1:

132

6.2:

135

6.3:

138

6.4:

142 144

6.5: 6.6:

146

xxv 6.7: Peroxidase, catalase and superoxide dismutase activities of regenerated plants from 0-160 mM NaCl-treated callus cultures of sugarcane (cv. HSF 240) Effect of medium and/or Polyethylene glycol (PEG) pretreatment on fresh weights of sugarcane callus cultures at day 90 and 120 Effect of medium and/or Polyethylene glycol (PEG) pretreatment on callus browning and necrosis in sugarcane (cvs. SPF 234 and HSF 240) callus cultures at day 90 and 120 Effect of medium and/or Polyethylene glycol (PEG) pretreatment on soluble protein contents of sugarcane (cv. SPF 234) callus cultures at day 90 and 120 Effect of medium and/or Polyethylene glycol (PEG) pretreatment on soluble protein contents of sugarcane (cv. HSF 240) callus cultures at day 90 and 120 Effect of medium and/or Polyethylene glycol (PEG) pretreatment on peroxidase activity of sugarcane (cv. SPF 234) callus cultures at day 90 and 120 Effect of medium and/or Polyethylene glycol (PEG) pretreatment on peroxidase activity of sugarcane (cv. HSF 240) callus cultures at day 90 and 120 Effect of medium and/or Polyethylene glycol (PEG) pretreatment on catalase activity of sugarcane (cv. SPF 234) callus cultures at day 90 and 120 Effect of medium and/or Polyethylene glycol (PEG) pretreatment on catalase activity of sugarcane (cv. HSF 240) callus cultures at day 90 and 120 Effect of medium and/or Polyethylene glycol (PEG) pretreatment on superoxide dismutase activity of sugarcane (cv. SPF 234) callus cultures at day 90 and 120 Effect of medium and/or Polyethylene glycol (PEG) pretreatment on superoxide dismutase activity of sugarcane (cv. HSF 240) callus cultures at day 90 and 120

147

7.1:

159

7.2:

165

7.3:

168

7.4:

169

7.5:

173

7.6:

174

7.7:

178

7.8:

179

7.9:

183

7.10:

184

xxvi 7.11: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on regeneration potential of 120-days-old NaCl-treated callus cultures of sugarcane (cv. SPF 234) Effect of medium and/or Polyethylene glycol (PEG) pretreatment on regeneration potential of 120-days-old NaCl-treated callus cultures of sugarcane (cv. HF 240) Effect of medium and/or Ascorbic acid pretreatment on fresh weights of sugarcane callus cultures maintained on MS medium supplemented with NaCl at day 90 Effect of medium and/or Ascorbic acid pretreatment on callus browning and necrosis in sugarcane callus cultures maintained on MS medium supplemented with NaCl at day 90 Effect of medium and/or Ascorbic acid pretreatment on soluble protein contents, peroxidase, catalase and superoxide dismutase activities of sugarcane (cv. SPF 234) callus cultures maintained on MS medium supplemented with NaCl at day 90 Effect of medium and/or Ascorbic acid pretreatment on soluble protein contents, peroxidase, catalase and superoxide dismutase activities of sugarcane (cv. HSF 240) callus cultures maintained on MS medium supplemented with NaCl at day 90 Effect of medium and/or Ascorbic acid pretreatment on morphological growth parameters of in vitro-grown sugarcane plants (cv. SPF 234) maintained on MS medium supplemented with different concentrations of NaCl at day 30 Effect of medium and/or Ascorbic acid pretreatment on morphological growth parameters of in vitro-grown sugarcane plants (cv. SPF 234) maintained on MS medium supplemented with different concentrations of NaCl at day 30 Effect of medium and/or Ascorbic acid pretreatment on soluble protein contents, peroxidase, catalase and superoxide dismutase activities of in vitro-grown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl at day 30

188

7.12:

190

8.1:

201

8.2:

202

8.3:

206

8.4:

210

8.5:

213

8.6:

214

8.7:

222

xxvii 8.8: Effect of medium and/or Ascorbic acid pretreatment on soluble protein contents, peroxidase, catalase and superoxide dismutase activities of in vitro-grown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl at day 30

224

CHAPTER 1 INTRODUCTION

CHAPTER 1 INTRODUCTION
Soil salinity occupies a prominent place among the soil problems that threaten the sustainability of agriculture over a vast area in the world (Flowers, 2004). It is one of the major abiotic stresses in plant agriculture worldwide (Mahajan and Tutea, 2005). Salinity and sodicity has affected over 6 % of the total world land. Over 800 million hectares of land throughout the world are salt affected, either by salinity (397 million hectares) or the associated condition of sodicity (434 million hectares; FAO, 2005). The soils with electrical conductivity of less than 4 dS m-1 are generally considered as salt-free, where almost all crops can be grown. Improper irrigation practices and lack of drainage have generally led to accumulation of salts in the soil in concentrations that are harmful to the crops. The capability of crops to grow on saline soils varies among species and depends on the concentration of salts present in the root zone and on various environmental and culture conditions (Maas, 1990). Pakistan has a total geographical land area of 79.61 million hectares (Khan et al., 2004). About 22.07 million hectares of this total land area is cultivated (Anonymous, 2006). Cultivated area of Pakistan has a canal irrigated system, about 62,400 km long and mainly confined to Indus plain covering an area of 19.43 million hectares (48 million acres). The salt-affected soils are also mainly situated in this plain. The reason for soil salinity in Pakistan is mainly due to an imbalance in the amount of salt entering and leaving the soils. Each year, about 120 million tones of salts are added to the land from canal and under ground water. Only about one-fifth of this salt finds its way to the sea. The remainder accumulates in the soil and continues to decrease the growth and survival of the crops. (Alam et al., 2000).

2 In Pakistan, out of the total cultivated area, about 6.30 million hectares of land are saltaffected. The magnitude of the problem can be gauged from the fact that the area of productive land is being damaged by salinity at an alarming rate of about 40,000 hectares annually (Javed et al., 2007). Another problem associated with soil salinity is waterlogging. Combined together, it may reduce the total yield by up to 25 % (Sandhu and Qureshi, 1986). Hence, soil salinity and water logging seriously affect the agricultural production in Pakistan. The country suffers an economic loss of about US $32 million annually in terms of the total yields of wheat, rice, cotton and sugarcane (Alam and Ansari, 2001). Attempts to enhance tolerance have involved conventional breeding programmes, the use of in vitro selection, pooling physiological traits, interspecific hybridization, using halophytes as alternative crops, the use of marker-aided selection, and the use of transgenic plants. Cultured plant cells have also been introduced as a convenient tool for research to elucidate the mechanisms operating on the cellular level by which plants survive under various abiotic stresses including salinity (Rains et al., 1980). This method supports the traditional breeding strategies by producing plants with improved salt tolerance through selection of salt-tolerant cells in culture and their regeneration. In vitro selection for salt tolerance commonly occurs as a result of a temporary adaptation; cells are able to compartment the excessive salts into vacuoles, and survive by adjusting the osmotic pressure. This adaptation causes reduction in cell division and elongation (Bressan et al., 1985). The mechanism of this relationship is not fully understood. However, tissue culture allows the control of stress homogeneity and the characterization of cell behavior under stress conditions, independently of regulatory systems that take place at the whole plant level (Lutts et al., 2004). Furthermore, cell and tissue culture techniques could prove to be good tools to

3 screen the plant materials obtained in different breeding programs due to the relatively small space and reduced time required for the selection (Dracup, 1991). Hence, in vitro selection appears to be the best methodological approach to evaluate salt tolerance in a breeding programme (Jain, 2001). Interestingly, it has also been reported that by using in vitro selection methods, some plants become so well-adapted to high salt levels that they are unable to grow in the absence of salt (Remotti, 1998). Sugarcane is an important industrial cash crop in Pakistan and in many countries of the world. It is grown in tropical and sub-tropical regions of the world in a range of climates from hot dry environment near sea level to cool and moist environment at higher elevations. Besides sugar production, sugarcane produces numerous valuable byproducts like alcohol used by pharmaceutical industry, ethanol used as a fuel, bagasse used for paper and chipboard manufacturing and press mud used as a rich source of organic matter and nutrients for crop production. Sugarcane accounts for approximately 65 % of world sugar production (Alam et al., 1995; Carson and Botha, 2002). Its predicament can be estimated by the fact that despite being the fifth largest country as regards area under sugarcane cultivation, Pakistan is the 11th in production and 60th in yield (Varzgani, 2007). Total cropped area in Pakistan is approximately 22 million hectares. Sugarcane is grown on over 966 thousand hectares in Pakistan (Anonymous, 2006). Its annual production in 2005 was 48.88 tons/hectare. In financial year 2006, the sugar production of the country declined to its lowest in the decade, to an estimated 40.95 million tons which was more than 16 per cent lower than previous years production. This national average cane yield is far below the existing potential (Varzgani, 2007). Soil salinity is one of the main factors affecting economic yield of sugarcane (Wahid, 2004).

4 Effect of soil salinity on sugarcane has been studied by different workers (Rizk and Normand, 1969; Syed and Swaify, 1973). All have suggested that sugarcane is a salt sensitive crop and exposure to salt stress deteriorates the quality of cane. Saline soils are also occasionally used for sugarcane cultivation. The salt-stressed plants show arrested growth of various parts (Maas and Niemann, 1978). Although there are considerable varietal

differences for tolerance of salinity but generally sugarcane has a threshold value of 1.4 dS m-1 (Maas, 1986). It has been observed that growth and productivity of sugarcane decline sharply with an increase in salinity (Rozeff, 1995; Wahid et al., 1997 a, b; Nasir et al., 2000; Akhtar et al., 2003). It has been calculated that yield loss of sugarcane due to salinity in Pakistan is about 62 % (Qureshi et al., 1993). Besides this, salinity in root zone of sugarcane decreases sucrose yield through its effect on both biomass and juice quality (Lingle and Weigand, 1996). Sugarcane plant exhibits enormous genetic variability in nature (Humbert, 1968), therefore, tremendous scope exists for the selection of desirable traits such as high yield and resistance against various stresses (Alexander, 1973). Due to a decline in sugarcane productivity with an increase in stress conditions and other environmental factors associated with sugarcane agriculture (Virupakshi et al., 2002), attempts have been made during the recent years to improve sugarcane plants by using tissue culture techniques (Khan et al., 2004; Gandonou et al., 2005 b; Errabii et al., 2006). Salinity affects the plant growth due to osmotic stress, specific ion (Na ) toxicity, nutritional imbalance, hormonal imbalance or production of reactive oxygen species (Greenway and Munns, 1980; Ashraf, 1994; Tester and Davenport, 2003; Ashraf and Harris, 2004; Chinnusamy et al., 2005; Bartels and Sunkar, 2005). Reactive oxygen species (ROS)
+

5 are molecules like hydrogen peroxide (H2O2), ions like the hypochlorite ion, radicals like the hydroxyl radical (OH) and the superoxide anion (O2-), which is both an ion, and a radical. Reactive oxygen species participate in multiple processes in plants and can damage other molecules and the cell structures of which they are a part (Mittler, 2002). They also participate in plant defense against pathogens and have signaling roles (Inze and Montagu, 1995; Bolwell, 1999). Cells have a variety of defenses against the harmful effects of reactive oxygen species. The antioxidant enzymes including peroxidase, catalase and superoxide dismutase are mainly intracellular scavengers of reactive oxygen species (Halliwell and Gutteridge, 1985; Fadzilla et al., 1997; Uchida et al., 2002). Peroxidase is a hexameric homoprotein catalyzing the oxidation by hydrogen peroxide of a number of substrates such as ascorbate, ferrocyanide, cytochrome C and the leuco form of many dyes. The enzyme plays an important role in physiological processes, like control of growth by lignification, cross-linking of pectins and structural proteins in cell wall and catabolism of auxins (Gaspar et al., 1982). Hence, plant peroxidases have been used as biochemical marker for various types of biotic and abiotic stresses (Ashraf and Harris, 2004). On the contrary, catalase is a tetrameric homoprotein that exists as multiple isozymes encoded by nuclear genes. This enzyme is located mostly in peroxisomes and glyoxisomes (Scandalios et al., 1997). Catalase catalyzes the conversion of H2O2 to oxygen and water. The enzyme uses H2O2 as a substrate as well as a hydrogen acceptor. It is an important antioxidant enzyme because it protects the cells by decomposing H2O2 into H2O and O2 (Ivanova and Ivanov, 2000). Another enzyme that plays important role during stress conditions in plants is superoxide dismutase (SOD). This enzyme converts two superoxide anions into a molecule of hydrogen peroxide and one of oxygen thus catalyzes the removal of

6 the O2- free radical. In this way, the enzyme protects oxygen-metabolizing cells against harmful effects of superoxide free-radicals. As salinity levels increase, plants extract water less easily from the soil resulting in water stress conditions. Hence, salt stress causes water deficit as a result of osmotic effects on a wide variety of metabolic activities of plants (Greenway and Munns, 1980). This water deficit results in oxidative stress because of the formation of reactive oxygen species (Bartoli et al., 1999; Loggini et al., 1999). The reactive oxygen species that are by-products of hyper osmotic and ionic stresses generally cause membrane dysfunction and may even cause cell death (Bohnert and Jensen, 1996). Polyethylene glycol (PEG) of higher molecular weights have long been used to stimulate water stress in plants (Ruf et al., 1967, Kaufman and Eckard, 1971). PEG is a non-penetrating inert osmoticum lowering the osmotic potential of nutrient solutions without being taken up or being phytotoxic (Lawlor, 1970). In sugarcane callus cultures, PEG has been used to induce osmotic stress (Patade et al., 2005). Exogenous application or pretreatment of pearl millet with PEG (Ashraf et al., 2003) and wheat seedlings with H2O2 (Wahid et al., 2007) has been considered as a short-cut method to enhance the tolerance against abiotic stresses as compared to plant breeding and genetic engineering techniques. However, to the best of our knowledge, no previous report describes the role of PEG in salinity tolerance of sugarcane. During the present investigation, a study was hence undertaken to determine a possible role of PEG pretreatment in improving salt tolerance of sugarcane. Plants possess both enzymatic and non-enzymatic mechanisms for scavenging ROS. Non-enzymatic factors include several small molecules that are antioxidant in nature such as alpha-tocopherol (vitamin E), ascorbic acid (vitamin C) and carotenoids (Sairam et al.,

7 2002). Ascorbic acid, also known as Vitamin C is an important antioxidant molecule, which is widely utilized in cell metabolism (Loewus and Helsper, 1982) and is needed to synthesize hydroxyproline-containing protein (Arrigoni et al., 1977). Ascorbic acid also acts as primary substrate in the cyclic pathway for enzymatic detoxification of hydrogen peroxide; hence, it plays an important role in activation of biological defense mechanisms (Arrigoni et al., 1979). Although ascorbic acid plays many important functions in plant biology (Noctor and Foyer, 1998; Arrigoni and De Tullio, 2000; Smirnoff and Wheeler, 2000), little is known about the factors that control its synthesis and accumulation in different tissues. This metabolite is present in plant species usually in millimolar concentrations ranging from 10 to 300 mM (Smirnoff, 2000). There are some reports indicating that externally applied ascorbic acid can be metabolized in tissues (Sapers et al., 1991, Gaddalah, 2002). No prior information is available on the role of ascorbic acid pretreatment in sugarcane tissue cultures. Considering the importance of ascorbic acid as an antioxidant compound as explained for other plants (Shalata and Neumann, 2001; Al-Hakimi and Hamada, 2001), an experiment was conducted to investigate the possible role of ascorbic acid pretreatment in improving salt tolerance of sugarcane callus cultures as well as in vitro-grown plants. The present study was undertaken to establish protocols for callus induction, maintenance and plant regeneration in two sugarcane cultivars (SPF 234 and HSF 240). This study also reports the morphological as well as biochemical parameters associated with the salt stress in in vitro cultures of sugarcane. Keeping in view the importance of antioxidant enzymes in salinity tolerance of plants, the effect of NaCl stress on peroxidase, catalase and superoxide dismutase enzymes was also investigated during the present study. Another objective of the present study was to further elucidate the relationship between salt and

8 osmotic stress. Hence, an experiment was also performed to determine the effect of osmotic stress induced by PEG in improving NaCl tolerance of sugarcane callus cultures. Role of ascorbic acid in alleviation of salt stress in sugarcane callus cultures/plants was also investigated during the present study.

CHAPTER 2 LITERATURE REVIEW

CHAPTER 2 LITERATURE REVIEW

Sugarcane is an important cash crop in many tropical and sub-tropical countries of the world. Pakistan occupies an important position in cane producing countries of the world. Total cropped area in Pakistan is 22.64 million hectares out of which 20.07 milllion hectares is cultivated. Sugarcane is grown on over 907 thousand hectares in Pakistan (Anonymous, 2006). Its annual production in 2006 was 40.95 million tones (Varzgani, 2007). Soil salinity is a major problem affecting the agricultural productivity in Pakistan because out of the total cultivated area, about 6.30 million hectares of land are salt-affected (Javed et al., 2007). Salt tolerance is a complex trait both physiologically as well as genetically. Therefore, attempts to improve the salt tolerance of crops through conventional breeding programmes have met with very limited success (Flowers, 2004). Furthermore, salinity tolerance is a developmentally regulated stage-specific phenomenon; tolerance at one stage of plant development is often not correlated with tolerance at other stages (Foolad, 2004). Plant tissue culture research is a multi-dimensional field that offers exciting prospects for crop productivity and improvement (Jain, 2001). If in vitro cultures are established from explants that did not contain a pre-organized meristem, or if cultures are maintained as callus prior to plant regeneration, the regenerated plants may exhibit variability (Larkin and Scowcroft, 1981). Therefore, plant tissue culture has an important role to play in the production of ornamental or agricultural plants and in the manipulation of plants for improved agronomic performance. Selecting disease, insect, or stress resistant plants through tissue culture techniques has become one of the most intensively studied areas of plant tissue culture during the recent

10 years (Jain, 2001). Current research in this area extends across many interests including attempts to select salt tolerant lines, freezing resistant plants, herbicide resistant agronomic crops, and developing resistance to heavy metals. Plant tissue culture has made it possible to understand various aspects of salt tolerance as well as the mechanisms adopted by plants to overcome the molecular damage due to salt.

2.1 Tissue Culture Studies in Sugarcane

Tissue culture techniques are of tremendous use in vegetatively propagated crops such as sugarcane. During the recent years, this technique has gained importance in sugarcane improvement and breeding programmes. Callus induction and regeneration from the callus cultures are two important aspects in in vitro studies of sugarcane because they serve as a tool for the production of plants with improved characters, such as salinity or drought tolerant, disease resistant etc. A brief review of work on these aspects is given below.

2.1.1 Callus Induction and Proliferation

Callus production is an essential step in the use of tissue culture studies for various physiological phenomena including resistance against various abiotic stresses. Callus is an unorganized, proliferative mass of predominantly parenchyma cells. The pioneering work on in vitro studies in sugarcane was conducted by Nickell (1964) who established first sugarcane callus cultures from mature internodal parenchyma tissue. Heinz et al., (1977) reported callus formation from parenchyma tissue of shoot apex and leaves of Saccharum spp. on MS (Murashige and Skoog, 1962) medium containing coconut water (10 %) and 2,4D.

11 Different plant parts like shoot and root apical meristems, pith parenchyma, cut edges of leaves, immature inflorescence etc. could be used as explants for callus induction in sugarcane (Liu et al., 1972). The innermost young leaves have been suggested to be the best explants for callus induction by many workers (Nadar et al., 1978, Chen et al., 1988, Fitch and Moore, 1990). Studies have revealed that better response of callus was obtained when callus cultures were kept in dark. Chen et al., (1988) reported that morphogenic callus could form most readily from the leaf explants with most proliferating callus when kept in dark. Aftab et al., (1996) have also reported that embryogenic callus could be obtained from young leaves of sugarcane on modified MS medium under dark conditions. Callus proliferation of sugarcane has also been reported by placing the calluses under dark conditions (Snyman et al., 2001). Studies have suggested that amongst all the media tested for callus induction and proliferation by different workers, the best medium was modified MS (Murashige and Skoog, 1962) medium (Liu and Chen, 1974; Guiderdoni, 1986; Aftab et al., 1996, Baksha et al., 2002). Role of auxins have also been studied for callus induction and proliferation. Nadar et al., (1978) found that embryogenic callus forms when auxin is added to the medium. On the other hand, no embryogenesis was observed in callus cultures on auxin-free media. Callus proliferation in modified MS medium with various levels of auxins and cytokinins in sugarcane was also reported by Bhansali and Singh (1982) and Zang et al., (1983). Studies have shown that amongst different auxins tested for callus induction, addition of 2, 4-D in the medium always produced better callus growth than any other growth regulator. Kulkarni (1989) reported that callus induction and proliferation from immature sugarcane leaves triggers on medium containing 2,4-D. Karim et al., (2002) while working on two sugarcane

12 varieties, (Isd-16 and Isd-28) also observed that highest percentage of callus induction was obtained on MS basal medium supplemented with 3.0 mg l-1 2,4-D and 10 % coconut milk. Similarly, Mamun et al., (2004) also found that among all the tested auxins (IAA, 2,4-D, IBA, and NAA), the best performance for callus induction was obtained on 3.0 mg l-1 2,4-D. Studies have further indicated that use of 2,4-D in MS medium not only results in an earlier (7-10 days) callus induction but it also improves the callus proliferation (Nagai et al., 1991; Islam et al., 1996). In a study by Snyman et al., (2000), it was found that a good mass of sugarcane callus could be obtained on MS medium containing 3.0 mg l-1 2,4-D after 3-4 weeks of inoculation. Ramanand et al., (2006) also found that when the young meristematic leaf sheath explants of sugarcane were aseptically inoculated on agar (7.5 g l-1) gelled Murashige and Skoog (MS) medium containing 20 g l-1 sucrose and different concentrations of NAA, IBA and 2,4-D for callus formation, maximum (67.3 %) explants showed callus initiation within 10-14 days at 4.0 mg l-1 2,4-D. The addition of kinetin and coconut water to the callus initiation medium was found to be inhibitory to embryogenesis (Fitch and Moore 1993). On the contrary, the role of 2,4-D in the medium has also been reported to be crucial in obtaining embryogenic calluses in sugarcane. Studies have suggested that the addition of 2,4-D in the medium favors the formation of embryogenic callus (Himanshu et al., 2000; Snyman et al., 2001). Foranzier et al., (2002) have also supported the observation that mostly embryogenic callus forms in the medium supplemented with 2,4-D. In another study by Marcano et al., (2002), efficient embryogenic callus formation (70 %) in Venezuelan sugarcane cultivars V78-1 and V75-6 was achieved using young leaf explants cultivated on modified MS medium containing 13 M 2, 4-D. Niaz and Quraishi (2002), however, found that the use of NAA in addition to 2,4-

13 D improves the callus and embryogenic response in sugarcane. They found that 1 mg NAA and 3 mg l-1 2,4-D was optimal for embryogenesis. Like many earlier findings, Gandonou et al., (2005 c) also reported that embryogenic callus could be obtained in sugarcane on MS medium supplemented with 3 mg l-1 2,4-D. Although 2,4-D has proven efficiency for good in vitro response, thidiazuron (TDZ) has also been used for this purpose. Gallo-Meagher et al., (2000) established embryogenic callus of sugarcane on MS basal medium having 2,4-D, kinetin and NAA. Various concentrations of thidiazuron were also tested for embryogenic callus induction. It was observed that TDZ has a positive effect on embryogenic callus induction. Use of TDZ for sugarcane callus induction is however scanty.

2.1.2 Plant Regeneration from Callus Cultures

Plant regeneration from callus is another important aspect during the tissue culture studies. Plant regeneration from sugarcane callus tissue was first reported by Heinz and Mee (1969). They observed that plant differentiation from callus was routinely achieved by withdrawing the auxin from the culture medium. Nadar and co workers (1978) showed all stages of embryoid development when the callus was cultured on modified MS medium containing 3.0 mg l-1 2,4-D. It was suggested that the process of differentiation and embryogenesis required relatively higher auxin concentration. Plantlet differentiation occurred only in the absence of auxin. Plant regeneration through somatic embryogenesis has been reported in sugarcane using young leaf rolls and immature inflorescences (Ho and Vasil, 1983; Chen et al., 1988; Brisibe et al., 1994; Guiderdoni, 1995). Anbalagan and co workers (2000) also worked on the regeneration of sugarcane callus tissue and found that MS

14 medium supplemented with NAA and Kinetin was efficient for regeneration of plantlets from callus tissue. In a study by Niaz and Quraishi (2002), the regeneration potential of calluses was observed under the effect of different growth regulators. They found that MS medium containing BA at the concentration of 1.0 mg l-1 performed better than other growth regulators. In an experiment by Karim et al., (2002) best response in terms of multiple shoot formation was observed on MS medium supplemented with 1.0 mg l-1 BAP and 0.5 mg l-1 IBA. However, Gandonou et al., (2005 c) accomplished plant regeneration from sugarcane callus cultures on hormone-free modified MS medium supplemented with casein hydrolysate. It was also suggested by these workers that regeneration potential of callus cultures is also genotype dependent. An interesting aspect in the tissue culture studies of sugarcane is direct somatic embryogenesis which offers several advantages in crop improvement, as cost-effective and large-scale clonal propagation is possible using bioreactors, ultimately leading to automation of somatic seed production and development of artificial seeds. Aftab and Iqbal (1999) observed that adventive somatic embryos were initiated from the cut edges of juvenile leaf explants of two cultivars of sugarcane (Saccharum spp. hybrid cv. CoL-54 and cv. CP43/33). This response was achieved using MS medium containing 9 M (2 mg l-1) 2,4-D and 500 mg l-1 CH under either continuous or 16-h photoperiod. Later on, many workers have studied this aspect and indicated the formation of somatic embryos directly from the explant, resulting in regeneration of the shoots. Desai et al., (2004) also observed direct somatic embryogenesis without an intervening callus phase in sugarcane using immature inflorescence segments. It was found that MS medium supplemented with 0.5 mg l-1 naphthalene acetic acid, 2.5 mg l-1 Kinetin, 100 mg l-1 L-glutamic acid and 4 % sucrose

15 resulted in high frequency of somatic embryo development. Gill et al., (2006) also observed that when the young leaf segments of sugarcane were cultured on different media composition based on MS salts, cultured explants exhibited swelling followed by direct shoot regeneration on media containing naphthalene acetic acid in all the three varieties. Apart from sugarcane callus cultures, cell suspension cultures are also important in in vitro studies on sugarcane. It involves incubation of friable callus in the liquid medium agitated on a shaker, which gives a suspension of cells. Nickell and Maretzki (1969) have reported that growth and dispersion of sugarcane cells in suspension cultures could be obtained in MS medium. Ho and Vasil (1983) also observed rapid growth and plant regeneration from embryogenic cell suspension cultures. During their study, embryogenic cell suspensions were established from calluses derived from young leaves of sugarcane by placing them in liquid medium containing 5 % coconut water, 2-3 mg l-1 2,4-D and 500 mg l-1 casein hydrolysate. Structures resembling early stages of embryo were formed when 2,4-D in the medium was lowered (0.1-1.0 mg). Transfer of embryoids to half-strength medium established plantlets. Regeneration from the embryogenic cell suspension cultures have also been reported in sugarcane by Aftab et al., (1996). Embryogenic calluses were achieved from young leaves of sugarcane using MS basal medium containing 0.5 mg l-1 picloram under dark conditions at 27 1 C. Fast growing homogenous cell suspension cultures were achieved in MS medium supplemented with 2 mg l-1 2,4-D and 50 mg l-1 casein hydrolysate. Recently, the role of TDZ in the medium for plant regeneration has also been investigated during different studies. Chengalrayan and Gallo-Meagher (2001) studied the effect of various growth regulators on shoot regeneration of sugarcane. Sugarcane

16 embryogenic calluses were induced from young leaves cultured on modified MS basal medium supplemented with 13.6 mM 2,4-D. Five concentrations, 0.5, 1.0, 2.5, 5.0 or 10.0 mM of four different growth regulators, BAP, kinetin, zeatin, and thidiazuron were tested with or without 22.5 mM NAA to asses their ability to induce regeneration from embryogenic calluses. After 4 weeks on medium, the percentage of shoots as well as the percentage of shoots greater than 1 cm in length, was recorded. Although it had the lowest percentage of elongated shoots, MS medium containing TDZ alone performed better than all other growth regulators tested, with the highest percentage of shoot induction and the largest number of shoots, particularly at a concentration of 2.5 mM. During their work, Dhawan et al., (2004) observed that TDZ has a positive effect on multiplication of shootlet cultures established from buds regenerated from apical domes of sugarcane. A set of two shootlets taken from these cultures was inoculated on media containing varying levels of TDZ. The number of multiple shootlets formed provided a simple, quantitative test system. TDZ concentrations, as low as 10-9 M and 10-7 M, produced the highest number of shootlets in var. CoH-92 and Co-7717, respectively. Greater multiplication of shootlets continued even after subculture on a medium devoid of TDZ for 24 and 48 days. This is suggestive of a high persistence of TDZ in the tissues. In another study, Chengalrayan et al., (2005) observed sugarcane seed germinated on MS basal medium alone or supplemented with different concentrations of TDZ, 2,4-D and Picloram. Maximum germination was observed on MS medium supplemented with 45 M TDZ. Callus induction occurred on 2,4-D and picloramcontaining media but not on TDZ medium. The greatest amount of callus was produced on 4.1 M picloram. Jain et al., (2007) have also reported regeneration of plantlets from callus cultures of sugarcane on MS basal medium supplemented with 2.5 M TDZ.

17 Rooting of sugarcane callus has also been reported in many studies. Some workers have reported that rooting of the regenerated shoots in sugarcane could be achieved on MS basal medium (Lal and Sing, 1994; Karim et al., 2002). Aftab et al., (1996) suggested that shifting of the regenerated plants to half-strength MS medium enhanced the rooting of sugarcane plants. Khan et al., (1998), however, suggested that the addition of IBA with 6 % sucrose to MS medium induced vigorous root development. Chengalrayan and GalloMeagher (2001) have also found that 19.7 M IBA plays an important role in rooting of regenerated sugarcane plants. Baksha et al., (2003) tried different growth regulators for rooting of the regenerated shoots of sugarcane. It was found that best rooting was obtained on MS medium supplemented with 5.0 mg l-1 NAA, 50 g l-1 sucrose at pH level 5.7. Mamun et al., (2004), however, observed that a combination of IBA and NAA is better in root formation of sugarcane plantlets. Furthermore, use of activated charcoal in MS medium supplemented with 0.5 mg l-1 IBA and 0.5 mg l-1 NAA showed better performance than those rooted on charcoal omitted medium. In a recent report, Jain et al., (2007) reported that rooting of the regenerated sugarcane plants could be obtained on MS medium supplemented with 19.7 M IBA, 20 g l1 sucrose, and 1.5 g l1 mannose. On the basis of the available literature, it is evident that protocols for sugarcane callus induction and regeneration are well documented. Use of sugarcane tissue culture in studies pertaining to its improvement hence remains a strong possibility.

2.2 Biochemical Aspects of Salinity Tolerance in Plants

Although salinity is one of the major problems limiting agricultural production around the world and a lot of work has actually been already done on salinity tolerance of

18 plants, the metabolic sites at which the salt stress damages plants or the adaptive mechanisms utilized by the plants to survive under saline conditions are mostly still elusive (Flowers and Flowers, 2005). Furthermore, the difference in growth response to salinity amongst various genotypes is still not clearly understood (He and Cramer, 1992). However, it is now known that salt tolerance operates at the cellular level (Mansour et al., 2003). There are many reviews, which highlight the diversity of plant responses to salinity and the complexity of the resistance mechanisms (Greenway and Munns, 1980; Flowers, 2004). Biochemical aspects of antioxidants and protective pigments have successfully been introduced as markers of environmental stress (Tausz et al., 2003). Understanding biochemical indicators for individual species rather than generic indicators could prove to be quite helpful to understand the physiology of salt tolerance (Ashraf and Harris, 2004). With our ever-increasing understanding of biochemical pathways and mechanisms that participate in plant stress responses, it has also become evident that many of these responses are common protective mechanisms that can be activated by salt, drought and cold, albeit sometimes through different signaling pathways (Winicov, 1998).

2.2.1 Proteins as Biochemical Markers of Salinity Tolerance

Biochemical and physiological changes in tissues in response to several kinds of stresses can be verified through alterations in proteins. Kogan et al., (2000) found that the accumulation of compatible solutes is one of the strategies that plants have developed to tolerate salt stress. Compatible osmolytes and proteins can therefore be used as potential biochemical markers useful in the identification and genetic manipulation of salt-resistant plants and plant cells (Shonjani, 2002). Many reports are available where cell proteins are

19 used as markers during differentiation of tissues and organs under stress conditions (Iqbal and Schraudolf, 1977; Ashraf and Harris, 2004). However, data do not always indicate a positive correlation between the osmolyte accumulation and the adaptation to stress (Mc Cue and Hanson, 1990; Ashraf, 1994; Lutts et al., 1996 a; Mansour, 2000). It has been revealed from various studies that soil salinity affects the growth and photosynthetic activity of plants (Shabala et al., 1998). The reason is that salt and other abiotic stresses result in the generation of reactive oxygen species that can cause oxidative damage to many cellular components including proteins and nucleic acids (Halliwell and Gutteridge, 1989). As proteins and nucleic acids are the important constituent of cells, their damage could result in stunted growth of plants. Levine et al., (1990) has also reported inhibition of growth and development, reduction in photosynthesis, respiration, and protein synthesis and disturbed nucleic acid metabolism in response to salt stress. Salt tolerant cell lines, when compared with normal sensitive cells, can provide a useful means of measuring the capacity and range of salt stress tolerance. As an initial attempt to study the biochemical mechanism to salt adaptation, Ericson and Alfinitio (1984) and Singh et al., (1985) compared the NaCl-adapted and non-adapted cell line of tobacco. It was observed that calluses showed stunted growth when subjected to salt stress by adding salt in the medium. The physiological effects of NaCl on callus cultures of Brassica campestris (Chinese cabbage) were studied by Paek and co workers (1988). Hypocotylderived callus cultures of Brassica campestris were grown on MS medium with varying salt concentrations. When growth and fresh:dry weight ratios of established calluses were measured, it was observed that NaCl was more than twice as inhibitory in comparison to the same medium without salt. The same response to salt stress on calluses derived from rice

20 embryos was observed by Reddy and Vaidyanath (1986). Callus cultures were grown on agar-solidified media containing 0, 1 or 2 % (w/v) NaC1 for 24 days. It was observed that salt stress negatively affected the growth of callus cultures. Callus growth decreased with an increasing concentration of sodium chloride. Although there are some reports showing non-significant changes in the levels of protein, starch, sucrose and -amino nitrogen in salt-grown callus (Paek et al., 1988) but most of the in vitro studies indicate that salt stress may result in varying levels of proteins (Lutts et al., 1999; Muthukumarasamy et al., 2000). It could also cause protein alterations in callus cultures of different plants (Niknam et al., 2006). Singh et al., (1985), for the first time, compared NaCl-adapted and non-adapted cell lines of tobacco for various proteins. Several proteins were found to be more abundant in the cells adapted to salt whereas a protein of 26 KD (Kilo Dalton) was quite unique to salt-adapted cells. Olmos and Hellin (1996) studied the mechanisms of adaptation to salt tolerance by selecting cell cultures of two lines of Pisum sativum (one salt sensitive and the other adapted to 85.5 mM NaCl). It was observed that calluses adapted to NaCl differed from unadapted ones in their accumulation of organic solutes such as proteins, sugars, amino acids, organic acids and ascorbic acid. Therefore, the adaptation of calluses to NaCl might depend upon modification of the osmotic adjustment through organic solutes including proteins, together with modification of physiological and biochemical parameters. It was also suggested by these workers that identification of the specific gene synthesizing proteins would open up the way to the genetic engineering of salt resistance in plants. A comparison of simple biochemical parameters such as soluble protein and proline content between control and callus cultures grown under varying sodium chloride

21 concentrations revealed that salt stress affects both these parameters (Reddy and Vaidyanath, 1986). It was observed by them that soluble protein contents decline with increase in salt stress. In control callus cultures, soluble protein content was 89 mg/g fresh weight, whereas it was 76.5 mg/g fresh weight at 1 % NaC1 and 71 mg/g fresh weight at 2 % NaC1. Shankhdhar et al., (2000) also observed that total protein content decreased markedly with increase in salt concentration at day 15 and 30 after inoculation in rice callus cultures. Similarly, Khedr et al., (2003) also reported a decrease in growth and protein content due to salt stress signaling in the desert plant Pancratium maritimum L. Studies have indicated that in vitro screening process by using proteins as a biochemical marker could be used for ranking the genotypes for salinity tolerance as well as the selection of salt tolerant lines (Singh et al. 2000). Bekheet et al., (2000) selected two cultivars of Asparagus officinalis by culturing shoot segments on callus induction medium supplemented with salt mixture. The cultivars showed better growth, high protein content, fresh and dry weight as salt concentration increased up to 6000 ppm. Similarly, in a study by Elavumoottil et al., (2003) salt-tolerant callus and cell suspension cultures of Brassica oleraceae L. var. Botrytis were obtained by the selection of cells from cultures growing in medium supplemented with 85, 170 or 255 mM NaCl. It was found that both salt-adapted calluses as well as cell suspensions differed in their RNA and protein concentrations. Like callus cultures, variation in the protein content have also been reported in plants subjected to salt stress (Skriver and Munday, 1990; Streb and Feierabend, 1996; Amini and Ehsanpour, 2006). It has been observed that salinity drastically inhibit the protein biosynthesis in plant tissues (Lutts et al., 1999). The reason might be that salinity retards nitrate reduction into ammonia, which is further incorporated into amino acids and hence

22 diminishes the rate of protein synthesis in plants. Studies are available in agreement with this observation (Ashraf and Waheed, 1993; Lutts et al., 1996 b; Streb and Feierabend, 1996). Evers et al., (1997) observed the effect of NaCl (up to 300 mM) on in vitro-grown poplar shoots. It was found that the soluble protein content of the plants decreased dramatically on day 1 but stabilized to a constant value thereafter, except for the 300 mM NaCl condition. In an experiment, the effects of long-term (30 days) NaCl treatments (100-200 mM) on proline and protein content in potato leaves were studied by Fidalgo et al., (2004). It was observed that proline contents enhanced in the treated plants whereas a significant decrease in protein was noted under salt stress. In an experiment by Khodary (2004), the effect of NaCl salinity, on nitrogen assimilation and ion uptake in the seeds of lupine (Lupinus termis L.) was investigated. Proteins, amino acids, nucleic acids, and nitrate (NO3), potassium and phosphorus uptake was determined. Significant decrease in protein, amino acid and nucleic acid contents was observed upon NaCl exposure (0, 500, 1000, 2000 or 3000 ppm). Likewise, Niknam et al., (2004) also observed that NaCl affects in vitro growth parameters as well as sugars, free proline and proteins in the seedlings and leaf explants of Nicotiana tabacum cv. Virginia. The fresh and dry mass of the seedlings decreased under salinity. Free proline content in both seedlings and leaf explants increased and polysaccharide content decreased continuously with increase in NaCl concentration. Reducing sugars,

oligosaccharides, soluble sugars and total sugar contents in both seedlings and leaf explants decreased up to 150 mM NaCl and then increased at higher concentrations of NaCl. Some workers are of the view point that some new proteins may also be synthesized as a result of salt stress. Reports also indicate an increase in protein contents in the plant tissues under salt stress (Munns et al., 1979; Skriver and Munday, 1990). These reports

23 suggest that stress-induced proteins play major role in salt tolerance. In one such study, Ashraf and OLeary (1999) observed changes in soluble proteins in spring wheat stressed with sodium chloride. It was found that there is an increase in soluble protein contents in response to salt stress in all the tested wheat cultivars. Amini et al., (2007) have also reported that new proteins are induced in tomato seedlings subjected to NaCl stress. It was thus suggested that tomato cells respond to salt stress by changes in different physiological processes. On the basis of the above information, it may be concluded that proteins have an important role in stress tolerance at the cellular level. Therefore, proteins can be used as markers for understanding salinity tolerance.

2.2.2 Antioxidant Enzymes as Biochemical Markers of Salinity Tolerance

The response of cultivated species to salinity in terms of growth and yield are the ultimate expression of several interacting physiological and biochemical parameters. Because of the complex nature of salt tolerance as well as the difficulties in maintaining long-term growth experiments, physiological parameters as selection criteria are recommended for screening (Yeo et al., 1990; Noble and Rogers, 1992, Munns and James, 2003). During the stress condition, free radicals mostly as reactive oxygen species (ROS) are constantly produced. Reactive oxygen species (ROS) control many different processes in plants (Mittler et al., 2004). ROS are now considered signal molecules at non-toxic concentrations but at toxic concentrations they are also capable of injuring cells (Gamaley and Klyubin, 1999). Plant cells have evolved defense antioxidant mechanisms to combat the danger posed by the presence of ROS. These include enzymatic mechanisms involving antioxidant enzymes such

24 as superoxide dismutases, peroxidases, and catalases (Landberg and Greger, 2002; Meloni et al., 2003). In different experiments, all of these enzymes have usually been studied together so the relevant literature regarding these enzymes is reproduced collectively.

2.2.2.1 Studies on Antioxidant Enzymes in Callus Cultures under Salt Stress Some evidence suggests that resistance to oxidative stress may, at least in part, be involved in salt stress tolerance (Gueta-Dahan et al., 1997; Hernandez et al., 2000; Mittova et al., 2002; Badawi et al., 2004). However, most of these studies were performed with plants and only a little information is hence available on the response of antioxidant enzymes in callus cultures where the cells are directly exposed to salt in the medium. Recently, however, more attention has been focused on this aspect. Salt-tolerant cell lines, when compared with normal sensitive cells, can provide a useful means of measuring the capacity and range of stress tolerance and may be used in order to elucidate tolerance mechanisms at the level of the cell (Kumar and Sharma, 1989). Studies have suggested that the levels of antioxidant enzymes in callus tissue exposed to NaCl increase with salt stress (Rajguru et al., 1999). High levels of antioxidant enzymes in NaCl-tolerant plants could thus be related to salt adaptation process (Piqueras et al., 1996). Gossett et al., (1994 a, b) have also observed that the NaCl-tolerant cultivars and calluses of cotton acclimated to grow on media supplemented with 150 mM NaCl had higher antioxidant enzyme activities. Garrat et al., (2002) also investigated the relationship between salinity tolerance and antioxidant status in callus cultures of different cotton cultivars. For all the cultivars, there was a reduction in mean fresh weight in media with more than 100 mM NaCl. A strong correlation existed between antioxidant status and growth of cells exposed to NaCl. Superoxide dismutase activities

25 increased with increasing salinity in the cv. TOL to a maximum value of 26.3 1.1 units/ mg protein at 150 mM NaCl; for the cultivars MED and SEN, there were no changes in enzyme activities between control and salt treatments. Catalase activity decreased progressively with increasing salt concentration in all cultivars except for SEN with 100 mM NaCl, where mean catalase activity was greater than control. Salt tolerance was also studied in callus cultures of Suaeda nudiflora, a dicotyledonous succulent halophyte, by Cherian and Reddy (2003). It was observed that growth was significantly inhibited at 50, 100, 150 or 200 mM NaCl. Salt stress enhanced the activity of peroxidase, whereas it decreased activities of superoxide dismutase and catalase. Involvement of an enzymatic antioxidant defense system in the adaptive response to salt stress was also reported by Davenport (2003) in two types of sunflower calluses differing in salt sensitivity. No reduction in growth occurred in the NaCladapted cell line when grown on 175 mM NaCl but growth of salt-stressed cell line was reduced by 83 %. The antioxidant defense system of callus adapted to growth under NaCl responded differently to 175 mM of salt compared with the corresponding controls under shock treatment. Except for catalase, the antioxidant enzymes were elevated constitutively in adapted calluses as compared to stressed cells, when both were grown in the absence of NaCl (time 0), and remained unaltered until 28 days after the beginning of the experiment. Similarly, Rahnama et al., (2003) reported antioxidant enzyme responses to NaCl stress in callus cultures of four potato cultivars. They used internode cuttings of potato on callus inducing medium with 0, 50, 100 or 150 mM NaCl. It was observed that the callus growth of all the cultivars significantly decreased under salt stress. Superoxide dismutase activity also decreased in all the cultivars when callus cultures were grown in the presence of NaCl. Catalase activity increased in potato cv. Agria and Diamant and declined in cv. Kennebe and

26 Ajax. The results showed that antioxidant enzymes had an important role in plant defence systems against oxidative stress. Later, Niknam et al (2006) studied the effects of NaCl on growth, proteins and proline contents, and catalase, peroxidase and polyphenol oxidase activities in seedlings and callus cultures of Trigonella foenum-graecum L. and T. aphanoneura Rech. f. Seeds and hypocotyl explants were cultured on MS medium supplemented with 0, 50, 100, 150 or 200 mM NaCl. Seed germination and the fresh and dry mass of the seedlings decreased significantly under salinity. In both species, significant increase in protein content of seedlings over that of control were observed at 150 and 200 mM NaCl. Protein content in callus cultures decreased at 200 mM NaCl over that of control. Protein content was higher in seedlings than in callus cultures at all NaCl concentrations. NaCl also caused changes in the activities of peroxidase, catalase and polyphenol oxidase in seedlings and callus cultures. The harmful effects of salinity on the crop performance may be attributable to the ionic effect, osmotic effect and alteration in ionic composition leading to deficiency of nutrient ions and excess of salt ions. Molassiotis et al., (2006) during their study worked on this aspect to determine whether the major influence of high salinity is caused by osmotic component or by salinity-induced specific ion toxicity. They treated shoot cultures of apple root stock with mannitol, sorbitol, NaCl and potassium chloride. It was found that mannitol and salts increased proline content, superoxide dismutase, peroxidase and non-enzymatic antioxidant activities. H2O2 content was also increased in the leaves and stems. However, reduced catalase activity was observed in the salt-treated leaves.

27 2.2.2.2 Studies on Antioxidant Enzymes in Seedlings under Salt Stress Salinity affects growth, vigour and area of seedling leaves (Rawson et al., 1988). ROS produced as a result of various abiotic stresses need to be scavenged for maintenance of normal growth. Superoxide dismutase, catalase and peroxidase have been viewed as a defensive team, whose combined purpose is to protect cells from active oxygen damage (Fridovich, 1988). In an experiment, Bandeoglu (2004), observed the effect of salt stress (100 mM and 200 mM NaCl) on antioxidant responses in shoots and roots of 14-days-old lentil (Lens culinaris M.) seedlings. A significant decrease in length, dry weight and an increase in proline content of both shoot and root tissues was observed as a result of salt stress. In leaf tissues, high salinity treatment resulted in a 4.4 fold increase in H2O2 content. Root tissues were, however, less affected with respect to these parameters and no significant change in the activity of ascorbate peroxidase, catalase and glutathione reductase was observed in root tissues. On the other hand, in leaf tissues, with the exception of catalase, salt stress caused significant enhancement in the activity of other antioxidant enzymes. Increase in the antioxidant enzyme activity was also reported by Agarwal and Pandey (2004) in seeds of Cassia angustifolia. During the experiment, seeds were treated with 0, 20, 50 or 100 mM NaCl for 7 days. It was found that salinity caused a great reduction in plant biomass. The root and shoot length, fresh and dry mass and germination percentage were inhibited by NaCl treatments. An increase in the activities of superoxide dismutase, catalase and peroxidase was recorded. Similarly, Rahnama and Ebrahimzadeh (2005) investigated the effect of NaCl on the growth and activity of antioxidant enzymes in the seedlings of four potato cultivars. It was observed that the shoot fresh mass of the salt-tolerant potato cultivars did not change at 50 mM NaCl, whereas salt-sensitive cultivars showed a decrease in shoot fresh mass as

28 compared to the controls. It was also observed that at higher NaCl concentration, superoxide dismutase activity was reduced in all cultivars while catalase and peroxidase activities increased in all cultivars under salt stress. On the basis of these observations, it was suggested that salt-tolerant cultivars may have a better protection against reactive oxygen species by increasing the activity of antioxidant enzymes (especially superoxide dismutase) under salt stress. It has been observed that the antioxidative response of the seedlings growing under salt stress is well correlated with sensitivity and tolerance of the cultivars under investigation. This is supported by many reports about the effect of salinity on antioxidant enzymes during growth of seedlings in different plants. Sreenivasulu et al., (1999) reported an increase in total peroxidase activity under NaCl stress in 5-days-old seedlings of two contrasting genotypes of Setaria italica L. (Prasad, a salt-tolerant cultivar and Lepakshi, a saltsusceptible cultivar). In another study, Bor et al., (2003) subjected 40-days-old seedlings of two sugar beet varieties B. maritima and B. vulgaris to 0, 150 or 500 mM NaCl for 12 days. It was observed that the activities of superoxide dismutase, peroxidase, ascorbate peroxidase, catalase and glutathione reductase were higher in B. maritima as compared to B. vulgaris. These results possibly suggested that the wild salt-tolerant beet, B. maritima could have a better protection mechanism against oxidative damage by maintaining a higher inherited and induced activity of antioxidant enzymes than the relatively sensitive plants of the sugar beet, B. vulgaris. Similarly, Vaidyanathan et al., (2003) compared the antioxidant enzyme response of two cultivars of rice by subjecting the seedlings of both cultivars to NaCl stress (100300 mM) for 42 h. They found that under NaCl stress, the salt-tolerant cultivar showed higher activity of the ROS scavenging enzyme, catalase and enhanced levels of antioxidants

29 like ascorbate and glutathione than the sensitive cultivar. These results thus indicated that the role of antioxidant enzymes is vital to overcome salinity-induced oxidative stress in rice.

2.2.2.3 Studies on Antioxidant Enzymes in Plants under Salt Stress Salinity alters the physiological and biochemical activities by inhibiting the anabolic and stimulating the catabolic processes (Corchete and Guerra, 1986). The effects of salt (NaCl) stress on antioxidant responses have been studied in a number of plant species including wheat, rice, potato, mulberry, tomato, citrus, cotton, pea, foxtail millet and lupines. These studies indicate that the degree of oxidative cellular damage in plants exposed to abiotic stress is controlled by the capacity of the antioxidative systems (Dhindsa, 1991, PerlTreves and Galun, 1991; Zhang and Kirkham, 1994; Zhu and Scandalios 1994, Mc Kersie et al., 1996, Noctor and Foyer, 1998). A correlation between the antioxidant capacity and NaCl tolerance was demonstrated in several plant species, e.g. Gossypium hirsutum cultivars (Gossett et al., 1994 a, b) and Oryza sativa (Dionisio-Sese and Tobita, 1998). Bueno et al., (1998) also showed the upregulation of antioxidants, superoxide dismutase and ascorbate peroxidase in response to salt stress at the transcriptional and translational level. However, comparative analyses of NaCl-dependent antioxidant modulation between tolerant and sensitive cultivars of the same plant species are still quite rare. Halophytes are the plants that can tolerate high salt levels. To understand the underlying mechanisms of salt tolerance in halophytes, Takemura et al., (2000) studied the physiological and biochemical responses induced by salt stress in laboratory-grown young plants of the mangrove, Bruguiera gymnorrhiza. The growth rates and leaf areas were highest in the cultures with 125 mM NaCl. The activities of the antioxidant enzymes,

30 superoxide dismutase and catalase, showed an immediate increase after the plants were transferred from water to high salinity, reaching in 10 days five to eight times those of initial activities, respectively. The activities of these two enzymes were not affected by salt concentrations up to 1000 mM NaCl. The mechanism that imparts NaCl tolerance to non-halophytic plants is not clearly understood. However, resistance to oxidative stress has been implicated in severa1 studies involving NaCl stress to plants. Sreenivansula et al., (2000) observed that plants show a strong correlation between salt stress and antioxidant enzymes. They analyzed the modulation of antioxidant components in salt-tolerant or salt-sensitive cultivars of foxtail millet (Setaria italica) under different NaCl concentrations. Under conditions of salt stress, salt-tolerant cultivar exhibited increased total superoxide dismutase and ascorbate peroxidase activity whereas both enzymes decreased acutely under salt stress in the sensitive cultivar. They concluded that salt-induced oxidative-tolerance was conferred by an enhanced compartment-specific activity of antioxidant enzymes. In rice plants, Lin (2000) also reported elevation in the activities of peroxidase, ascorbate peroxidase, superoxide dismutase and glutathione reductase as a result of NaCl (200 mM) treatment as compared to control leaves. There was no difference in catalase activity between NaCl and control treatments. These results suggested that some antioxidant enzymes could be activated in response to oxidative stress induced by NaCl. In other cereals, the response of antioxidant enzymes in response to salinity has also been observed. In wheat, Sairam et al., (2002) demonstrated that salinity stress decreased relative water content, chlorophyll, carotenoids, membrane stability index, biomass and grain yield, and increased hydrogen peroxide (H2O2), proline, glycinebetaine, soluble sugars, superoxide dismutase, catalase and glutathione reductase activity.

31 During their work, Kukreja et al., (2005) studied the effect of salinity on H2O2 scavenging enzymes in Cicer arietinum. The chickpea plants were exposed to single saline irrigation (0, 2.5, 5.0 or 10.0 dS m-1). The defense mechanism activated in roots was confirmed by the increased activities of superoxide dismutase, peroxidase, ascorbate peroxidase, glutathione transferase, glutathione reductase and catalase. Upon desalinization, a partial recovery was observed in most of the parameters studied. Cavalcanti et al., (2004), however, reported that leaf catalase activity decreased twofold only after one day NaCl treatment. Peroxidase activity on the other hand increased after NaCl treatment. It was concluded by these workers that superoxide dismutase, catalase and peroxidase activities do not confer protection against oxidative damage in salt-stressed cow-pea leaves. Salt stress can affect the plant at all the developmental stages. In an experiment, Swapna (2003) studied the activity of superoxide dismutase and peroxidase and catalase during different developmental stages such as embryo, 14-days-old seedling, tillering and flowering stage, and in undifferentiated embryo-derived callus after giving NaCl stress. It was observed that a 100 mM NaCl stress increased the activities of superoxide dismutase and peroxidase enzyme at different developmental stages of rice (Oryza sativa L.). Usually the salt-tolerant cultivars had more antioxidant enzyme activities as compared to salt-sensitive cultivars. Harinasut et al., (2003) investigated the salt stressinduced changes of antioxidant enzymes in the leaves of a salt-tolerant mulberry cultivar. It was found that activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase slightly increased at 150 mM NaCl. Hence, these enzymes play an active role in scavenging ROS in this cultivar. Likewise, Meloni et al., (2003) observed the effect of salinity on the activity of antioxidant enzymes (superoxide dismutase, peroxidase and

32 glutathione reductase), in two cotton cultivars namely Guazuncho and Pora. Plants were treated with three salt concentrations (50, 100 or 200 mM NaCl) for 21 days. The superoxide dismutase activity in Pora increased with an increase in the intensity of NaCl stress, but salt treatment had no significant effect on this enzyme activity in Guazuncho. In an interesting study, Benavides et al., (2004) observed the relationship between antioxidant defense systems and salt tolerance in two clones of Solanum tuberosum differing in salt tolerance. The antioxidant defense system of the sensitive clone responded differently to 100 and 150 mm NaCl. At 100 mM, growth, dehydroascorbate reductase and catalase activities remained unaltered while increase in superoxide dismutase activity was observed. The superoxide dismutase increment was higher under 150 mM NaCl stress while a general decrease in other enzymes was observed. All the antioxidant enzymes were significantly elevated in salttolerant clone as compared to sensitive one when both were grown on NaCl-free medium. No changes in antioxidant stress parameters were detected in the tolerant clone at both salt concentrations. Sairam et al., (2005) while studying the effects of long-term sodium chloride salinity (100 and 200 mM NaCl) in tolerant (Kharchia 65, KRL 19) and susceptible (HD 2009, HD 2687) wheat genotypes found almost similar results. It was observed that the salttolerant genotypes showed less decline in relative water content, chlorophyll content, and ascorbate peroxidase content and higher increase in superoxide dismutase and its isozymes while susceptible genotypes showed the highest decrease in ascorbic acid content, highest increase in H2O2 and smallest increase in activities of antioxidant enzymes. Recently, Liu et al., (2007 b) reported that the stronger salt tolerance of grafted eggplant seedlings was related to their higher antioxidant enzyme activities and less oxidative damage. Kusvuran et al., (2007) studied the changes in ion acumulation and the possible involvement of the

33 antioxidant system in relation to the tolerance of salt stress in melon (Cucumis melo L.). They observed that activities of superoxide dismutase and catalase were inherently higher than in salt-tolerant cultivars of melon. These results possibly suggested that some cultivars exhibit a better protection mechanism against oxidative damage by maintaining a higher inherited and induced activity of antioxidant enzymes than the relatively sensitive plants.

2.2.3 Non-Enzymatic Antioxidants as Markers of Salinity Tolerance

As already mentioned above, common consequence of most abiotic and biotic stresses is an increased production of reactive oxygen species (ROS) (Polle and Rennenberg 1993). In addition to antioxidant enzymes, plants also protects cell and sub-cellular systems from the cytotoxic effects of ROS by increasing non-enzymatic substances such as glutathione, ascorbic acid, -tocopherol and carotenoids (Elstner, 1986; Bowler et al., 1992, Menconi et al., 1995; Alscher et al., 1997). Amongst all these antioxidants, ascorbic acid has been considered very important because it plays a central role in plant defense by reacting directly with hydrogen peroxide, superoxide ion and singlet oxygen as well as by recovering -tocopherol from its oxidized form (Noctor and Foyer, 1998). The role of this compound under salt stress has been investigated by some workers. The available literature on this aspect is given below. Some workers are of the view point that ascorbic acid is synthesized in the cells subjected to salt stress. Menneguzzo et al., (1999) studied the effect of NaCl in seedlings of two wheat cultivars. The seedlings were grown for 9 days in Hoagland's solution, supplemented with increasing NaCl concentrations (0, 50 or 100 mM). Comparisons of control and salt-stressed plants for ascorbic acid and glutathione contents indicated an

34 involvement of activated oxygen species in the mechanism of cellular toxicity of NaCl and pointed out differences in the induction of antioxidant defenses among the two cultivars. It was thus suggested that the synthesis of ascorbic acid is induced when subjected to salt stress. Higher levels of NaCl also resulted in increasing glutathione contents in the roots of both cultivars likely for an increased requirement of antioxidants in the organs that firstly suffer stress. Bartoli et al., (2004) during their work studied the oxidative damage to various subcellular compartments in two wheat cultivars differing in ascorbic acid content. It was observed that in general, oxidative damage to proteins was more intense in the cultivar with the lower content of ascorbic acid. Similarly, Jaleel et al., (2007) also observed that the nonenzymatic antioxidants ascorbic acid and glutathione were affected under NaCl stress in Catharantus roseus. In contrast, Sairam et al., (2002) by comparing the ascorbic acid content of two wheat genotypes reported that NaCl salinity caused decrease in relative water content, chlorophyll, membrane stability index and ascorbic acid content of both genotypes. However, less decline in ascorbic acid content was recorded in salt-tolerant genotype. There are some reports, which indicate that application of L-ascorbic acid may help in improving germination by neutralizing the excessive superoxide radicals or singlet oxygen. Shalata and Neumann (2001) reported that the addition of 0.5 mM ascorbic acid to the root medium, prior to and during salt-treatment for 9 h, facilitated the subsequent recovery and long-term survival of 50 % of the wilted tomato seedlings. Other organic solutes without known anti-oxidant activity were not effective. Salt-stress increased the accumulation in roots, stems and leaves, of lipid peroxidation products produced by interactions with damaging active oxygen species. Additional ascorbic acid partially inhibited this response but did not significantly reduce sodium uptake or plasma membrane leakiness. Similarly, Al-

35 Hakimi and Hamada (2001) studied the interactive effect of salinity stress (40, 80, 120 or 160 mM NaCl) and ascorbic acid (0.6 mM), thiamin (0.3 mM) or sodium salicylate (0.6 mM) in wheat. They also observed that soaking of wheat grains in ascorbic acid, thiamin or sodium salicylate could counteract the adverse effects of NaCl salinity on wheat seedlings by suppression of salt stress-induced accumulation of proline. In another study on wheat, Afzal et al., (2005) found that hormonal priming with salicylic acid and ascorbic acid reduced the severity of the effect of salinity. Highest salt tolerance was obtained in seeds subjected to 50 ppm salicylic acid and 50 ppm ascorbic acid as indicated by shoot and root length, shoot fresh and dry weight of the plants. Khan et al., (2006) observed the effects of L-ascorbic acid and sea salt solutions on the seed germination of different halophytes. It was found that increasing concentration of sea salt inhibited seed germination of all species. Pretreatment of seeds with L-ascorbic acid alleviated the sea salt effects only in some halophytes while it had no effect on other species. It was thus concluded that the variability of metabolic responses to salinity depends on a particular species.

2.3 Effect of Salt Stress on Graminaceous Crops

The literature survey thus far indicates that plants are exposed to a variety of abiotic stresses such as drought and salinity that influence their growth and productivity. Salinity as explained earlier is one of the major abiotic stresses, which decrease the productivity of different crops. Plant tissue culture has gained importance in the solution of salinity tolerance of plants. In vitro cultures have shown to be useful for the evaluation of salt tolerance in many species (Emilio et al., 1996, Liu and Staden, 2000). It involves selection of a desired trait in a cell line and ultimately the recovery of whole plants. In vitro selection is based on

36 subjecting a population of cells to a suitable selection pressure and recovering any variant lines that have developed resistance or tolerance to the stress, and to regenerate whole plants from such resistant cell lines. Initially, salt tolerant cell lines have been selected in callus cultures of Nicotiana tabaccum (Nabros et al., 1980) and flax (Mc Hughen and Swartz, 1984). During recent years, the potential of the in vitro selection techniques for the production of salt-tolerant plants has gained considerable attention and there are number of reports available on the selection of salt-tolerant cell lines in various crop plants. Tissue culture selection for salt tolerance has been successful with diverse plant species (Stavarek and Rains, 1984). In vitro selection of salt-tolerant cell lines and regenerated plants has been reported in several species such as potato (Sabbah and Tal, 1990), rice (Lutts et al, 1999), Hordeum (Sibi and Fakiri, 2000), wheat (Barakat and Abdel-Latif, 1996) and sunflower (Alvarez et al., 2003). This suggests that tissue culture selection may be used in improving salt tolerance of many plants. Salinity tolerance of different graminaceous plants is different. In the field, where the salinity rises to 100 mM NaCl, rice (Oryza sativa) will normally die before maturity, while wheat will produce a reduced yield. Even barley (Hordeum vulgare), the most tolerant amongst cereals (Mass and Hoffman, 1977), dies after extended periods at salt concentrations higher than 250 mM NaCl (equivalent to 50 % seawater). Durum wheat (Triticum turgidum ssp. durum) is less salt tolerant than bread wheat, as are maize (Zea mays) and sorghum (Sorghum bicolor) (Maas and Hoffman, 1977).

37

2.3.1 Selection of Salt-Tolerant Callus Cultures

Tissue culture system is useful for the evaluation of tolerance to environmental stresses because the stress conditions can be easily controlled in vitro. Moreover, in vitro cultures provide a uniform population of synchronously-developing plant cells without involving regulatory mechanisms that naturally operate at the whole plant level (Tal, 1983). The saline growth medium causes many adverse effects including osmotic stress, nutritional imbalance etc. on both callus, as well as, plant growth at physiological and biochemical levels (Levitt, 1980; Hernandez et al., 1985; Mansour, 2000). Studies have revealed that presence of salt in the medium affects the callus induction frequency of different plants. Subhashini and Reddy (1989) screened five genotypes of rice plants for salt tolerance in callus cultures, not only with NaCl but also with different concentrations of seawater directly, and the regeneration of plants from these salt-tolerant callus cultures. Profuselygrowing calluses were inoculated onto media containing 1, 2 or 3 % NaCl, or 25, 50 or 75 % seawater with 2 mg l-1 2,4-D. Salt-exposed calluses usually turned brown, indicating necrosis. There was a decrease in callus growth with increasing salt concentrations. Though fresh weights were generally reduced under saline conditions, the fresh weight reduction was different in different genotypes. Arzani and Mirodjagh (1999) in their study evaluated the response of twenty-eight cultivars of durum wheat (Triticum turgidum var. durum) to immature embryo culture, callus production and in vitro salt tolerance. Comparison of cultivars for callus induction from immature embryo was based on callus induction frequency and fresh weight growth of callus. For salt tolerance, the relative fresh weight growth and necrosis of callus were used as parameters of study. There were significant differences among cultivars for potential of regeneration from immature embryo, and `Shahivandi' a

38 native durum wheat cultivar was found superior among the cultivars tested. In other cereals, salinity has also been reported to cause decrease in fresh weight. Chauhan et al., (2000) reported that rice varieties when cultured on LS (Linsmaier and Skoog, 1965) medium containing NaCl showed decrease in fresh weight. Lee et al., (2003) observed similar effect of salt stress in anther culture of rice. It was found that the efficiency of callus induction and plant regeneration were decreased by increasing NaCl concentration in the medium. The percentages of callus induction in two rice varieties, i.e., Gyehwa 5 (japonica, tolerant) and IR61633-B-2-2-1 (japonica, sensitive) were 21.1 and 13.5 % on agar medium containing 0.3 % NaCl, respectively. Many workers have studied the response of callus shifted to salt-containing medium and concluded that when the callus is shifted to NaCl-containing medium, usually a reduction is observed in the fresh weight of callus cultures. It has been observed during different studies that the soil salinity reduces the productivity of plants in the soil (Flowers, 2004; Wahid and Ghazanfar, 2006). Similarly, whenever callus cultures are subjected to in vitro salt stress, there is a reduction in fresh weight or other growth parameters of callus cultures (Barakat and Abdel-Latif, 1996; Rahnama et al., 2003). Cell growth rate is the product of cell wall extensibility and growth-effective turgour (Lockhart, 1965). Osmotic balance is essential for plants growing in saline medium. Therefore, if this balance is non-existant, loss of turgidity and cell dehydration occurs and ultimately the death of cells takes place (Gorham, 1995). The effect of NaCl on selected and unselected wheat cell lines was investigated by Barakat and Abdel-Latif (1996). Embryogenic calluses isolated from immature embryos of four wheat cultivars were subjected to three in vitro selection methods for salt tolerance. The results indicated that the relative growth rate of callus decreased as the

39 concentration of NaCl increased in both callus lines. The selected callus line had a higher growth potential in the presence of NaCl that was highly significant as compared to unselected callus line across medium protocols in all wheat cultivars. In another study, Babu et al., (2007) observed the response of plant cell to salt stress and the feasibility of selecting salt-tolerant callus in rice. Callus was grown on agar-solidified media containing 0.2, 0.4, 0.6, 0.8 or 1.0 % NaCl. Parameters such as fresh weight of callus, callus morphology and proline content were also studied. It was observed that the callus growth decreased with increasing NaCl concentration in the medium. It is interesting to note that salt stress also influences other characteristics of callus tissue. NaCl has been reported to influence somatic embryogenesis in maize (Bezerra et al., 2001). During their work, calluses were induced using immature embryos aseptically removed from the seeds and grown on N6 medium supplemented with 2 mg l-1 2,4-D. To study the effect of salinity in vitro, embryogenic calluses were subjected to different concentrations of sodium chloride (0, 50, 100, 150 or 200 mM NaCl), in three replicates, during 60 days. Callus cultures subjected up to100 mM NaCl showed 37 % higher proline concentration than calluses treated with 50 mM NaCl. This suggested proline accumulation under salt stress conditions. However, at the highest concentration tested (150-200 mM NaCl), lower growth rate and proline accumulation was observed probably due to an increase in non-embryogenic fraction of callus cultures. Similarly, Singh et al., (2003) successfully developed NaCl-tolerant embryogenic calluses via somatic embryogenesis in Bamboo (Dendrocalamus strictus). The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0-200 mM) concentrations of NaCl on callus

40 induction medium. The tolerant embryogenic callus had high levels of Na+, sugar, free amino acids and proline content.

2.3.2 Regeneration of Salt-Tolerant Cell Lines

Various factors such as genotype (Abe and Futsuhara, 1986; Kamiya et al., 1988; Oard and Rutger, 1988), nutrient media (Kamiya et al., 1988; Raval and Chattoo, 1993), growth regulators (Bhaskaran and Smith, 1990), age of explants (Koetje et al., 1989) and passage in culture (Raval and Chattoo, 1993) influence the potential for regeneration. Presence of salt in regeneration medium also results in lowering of the rate of regeneration (Binh et al., 1992). This is due to the loss of regeneration potential during the long periods required for selection or due to the use of non-embryogenic cells or the presence of high concentrations of NaCl in the regeneration medium. Progress of work with salt-adapted calluses has been quite limited mainly due to non-regenerable calluses. Many workers have observed the reduction in the regeneration potential of salt-stressed callus cultures. Lutts et al., (1999) studied the effects of abscisic acid (37.8 M), polyethylene glycol (5 %), proline (10 mM), tryptophan (490 M) and indoleacetic acid (5.7 M) on rice callus regeneration at various doses of NaCl (0, 50 or 100 mM) on three-months-old mature embryo-derived callus cultures of rice. It was observed that NaCl strongly decreased the regeneration frequency of all the cultivars but slightly increased the survival of regenerated plantlets. Tryptophan stimulated regeneration and increased subsequent survival rates of regenerated plantlets in all the cultivars at all NaCl doses. Abscisic acid and polyethylene glycol, though not affecting the final regeneration percentages, delayed regeneration and reduced the mean number of plantlets produced per regenerating callus in all cultivars, as well as rooting ability and survival of regenerated plantlets in indica genotypes. Proline had no marked effect on

41 regeneration while indoleacetic acid reduced shoot regeneration but increased root regeneration. In an experiment, Shankhdhar et al., (2000) studied embryogenic callus growth and plant regeneration under salt stress in six cultivars of rice (Oryza sativa L.). Four weeks after callus inoculation, the fresh mass decreased with increasing salt concentration in all the six cultivars. The regeneration frequency in salt-stressed callus was also lower as compared to control. Proline content increased several fold whereas total protein content decreased markedly with increase in salt concentration 15 and 30 days after inoculation. In a study by Abdul-Hady and co-workers (2001), salt-tolerant plants were selected in three cultivars of barley by exposing immature embryos on MS medium supplemented with different NaCl concentrations, i.e., 4000, 8000 or 12000 ppm, respectively. They found that cultivars survived best at 4000 ppm while at 12000 ppm, regeneration percentage of the tested cultivars scored high reduction. It has been reported by many workers that salt pretreatment may have a positive effect on plant regeneration of selected lines that were developed on salt-free medium (Yoshida et al., 1983; Galiba and Yamada, 1988). There are also some reports of the regeneration of plants on NaCl-containing medium (Beloualy and Bouharmont, 1992).

These salt tolerant studies are based on the observation that when the callus is exposed to different salt concentrations for a specific period of time, the cells are adapted to that environment. Since the presence of salt (NaCl) is inhibitory to regeneration, the salt-tolerant cell lines (even when verified for salt tolerance) have been regenerated on salt-free medium in most of the cases (Li and Heszky, 1986; Ben-Hayyim and Goffer, 1989). This has enabled regeneration from not only tolerant cells, but also from unadapted/non-tolerant cells freed from the constraints of salt in the medium.

42 In rice, selection of salt-tolerant callus line under a highly saline condition has been reported by a number of workers (Yoshida et al., 1983; Li and Heszky, 1986). In one such study, Rains et al., (1980) selected cells of rice in the presence of about 2-3 % NaCl, a concentration lethal for the unselected cells. The selected cells required the presence of sodium chloride for optimal growth. Salt-tolerant plants, however, have been regenerated only by few of them. Yano et al. (1982) regenerated salt-tolerant plants from salt-tolerant callus cultures formed on immature embryos cultured on medium containing 17.5 to 67.5 % seawater. Vajrabhaya et al., (1989) regenerated salt-tolerant plants (with reduced frequency of 0.075 % or less as against 8.3-30 % in control callus) from salt-tolerant embryogenic calluses of indica rice selected on media containing 1 or 2 % NaCl. The regenerants showed increased salt tolerance for several generations. In the third generation of the tolerant plants, there was 94.3% survival compared to 2 % in the control under salt stress and hence a marked increase in salt tolerance was achieved during the successive generations. Increase in salt tolerance in advance generations was thought to be due to the selection of suitable gene combinations for salt tolerance. Basu et al., (1997) also reported that NaCl-adapted callus of a salt-sensitive scented indica variety of rice (Oryza sativa var. Basmati 370) showed 55 % regeneration in culture medium supplemented with IAA and kinetin. Regeneration was low in 85 mM NaCl but a concentration of 128 mM was inhibitory to regeneration. SEM studies revealed organogenesis and somatic embryogenesis from the same callus. The rate of survival and endogenous free proline content of the plants regenerated from the NaCladapted callus was significantly higher than for those obtained from unadapted callus in liquid maintenance media supplemented with NaCl. Likewise, Zhu et al., (2000), during their work, observed that plants regenerated from a salt resistant callus exhibited higher yield

43 performances. This improvement in salinity resistance, however, was not transmitted to the following generations. Regeneration from salt-stressed callus cultures has also been reported in maize by Zhang and Kirkham (1994). The embryonic callus cultures produced from immature embryos of maize that had been maintained for half a year were transferred to media supplemented with different NaC1 concentrations (5, 10, 15, 20, 25, or 30 g l-1) for callus selection. NaCltolerant callus cultures were established through three generations of selections. The growth and frequency of surviving callus cultures were affected significantly by NaCl concentration. The proliferation of NaCl-tolerant callus cultures was relatively good on medium containing 10 g l-1 NaCl. From these callus cultures, plantlets could be produced on differentiation medium. On medium supplemented with 10 g l-1 of NaCl, the plantlets could normally grow. In another study on maize, Pesqueira et al., (2006) observed the effects of NaCl on callus cultures. Organogenic calluses were induced using immature embryos. The callus cultures were exposed to different NaCl concentrations and the survival and regeneration percentages were calculated. Plants obtained from organogenic calluses were then exposed to NaCl concentrations considered harmful for maize. The shoot dry weights of plants exposed to 250 mM NaCl did not show significant differences with respect to the control ones. Although sodium content in shoots was incremented 2-5 fold, it was not toxic for this material. Since it has been established that salt tolerance could be improved in wheat cultivars after application of in vitro selection pressure (Zair et al., 2003), hence, it was suggested by these workers that regeneration from callus initiated on high NaCl levels might be a valid method of selection for salt tolerance. In an experiment by Pellegrineschi et al., (2004), effect of 2,4-D and NaCl on the establishment of callus and plant regeneration in bread and durum

44 wheat was observed. Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the medium. Immature embryos were cultured on MS medium supplemented with 2.5 mg l-1 2,4-D, 2 % sucrose and 0.9 % Bacto Agar. The treated embryos were transferred to liquid medium supplemented with various levels of 2,4-D and NaCl. It was found that both varieties responded differently towards NaCl in the medium. Callus yield and regeneration frequencies were higher in durum wheat in medium containing NaCl than bread wheat. Bhaskaran et al., (1983) used a hydroponic system to test progeny of a sorghum plant regenerated from salt-tolerant cell line in vitro and observed superior agronomic performance over that of the parent. Mac Kinnon and co workers (1987) have also reported regeneration of selected NaCl-tolerant cell lines of Sorghum.

2.4 Salt vs. Water Stress

An important aspect of salt tolerance studies in different plants is the relationship between salt and water stress. Soil salinity is sometimes a key factor in determining the ecological distribution of drought-adapted species (Gucci et al., 1997). Salt in the soil water inhibits plant growth for two reasons. Firstly, the presence of salt in the soil solution reduces the ability of plant to take up water, and this leads to slower growth. This is the osmotic or water-deficit effect of salinity. Secondly, an excessive amount of salt entering the transpiration stream will eventually injure cells in the transpiring leaves and this may further reduce growth. Upon exposure to water deficit, plants exhibit physiological, biochemical and molecular responses at both the cellular and whole plant levels (Greenway and Munns, 1980; Hasegawa et al., 2000). Generally, plants accumulate some kind of organic and/or inorganic

45 solutes in the cytosol to raise osmotic pressure and thereby maintain both turgour and the driving gradient for water uptake Drought-induced osmotic stress triggers a wide range of perturbations ranging from growth and water status disruption to the modification of ion transport and uptake systems (Lutts et al., 1996 a; Bajji et al., 2000; Santos-Diaz and Ochoa-Alejo, 1994). In order to determine the relative importance of ionic toxicity versus the osmotic component of salt stress on germination in durum wheat (Triticum durum Desf.), seeds of three cultivars differing in their salt and drought resistance (Omrabi-5, drought-resistant; Belikh, saltresistant and Cando, salt-sensitive), were incubated in various iso-osmotic solutions of NaCl, mannitol and polyethylene-glycol (PEG) (osmotic potential of 0.15 (control solution) 0.58, 1.05 or 1.57 MPa). It was observed that moderate stress intensities only delayed germination, whereas the highest concentration of NaCl and PEG reduced final germination percentages. PEG was the most detrimental solute, while mannitol had no effect on final germination percentages. The effect of PEG-induced drought and NaCl-induced salt stresses were also observed by Keripesi and Galiba (2000) in wheat seedlings. It was found that both stresses increase the soluble carbohydrate content in wheat seedlings. In a similar study, Alam et al., (2002) observed the effect of NaCl and PEG-induced osmotic potentials on germination and early seedling growth of rice cultivars differing in salt tolerance. It was observed that the onset of germination, germination rate and seedling growth all declined with increasing concentration of both NaCl and PEG. Salt stress appeared to be lethal than equivalent osmotic potentials of PEG. The results suggested that salt tolerance of rice cultivars was probably determined by their ability to withstand excessive Na+ and Cl- ions rather than their ability to tolerate water stress. Castillo et al., (2007), during their work

46 examined the effects of the osmotic component of salt stress on rice cultivar IR64. It was observed that both PEG and NaCl reduced the total above-ground biomass and delayed flowering and maturity, with a longer delay observed with the high-level stress. Likewise, Liu et al., (2007 a) assayed the cell death in the roots of two rice ecotypes under PEGinduced osmotic and NaCl stresses. It was found that although both ecotypes exhibited cell death in response to the osmotic and salt stresses, they appeared to adapt to the two types of stresses through different timing and tissue specificity of cell death. Under the salt stress, cell death progressed successively in a well regulated manner, starting from the outer layer cells in the epidermis and exodermis of roots and subsequently to the endodermis and stele, suggesting a possible function of the dead cells in preventing the influx of excess Na+ ions into the inner parts of roots and into shoots, leading to salt exclusion. In contrast, cell death induced by PEG-induced osmotic stress occurred randomly in roots, allowing a better ability to recover after stress. It has been reported that tolerance to one kind of stress often show tolerance to other stresses (Ryu et al., 1995). There are few other reports indicating that osmotic stress (induced by PEG pretreatment) can also be helpful in improving the NaCl stress tolerance of the plants or vice versa. Ben-Hayyim (1987) examined the response of cells of two citrus species, isolated for better tolerance to NaCl, for their response to PEG-induced water stress. It was found that NaCl-tolerant cell lines of citrus also grew better under osmotic stress conditions induced by PEG as compared to the non-selected cell lines. Gonzalez-Fernandez (1996) reported that tomato plants, which had previously been subjected to a drought stress pretreatment, were able to grow better than non-pretreated plants after 21 days of salt treatment. Likewise, Balibrea et al., (1999) also found that pretreatment of the tomato

47 seedlings with PEG has a positive effect in salt tolerance of the tomato plants. Ashraf et al., (2003) carried out a study to assess whether salt tolerance could be improved in pearl millet at the germination stage and vegetative stages by soaking the seeds of two cultivars, IC-8206 and 18-BY, for 8 h in distilled water, 150 mM NaCl , or polyethylene glycol (PEG- 8000, 0.672 MPa). It was observed that PEG pretreatment increased the final germination percentage. However, the germination rate of both the cultivars under both saline and nonsaline conditions was unaffected. During their study, Chen et al., (2005) pretreated the transgenic tobacco plants with Hoaglands solution containing 2 % PEG and then were transferred to Hoaglands solution containing 100 mM NaCl, and the concentration of NaCl was increased 50 mM every day with the result that the final concentration after five days was 300 mM. It was found that PEG pretreatment was able to promote tolerance of the transgenic plants to the salt stress as compared to controls. In contrast to the above-cited reports, Katemb et al., (1998), observed that priming with NaCl improved the seed performance of two Atriplex species more than iso-osmotic PEG solution.

2.5 Effect of Salt Stress on Sugarcane

Tissue culture generates a wide range of genetic variation in plant species which may also be of potential benefits in plant breeding programmes in sugarcane. Sugarcane plant is affected by variety of abiotic stresses like freezing (Moore, 1987), drought (Ramesh and Mahadevaswamy, 1999) and salinity (Mass, 1987). By in vitro selection, mutants with useful agronomic traits and resistance to above-mentioned stresses can be isolated in a short duration. Salinity is due to the excess of Na+, Ca++, Mg++, SO4 and Cl ions. These ions cause both hyperionic and hyperosmotic stress with serious consequences. The relative toxicity of

48 different ions on sugarcane has been observed in an experiment by Joshi and Naik (1980). It was observed that all these salts cause inhibition of growth and chlorophyll synthesis. The most common stress is, however, caused by high Na+ and Cl- concentrations in soil solution (Hasegawa et al., 2000). Sodium toxicity represents the major ionic stress associated with high salinity. Additionally, some plant species are also sensitive to chloride, the major anion found in saline soils. It is with this reason that in most of the in vitro studies, NaCl has been used in the medium. Sugarcane, one of the two important sugar crops (the second being sugarbeet) in the world, has been ranked as moderately salt-sensitive plant (Shannon, 1997). Published reports suggest that a saturated-extract electrical conductivity threshold for yield reduction in sugarcane is 1.4 dS m-1 (Maas, 1986). Understanding salt tolerance in this crop is economically important because salinity apparently affects both its growth and sugar yield (Bernstein et al., 1966). A brief overview of the available literature on the salt stress studies in sugarcane is given below:

2.5.1 Effect of Salt Stress on Sugarcane Plants

It has been observed that although sugarcane, being a typical glycophyte, grows poorly on saline lands but genotypic differences are present in its species for salinity tolerance. Susceptible plants show signs of arrested growth of various parts (Mass and Niemann, 1978) while resistant plants cope with the saline environments by possessing inherent genetic abilities. In an experiment by Wahid et al., (1997 a), nine cane lines were selected out of a vast gene pool, based upon contrasting morphological characters. The plants were screened in pots and plots at germination, formative, grand growth and maturity stages of growth under 0, 2.5, 7, 14 or 21 dS m-1 levels of sodium chloride. Significant differences were seen amongst the lines and growth stages. Lines showing xeric characters were better

49 able to tolerate high salinity. Ahmed et al., (2003) have also reported that developmental shifts, in relation to salinity tolerance, vary according to the genotypes in sugarcane. Sensitivity of sugarcane to salinity is mainly manifested by reduced photosynthetic activity. Studies have also shown that with increasing salinity levels, all photosynthetic parameters are affected. At the leaf level, the anatomical changes induced by salinity are smaller leaves, reduced stomatal frequency and changes in the mesophyll area (Javed et al, 2000). All of these factors indicate a close relationship with each other, and hence play an important role in reduction in final yield and productivity of a crop. Different workers have observed similar effect of salinity on sugarcane. Meinzer et al., (1994) evaluated the physiological features associated with differential resistance to salinity in two sugarcane (Saccharum spp. hybrid) cultivars over an 8-week period during which greenhouse-grown plants were drip-irrigated with water or NaCl solutions of 2, 4, 8, or 12 dS m-1 electrical conductivity. The CO2 assimilation rate, stomatal conductance, and shoot growth rate began to decline as electrical conductivity of the irrigation solution increased above 2 dS m-1. Assimilation rate, stomatal conductance, and shoot growth rate of a salinity-resistant cultivar (H69-8235) were consistently higher than those of a salinity-susceptible cultivar (H65-7052) at all levels of salinity and declined less sharply with increasing salinity. Because photosynthesis of sugarcane plant is affected by salinity, thus salt stress affects the growth rate and yield of sugarcane. It has been observed that under saline conditions, sugarcane yield falls to 50 % or even more of its true potential (Subbarao and Shaw, 1985). Similarly, Rozeff (1995) reported a 50 % reduction in the sprouting of sugarcane at 13.3 dS m-1 while the same reduction of yield occurred at 9.5 dS m-1 level of salinity. Wahid (2004) worked on analysis of toxic and osmotic effects of sodium chloride on

50 leaf growth and economic yield of sugarcane. It was found that clones indicated significant differences in terms of reductions in dry weight and area of leaves under salinity at the grand growth stage. Leaf dry weight was more affected than leaf area, resulting in reduced specific leaf weight. Clones indicated significant difference with regard to increase in leaf Na+ and Cl- and decrease in K+, but no difference in K+:Na+ ratio. Na+ and Cl- were negatively correlated while K+ was positively correlated with leaf growth parameters, indicating an adverse effect of Na+ and Cl- and importance of K+ to salt tolerance. The tolerant clone displayed higher water content, water, and turgor potentials of leaf than sensitive clone, but the osmotic potential did not significantly change. The incidence of salinity may affect any of the growth stages and may lead to drastic reduction in crop yield or even may lead to crop failure. Therefore, an important aspect is the selection of salinity tolerant plants at different growth stages. Sugarcane is mostly propagated from sets. It has also been observed that like many other crops, sugarcane sprouting and early growth is considerably more resistant to salinity than at later developmental stages (Mass, 1986). In a study, Wahid et al., (1997 b) reported that sugarcane germination is also affected by salinity. They compared the germination response of two sugarcane lines CP-43/33 and CP-71-3002. It was observed that CP-43/33 had a substantial potential for salt tolerance as it had high rate and percentage of germination, more elongated roots and shoots along with a higher dry weight and water content under NaCl stress. In another study, Wahid et al., (1999) also highlighted that emergence and establishment of sugarcane seedlings is crucial under salinity as they determine final crop productivity. Studies are available to reveal the fact that sugarcane plant, when subjected to salt stress, displays signs of salt damage and arrested growth at different developmental stages. Akhtar et al., (2001) has reported that in sugarcane,

51 salt sensitivity varies during different developmental stages of plant. In another experiment by Akhtar et al., (2003), a comparison was made in changes induced by 80 and 120 mM NaCl in salt-tolerant (CPF-213) and sensitive (L-116) genotypes of sugarcane during emergence and growth of sprouts. It was found that the rate and percentage of emergence of sprouts, length and dry mass of shoot and root, and number of nodal roots decreased under salinity. A greater salinity tolerance ability of CPF-213 than L-116 was attributable to greater root mass and higher nutrient concentrations in the sprouts of the former genotype. There is a consensus that salinity in the root zone of sugarcane interferes with the sugar production by affecting the sucrose content of the stalk through its effect on both biomass and juice quality (Lingle and Wiegand, 1996). Sugarcane sucrose accumulation balances increased salt concentration in the juice and maintain water potential of the tissue (Rozeff 1995; Lingle, et al., 2000), therefore, sugarcane from saline soils also shows reduced soluble sugars and thus sucrose in the whole stalk. This may be due to a salt-induced stimulation of the sucrolytic activities of acid and neutral invertases (Balibrea et al., 2000; Tazuke and Wada, 2002). It can be thus suggested that at maturity, increased salinity reduces the millable cane yield, extractable juice and juice-brix percentage, but increased juice osmolality. Akhtar et al., (2001) also had similar findings during their experiment on three differentially salt tolerant genotypes i.e., CPF-213, CP-43/33 (tolerant) and L-116 (sensitive) under 2.5 (control), 8 and 12 dS m-1 levels of salinity. It was observed that at high levels of salinity, i.e., 12 dS m-1, the genotypes CPF-213 and CP-43/33 exhibited higher yield of juice, sucrose and cane yield while L-116 responded poorly for these parameters. The plants when subjected to salt stress accumulate toxic ions. Salt tolerant plants have adopted certain strategies of ion regulation at root stem or leaf level (Kumar et al, 1994;

52 Wahid et al., 1999). One such strategy is the production of secondary metabolites in the plant cells. The possible involvement of secondary metabolites in salt tolerance in sugarcane plants was highlighted by Wahid and Ghazanfar (2006). In their study, two sugarcane somaclones were exposed to salinity levels at the formative stage and evaluated 3 times at 10 days interval. Both clones showed a general tendency to accumulate Na+ and Cl- and little K+. The carotenoid content remained steady, while total chlorophyll was slightly reduced in CP-43/33 and significantly reduced in HSF 240. In contrast, anthocyanins, soluble phenolics and flavones were greater after salt stress. These results suggest the role of secondary metabolites to protect sugarcane from ion-induced oxidative stress.

2.5.2 Effect of Salt Stress on Sugarcane Callus Cultures

Environmental and abiotic stresses have also been studied for their effects on callus growth and antioxidant enzyme/non-enzyme systems in sugarcane. For example, Errabii et al., (2006) studied the growth, proline and ion accumulation in sugarcane callus cultures under drought-induced osmotic stress. The effect of Fe stress in sugarcane callus cultures had been observed by Naik et al., (1990) who obtained Fe-efficient lines through in vitro selection on Fe-stress MS medium. The published literature on the effect of NaCl stress on sugarcane under in vitro conditions is very scanty. However, few attempts have been made for the selection of salt-tolerant cell lines in sugarcane (Liu and Yeh, 1982; Yasuda et al., 1982; Gandonou et al., 2005 a,b). It has been observed that the sugarcane callus cultures show inhibited growth when subjected to salt stress. In a study, the effect of various levels of NaCl on growth of the unadapted parental sugarcane cultures was observed by Ramagopal and Carr (1991). They found that untreated cultures grew seven-to-ten fold in a 3-week period. Cell growth was

53 inhibited significantly when NaCl was added to the medium. Osmotic pressure of cells increased along with increase in salt level in the medium. Similarly, Gandonou et al., (2005 a) studied the response of three varieties of sugarcane to callus induction, embryogenic callus production and in vitro salt tolerance. Leaf segments cultured on MS medium were supplemented with 3mg l-1 2,4-D for callus induction and embryogenic callus. These growing callus cultures were exposed after two subsequent subcultures to different concentration of NaCl (0, 17, 34, 68 or 102 mM). It was observed that addition of NaCl to the medium resulted in callus necrosis and growth reduction. In another study, Gandonou et al., (2006) obtained stable callus cultures tolerant to NaCl (68 mM) from salt-sensitive sugarcane cultivar CP65-357 by in vitro selection process. Both salt-tolerant and non-selected callus cultures showed similar relative fresh weight growth in the absence of NaCl. No growth reduction was observed in salt-tolerant callus cultures while a significant reduction (about 32 %) was observed in non-selected ones when both were cultivated on 68 mM NaCl. Similarly, Errabii et al., (2006) investigated the effects of NaCl and mannitol iso-osmotic stresses on calluses of sugarcane cultivars (cvs.) R570, CP59-73 and NCo310 in relation to callus growth, water content, ion and proline concentrations. Callus growth and water content decreased under both stresses with the highest reduction in above-mentioned parameters under mannitol-induced osmotic stress. Plant regeneration has also been reported in sugarcane from the salt-stressed callus cultures in sugarcane. Yasuda et al., (1982) observed plant regeneration from the callus grown in NaCl-containing medium. Tanvir et al., (2001) worked on the development of salttolerant plants of sugarcane through tissue culture. Ten sugarcane varieties were cultured on MS medium supplemented with varying levels of NaCl, i.e., 0, 0.3, 0.6, 0.9 or 1.2 g l-1.

54 Callus formation was initiated on basal medium and it was adversely affected by progressive increase in salt concentration. Response of genotypes to callus formation also varied considerably under salt stress. After 3-4 subcultures, active callus masses of four genotypes indicating high score for callus formation were subjected to regeneration medium for organ differentiation. Difference among varieties was observed in their ability to regenerate from salt stressed calluses. Tissue culture techniques were also used to develop somaclones in sugarcane to screen salt-tolerant clones. Tanvir et al., (2002) selected three sugarcane varieties, i.e., CP 43/33, BF-162 and IM-61 that had exhibited salt tolerance potential for callus formation and plant regeneration at 0.3, 0.6 or 0.9 % NaCl. These plantlets were multiplied in non-salinized field. Progeny of these plants were planted on saline sodic soil having pH 8.5, electrical conductivity 4.9 dS m-1 and sodium absorption ratio 8.6. The response of genotypes towards salt tolerance was observed and compared for cane yield, height, girth and brix indicating high salt tolerance. Similarly, Khan et al (2004), obtained callus masses from young leaves of high-yielding cultivar of sugarcane (CP-43/33). The plantlets were regenerated from onemonth-old callus. The plantlets were transplanted to pots after hardening. The grown-up plants were transplanted to well-prepared soil. The sets of well developed plants were planted in saline sodic soil. Screening of salt-tolerant somaclones was made. The salt-tolerant somaclones performed better in terms of number of tiller/plant, stem height, number of nodes/stem, root band width with variability of 20, 50, 8.33 and 14.3 %, respectively. The somaclones performed less in the characteristics of girth of stem and brix (%) of 12.5 and 5.2, respectively.

55 Although the studies related to the selection and the physiological characterization of cell lines tolerant to salt stress are abundant in the literature, none of them was focused on sugarcane. In a study by Gandonou et al., (2005 b), the effect of salt on growth, ion and proline accumulation were investigated in vitro in two sugarcane culture NCO310 and CP65357. Leaf explants were cultured on four concentrations of NaCl (0, 34, 68 or 102 mM). Relative growth rate of callus, ion concentration and proline were quantified. The accumulation of both inorganic (Na+, Cl and K+) and organic (proline and soluble sugars) solutes was determined in selected and non-selected callus cultures after an NaCl shock in order to evaluate their implication in in vitro salt tolerance of the selected lines. Accumulation of Na+ was similar in both salt-tolerant and non-selected callus cultures in the presence of NaCl. Accumulation of Cl was lower in NaCl-tolerant than in non-selected callus cultures while proline and soluble sugars were more accumulated in salt-tolerant than in non-selected callus cultures when both were exposed to salt. In a similar study, Errabii et al, (2007) investigated the effects of NaCl and mannitol iso-osmotic stresses on calluses issued from sugarcane cultivars in relation to callus growth, water content, ion and proline concentrations. It was observed that callus growth and water content decreased under both stresses with the highest reduction under mannitol-induced osmotic stress. The ion concentration was drastically affected after exposure to NaCl and mannitol. To the best of our knowledge to date, no study had been carried out on antioxidant response of sugarcane under NaCl stress, but there are few reports, indicating the response of sugarcane callus cultures to other abiotic stresses. In a study by Foranzier et al., (2002), the effect of cadmium was observed on sugarcane callus cultures. During the experiment, sugarcane (Saccharum officinarum L.) in vitro callus cultures were exposed to CdCl2 and the

56 activities of catalase and superoxide dismutase were analyzed. It was observed that lower concentrations of CdCl2, such as 0.01 and 0.1 mM caused a significant increase in callus growth, whereas 0.5 and 1 mM CdCl2 strongly inhibited growth of the callus cultures, but only after 9 days of CdCl2 treatment. Red-brown patches were also observed in calluses exposed to 0.5 and 1 mM CdCl2. Calluses grown in 0.01 and 0.1 mM CdCl2 did not exhibit any changes in catalase activity even after 15 days of growth in the presence of CdCl2. However, for calluses grown in higher concentrations of CdCl2 (0.5 and 1 mM), a rapid increase in catalase activity was detected, which was 14-fold after 15 days. Furthermore, up to five catalase isoforms were observed in callus tissue. Total superoxide dismutase activity did not exhibit any major variation. One Mn-SOD and two Cu/Zn-SOD isozymes were observed in callus cultures and none exhibited any variation in response to the CdCl2 treatments. Hence, it was suggested that in sugarcane callus cultures, catalase might be the main antioxidant enzyme metabolizing H2O2. On the basis of above review, it may be suggested that in vitro studies mainly using tissue culture techniques, might prove to be very useful in understanding not only the salt tolerance in sugarcane plants but also to elucidate the correlation between plants organizational level and salt tolerance.

CHAPTER 3 MATERIALS AND METHODS

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CHAPTER 3 MATERIALS AND METHODS 3.1 Media Preparation 3.1.1 Preparation of Stock Solutions
MS basal medium (Murashige and Skoog, 1962; Annexure 1) supplemented with various growth regulators was tested for callus induction. MS medium supplemented with various salt levels was also used for stress treatments. Stock solutions of nutrients used in the culture medium were prepared in advance for the sake of convenience and accuracy. All stock solutions were prepared by using analytical grade chemicals and double distilled water as detailed in Annexure 2.

3.1.2 Growth Regulators

Stock solutions of growth regulators were either prepared in mM or M concentrations and were used according to the requirement of the medium. Details are given in Annexure 3.

3.1.3 Preparation of Medium from the Stocks

The steps for the preparation of 1 liter MS medium as used in the present study for callus induction and proliferation are detailed in Annexure 4. All the stock components of the medium were mixed in an appropriate quantity. The final volume of the solution was made by the addition of distilled water. The pH of the medium was then adjusted to 5.8 with 1 N NaOH or 1 N HCl. The agar (7g; Oxoid, Hampshire, England) was added and the medium was heated till boiling to melt agar. The medium was then

58 poured in pre-sterilized culture vessels (145 x 25 mm). Culture vessels were wrapped individually with polypropylene sheets of appropriate size.

3.2 Sterilization 3.2.1 Sterilization of Glassware

The glassware was usually washed using a household detergent followed by a rinse with distilled water. Before washing with a detergent, new glassware was, however, first dipped in chromic acid for 8-10 hours. Chromic acid was prepared by mixing potassium dichromate (10 %) and concentrated sulphuric acid in 2:1 (v/v) ratio. After this treatment, glassware was washed with running tap water for the removal of all the traces of chromic acid and then rinsed with distilled water. The final step of sterilization was the placing of cleaned glassware in an oven at 180C for two hours. Glassware was then stored in dust proof cupboard till its use.

3.2.2 Sterilization of the Media

Media was poured in already cleaned and sterilized culture vessels as detailed above. Afterwards, openings of culture vessels were wrapped with piece of polypropylene sheet and fastened with rubber bands. The medium was sterilized by autoclaving at 121C and 15 lbs inch-2 for 15-20 minutes. The sterilized medium was allowed to cool at room temperature.

3.2.3 Sterilization of Working Area of Laminar Airflow Cabinet

Laminar airflow cabinet, the main working area for aseptic manipulation was first thoroughly scrubbed with 70 % ethanol. UV light was switched on one hour before the inoculation. The UV light was switched off at least 15 minutes before inoculation.

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3.2.4 Sterilization of Surgical Tools

The surgical tools were sterilized by putting them in a glass bead sterilizer at a temperature of 250 C. The hot forceps and other tools were allowed to cool down for some time and then used for culture manipulation.

3.3 Plant Material 3.3.1 Source of Plant Material

The plant material of two sugarcane cultivars (SPF 234 and HSF 240) used in the present research work was collected from the sugarcane fields established at Botanical Gardens, Punjab University, Lahore.

3.3.2 Explant Preparation and Disinfection

The young inner 2-3 whorls of sugarcane leaves, wrapped deeply within the mature ones were used as explants. The inner leaves were surrounded by many layers of leaves and therefore, did not require any particular disinfection. However, to minimize the chances of contamination, during explant preparation, 20-30 cm long field-grown plant material was initially surface sterilized with 95 % ethyl alcohol. Uniform-sized leaf discs, 5-8 mm in diameter and 2-3 mm in thickness were prepared for inoculation under laminar airflow cabinet. Before inoculation, hands and arms were washed with soap and then sprayed with 70 % ethanol. Polypropylene wrapper was removed from each culture vessel for inoculation and with the help of forceps the explant was transferred to the agar medium. The culture vessel was then wrapped again by polypropylene sheet after briefly heating the opening of culture vessel. The same procedure was repeated for each culture vessel.

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3.4 Culture Conditions

Already standardized light and temperature conditions for callus induction and regeneration were managed. It has been observed that for sugarcane callus formation total darkness is favorable (Ahmed, 1991; Aftab et al., 1996), therefore, the cultures were kept under dark conditions. For regeneration, the cultures were placed under a 16-h photoperiod (35 mol m-2 s-1) provided by cool fluorescent tube lights. For both callus formation and regeneration the cultures were maintained at 27 2 C.

3.5 Biochemical Studies

Quantitative analyses were performed for total soluble proteins and antioxidant enzymes as detailed below:

3.5.1 Quantitative Estimation of Soluble Protein Contents

3.5.1.1 Extraction of Protein Callus (2.0 g) was taken and crushed in mortar with 4 ml of 0.1 M phosphate buffer (pH 7.2) (Annexure 9) and 0.1 g polyvinyl polypyrrolidone (PVP). The ratio of callus: buffer was kept at 1:2 (w/v). The slurry so obtained was centrifuged at 14000 rpm for 10 minutes at 4 C. The supernatant obtained was used for quantitative analysis. 3.5.1.2 Estimation of Soluble Protein Contents For the estimation of soluble protein contents, Biuret method (Racusen and Johnstone, 1961) was employed. Protein produced violet color due to a complex formed by peptide of protein with copper ions of Biuret reagent in solution. The following two samples (experimental and the control) were prepared: Constituents Protein extract Experimental 0.2 ml Control _

61 Biuret reagent 2.0 ml 2.0 ml

(Composition given in Annexure 10) Distilled H2O _ 0.2 ml

Both tubes were shaken and kept for half an hour at room temperature for the completion of reaction. The optical density was measured at 545nm (visible range) on HITACHI U1100 spectrophotometer. The amount of protein was calculated using an already prepared standard curve for protein estimation. It was drawn by using Bovine Albumin Serum against Biuret reagent following method of Racusen and Johnstone (1961). The following formula was employed for the estimation of protein contents. Protein content = CVTE / EUWt 1000 CV= Curve Value TE = Total Extract EU = Extract Used Wt = Fresh weight of sample tissue

3.5.2 Quantitative Estimation of Peroxidase, Catalase and Superoxide Dismutase

3.5.2.1 Enzyme Extraction For the analysis of all the enzymes, following extraction procedure was adopted. Exactly 2 g callus was crushed using an ice-chilled pestle and mortar with 4ml 0.1 M phosphate buffer (pH 7.2) (Composition given in Annexure 9). PVP (0.1 g) was also

62 added. The slurry so obtained was centrifuged at 14000 rpm for 10 minutes at 4C. The supernatant obtained was used for enzyme analysis. 3.5.2.2 Estimation of Enzymes 3.5.2.2.1 Estimation of Peroxidase For quantitative analysis of peroxidase, the method proposed by Racusen and Foote (1965) was employed. In this method, guaiacol is used as substrate for the assay of peroxidase enzyme. Guaiacol and H2O2 solutions used in the analysis should be freshly prepared (Composition given in Annexure 11). For the enzyme estimation, sample and blank were prepared as follows: Constituents Enzyme extract 0.1 M phosphate buffer 1 % Guaiacol Distilled H2O Experimental 10 l 2.5 ml 0.2 ml _ Control 10 l 2.5 ml _ 0.2 ml

Both tubes were kept as such for 30 minutes and then 0.1 ml H2O2 was added to both tubes. Optical density was measured at 470 nm using spectrophotometer. The activity of enzyme was then calculated as follows: Peroxidase activity (mg/g protein) = A x df / EU x Wt x 1000 Where, A = Absorbance df = dilution factor

EU = Extract Used Wt = Fresh weight of the sample tissue

63 3.5.2.2.2 Estimation of Catalase For the estimation of catalase activity, method of Beers and Sizer (1952) was employed with some modifications. The disappearance of hydrogen peroxide is followed spectrophotometrically at 240 nm. One unit decomposes one micromole of H2O2 per minute at 25 C and pH 7.0 under the specified conditions. The methodology is described as follows: Pipetted (in milliliters) the following reagents into suitable cuvettes. Test Reagent A (50 mM Phosphate Buffer) (Preparation given in Annexure 12 under a) Reagent B (0.036 % H2O2 solution prepared in reagent A) (Preparation given in Annexure 12 under b) 2.9 ml -------------Blank 3.0 ml

The cuvettes were equilibrated at 25 C. Monitored the absorbance until constant and recorded the absorbance at 240 nm on spectrophotometer. Then added Reagent C (Catalase extract) (Preparation given in Annexure 12, under c) 0.1 ml ---------

The reagents were immediately mixed by inversion and recorded the decrease in absorbance in one minute by taking mean of five consecutive readings at 240 nm. From

64 this, calculated the time required for the A240nm to decrease from 0.45 to 0.40 absorbance units. The catalase activity was calculated as follows: Catalase activity (units/ml enzyme) = (3.45) (df) / (Min)(0.1)

where , 3.45 corresponds to the decomposition of 3.45 micromoles of hydrogen peroxide in a 3.0 ml of reaction mixture producing a decrease in the A240nm from 0.45 to 0.40 absorbance units. df = dilution factor Min= Time in minutes required for the A240nm to decrease from 0.45 to 0.40 absorbance units 0.1= Volume of enzyme used (in milliliters) 3.5.2.2.3 Estimation of Superoxide Dismutase Superoxide dismutase (SOD) activity during the present study was assayed by slight modification in the methods proposed by Maral et al., (1977). The method is based on ability of SOD to inhibit photochemical reduction of nitroblue tetrazolium (NBT). One Unit of SOD inhibits by 50 % the maximum reduction of NBT under the specified conditions. The enzyme activity was estimated as follows: Constituents Reaction mixture Experimental 2 ml Control 2ml

(Composition given in Annexure 13) Enzyme extract 5 l -----

65 Covered both tubes with black paper. The experimental tubes were then immediately illuminated for 10 minutes by placing them below two 40 W fluorescent lamps. The absorbance of both samples was measured at 560 nm by using a spectrophotometer. Superoxide dismutase activity was determined by calculating the percentage inhibition of NBT as follows: % inhibition = Absorbance of experimental sample - Absorbance of control sample x 100
Absorbance of control sample

The SOD activity was then calculated based on the fact that one unit of SOD caused 50 % inhibition.

3.6 Experimental Plan 3.6.1 Callus Formation and Proliferation

MS (Murashige and Skoog, 1962) basal medium was supplemented with various growth regulators for callus induction. Ten culture vessels (145 x 25 mm) were used for each medium. In total, twenty four media (C1-C24; Annexure 5) were used. Four different concentrations of 2,4-D (4.52-18.08 M) supplemented to MS medium were tested. The other media consisted of combination of 2,4-D with BAP or Kinetin. Details of different media tried for callus induction and proliferation are given in Annexure 5. Data were recorded at day 30 after inoculation. In vitro agar-solidified cultures were transferred periodically to fresh nutrient medium for continuous supply of inorganic salts, vitamins, sucrose and growth regulators. At day 30 of initial culture, calluses were shifted to fresh MS media without NaCl and further proliferated for 30 days.

66

3.6.2 Plant Regeneration from the Callus Cultures

As it was observed that the callus cultures become too necrotic after second subculture on salt-containing media, i.e., at day 150, therefore, the callus cultures were shifted to the regeneration medium at day 120. Twelve different combinations of growth regulators supplemented to MS medium were tested to study the regeneration potential of sugarcane callus. The best regeneration response was observed on R7 regeneration medium. This medium consisted of 8.87 mM BAP and 0.5 M TDZ. The details of other media tried for regeneration are also given in Annexure 6. Regeneration frequencies, number of shoots per culture vessel and shoot lengths were recorded from 30 culture vessels at day 30. At day 30 from the initial culture on regeneration medium, the regenerated shoots were shifted to the MS basal medium for 30 days for further development of shoots. At day 60 of the initial culture on regeneration medium, the shoots were then shifted to the rooting medium (details given in Annexure 7) for the development of roots. The data for percentage root regeneration, number of roots per culture vessel and root lengths were recorded from 30 culture vessels for each medium at day 30. The fully developed plants were maintained on MS basal medium. After one month, in vitro plants were shifted to pots for hardening. Roots of plants were kept in water for 10-15 minutes in order to remove the remnant of sugar and agar before transplantation to ex vitro condition. Roots were then dipped in 0.8 % (w/v) Dithane M45, WP 80 fungicide [Magnese ethylene bis (Dithiocarbonate Polymeric) complex with zinc salt 80 % w/w; Dow AgroSciences, SAS France] for 5 minutes. Thoroughly cleaned pots were filled with sand. Each pot was watered simultaneously. After removing from fungicide, plantlets were planted in pots and watered according to requirement.

67

3.6.3 Effect of Various Salt Treatments on Callus Cultures

Different concentrations of NaCl were used in the present study and added directly into already prepared MS medium. MS medium was supplemented with 9 different salt concentrations (0-160 mM) to observe the effect of salt stress on sugarcane callus cultures. Each treatment was replicated 20 times and the experiment was repeated thrice. The details of different formulations of MS medium supplemented with different salt concentrations used in salt stress experiments are given in Annexure 8. Callus necrosis being a qualitative parameter was difficult to express in quantitative terms. However, five categories (Scales A-E) as mentioned below were arbitrarily selected. Callus necrosis scales; A: 81-100 %, B: 61-80 %, C: 41-60 % D: 21-40 %, E: 0-20 % callus necrosis. The amount of soluble protein contents, peroxidase, catalase and superoxide dismutase activities was calculated by using spectrophotometer before every subculture at day 90, 120 and 150 (methodology described in section 3.5). The 120days-old NaCl-treated callus cultures were then shifted to regeneration medium. The regeneration medium consisted of MS (Murashige and Skoog, 1962) basal salts supplemented with 13.31 M BAP and 0.5 M TDZ. The experiment was planned with 15 culture vessels per NaCl treatment and it was repeated three times. At day 30, regeneration frequency, number of shoots per culture vessel and mean shoot length was recorded. The regenerated shoots were shifted to MS basal medium for further shoot development for 30 days and then were transferred to the already standardized rooting medium for each cultivar. The experiment was planned with 15 replicates per

68 treatment in one experiment and the experiment was repeated thrice. Data for root formation percentage, number of roots per culture vessel and root length were recorded at day 30, after shifting the regenerated plants (60-days-old) to the rooting medium.

3.6.4 Polyethylene Glycol (PEG) Pretreatment Experiment

Polyethylene glycol is a metabolically inactive osmoticum. In an attempt to see the effect of PEG pretreatment on salt tolerance characteristics of callus cultures, an experiment was conducted. The callus cultures of cv. SPF 234 and cv. HSF 240 had shown category A necrosis at day 30 after first subculture on MS medium containing 140 and 120 mM NaCl respectively. Therefore, 120 and 100 mM NaCl was considered as sublethal concentrations for SPF 234 and HSF 240, respectively. For this experiment, MS medium was supplemented with 13.5 M 2,4-D and 1 % PEG. This experiment was planned to have NaCl level one above and one below from the sublethal salt concentration for each cultivar. The calluses were treated with this medium for five days and then shifted to MS medium with different salt concentrations (0, 80, 100, 120 or 140 mM NaCl) for two subcultures. For each treatment, 20 culture vessels were selected. A set of 20 x 4 non-treated culture vessels was used as control. PEG-pretreated and nonpretreated callus cultures were then analyzed for fresh weights, callus necrosis, soluble protein contents, peroxidase, catalase and superoxide dismutase activities at day 90 and 120. The callus cultures were then shifted to regeneration medium to study the effect of PEG pretreatment on regeneration potential.

3.6.5 Ascorbic acid Pretreatment Experiment

Ascorbic acid is a small water-soluble antioxidant water molecule, which acts as a primary substrate in the cyclic pathway for enzymatic detoxification of hydrogen

69 peroxide (Noctor and Foyer, 1998). An experiment was designed to observe effect of exogenous supply of ascorbic acid on the tolerance of callus cultures to salt stress. Ascorbic acid was added to MS medium at the concentration of 0.5 mM. The calluses were treated for 24 or 48 hours and then shifted to MS medium with 0, 80, 100, 120 or 140 mM NaCl. The experiment was performed with 20 replicates per treatment. A control experiment of 20 replicates for each treatment was also run. Ascorbic acidpretreated and non-pretreated callus cultures were then analyzed for fresh weight, callus necrosis, soluble protein contents, peroxidase, catalase and superoxide dismutase activities at day 90. The effect of ascorbic acid pretreatment was also observed on in vitro growth of plants. To analyze the effect of ascorbic acid pretreatment on in vitro-grown plants, 60-days-old regenerated plants of cv. SPF 234 and cv. HSF 240 were pretreated with 0.5 mM ascorbic acid for 24 hours and then were shifted to MS medium supplemented with various NaCl concentrations (0-160 mM) for 30 days. The data for fresh weights of plants, number of shoots per culture vessel, shoot length, numbers of roots per culture vessel and root length, soluble protein contents and antioxidant enzyme activities were recorded at day 30.

3.7 Statistical Analysis

The data were analyzed using Univariate analysis of variance (SPSS Version 11). Standard error of the mean values was calculated for each treatment in the callus induction experiments. F test was applied to the data to analyze the data for significant differences. To apply the F test, data were transformed where required. The values were also compared for significant difference using Duncans multiple range test.

ANNEXURES

70

Annexure 1 Formulation of MS Medium (Murashige and Skoog, 1962) for the Preparation of Stock Solutions
Ingredients a) Macronutrients (NH4) NO3 KNO3 CaCl2.2H2O MgSO4.7H2O KH2PO4 Stocks mg l-1 (20x) 20 x 1650=33000 38000 8800 7400 3400 Final concentration in MS Medium mg l-1 1650 1900 440 370 170

b) Micronutrients MnSO4.4H2O ZnSO4.7H20 H3BO3.7H2O KI Na2MoO4.2H2O CuSO2.5H2O CoCl2.6H2O 22.3 x 100=2230 860 620 83 25 2.50 2.50

mg l-1 (100x) 22.3 8.6 6.2 0.83 0.25 0.025 0.025

c) Vitamins Glycine

mg l-1 (200x) 2 x 200=400 2.0

71 Nicotinic acid Pyridoxine HCl Thiamine HCl 100 100 20 0.5 0.5 0.1

d) Iron

mg l-1 (200x) 36.2

Na2EDTA.2H2O FeSO4.7H2O e) Myo-inositol

33.6 x 200=6720 5560 mg l-1 (100x) 100 x 100=10000 100 27.8

Annexure 2
Preparation of Stock Solutions for MS (Murashige and Skoog, 1962) Medium a) Macronutrients Macronutrients stock for MS medium was prepared at the final concentration of 20X (Annexure 1, section a). All the salts were weighed individually and dissolved separately in distilled water. Separately dissolved salts were mixed together in a conical flask already containing an appropriate amount of distilled water so as to avoid precipitation. The solution was then transferred to a 1000 ml capacity volumetric flask to make up the final volume. b) Micronutrients Stock solution of micronutrients was prepared 100 times more concentrated than the final volume (100X). All the salts of micronutrients as given in Annexure 1, under section b

72 were weighed and dissolved separately and made up to the final volume as described above in section a. c) Fe EDTA Iron EDTA stock solution was prepared at a concentration of 200X. The salts for this stock solution are given in Annexure 1, section c. The prepared 200X stock was poured in an amber-colored bottle and stored in refrigerator. For the preparation of 1liter of MS medium, 5 ml of this stock solution was used. d) Vitamins Vitamins of MS medium were prepared as 200X. Separately dissolved vitamins (as given in Annexure 1, section d) were transferred to a 500 ml volumetric flask and final volume was made with distilled water. For the preparation of 1 liter medium, 5 ml of vitamin stock was used. e) Myo-inositol Stock solution of myo-inositol was prepared separately as 100X. It was prepared by dissolving 10 g of myo-inositol in 1000 ml of distilled water and 10 ml of this stock was taken for 1 L MS medium

Annexure 3
Preparation of Stock Solutions of Growth Regulators Auxins (2,4-D, NAA, IBA etc.) were dissolved initially in a little quantity of 0.1 N NaOH while the initial solvent for cytokinins (BAP, TDZ, Kinetin etc.) was 0.1 N HCl. Once dissolved, the final volume was made up with distilled water in an appropriate volumetric flask and stored at 4C in refrigerator till use.

73

Annexure 4
Preparation of 1 liter MS Medium One liter MS medium for callus induction and proliferation was prepared in a manner given below. Medium Components 1) Macronutrients 2) Micronutrients 3) Vitamins 4) Myo-inositol 5) Iron-EDTA 6) Sugar Volume of Stock solution 50 ml l-1 10 ml l-1 05 ml l-1 10 ml l-1 05 ml l-1 30 g l-1

7) Agar (Oxoid, Hampshire, England) 7 g l-1 8) pH 9) Growth regulators (2,4-D, BAP, TDZ, IBA) 5.8 According to the requirement of a specific medium as illustrated in Annexure 5.

Annexure 5
Composition of Different Media Used for Callus induction/Maintenance Medium C1 C2 C3 C4 Medium composition MS + 4.52 M 2,4-D MS + 9.04 M 2,4-D MS + 13.52 M 2,4-D MS +18.08 M 2,4-D

74 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 MS + 4.52 M 2,4-D+ 4.43 M BAP MS + 9.04 M 2,4-D + 4.43 M BAP MS +13.52 M 2,4-D + 4.43 M BAP MS + 18.08 M 2,4-D + 4.43 M BAP MS + 4.52 M 2,4-D+ 8.87 M BAP MS + 9.04 M 2,4-D + 8.87 M BAP MS + 13.52 M 2,4-D + 8.87 M BAP MS + 18.08 M 2,4-D + 8.87 M BAP MS + 4.52 M 2,4-D + 2.32 M Kinetin MS + 9.04 M 2,4-D+ 2.32 M Kinetin MS + 13.52 M 2,4-D +2.32 M Kinetin MS + 18.08 M 2,4-D + 2.32 M Kinetin MS + 4.52 M 2, 4-D+ 4.64 M Kinetin MS+ 9.04 M 2,4-D + 4.64 M Kinetin MS + 13.52 M 2,4-D + 4.64 M Kinetin MS +18.08 M 2,4-D + 4.64 M Kinetin MS + 4.52 M 2,4-D + 13.92 M Kinetin MS + 9.04 M 2,4-D + 13.92 M Kinetin MS + 13.52 M 2,4-D +13.92 M Kinetin MS +18.08 M 2,4-D+13.92 M Kinetin

75

Annexure 6
Composition of Different Media Used for Plant Regeneration from Callus Cultures Medium R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 Medium composition MS + 2.22 M BAP MS + 4.43 M BAP MS + 8.87 M BAP MS + 13.31 M BAP MS + 0.5 M TDZ MS + 1.0 M TDZ MS + 8.87 M BAP + 0.5 M TDZ MS + 8.87 M BAP + 1.0 M TDZ MS + 13.31 M BAP + 0.5 M TDZ MS + 13.31 M BAP + 1.0 M TDZ MS + 8.87 M BAP + 1.0 M NAA MS + 8.87 M BAP + 2.0 M NAA

Annexure 7
Composition of Different Rooting Media Medium RT1 RT2 RT3 RT4 Medium composition MS Basal MS+ 1.0 M IBA MS+ 2.0 M IBA MS + 4.43 M BAP + 2.0 M IBA

76

Annexure 8
Composition of Different MS Media Used in Salt Stress Experiments Medium S1 S2 S3 S4 S5 S6 S7 S8 S9 Medium composition MS + 13.52 M 2,4-D + 0 mM NaCl MS + 13.52 M 2,4-D + 20 mM NaCl MS + 13.52 M 2,4-D + 40 mM NaCl MS +13.52 M 2,4-D + 60 mM NaCl MS + 13.52 M 2,4-D + 80 mM NaCl MS +13.52 M 2,4-D + 100 mM NaCl MS + 13.52 M 2,4-D + 120 mM NaCl MS +13.52 M 2,4-D + 140 mM NaCl MS + 13.52 M 2,4-D + 160 mM NaCl

Annexure 9
0.1 M Phosphate Buffer (pH 7.2) for Extraction of Proteins and Enzymes

Components KH2PO4 K2HPO4

Amount 13.61 g 17.42 g

Distilled water was added to make up final volume, i.e., 1000 ml.

77

Annexure 10
Composition of Biuret Reagent for Protein Estimation. Components CuSO4.5H2O KI Na-EDTA 5N NaOH Distilled water Amount 3.8 g 1.0 g 6.7 g 200 ml 700 ml

Annexure 11
Reagents for Peroxidase Estimation a) 1 % Guaiacol Components Guaiacol Distilled H2O b) 0.3 % H2O2 Components H2O2 (35 %) Distilled H2O Amount 0.86 ml 99.14 ml Amount 1 ml 99 ml

78

Annexure 12
Reagents for Catalase Estimation a) Reagent A (50 mM Potassium Phosphate Buffer, pH 7.0 at 25C) Prepared 200 ml in deionized water using Potassium Phosphate. Adjusted to pH 7.0 at 25C using 1 M KOH. b) Reagent B [Substrate Solution: 0.036 % (w/w) Hydrogen Peroxide (H2O2) Solution] Prepared in Reagent A using Hydrogen Peroxide, 35 % (w/w). Determined the A240nm of this solution using Reagent A as a blank. The A240nm should be between 0.550 and 0.520 absorbance units. Added hydrogen peroxide to increase the absorbance and Reagent A to decrease the absorbance. c) Reagent C (Catalase Solution) Immediately before use, prepared a solution containing 50-100 units per ml in cold Reagent A.

Annexure 13
A. Reagents for Superoxide Dismutase Estimation 1.Phosphate buffer (pH 7.8): Dissolved 6.9 g NaH2PO4.H2O in 900 ml distilled water and adjusted to pH 7.8 by 10 % NaOH. Final volume was made up to 1 liter with distilled water. 2. Riboflavin solution: (prepared fresh and kept in darkness) Dissolved 7.5 mg of riboflavin in 100 ml distilled water.

79 3. Sodium cyanide: Dissolved 13 g sodium cyanide in 1 liter distilled water 4. Nitroblue tetrazolium (NBT): (prepared fresh and kept in darkness). Dissolved 137 mg NBT in 10 ml distilled water 5. Methionine: (prepared fresh and kept in darkness). Dissolved 14.9 mg methionine in 10 ml phosphate buffer. 6. EDTA : Dissolved 245 mg of di-sodium salt of EDTA in 10 ml buffer solution.

B. Preparation of Reaction Mixture The reaction mixture was prepared as follows. 1. 1 ml NaCN 2. 10 ml methionine 3. 10 ml EDTA 4. 1 ml NBT 5. 1 ml Riboflavin The final volume was made up to 100 ml with buffer solution. This mixture was prepared away from a direct light source and kept in a dark bottle.

CHAPTER 4 CALLUS INDUCTION, MAINTENANCE AND REGENERATION OF SUGARCANE (cv. SPF 234 and cv. HSF 240)

80

CHAPTER 4 CALLUS INDUCTION, MAINTENANCE AND REGENERATION OF SUGARCANE (cv. SPF 234 and cv. HSF 240) RESULTS 4.1 Callus Induction and Proliferation of Sugarcane (cv. SPF 234 and cv. HSF 240)
During the present work, callus induction and proliferation was the first step in order to raise experimental plant material and proceed further towards the main objectives. For callus induction, 2-3 whorls of inner young leaves were used as explants. Different growth regulators either singly or in combination were tested to obtain better callus response. Cultures were placed in dark at 27 2 C according to already standardized culture conditions in this lab for other sugarcane cultivars (Aftab and Iqbal, 1999). The data pertaining to callus induction, fresh weight and morphological characteristics of cv. SPF 234 and cv. HSF 240 are given in Table 4.1 and 4.2 respectively. It was observed that for both the sugarcane cultivars (SPF 234 and HSF 240), MS medium supplemented with 13.5 M 2,4-D (C3) proved to be the best combination both in terms of callus induction as well as fresh weight of callus at day 30. Although the best medium for both cultivars was MS + 13.5 M 2,4-D, however, the fresh weight of cv. HSF 240 was greater as compared to cv. SPF 234. Comparison of the callus characteristics indicated that both friable and compact calluses were obtained. During the experiments, it was further observed that occasionally both compact and friable calluses were obtained on the same medium. However, mostly greenish-yellow friable calluses were obtained during the present work (Fig. 4.1-4.6). C3 medium (MS + 13.5 M

81 2,4-D) was thus selected for further experimental work on the basis of better callus induction on this medium for both the cultivars. To get a good callus mass, the calluses were subcultured on the same medium for another 30 days. Figure 4.1-4.6: Callus morphology of sugarcane (Saccharum spp. hybrid cvs. SPF 234 and HSF 240) raised from inner 2-3 young leaf whorls on MS basal medium supplemented with 13.5 M 2,4-D at day 10, 30 and 60 under dark conditions at 27 2 C

Fig. 4.1

Fig. 4.2

Fig. 4.3

Fig. 4.4

Fig. 4.1: A 10-days-old callus of cv. SPF 234 (1x) Fig. 4.2: A 10-days-old callus of cv. HSF 240 (1x) Fig. 4.3: A callus culture of cv. SPF 234 at day 30 (1x) Fig. 4.4: A 30-days-old callus of cv. HSF 240 (1x)

Fig. 4.5 Fig. 4.5: A 60-days-old callus of cv. SPF 234 (2x) Fig. 4.6: A 60-days-old callus of cv. HSF 240 (2x)

Fig. 4.6

82 Table 4.1: Effect of MS medium supplemented with various levels of growth regulators on callus induction, fresh weight and morphological characteristics in sugarcane (cv. SPF 234) under dark conditions at 27 2 C

Medium

Medium composition

Callus induction
(days)

Fresh weight a (g) 0.07 0.03 0.33 0.04 0.58 0.28 0.45 0.07 0.16 0.04 0.14 0.02 0.17 0.02 0.15 0.03 0.03 0.01 0.07 0.01 0.19 0.02 0.06 0.01 0.06 0.01 0.11 0.01 0.19 0.02 0.17 0.03

Morphological Characteristics
Greenish-yellow, friable, granular Greenish-yellow, friable, granular Greenish-yellow, friable, granular Greenish-yellow, friable, granular

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16

MS + 4.52 M 2,4-D

10 8 8 8 15 12 12 14 14 12 9 12 12 10 10 10

MS + 9.04 M 2,4-D

MS + 13.52 M 2,4-D

MS + 18.08 M 2,4-D MS + 4.52 M 2, 4-D+ 4.43 M BAP MS + 9.04 M 2,4-D + 4.43M BAP MS + 13.52 M 2,4-D + 4.43 M BAP MS + 18.08 M 2,4-D + 4.43 M BAP MS + 4.52 M 2,4-D + 8.87 M BAP MS + 9.04 M 2,4-D + 8.87 M BAP MS + 13.52 M 2,4-D + 8.87 M BAP MS + 18.08 M 2,4-D + 8.87 M BAP MS + 4.52 M 2,4-D + 2.32 M Kinetin MS + 9.04 M 2,4-D + 2.32 M Kinetin MS + 13.52 M 2,4-D + 2.32 M Kinetin MS + 18.08 M 2,4-D + 2.32M Kinetin

Yellowish-white, friable Yellowish-white, friable

Greenish-yellow, friable Translucent

Yellowish-white, friable Yellowish-white, friable Yellowish-white, friable Yellowish-white, friable Yellowish-white, friable Yellowish-white, friable Yellowish-white, friable Yellowish-white, friable, translucent Yellowish-white, friable

83
MS + 4.52 M 2, 4-D + 4.64 M Kinetin MS + 9.04 M 2,4-D + 4.64 M Kinetin MS + 13.52 M 2,4-D + 4.64 M Kinetin MS+ 18.08 M 2,4-D + 4.64M Kinetin MS + 4.52 M 2,4-D + 13.92 M Kinetin MS + 9.04 M 2,4-D + 13.92 M Kinetin MS + 13.52 M 2,4-D + 13.92 M Kinetin MS + 18.08 M 2,4-D + 13.92 M Kinetin

C17 C18 C19 C20 C21 C22 C23 C24

10 12 10 12 12 8 17 10

0.08 0.01 0.13 0.02 0.36 0.04 0.14 0.09 0.03 0.01 0.20 0.02 0.35 0.04 0.26 0.08

Yellowish-white, friable Yellowish-white, compact Yellowish-white,

friable

Yellowish-white, compact Yellowish-white, compact Yellowish-white, compact Yellowish-white, compact Yellowish-white, compact

Data for fresh weights were recorded from 10 culture vessels for each medium at day 30.

Table 4.2: Effect of MS medium supplemented with various levels of growth regulators on callus induction, fresh weight and morphological characteristics in sugarcane (cv. HSF 240) under dark conditions at 272 C Callus induction
(days)

Medium C1 C2 C3 C4 C5 C6 C7 C8

Medium composition
MS + 4.52 M 2,4-D MS + 9.04 M 2,4-D MS + 13.52 M 2,4-D MS + 18.08 M 2,4-D MS + 4.52 M 2, 4-D + 4.43 M BAP MS + 9.04 M 2,4-D + 4.43 M BAP MS + 13.52 M 2,4-D + 4.43 M BAP MS + 18.08 M 2,4-D + 4.43 M BAP

Fresh weight a (g) 0.05 0.02 0.05 0.01 0.68 0.05 0.28 0.04 0.05 0.01 0.28 0.05 0.24 0.01 0.10 0.04

Morphological Characteristics
Greenish-yellow, friable, granular Greenish-yellow, friable, granular Greenish-yellow, friable, granular Greenish-yellow, friable, granular

14 12 8 9 10 10 12 12

Yellowish-white, friable Yellowish white, friable

Greenish-yellow, friable translucent

Yellowish-white, friable

84 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24
a

MS + 4.52 M 2,4-D + 8.87M BAP MS + 9.04 M 2,4-D + 8.87 M BAP MS + 13.52 M 2,4-D + 8.87M BAP MS + 18.08 M 2,4-D + 8.87M BAP MS + 4.52 M 2,4-D + 2.32 M Kinetin MS + 9.04 M 2,4-D + 2.32 M Kinetin MS + 13.52M 2,4-D + 2.32 M Kinetin MS + 18.08M 2,4-D + 2.32 M Kinetin MS + 4.52M 2, 4-D + 4.64 M Kinetin MS + 9.04 M 2,4-D + 4.64 M Kinetin MS + 13.52 M 2,4-D + 4.64 M Kinetin MS +18.08 M 2,4-D + 4.64 M Kinetin MS + 4.52M 2,4-D + 13.92 M Kinetin MS + 9.04 M 2,4-D + 13.92 M Kinetin MS + 13.52 M 2,4-D + 13.92 M Kinetin MS + 18.08 M 2,4-D + 13.92 M Kinetin

12 10 8 10 12 10 10 15 10 15 8 10 14 12 12 14

0.04 0.01 0.10 0.01 0.16 0.02 0.09 0.02 0.03 0.01 0.17 0.03 0.19 0.03 0.04 0.01 0.05 0.02 0.20 0.13 0.57 0.42 0.14 0.12 0.04 0.02 0.16 0.08 0.44 0.31 0.21 0.15

Yellowish-white, friable Yellowish-white, friable Greenish-yellow, friable Yellowish-white, friable Yellowish-white, friable Yellowish-white, friable Yellowish-white, friable, translucent Yellowish-white, friable Yellowish-white, friable Yellowish-white, compact Yellowish-white, compact Yellowish-white, compact Greenish-yellow, friable Yellowish-white, compact Yellowish-white, friable, granular Yellowish-white, friable

Data for fresh weights were recorded from 10 culture vessels for each medium at day 30.

85

4.2 Plant Regeneration from Callus Cultures of Sugarcane (cv. SPF 234 and cv. HSF 240)

In an attempt to study the regeneration potential of callus cultures of sugarcane, proliferated calluses (60-days-old) were shifted to the regeneration media. The effect of different growth regulators (BAP, TDZ and NAA) was tested on the regeneration potential of sugarcane callus cultures. Table 4.3 and 4.4 show the regeneration frequency, number of shoots per culture vessel and shoot length recorded at day 30 for cv. SPF 234 and cv. HSF 240, respectively. It was observed that R7 medium, i.e., MS medium supplemented with 8.87 M BAP and 0.5 M TDZ was the best in terms of all the growth parameters studied for both the cultivars. Shoot initiation on this medium from 60-days-old callus cultures of cv. SPF 234 and cv. HSF 240 is shown in Fig. 4.7 and 4.8, respectively, while Fig. 4.9 and 4.10 show the stereomicographic representation of shoot initiation for both cultivars. It was also noted that the maximum regeneration frequency in cv. SPF 234 was greater (85 %) as compared to cv. HSF 240 (76 %) in medium R7. The same was observed for the other growth parameters. The mean number of shoots in cv. SPF 234 was 70 with a mean shoot length of 0.97 cm while in HSF 240, the mean number of shoots and shoot lengths were 66.33 cm and 0.95 cm respectively. The regenerated shoots were shifted to MS basal medium and maintained for 30 days for further development (Fig. 4.11-4.12).

86 Fig. 4.7-4.8: Shoot initiation from 60-days-old callus cultures of sugarcane (cv. SPF 234 and cv. HSF 240) on MS basal medium supplemented with 8.87 M BAP + 0.5 M TDZ at day 30 under 16 h photoperiod at 27 2 C Fig. 4.9-4.10: Stereomicrographic representation of shoot initiation from 60-days-old callus cultures of sugarcane (cv. SPF 234 and cv. HSF 240) on MS basal medium supplemented with 8.87 M BAP + 0.5 M TDZ at day 30 under 16 h photoperiod at 27 2 C

Fig. 4.7

Fig. 4.8

Fig. 4.9

Fig. 4.10

Fig. 4.7: Soot initiation (arrows) in callus culture of cv. SPF 234 (1.5x ) Fig. 4.8: Soot initiation (arrows) in callus culture of cv. HSF 240 (2x ) Fig. 4.9: Stereomicrograph showing shoot initiation in callus culture of cv. SPF 234 (20x ) Fig. 4.10: Stereomicrograph showing shoot initiation in callus culture of cv. HSF 240 (20x ) Fig. 4.11-4.12: Further growth and shoot development in sugarcane (cv. SPF 234 and cv. HSF 240) on MS basal medium under 16 h photoperiod at 27 2C. The photographs were taken at day 30 of subculture on MS basal medium

Fig. 4.11

Fig. 4.12

Fig. 4.11: Regenerated shoots of cv. SPF 234 at day 30 after subculture on MS basal medium (1.4x) Fig. 4.12: Regenerated shoots of cv. HSF 240 at day 30 after subculture on MS basal medium (1.6x)

87 Table 4.3: Effect of BAP, TDZ and NAA supplemented to MS medium on regeneration frequency of 60-days-old callus cultures of sugarcane (cv. SPF 234) under 16 h photoperiod at 272 C a Medium Medium composition Regeneration Frequency a (%)
14 35 75 73 3 2 85 72 67 69 82 65

Number of shoots /culture vessel b

31.33 6.83 23.67 6.64 29.66 2.33 18.33 3.75 2.33 0.33 4.0 1.15 70.0 5.77 69.67 2.91 55.33 8.1 42.33 8.88 33.66 0.88 29.0 1.12

Shoot length b (cm)

0.77 0.23 0.92 0.12 1.37 0.03 1.2 0.06 0.23 0.03 0.20 0.06 0.97 0.09 0.93 0.18 0.60 0.21 0.83 0.12 1.17 0.08 1.0 0.05

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12

a

MS+ 2.22M BAP MS+ 4.43M BAP MS+ 8.87 M BAP MS+ 13.31 M BAP MS + 0.5 M TDZ MS + 1.0 M TDZ MS + 8.87M BAP + 0.5 M TDZ MS + 8.87M BAP + 1.0 M TDZ MS + 13.31M BAP + 0.5 M TDZ MS + 13.31 M BAP + 1.0 M TDZ MS + 8.87 M BAP + 1.0 M NAA MS + 8.87M BAP + 2.0 M NAA

Regeneration frequency is calculated from 30 culture vessels for each medium at day 30. Number of shoots per culture vessel and shoot length represented here are the mean values ( S.E) taken from 30 culture vessels at day 30.

88 Table 4.4: Effect of BAP, TDZ and NAA supplemented to MS medium on regeneration frequency of 60-days-old callus cultures of sugarcane (cv. HSF 240) under 16 h photoperiod at 27 2 C a

Medium

Medium composition

Regeneration Frequency a (%)

8 56 72 70 0 0 76 71 67 72 70 55

Number of shoots /culture vessel b

7.66 3.71 24 6.65 16.33 0.88 19.33 3.84 0 0 66.33 6.84 48.66 8.57 16.33 4.33 39.33 13.22 34.33 4.6 35 2.21

Shoot length b (cm)

1.03 0.08 1.33 0.03 1.23 0.03 1.13 0.14 0 0 0.95 0.02 0.6 0.15 0.86 0.20 0.97 0.03 0.83 0.15 1.3 0.12

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12

a

MS + 2.22 M BAP MS + 4.43 M BAP MS + 8.87M BAP MS+ 13.31M BAP MS + 0.5M TDZ MS + 1.0M TDZ MS + 8.87M BAP + 0.5 M TDZ MS + 8.87M BAP + 1.0 M TDZ MS +13.31 M BAP+ 0.5M TDZ MS + 13.31M BAP + 1.0 M TDZ MS + 8.87M BAP +1.0M NAA MS + 8.87M BAP +2.0 M NAA

Regeneration frequency is calculated from 30 culture vessels for each medium at day 30. Number of shoots per culture vessel and shoot length represented here are the mean values ( S.E) taken from 30 culture vessels at day 30.

89

4.3 Rooting of the Regenerated Shoots of Sugarcane (cv. SPF 234 and cv. HSF 240)
At day 60 of the initial cultures, the regenerated shoots acquired sufficient length (5-6 cm) and were ready for transfer to the rooting media. Four different media were tested for rooting. The results of the experiment for rooting of sugarcane plants are given in Table 4.5 and 4.6. It was interesting to note that in both the cultivars, none of the regenerated shoots rooted in MS basal medium under the specific experimental conditions. Best rooting of the regenerated shoots in cv. SPF 234 (Fig. 4.13) was obtained on MS medium supplemented with 2 M IBA (medium RT3). On this medium, 95 % rooting was observed at day 30. The average number of roots per culture vessel in this medium was 9 with an average root length of 1.01 cm. The shoots shifted to MS medium with 1 M IBA (medium RT2) resulted in 80 % rooting with 6.67 roots/culture vessel after 30 days. On RT4 medium, i.e., MS medium supplemented with 4.43 M BAP + 2 M IBA, the rooting percentage was 45 with 7.67 roots per culture vessel. Mean root length in this case was 0.833 cm. Root development of the regenerated shoots of cv. HSF 240 is shown in Fig. 4.14. For this cultivar, almost similar results for percentage root regeneration were observed either on RT2 or RT3 medium with 8.33 and 7 roots per culture vessel respectively. However, greater root length was obtained on MS medium supplemented with 4.43 M BAP and 2.0 M IBA (medium RT4). Fig. 4.15 and 4.16 show well developed in vitro-grown plants of cv. SPF 234 and cv. HSF 240, respectively.

90 Fig. 4.13-4.14: Root initiation and development in regenerated shoots of sugarcane (cv. SPF 234 and cv. HSF 240) on MS basal medium supplemented with IBA at day 30 under 16 h photoperiod at 27 2 C

Fig. 4.13

Fig. 4.14

Fig. 4.13: Root development of regenerated shoots of cv. SPF 234 on MS basal medium supplemented with 2 M IBA (1.8x) Fig. 4.14: Root development of regenerated shoots of cv. HSF 240 on MS basal medium supplemented with 1 M IBA (2x)

Fig. 4.15-4.16: Well-developed in vitro-grown plants of sugarcane cv. SPF 234 (Fig. 4.15) and cv. HSF 240 (Fig. 4.16) maintained on MS basal medium under 16 h photoperiod at 27 2 C

Fig. 4.15

Fig. 4.16

Fig. 4.15: Well-developed plants of cv. SPF 234 on MS basal medium (1x) Fig. 4.16: Well-developed plants of cv. HSF 240 on MS basal medium (1x)

91 Table 4.5: Effect of IBA and BAP on the rooting potential of regenerated shoots of sugarcane (cv. SPF 234) under 16 h photoperiod at 27 2 C Medium Medium composition Root regeneration a (%)
0 80 95 45

Number of roots /culture vessel b

0 6.67 0.88 9.0 0.577 7.67 0.33

Root length b (cm)

0 0.99 0.02 1.01 0.01 0.833 0.09

RT1 RT2 RT3 RT4

MS Basal MS+ 1.0 M IBA MS+ 2.0 M IBA MS + 4.43 M BAP + 2.0 M IBA

a b

Regeneration frequency is calculated from 30 culture vessels for each medium at day 30. Number of roots per plant and root length represented here are the mean values ( S.E) taken from 30 culture vessels at day 30

Table 4.6: Effect of IBA and BAP on the rooting potential of regenerated shoots of sugarcane (cv. HSF 240) under 16 h photoperiod at 27 2 C Medium Medium composition Root regeneration a (%)
0 94 93 51

Number of roots /culture vessel b

0 8.33 1.30 7.0 0.57 5.67 0.33

Root length b (cm)

0 0.80 0.09 0.84 0.08 0.86 0.10

RT1 RT2 RT3 RT4

MS Basal MS+ 1.0 M IBA MS+ 2.0 M IBA MS + 4.43 M BAP + 2.0 M IBA

Regeneration frequency is calculated from 30 culture vessels for each medium at day 30. Number of roots per plant and root length represented here are the mean values ( S.E) taken from 30 culture vessels at day 30.

92

4.4 Hardening and Acclimatization of in vitro-grown Plants of Sugarcane (cv. SPF 234 and cv. HSF 240)
The usual protocols (as described in Materials and Methods, section 3.6.2) were employed for hardening of the well-established in vitro-grown sugarcane plants. Hardening of sugarcane plants was successfully accomplished for both the sugarcane cultivars. More than 95 % in vitro-raised plants of both the cultivars were successfully acclimatized to the glasshouse conditions. The plants established under ex-vitro conditions were also healthy and showed vigorous growth (Fig. 4.17-4.18).

Fig. 4.17-4.18: Hardening and acclimatization of in vitro-grown plants of sugarcane (cv. SPF 234 and cv. HSF 240) under glasshouse conditions (natural light, 27 2 C)

Fig. 4.17

Fig. 4.18

Fig. 4.17: Plants of cv. SPF 234 grown under ex vitro condition (1.5x) Fig. 4.18: Plants of cv. HSF 240 grown under ex vitro condition (1.5x)

93

DISCUSSION
Sugarcane has been extensively investigated for callus induction, proliferation, maintenance and regeneration (Heinz and Mee, 1969, 1971; Liu and Chen, 1974; Bhansali and Singh, 1982; Zang et al., 1983; Flick et al., 1983; Lago and Baretto, 1987; Taylor et al., 1992, Brisibe et al., 1994). As far as the explant material is concerned, innermost or two to three inner whorls of leaves, pith parenchyma, immature inflorescence, apical meristem and sugarcane buds have routinely been used for callus induction and maintenance purposes in sugarcane. In the present study, young inner 2-3 whorls of leaves were used as an explant source. Young sugarcane leaves are known to be a good explant source for callus induction (Ahloowalia and Maretzki, 1983; Ho and Vasil, 1983; Chen et al., 1988; Brisibe et al., 1994; Aftab and Iqbal., 1999). During the present work, the effect of different growth regulators and their concentrations were investigated. It was observed that amongst all the growth regulators tested (2,4-D, BAP and Kinetin) for callus induction, 2,4-D proved to be the best growth regulator. These results supported the earlier findings indicating a significant role of 2,4-D in the establishment of in vitro cultures of sugarcane (Nickell, 1977; Liu, 1983; Vasil, 1987; Chen et al., 1988; Fitch and Moore, 1990; Iqbal et al., 1991; Aftab et al., 1996 and Ramanand, 2006). The results of the present study highlighted the fact that the growth potential of callus cultures was affected not only by the type but also the concentration of growth regulator in use. It was observed in this study that for both the sugarcane cultivars (SPF 234 and HSF 240), 13.5 M proved to be the best concentration of 2,4-D both in terms of callus induction as well as fresh weight. The same medium was also the best for further callus maintenance. Earlier workers have also reported maximum callus formation in

94 different sugarcane varieties on MS medium supplemented with 3 mg l-1 (13.5 M) 2,4-D (Aftab et al., 1996; Snyman et al., 2000, 2001; Mamun et al., 2004; Gandonou et al., 2005 c). The same concentration seems to work well for the present cultivars being investigated. The present investigation also revealed different morphological characteristics of calluses in both the cultivars. Callus formation with different morphological characteristics have also been reported by Liu et al., (1972) and Fitch and Moore (1983). Similarly, Shaheen and Mirza (1989) and Khan et al., (1998) also reported that in sugarcane, two types of calluses, i.e., Type A (compact and dry) or Type B (non-compact and globular) were obtained. It was also observed during the present investigation that friable callus showed better growth and proliferation response. According to Orton (1979), the same trend was observed in callus cultures of Hordeum vulgare in which friable calluses had more growth as compared to compact calluses. It was observed during the present study that none of the callus cultures showed regeneration on MS medium supplemented with 2,4-D. It has been reported previously that 2,4-D has an important role in stimulating somatic embryogenesis in sugarcane and shifting of embryogenic calluses to the half-strength MS medium can result in regeneration of plants (Ho and Vasil, 1983; Aftab and Iqbal, 1999). This was, however, not possible for the two cultivars under study. Although 2,4-D promoted best callus proliferation but somatic embryogenesis as observed in other cultivars reported earlier (Aftab et al., 1996; Aftab and Iqbal, 1999) could not be obtained. Both modes of regeneration, i.e., somatic embryogenesis and/or organogenesis have been well documented in the literature. In the present investigation, organogenic mode of regeneration was the dominating one.

95 It has already been reported that a high level of cytokinin in combination with a low auxin level was essential for the differentiation of adventitious shoots in sugarcane leaf sheath callus (Islam et al., 1982; Karim et al., 2002). Cell differentiation and shoot regeneration are the processes regulated by cytokinins, therefore, the addition of cytokinins to the medium were found to cause differentiation of the sugarcane callus tissue to form shoots (Irvine and Benda, 1987; Irvine et al., 1991). During the present work, BAP was found to be an important cytokinin in the development of shoots in line with some earlier findings (Islam et al., 1982, Niaz and Quraishi, 2002) but an interesting aspect of shoot regeneration from sugarcane callus during the present study was that the use of thidiazuron (TDZ) in combination with BAP significantly increased the number of shoots per culture vessel. TDZ has been shown to be highly effective in shoot induction for a variety of plants (Murthy et al., 1998). Some workers believe that TDZ is superior to other growth regulators as it can promote growth directly through its own biological activity (Mok and Mok, 1985). Most of the studies regarding role of TDZ in regeneration of callus cultures are on woody plants, however, there are a few recent reports which indicate the role of TDZ in shoot regeneration from sugarcane callus cultures as well. The role of TDZ on sugarcane regeneration was reported by Gallo-Meagher et al., (2000) who observed that sugarcane callus derived from inflorescence result in greater number of regenerated shoots when cultured on 1 mM or higher TDZ. Similar results were also reported by Chengalrayan and Gallo-Meagher (2001) who found that TDZ was superior to the other growth regulators in producing more number of shoots per callus. Recently, Jain et al., (2007) have also reported regeneration of plantlets from callus cultures of sugarcane on MS basal medium supplemented with 2.5 M TDZ.

96 Present study also indicates that the mean shoot length of the regenerated shoots was less in MS medium supplemented with BAP and TDZ as compared to the medium in which only BAP was added. Thus it can be suggested that TDZ might have reduced shoot elongation in the two sugarcane cultivars under study. Earlier workers have also reported reduced shoot elongation as a result of TDZ in different plants (Murthy et al., 1998). In one such study on white ash, reduction in shoot length was reported by Bates et al., (1992). Charleson et al., (2006) found that TDZ treatment inhibited the shoot elongation of the regenerated spearmint shoots from callus cultures. On the basis of the results of the present study, it may be suggested that TDZ is an important growth regulator in the regeneration of sugarcane callus cultures. Although the addition of TDZ resulted in reduction of shoot length but the number of shoots regenerated from callus cultures was considerably higher. Thus large number of plants could be obtained by the addition of TDZ in the medium that could be proliferated further in a medium without TDZ. Furthermore, it has also been reported that TDZ also has a positive effect on multiplication of shootlet cultures established from buds regenerated from apical domes of sugarcane (Dhawan et al., 2004). There are some reports on rooting of the regenerated shoots in sugarcane on MS basal medium (Lal and Singh, 1994; Karim et al., 2002). The results of experiments during the present study indicated that none of the regenerated shoots showed root initiation on MS basal medium. It was observed during the rooting experiments that rooting of regenerated shoots could be obtained on MS medium supplemented with either 2.0 M (for cv. SPF 234) or 1.0 M IBA (for cv. HSF 240), respectively. Chengalrayan and Gallo-Meagher (2001) and Jain et al., (2007) have also found that 19.7 M IBA plays an important role in rooting of the regenerated sugarcane plants.

97 So during the present work, well-developed callus masses were produced from sugarcane explants of both the cultivars (cv. SPF 234 and cv. HSF 240). Different aspects of in vitro plant development for both sugarcane cultivars were also successfully accomplished. It can also be concluded from the results of this study that although TDZ alone could not result in regeneration of callus cultures but when used in combination with BAP produced better results both in terms of regeneration potential as well as number of shoots per culture vessel. Thus it has an important role in improvement of regeneration potential of sugarcane callus cultures.

CHAPTER 5 EFFECT OF SALT STRESS ON CALLUS CULTURES OF SUGARCANE (cvs. SPF 234 and HSF 240)

98

CHAPTER 5 EFFECT OF SALT STRESS ON CALLUS CULTURES OF SUGARCANE (cvs. SPF 234 and HSF 240) RESULTS 5.1 Effect of Salt Stress on Morphological Characteristics of Sugarcane Callus Cultures
During the present study, it was observed that sugarcane callus cultures underwent several morphological changes with the addition of salt in the medium over successive subcultures. Morphology of sugarcane callus cultures (cvs. SPF 234 and HSF 240) at various NaCl levels is shown in Table 5.1 and 5.2, respectively. It is evident from the data given in Table 5.1 that 60-days-old callus cultures of cv. SPF 234 maintained on MS basal medium supplemented with 13.5 M 2,4-D were greenish-yellow, friable and granular (Fig. 5.1). Fig. 5.1: Callus morphology of sugarcane (Saccharum spp. hybrid cv. SPF 234) on MS basal medium supplemented with 13.5 M 2, 4-D at day 60 under dark conditions at 27 2 C (2x)

Fig. 5.1

99 When 60-days-old calluses were shifted to the same medium containing various NaCl levels (0-160 mM), variation in callus morphology of both the cultivars was observed. The data recorded at day 90, 120 or 150 for cv. SPF 234 is given in Table 5.1. The morphology of callus cultures of cv. SPF 234 maintained on the control medium (0 mM NaCl) is shown in Fig. 5.2-5.4. It is evident from the figures that calluses during different subcultures (at day 90, 120 or 150) had no signs of necrosis in the absence of salt in the medium. In the figures 5.5 to 5.25 are shown the effect of 20-140 mM NaCl on callus morphology at day 90, 120 and 150. For this cultivar, callus cultures at 160 mM NaCl concentration turned completely brown in appearance at day 90 and further maintenance was not possible due to necrosis (Fig. 5.26).

Fig. 5.2-5.4: Callus morphology of sugarcane (cv. SPF 234) at 0 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.2

Fig. 5.3

Fig. 5.4

Fig. 5.2: A well proliferating callus culture at day 90 (1.2x) Fig. 5.3: Callus morphology at day 120 (1.5x) Fig. 5.4: A callus culture at day150 (1.2x)

100 Fig. 5.5-5.7: Callus morphology of sugarcane (cv. SPF 234) at 20 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.5

Fig. 5.6

Fig. 5.7

Fig. 5.5: First subculture at 20 mM NaCl concentration at day 90 (1.2x) Fig. 5.6: Greenish callus with somewhat whitish portions undergoing browning (farthest from the viewer; indicated by an arrow) at day 120 (1.2x) Fig. 5.7: Brownish-yellow callus at day 150 (1x) Fig. 5.8-5.10: Callus morphology of sugarcane (cv. SPF 234) at 40 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

. Fig. 5.8 Fig. 5.9 Fig. 5.10

Fig. 5.8: A callus culture (day 90) transferred to 40 mM NaCl concentration (1.2x) Fig. 5.9: Greenish-yellow callus at day120 (1.2x) Fig. 5.10: A decreased callus growth potential at day150 (1x)

101 Fig. 5.11-5.13: Callus morphology of sugarcane (cv. SPF 234) at 60 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2C

Fig. 5.11

Fig. 5.12

Fig. 5.13

Fig. 5.11: A 90-days-old greenish-yellow callus with signs of necrosis at 60 mM NaCl concentration (1.4x) Fig. 5.12: A callus culture with some brown and greenish-yellow portions at day 120 (1.2x) Fig. 5.13: Brownish-yellow callus at day 150 (1.2x) Fig. 5.14-5.16: Callus morphology of sugarcane (cv. SPF 234) at 80 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.14

Fig. 5.15

Fig. 5.16

Fig. 5.14: Callus (day 90) at 80mM NaCl concentration showing signs of necrosis (1.2x) Fig. 5.15: A 120-day-old off-white callus with patches of reddish-brown color (1.2x) Fig. 5.16: A callus culture showing blackish-brown portion (on the lower side) at day150 (1.4x)

102 Fig. 5.17-5.19: Callus morphology of sugarcane (cv. SPF 234) at 100 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.17

Fig. 5.18

Fig. 5.19

Fig. 5.17: Callus necrosis at day 90 (1.2x) Fig. 5.18: A 120-day-old callus showing category A necrosis (1.3x) Fig. 5.19: Yellowish-brown callus with somewhat translucent appearance at day 150 (1.2x) Fig. 5.20-5.22: Callus morphology of sugarcane (cv. SPF 234) at 120 mM NaCl level supplemented to MS medium at day 90, 120 or 150 under dark at 27 2C

Fig. 5.20

Fig. 5.21

Fig. 5.22

Fig. 5.20: Callus at 120 mM concentration at day 90 on MS basal medium (1x) Fig. 5.21: Blackish-brown callus at 120 mM NaCl concentration showing category A necrosis at day 120 (1.2x) Fig. 5.22: Callus with retarded growth (day 150) at 120 mM NaCl concentration (1.2x)

103 Fig. 5.23-5.25: Callus morphology of sugarcane (cv. SPF 234) at 140 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.23

Fig. 5.24

Fig. 5.25

Fig. 5.23: Callus at day 90 showing browning (arrows) at 140 mM NaCl (1x) Fig. 5.24: Callus with lower blackish portion at day 120 (1x) Fig. 5.25: A necrotic callus culture at day 150 showing category A necrosis at 140 mM NaCl (1x)

Fig. 5.26: Callus morphology of sugarcane (cv. SPF 234) at 160 mM NaCl level supplemented to MS medium at day 90 under dark at 27 2 C (1.2x)

Fig. 5.26

104 Table 5.1: Morphology of sugarcane callus cultures (cv. SPF 234) at different NaCl levels (0-160 mM) supplemented to MS medium under dark conditions at day 90, 120 and 150 a
Callus morphology before NaCl treatment At day 60 Greenish-yellow, friable, granular Greenish-yellow, friable, granular

Salt concentration (mM) 0

Morphology of sugarcane callus cultures

after NaCl treatment At day 90 Greenish-yellow, friable, granular Greenish-yellow, friable, granular At day 120 Greenish-yellow, friable, granular Greenish-yellow with some whitish portion, granular Greenish-yellow, friable, granular Greenish brown, friable, granular Off-white with some reddishbrown portion, granular Yellowishbrown, friable, granular Blackish-brown, necrotic Blackish-brown At day 150 Whitish-yellow, friable, granular Brownish-yellow, friable, granular Greenish-brown, smooth surfaced Yellowish-brown, friable, granular Blackish-brown, friable, granular Yellowish-brown, translucent

20

40

Greenish-yellow, friable, granular Greenish-yellow, friable, granular Greenish-yellow, friable, granular

Brownish-yellow, friable, granular Greenish-yellow granular Off-white with some brown portion, granular Brownish-off white, friable, granular Brownish-off white, friable, granular Brownish-off white, friable, granular Brownish-black

60

80

100

Greenish-yellow, friable, granular

120

Greenish-yellow, friable, granular

Blackish-brown, necrotic Brownish-black, necrotic

140

Greenish-yellow, friable, granular Greenish-yellow, friable, granular

160
a

ND b

ND b

Callus morphology is based on 60 culture vessels per NaCl treatment ND: Not determined because callus cultures could not be maintained at these salt levels over successive subcultures.
b

105 For cv. HSF 240, the callus color and morphology was also found to be affected with increasing salt concentration. The callus cultures before NaCl treatment at day 60 were greenish-yellow and friable (Table 5.2, Fig. 5.27). Like the other cultivar, most of the callus cultures of cv. HSF 240 maintained at 0 mM NaCl level did not show any sign of necrosis during different subcultures at day 90, 120 or 150 (Fig. 5.28-5.29). Fig. 5.27: Callus morphology of sugarcane (Saccharum spp. hybrid cv. HF 240) on MS basal medium supplemented with 13.5 M 2, 4-D at day 60 under dark at 27 2 C (1.4x)

Fig. 5.27 Fig. 5.28-5.30: Callus morphology of sugarcane (cv. HSF 240) at 0 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.28

Fig. 5.29

Fig. 5.30

Fig. 5.28: A greenish-yellow 90-days-old callus culture at 0 mM NaCl (1.1x) Fig. 5.29: Proliferating callus (day 120) at 0 mM NaCl concentration (1.3x) Fig. 5.30: Callus with translucent appearance at day150 at 0 mM NaCl concentration (0.8x)

106 Upon shifting of these callus cultures to MS medium with various salt concentrations, changes in morphological characteristics were recorded. It was observed that with an increase in salt concentration as well as with the passage of time (during successive subcultures), greenish-yellow callus started turning brownish in color (Fig. 5.31-5.50). For this cultivar (unlike SPF 234), complete browning of the callus cultures, however, was observed when MS medium was supplemented with 140 mM NaCl level or above. Fig. 5.49 and 5.50 show the complete callus necrosis at 140 and 160 mM NaCl level.

Fig. 5.31-5.33: Callus morphology of sugarcane (cv. HSF 240) at 20 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.31

Fig. 5.32

Fig. 5.33

Fig. 5.31: Callus morphology at 20 mM NaCl concentration at day 90 (1x) Fig. 5.32: Off-white callus culture with a little brown portion at day 120 (1.1x) Fig. 5.33: Callus at day150 at 20 mM NaCl concentration showing some browning (1.2x)

107 Fig. 5.34-5.36: Callus morphology of sugarcane (cv. HSF 240) at 40 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.34

Fig. 5.35

Fig. 5.36

Fig. 5.34: Initiation of callus necrosis (category E) in 90-days-old callus at 40 mM NaCl concentration (1x) Fig. 5.35: Callus browning (day 120) at 40 mM NaCl concentration (1.1x) Fig. 5.36: Callus culture with smooth appearance having brown patches at day150 (1.2x)

Fig. 5.37-5.39: Callus morphology of sugarcane (cv. HSF 240) at 60 mM NaCl level supplemented to MS medium at day 90, 120 or 150 under dark at 27 2 C

Fig. 5.37

Fig. 5.38

Fig. 5.39

Fig. 5.37: A 90-days-old callus upon transfer to 60 mM NaCl concentration (1.1x) Fig. 5.38: Callus culture (day 120) with reddish-brown patches (1.22x) Fig. 5.39: Brownish-yellow callus with reduced proliferation at day 150 (1.1x)

108 Fig. 5.40-5.42: Callus morphology of sugarcane (cv. HSF 240) at 80 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.40

Fig. 5.41

Fig. 5.42

Fig. 5.40: A 90-days-old callus culture transferred to 80 mM NaCl concentration (1.1x) Fig. 5.41: Brownish-yellow callus with some off-white portion at day120 (1.2x) Fig. 5.42: A 150-days-old yellowish-brown necrotic (category A) callus (1.1x) Fig. 5.43-5.45: Callus morphology of sugarcane (cv. HSF 240) at 100 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.43

Fig. 5.44

Fig. 5.45

Fig. 5.43: Translucent callus showing signs of necrosis at 100 mM NaCl concentration (1x) Fig. 5.44: A callus culture with some black portion due to necrosis at day 120 (1.2x) Fig. 5.45: Category A necrosis observed in a callus culture at day 150 (0.8x)

109 Fig. 5.46-5.48: Callus morphology of sugarcane (cv. HSF 240) at 120 mM NaCl level supplemented to MS medium at day 90, 120 and 150 under dark at 27 2 C

Fig. 5.46

Fig. 5.47

Fig. 5.48

Fig. 5.46: Brownish off-white callus at day 90 (1.2x) Fig. 5.47: A 120-days-old category A necrotic callus with a small whitish portion (1.4 x) Fig. 5.48: A callus culture at 120 mM NaCl (day 150) that has already turned dark brown to black in color (1x)

Fig. 5.49-5.50: Callus morphology of sugarcane (cv. HSF 240) at 140 and 160 mM NaCl level supplemented to MS medium at day 90 under dark at 27 2 C

Fig. 5.49

Fig. 5.50

Fig. 5.49: Callus necrosis at 140 mM NaCl level at day 90 (1.1x) Fig. 5.50: A 90-days-old dry and necrotic callus culture at 160 mM NaCl (1x)

110 Table 5.2: Morphology of sugarcane callus cultures (cv. HSF 240) at different NaCl levels (0-160 mM) supplemented to MS medium under dark conditions at day 90, 120 and 150 a
Callus morphology before NaCl treatment

Salt concentration (mM) 0

Morphology of sugarcane callus cultures after NaCl treatment

At day 90 Greenish-yellow, friable, granular Greenish-yellow, friable, granular Brownishyellow, friable, granular Brownishyellow, friable, granular Brownishyellow, friable, granular Brownish-off white, translucent Brownish offwhite, friable, granular Blackish-brown, necrotic Brownish-black, necrotic At day 120 Greenishyellow, friable, granular Off white with some brown portion, friable, granular Greenishbrown, friable, granular Brownish off white, friable, granular Brownish offwhite, friable, granular Blackishbrown, friable, granular Blackishbrown, necrotic ND b At day 150 Translucent, yellowish-green Yellowish-brown, friable, granular Greenish-brown, smooth Yellowish-brown, smooth Brownish with some off-white portion, friable, granular Brownish-black, smooth surfaced

At day 60
Greenish-yellow, friable, granular Greenish-yellow, friable, granular Greenish-yellow, friable, granular Greenish-yellow, friable, granular Greenish-yellow, friable, granular

20

40

60

80

100

Greenish-yellow, friable, granular

120

Greenish-yellow, friable, granular Greenish-yellow, friable, granular Greenish-yellow, friable, granular

Brownish-black, necrotic ND b

140

160

ND b

ND b

Callus morphology is based on 60 culture vessels per NaCl treatment. ND: Not determined because callus cultures could not be maintained at these salt levels over successive subcultures.
b

111

5.2 Effect of Salt Stress on Fresh Weights, Callus Browning and Necrosis in Sugarcane Callus Cultures
To study the effect of different concentrations of NaCl on callus cultures of sugarcane, 60-days-old proliferating callus cultures were shifted to an already standardized callus maintenance medium (C3; MS medium + 13.52 M 2,4-D) supplemented with different NaCl concentrations (0-160 mM NaCl; 9 combinations) as explained in Materials and Methods (Section 3.6.3). The effect of salt stress in terms of fresh weight (g), browning and necrosis of the callus cultures of the two cultivars, i.e., cv. SPF 234 and cv. HSF 240 is shown in Table 5.3 and 5.4, respectively. A significant effect of the tested media was recorded on fresh weights of callus cultures at day 90, 120 and 150 after NaCl treatment. It was observed that callus cultures of both the cultivars showed a gradual decrease in fresh weights with an increase in salt concentration at day 90, 120 or 150 as compared to the control. During different subcultures, fresh weights of the calluses of both sugarcane cultivars maintained at 0 mM NaCl level (control), increased slightly up to day 150, i.e., the final day of data collection. The callus cultures of cv. SPF 234 at day 120 up to 60 mM NaCl level had slightly greater fresh weights as compared to callus cultures maintained on the same NaCl level at day 90. At day 150, once again reduction was observed in fresh weights of callus cultures subjected to various NaCl concentrations. Almost similar growth trend was observed in callus cultures of cv. HSF 240. However, at 40 or 60 mM NaCl levels, fresh weight of callus cultures at day 120 had similar values (1.06 g). For this cultivar, once again reduction was observed in fresh weights of NaCl-treated callus cultures as compared to the control at day 150. The overall significance thus depicts reduced callus growth with an increasing salt level.

112 Table 5.3: Effect of different NaCl levels (0-160 mM) supplemented to MS medium on fresh weights of sugarcane (cvs. SPF 234 and HSF 240) callus cultures under dark conditions at day 90, 120 and 150 Fresh weight of callus cultures A (g) Cultivars SPF 234 Days 120 1.29 a 1.20 b 1.17 bc 1.19 c 1.07 d 1.05 d 1.02 e 1.02 e ND B * 7 and 232 HSF 240 Days 120 1.26 a 1.18 b 1.06 c 1.06 c 1.07 c 0.99 d 1.02cd ND B ND B * 6 and 203

Medium

Medium Composition 90

150 1.29 a 1.17 b 1.04 c 1.05 c 1.02 d 1.01 d 1.01 d 0.98 e ND B * 7 and 232

90 1.22 a 1.12 b 1.13 b 1.12 b 1.04 c 1.01cde 1.02cd 0.90de 0.97 e * 8 and 261

150 1.30 a 1.02 c 1.0 d 1.0 d 1.05 b 0.98d 0.98d ND B ND B * 6 and 203

S1 S2 S3 S4 S5 S6 S7 S8 S9

C3 + 0 mM NaCl C3 + 20 mM NaCl C3 + 40 mM NaCl C3 + 60 mM NaCl C3 + 80 mM NaCl C3 +100 mM NaCl C3 +120 mM NaCl C3 +140 mM NaCl C3 +160 mM NaCl Significance df

1.24 a 1.14 b 1.13 b 1.13 b 1.10 c 1.04 d 1.04 d 1.03 d 0.93 e * 8 and 261

Data presented here are the means of 60 culture vessels per NaCl treatment (20 replicates per treatment and the experiment was repeated thrice). Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. Data were transformed using 3y (where y is the fresh weight) to normalize the data. Non-transformed mean values are presented. B ND represents that the value could not be determined due to complete callus necrosis. *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

113 The effect of salt stress on browning and necrosis of callus cultures was also recorded at different salt treatments (Fig 5.1-5.50). The callus cultures maintained on salt-free S1 medium showed very little or no necrosis (category E). A gradual transition towards category A or B necrosis was quite evident at higher salt concentrations. A clear difference in salt tolerance level of both the cultivars was also observed. Browning and necrosis equivalent to category A initiated at salt concentration 120 mM and 100 mM at day 150 for cv. SPF 234 and HSF 240, respectively. For cv. SPF 234, category A necrosis was observed at day 90, i.e., after first subculture to the salt-containing medium at 140 mM NaCl while for cv. HSF 240, it took only 30 days or less for complete browning of callus cultures (category A necrosis) at 120 mM NaCl level. Complete callus necrosis was observed for both cultivars at 160 mM salt concentration at day 90. During the present work, callus necrosis was quantified according to the details given in Materials and Methods (section 6.3). Although this parameter was explained under different categories (A-E Scales), qualitative characters (such as color, morphology, etc) were found difficult to quantify. During the experiments, callus cultures with unique morphological characters were also commonly observed (Fig. 5.38) which were very difficult to place in a particular category. It was also noted that callus necrosis and fresh weights were also affected during different subcultures. Salt-treated calluses of both the cultivars showed an increase in callus necrosis and decrease in fresh weight with the passage of time (at day 120 and 150). At day 150, the callus cultures became too necrotic (category A; especially at higher salt concentrations) that they could not be maintained any longer.

114 Table 5.4: Effect of different concentrations of NaCl (0-160 mM) supplemented to MS medium on callus browning and necrosis in sugarcane under dark conditions at day 90, 120 and 150 a Callus necrosis (Scales A-E) b Medium Medium Composition SPF 234 Days 90 S1 S2 S3 S4 S5 S6 S7 S8 S9
a

Cultivars HSF 240 Days 150 E D C C B B A A ND c 90 E E D C B B A A A 120 E D D D C B A ND c ND c 150 E C C B B A A ND c ND c

120 E D D C C B B A ND c

C3 + 0 mM NaCl C3 + 20 mM NaCl C3 + 40mM NaCl C3 + 60mM NaCl C3 + 80 mM NaCl C3 +100 mM NaCl C3 +120 mM NaCl C3 +140 mM NaCl C3 +160 mM NaCl

E E E D C C B A A

Data on callus browning and necrosis were based on 60 culture vessels per NaCl treatment (20 replicates per treatment and the experiment was repeated thrice). b Callus necrosis ( Scales A-E); A: 81-100 %, B: 61-80 %, C: 41-60 % D: 21-40 %, E: 0-20 % callus necrosis. c ND: Data could not be recorded due to complete callus necrosis.

115

5.3 Effect of Salt Stress on Regeneration Potential of Sugarcane Callus Cultures

The callus cultures after two subcultures on MS medium supplemented with various NaCl concentrations were shifted to the regeneration medium to determine the regeneration potential of the salt-treated calluses. The data to this effect are given in Table 5.5 and 5.6. No regeneration was recorded on salt-containing medium during the present work. It was, however, observed that in both the sugarcane cultivars under study (cv. SPF 234 and cv. HSF 240), shoot regeneration was possible on salt-free MS regeneration medium. The regeneration frequency and number of shoots per culture vessel was less in the plants regenerated from NaCl-treated callus cultures than the control (calluses grown on 0 mM NaCl concentration). It is evident from Table 5.5 that callus cultures of cv. SPF 234 showed 82 % regeneration after treatment with 100 mM salt concentration but this frequency decreased sharply at 120 mM salt concentration and only 10 % callus cultures retained regeneration potential. In Table 5.6 is given the regeneration potential of 120-days-old NaCltreated callus cultures of cv. HSF 240. It is evident from the data that 75 % of the callus cultures were capable of plant regeneration after 100mM NaCl treatment. Regeneration after a months treatment with 120 mM NaCl supplemented to MS medium was not possible in this cultivar. Shoot initiation and development from the callus culture of cv. SPF 234 treated with different NaCl levels is shown in Fig. 5.51-5.54 while Fig. 5.55-5.58 shows the initiation and development of shoots from a callus culture of cv. HSF 240 treated with various NaCl concentrations. It was also observed during the present study that the number of regenerated shoots from callus cultures treated with various NaCl concentrations was generally greater as

116 compared to the control calluses maintained at 0 mM NaCl. For cv. SPF 234, the number of regenerated shoots from the callus cultures without NaCl was 42 per culture vessel (Table 5.5). At all the NaCl levels tested (where plant regeneration was possible), the value of regenerated shoots was significantly greater than the control. The number of regenerated shoots for this cultivar after 60 or 80 mM NaCl treatment were almost identical, i.e., 57.67 and 57.78, respectively. The maximum number of shoots was recorded after 100 mM NaCl treatment (60.38). This was indeed a very high number of shoots arising from the callus mass within a single culture vessel. Table 5.6 indicates that for cv. HSF 240, the number of shoots regenerated from callus cultures at 0 mM NaCl level was 44.33. The maximum number of regenerated shoots (65) for this cultivar was observed at 60 mM NaCl level. At the highest salt level showing regeneration potential (100 mM), the number of shoots per culture vessel was recorded to be 63. Except for 20 mM NaCl level, the number of shoots per culture vessel was significantly more as compared to the control. For cv. SPF 234, although there was a significant effect of different salt treatments on the mean number of shoots, but the mean shoot lengths of the regenerated plants from callus cultures treated with different salt concentrations (0-100 mM) were closely related (Table 5.5). The overall effect of NaCl, however, was significant in statistical terms which was perhaps due to higher NaCl levels (120 mM or above) that either tremendously influenced the shoot length or resulted in callus necrosis. A significant reduction was observed in the mean shoot length (0.23 cm) in callus cultures treated with 120 mM NaCl. A significant effect of different NaCl levels on mean shoot length of the regenerated plants was also observed in callus cultures of cv. HSF 240 (Table 5.6). The effect of different salt levels was

117 more prominent on shoot length in this cultivar since almost all values were statistically significantly different from one another. Fig. 5.51-5.54: Shoot regeneration from sugarcane callus (cv. SPF 234) treated with different salt concentrations (0-120 mM)

Fig. 5.51

Fig. 5.52

Fig. 5.53

Fig. 5.54

Fig. 5.51: Initiation of shoot regeneration from a callus treated with 20 mM NaCl at day 25 after shifting to regeneration medium (1.2x) Fig. 5.52: Shoot regeneration at day 30 from a callus treated with 60 mM NaCl (1 x) Fig. 5.53: Transfer of shoots to MS basal medium (1x) Fig. 5.54: Plants regenerated from callus treated with 100 mM NaCl medium shifted to MS basal medium at day 30 (1x) Fig. 5.55-5.58: Shoot regeneration from sugarcane callus (cv. HSF 240) treated with different salt concentrations (0-100 mM)

Fig. 5.55

Fig. 5.56

Fig. 5.57

Fig. 5.58

Fig. 5.55: Shoot initiation from a 40 mM NaCl treated callus culture at day 30 (1.2x) Fig. 5.56: Somewhat elongated shoots at day 15 after transfer to MS basal medium (1x) Fig. 5.57: Regenerated shoots from a callus culture treated with 80 mM NaCl (1.2x) Fig. 5.58: Shoots regenerated from a callus treated with 100 mM NaCl at day 30 (0.8x)

118 Table 5.5: Regeneration potential of 120-days-old NaCl-treated callus cultures of sugarcane (cv. SPF 234) A Salt concentration (mM) 0 20 40 60 80 100 120 140 160 Significance df
A

Regeneration Frequency B (%) 85 74 83 77 75 82 10 0 NDE * 7 and 16

Number of shoots /culture vessel C 42 d 52.69 bc 47.53 c 57.67 ab 57.78ab 60.38 a 48.11 bc 0e NDE * 7 and 352

Shoot length D (cm) 0.97 a 0.85 a 0.93 a 0.99 a 0.93 a 0.89 a 0.23 b 0b NDE * 7 and 352

Data were recorded at day 30, after shifting 120-days-old NaCl-treated callus cultures to the regeneration medium. The regeneration medium consisted of MS (Murashige and Skoog, 1962) basal salts supplemented with 8.87 M BAP and 0.5 M TDZ. The values represent means of 45 culture vessels per NaCl treatment (15 replicates per treatment in an experiment and the experiment was repeated thrice). Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. B Data for regeneration frequency were transformed using arcsin y (where y is the percentage value for regeneration frequency) to normalize the data. Non-transformed mean values are presented. C Data for number of shoots per culture vessel were transformed using log (y) (where y is the number of shoots/culture vessel) to normalize the data. Non-transformed mean values are presented. D Data for shoot length were transformed using 4y (where y is shoot length) to normalize the data. Non-transformed mean values are presented. E ND represents that a particular value could not be determined due to complete callus necrosis. *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

119 Table 5.6: Regeneration potential of 120-days-old NaCl-treated callus cultures of sugarcane (cv. HSF 240) A Salt concentration (mM) 0 20 40 60 80 100 120 140 160 Significance df
A

Regeneration Frequency B (%) 76 60 63 72 70 75 0 ND D ND D * 6 and 14

Number of shoots /culture vessel 44.33 d 45.33 d 49.66 c 65 a 53.66 b 63 a 0e ND D ND D * 6 and 307

Shoot length C (cm) 0.95 a 0.76 d 0.83 c 0.87 c 0.93 ab 0.88 bc 0e ND D ND D * 6 and 307

Data were recorded at day 30, after shifting 120-days-old NaCl-treated callus cultures to the regeneration medium. The regeneration medium consisted of MS (Murashige and Skoog, 1962) basal salts supplemented with 8.87 M BAP and 0.5 M TDZ. The values represent means of 45 culture vessels (15 replicates per treatment in an experiment and the experiment was repeated thrice). Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. B Data for regeneration frequency were transformed using arcsin y (where y is the percentage value) to normalize the data. Non-transformed mean values are presented. C Data for number of shoot length were transformed using arsin (y) (where y is shoot length) to normalize the data. Non-transformed mean values are presented. D ND represents that a particular value could not be determined due to complete callus necrosis *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

120

5.4 Rooting of the Regenerated Plantlets

During the present study, rooting of the regenerated plants from salt-treated callus cultures was also observed. At day 30 of the shifting of callus cultures to the regeneration medium, regenerated plants were shifted to MS basal medium for further development (Fig. 5.53, 5.56). At day 30 of the shifting of shoots to MS basal medium, they were transferred to already standardized rooting medium for both the cultivars (details given in section 4.3). Rooting of the regenerated plants of both the sugarcane cultivars is shown in Fig. 5.59 and 5.60. Fig. 5.59-Fig. 5.60: Rooting of the regenerated plants of sugarcane (cv. SPF 234 and cv. HSF 240) on MS medium supplemented with IBA at day 30 under 16 h photoperiod at 27 2 C

Fig. 5.59

Fig. 5.60

Fig. 5.59: Root initiation in in vitro-grown shoots of cv. SPF 234 at day 20 on MS medium supplemented with 2 M IBA (1.1x) Fig. 5.60: Well-developed roots of the sugarcane plants (cv. HSF 240) regenerated from a callus culture treated with 100 mM NaCl on MS medium supplemented with 1 M IBA at day 30 (1.2x) It is evident from the data given in Tables 5.7 and 5.8 that in both sugarcane cultivars (cv. SPF 234 and cv. HSF 240), no significant difference was observed either for % root formation or the mean number of roots per culture vessel. However, a significant difference

121 in both the cultivars was recorded for the mean root length of the plants regenerated from callus cultures treated with different NaCl levels (Tables 5.7, 5.8). It was observed that in both the cultivars, plants regenerated from calluses treated with higher NaCl concentrations were generally with greater root length as compared to the control plants (regenerated from callus cultures treated with 0 mM NaCl). Plants of cv. SPF 234 had maximum root length of 8.63 cm in regenerated plants from callus cultures treated with 80 mM NaCl concentration as compared to control plants with 7.73 cm root length. In cv. HSF 240, the plants regenerated after 100 mM NaCl treatment also had greater root length (7.82 cm) as compared to control plants with 6.94 cm root length. The maximum root length (8.14) for this cultivar was recorded in the plants regenerated from callus cultures treated with 40 mM NaCl level. The plants from the rooting medium were shifted to MS medium for further maintenance (Fig. 5.61-5.62). Fig. 5.61-5.62: Well-developed in vitro-grown sugarcane plants maintained on MS basal medium under 16 h photoperiod at 27 2 C

Fig. 5.61 Fig. 5.61: In vitro plants of sugarcane (cv. SPF 234) regenerated from callus culture treated with 120 mM NaCl (0.8x)

Fig. 5.62 Fig. 5.62: In vitro-grown plants of cv. HSF 240 regenerated from callus culture treated with 100 mM NaCl level (1x)

122 Table 5.7: Rooting potential of regenerated shoots of sugarcane (cv. SPF 234) under 16 h photoperiod A Salt concentration (mM) 0 20 40 60 80 100 120 140 160 Significance df Root formation (%) B 98 96 97 95 100 97 99 ND D ND D NS 6 and 14 Number of roots/culture vessel 8.80 a 8.84 a 8.80 a 8.22 a 9.17 a 9.48 a 9.44 a ND D ND D NS 6 and 307 Root length C (cm) 7.73 cd 7.61 d 8.33 abc 8.19 abc 8.63 a 8.51 ab 8.50 ab ND D ND ** 6 and 307

Data were recorded at day 30, after shifting the regenerated plants (60-days-old) to the rooting medium. The rooting medium consisted of MS (Murashige and Skoog, 1962) basal salts supplemented with 2.0 M IBA. The values represent means of 45 culture vessels (15 replicates per treatment in one experiment and the experiment was repeated thrice). Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. B Data for root formation (%) were transformed using arcsin y (where y is the percentage value) to normalize the data. Non-transformed mean values are presented. C Data for root length were transformed using log10 (y) (where y is the root length) to normalize the data. Non-transformed mean values are presented. D ND represents that a particular value could not be determined due to complete callus necrosis. **,NS Significant at 5% level (**) or non-significant (NS) according to F test with df mentioned against each.

123 Table 5.8: Rooting potential of regenerated shoots of sugarcane (cv. HSF 240) under 16 h photoperiod A Salt concentration (mM) 0 20 40 60 80 100 120 140 160 Significance df
A

Root formation B (%) 98 99 98 98 99 97 ND D ND D ND D NS 5 and 12

Number of roots /culture vessel 7.95 a 8.69 a 8.11 a 7.44 a 7.77 a 8.0 a ND D ND D ND D NS 5 and 260

Root length C (cm) 6.94 bc 7.34 ab 8.14 a 6.16 c 7.80 ab 7.82 ab ND D ND D ND D * 5 and 260

Data were recorded at day 30, after shifting the regenerated plants (60-days-old) to the rooting medium. The rooting medium consisted of MS (Murashige and Skoog, 1962) basal salts supplemented with 1.0 M IBA. The values represent means of 45 culture vessels (15 replicates per treatment in one experiment and the experiment was repeated thrice). Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. B Data for root formation (%) were transformed using y+1/2 (where y is the percentage value) to normalize the data. Non-transformed mean values are presented. C Data for root length were transformed using arcsin y (where y is the root length) to normalize the data. Non-transformed mean values are presented. D ND represents that a particular value could not be determined due to complete callus necrosis. *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each

124

DISCUSSION
It is evident from the results of this study that 60-days-old callus cultures on MS basal medium were greenish yellow, friable and granular. When 60-days-old calluses were shifted to various NaCl levels (0-140 mM), variation in callus morphology was quite distinct and data to that effect were recorded at day 90, 120 and 150. During the study, it was also observed that with an increase in salt concentration, callus necrosis also increased. Thus our results conform to the earlier findings of Gandonou et al., (2005 a, b) who have also reported that salt stress affect callus morphology. It is not surprising to note that these results are also true for many other members of the family Poaceae, e.g., wheat (Karadimova and Dambova, 1993). Arzani and Mirodjagh (1999) have also observed similar results and reported brown coloration, necrosis and inhibited growth of callus cultures of wheat at higher NaCl concentrations. Jaiwasal and Singh (2001) observed in chick pea that the control treatment (without stress) could establish green and friable callus while NaCl treatments resulted in varying degrees of browning and necrosis depending on the NaCl concentration used. During the present work, a decrease in fresh weight of callus cultures was observed with increasing salt concentration in the medium. Decline of callus growth due to NaCl stress as observed in the present study is a usual phenomenon in many plant tissues subjected to stress (Greenway and Munns, 1980; Reddy and Vaidyanath, 1986; Rains, 1989; Cushman et al., 1990). This retardation of growth may be due to the fact that certain amount of the total energy available for tissue metabolism is channeled to resist the stress (Cushman et al., 1990).

125 In sugarcane, Gandonou et al., (2005 a, b; 2006 ) and Errabii et al., (2006) studied the effect of salt on sugarcane callus cultures and found that fresh weight of calluses decreased with the corresponding increase in the concentration of NaCl in the culture medium. Reddy and Vaidyanath (1986) also reported similar reduction in growth and fresh weights of rice callus cultures in response to salt stress. Apart from sugarcane and other members of the family Poaceae, Andrade et al., (1998), El-Aref et al., (1998) and Farhatullah et al., (2002) also reported that NaCl damages cells and restrict the growth activities in potato at higher salt levels. A gradual decrease in fresh as well as dry weights under salt stress has also been reported by Rahnama et al., (2003) and Agarwal and Pandey (2004) in potato and cassia plants, respectively. Similarly, Nasir et al., (2000) also observed a same decrease in fresh weight of sugarcane seedlings under salt stress. Akthar et al., (2003) and Wahid (2004) reported the decrease in rate and percentage of dry mass of root and shoot under salinity in salt-tolerant and salt-sensitive genotypes of sugarcane. Limited seedling growth has also been found by increasing concentration of NaCl (Sreenivasulu et al., 2000). It was observed that the two sugarcane cultivars used during the present study (cv. SPF 234 and cv. HSF 240) were different in their salt tolerance level. This was quite evident by the data on fresh weight of callus cultures after salt stress treatments. Not only the callus cultures but also the plants growing under saline conditions showed different degrees of salt tolerance at varietal level (Qureshi et al., 1980; Rashid et al., 1999). Errrabii et al., (2006) have reported that significant differences in relative fresh weight growth were observed among different cultivars (R570, CP59-73 and NCo310) of sugarcane. Similar findings were reported for rice (Lutts et al., 1996 a; Basu et al., 2002), wheat (Barakat and Abdel-Latif, 1996) and sunflower (Alvarez et al., 2003) where NaCl stress was shown to reduce callus

126 growth and the fact that different genotypes responded differently. In a similar study, Pandey and Ganapathy (1984) compared callus cultures of two verities of Cicer arietinum (salttolerant and sensitive) at 0-200 mM NaCl concentration. They observed that as a result of salt stress, the cells become adapted to salt stress and show better growth on salt-containing medium as compared to the medium without salt. Relative shoot fresh weight under salinity compared with a control was considered to be the best criteria to determine the salt tolerance in wheat (Qureshi et al., 1990). In this study, despite a reduction in regeneration frequency, callus cultures, nonetheless were capable of regeneration after NaCl treatment. It has earlier been observed that the cells grown under stress (though with reduced cell division) retained the differentiation ability and hence were capable of plant regeneration (Kazuhirosuenga and Yoshida, 1982). So it was observed in this study that salt-treated sugarcane callus cultures had the ability to regenerate up till 120 and 100 mM NaCl treatment in cv. SPF 234 and cv. HSF 240, respectively. A sudden reduction in regeneration frequency at 120 mM NaCl in callus cultures of cv. SPF 234 probably indicated severely damaged cells at that salt concentration. The morphological parameters of the callus cultures were also in line with the reduced regeneration potential. There are reports indicating NaCl-induced decrease in plantlet regeneration in rice (Subhashini and Reddy, 1989; Binh et al., 1992). Vajrabhaya et al., (1989) have also reported regeneration of salt-tolerant rice plants with reduced frequency of 0.075 percent or less for calluses maintained on 1 or 2 % NaCl stress as compared to 8.330 % in control callus cultures. During the present study, regeneration of plants from salt-stressed callus cultures of both the sugarcane cultivars was possible on salt-free medium. It has been observed

127 previously by many workers that the presence of salt in the medium was usually inhibitory to plant regeneration. The plants, therefore, were regenerated on salt-free medium by several workers (Li and Heszky, 1986; Ben- Hayyim and Goffer, 1989). On the contrary, several reports are also available suggesting no effect of NaCl on the regeneration potentiality of cells (Heszky et al., 1986; Reddy and Viddianth, 1986; Beloualy and Bouharmont, 1992). Plant regeneration from salt-stressed calluses have generally been preferred on salt-free medium because in this case not only the salt-tolerant cells but also the other cells that had just been freed from the damaging effect of salt in the medium could also undergo an organized differentiation pattern thus leading to plant regeneration. Binh et al., (1992) have reported that a high regeneration potential (59.6 %) of rice suspension cultures on salt-free medium decreased rapidly with increasing concentration of salt in the regeneration medium. Similar observations have also been reported for other plant groups. For instance, Jaiwasal and Singh (2001) have reported plant regeneration from NaCl-tolerant callus/cell lines of chickpea on salt-free medium. Likewise, our results also indicate that the salt- treated callus cultures had shoot regeneration only in the salt-free medium. An interesting observation during the present study was that all the NaCl-treated sugarcane callus cultures had greater number of shoots per culture vessel when shifted to the regeneration medium as compared to the control. In literature, no such effect of salt stress on number of regenerated shoots has ever been reported. A decrease in shoot number per culture unit, however, is well documented. Working with rice callus cultures, Lutts et al., (1999) observed a decreased number of shoots produced per callus in rice at all salt levels. Results of the present work also indicated that root formation percentage and number of roots per culture vessel were not affected by salt treatments. Salt stress, however,

128 significantly influenced the root length. An increase in root length was recorded in plants regenerated from NaCl-treated callus cultures as compared to the corresponding controls. In contrast, Rodriguez et al., (1997) reported a rapid but transient reduction in growth rates of plant roots under salt stress. It was also observed during this study that the plants regenerated from salt-treated callus cultures were healthy and relatively vigorous. Though much on this aspect is not available on sugarcane, Winicov (1991), however, have also selected salt-tolerant cell lines from Medicago sativa, by a single-step selection process on tissue culture medium containing 1 % NaCl. The regenerated plants were quite vigorous as well as self-fertile. In another similar study in potatoes, Marconi et al., (2001) made a comparative growth analysis between salt-tolerant plants of potato obtained by in vitro recurrent selection methods. Although no difference was observed in growth fourteen days after the saline treatment, the comparative analysis between clones at day 28 revealed intra-specific differences in relative growth rate as well as biomass. In conclusion, results of the salt stress experiments demonstrated three important aspects. Firstly, callus cultures retained the ability to regenerate after salt treatment despite browning and reduction in fresh weights. Secondly, salt treatment resulted in more number of regenerated shoots per cultures vessel. Finally, the plants regenerated from salt-treated callus cultures had greater root lengths though the percentage root formation generally remained unchanged.

CHPATER 6 EFFECT OF SALT STRESS ON SOLUBLE PROTEIN CONTENTS AND ANTIOXIDANT ENZYME AVTIVITIES IN SUGARCANE CALLUS CULTURES AND REGENERATED PLANTS

129

CHPATER 6 EFFECT OF SALT STRESS ON SOLUBLE PROTEIN CONTENTS AND ANTIOXIDANT ENZYME AVTIVITIES IN SUGARCANE CALLUS CULTURES AND REGENERATED PLANTS RESULTS 6.1 Soluble Protein Contents (mg/g tissue) of Sugarcane (Saccharum spp. Hybrid, cv. SPF 234 and cv. HSF 240) Callus Cultures Maintained on 9 Different Salt Stress Treatments (0-160 mM NaCl) Supplemented to MS Medium at Day 90, 120 or 150 under Dark Conditions (27 2 C)
The changes in soluble protein contents (mg/g tissue) of the callus cultures maintained on MS medium containing various salt concentrations are shown in Table 6.1. It is evident from the data that there was a statistically significant difference in soluble protein contents of both the cultivars at various salt levels as compared to the control. For the callus cultures of cv. SPF 234, a gradual decrease in the soluble protein contents was observed at day 90 with a corresponding increase in salt level (Fig. 6.1). In the control callus cultures of this cultivar, the value of soluble protein contents was 3.86 mg/g tissue which decreased gradually and reached 0.93 mg/g tissue at 140 mM salt concentration. Fig 6.1 also indicates that nearly the same trend was observed in the values of protein contents at day 120. However, at day 150, a gradual decrease was observed up till 40mM NaCl (from 2.92 to 1.10 mg/g tissue) but then a slight increase (1.27 mg/g tissue) was recorded in protein contents at 60mM salt concentration. Thereafter, the values of protein contents again showed a decreasing trend till 140 mM NaCl concentration (0.12 mg/g tissue).

130 Fig. 6.2 shows the changing profile of the soluble protein contents of callus cultures of cv. HSF 240 after treatment with various NaCl concentrations at day 90, 120 or 150. Callus cultures of cv. HSF 240 also showed a significant decrease in soluble protein contents with increase in salt concentration at day 90 from 4.43 mg/g protein at 0 mM NaCl (control) to 0.33 at 120 mM NaCl concentration. For this cultivar at day 120, various salt levels had a statistically significant effect upon soluble protein contents of callus cultures. The difference amongst values was somewhat sharper than at day 90. Up till 60 mM salt level, the value of soluble protein contents gradually decreased from 4.06 mg/g tissue (at 0 mM NaCl) to 0.32 mg/g tissue (at 60 mM NaCl). A slight increase in soluble protein contents was then observed at 80 mM NaCl level (0.58 mg/g tissue). Afterwards, once again a decrease was recorded in the values of soluble protein contents. At day 150, it was observed that the value of soluble protein contents of the callus tissues was greatly reduced. Category A necrosis of the callus cultures was recorded at day 90 at 160 mM NaCl concentration for cv. SPF 234 and both at 140 and 160 mM for cv. HSF 240. Using callus cultures at 140 and 160 mM NaCl levels, soluble protein contents approached zero.

131

Fig. 6.1: Soluble protein contents in callus cultures of cv. SPF 234 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C
4 3.5 3 2.5 2 1.5 1 0.5 0 0 20 40 60 80 100 120 140 160

Soluble protein contents (mg/g tissue)

90 days 120 days 150 days

NaCl concentration (mM)

Fig. 6.2: Soluble protein contents in callus cultures of cv. HSF 240 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C
4.5

Soluble protein contents (mg/g tissue)

4 3.5 3 2.5 2 1.5 1 0.5 0 0 20 40 60 80 100 120 140 160

90days 120 days 150 days

NaCl concentration (mM)

132 Table 6.1: Soluble protein contents of sugarcane callus cultures treated with different NaCl concentrations (0-160 mM) at day 90, 120 and 150

Soluble protein contents (mg/g tissue) Days Medium composition 90 Cultivars SPF 234 MS + 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 20 mM NaCl MS + 13.5 M 2,4-D + 40 mM NaCl MS + 13.5 M 2,4-D + 60 mM NaCl MS + 13.5 M 2,4-D + 80 mM NaCl MS + 13.5M 2,4-D +100 mM NaCl MS + 13.5M 2,4-D +120 mM NaCl MS + 13.5M 2,4-D +140 mM NaCl MS +13.5 M 2,4-D +160 mM NaCl Significance df
A

120 Cultivars SPF 234 2.76 a 2.43 ab 2.09 b 2.0 b 1.43 c 0.98 c 0.91 c 0.88 c ND * 7 and 40 HSF 240 4.06 a 2.07 b 1.96 b 0.32 c 0.58 cd 0.26 cd 0.09 d ND ND * 6 and 35

150 Cultivars SPF 234 2.92 a 1.11 bc 1.10 bc 1.27 b 0.78 cd 0.62 d 0.44 de 0.12 e ND * 7 and 40 HSF 240 3.24 a 1.15 b 0.53 b 0.04 c 0.05 c 0.08 c 0.08 c ND B ND * 6and 35

HSF 240 4.43a 3.4 b 3.38 b 3.31 b 2.63 c 1.77 d 0.33 e 0e 0e * 8 and 45

3.86 a 3.37 b 3.06 bc 2.80 c 2.43 d 2.15 d 1.29 e 0.93 e 0f * 8 and 45

Data presented here are the means of 6 values per NaCl treatment. Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. Data were transformed using log (y) (where y is the value of protein content) to normalize the data. Nontransformed mean values are presented. B ND represents that a particular value could not be determined due to complete callus necrosis. *.NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each

133

6.2 Peroxidase Activity (mg/g tissue) of Sugarcane Callus Cultures Maintained on 9 Different Salt Stress Treatments Supplemented to MS Medium at Day 90, 120 or 150 Under Dark Conditions (27 2C)
Peroxidase activities (mg/g tissue) of the callus cultures of both the cultivars (SPF 234 and HSF 240) maintained on nine different salt stress treatments are shown in Table 6.2. For cv. SPF 234, the value of peroxidase activity at day 90 in control callus cultures was 0.023 mg/g tissue. There was an overall increase in peroxidase activity up till 120 mM NaCl (0.084 mg/g tissue). The peroxidase activity then somewhat decreased at 140 mM NaCl level and approached zero at 160 mM level (Fig. 6.3). For this cultivar, the value of peroxidase activity of the callus cultures on the control medium (MS containing 0mM NaCl level) at day 120 was 0.017 mg/g tissue which also showed a general increasing trend with increase in NaCl concentration but up till 80 mM NaCl level and afterwards a decrease was observed in peroxidase activity with an increase in NaCl concentration. Fig. 6.3 also indicates the response of peroxidase activity at day 120 and 150 with increasing salt concentration in the medium. At day 150, the callus cultures maintained at 0 mM NaCl level had peroxidase activity of 0.012 mg/g tissue. At day 150 also, a statistically significantly effect of NaCl treatment was recorded on peroxidase activities of callus cultures. It is also evident from the results given in Table 6.2 that peroxidase activities of the callus cultures of cv. HSF 240 were significantly affected by the addition of salt in the medium. Fig. 6.4 indicates the changes in peroxidase activities in callus cultures of cv. HSF 240 at day 90, 120 and 150. It was also interesting to note that in both the cultivars, the respective value of peroxidase activity at 0 or 20 mM NaCl were exactly the same at day 90. Table 6.2 also indicates that the peroxidase activity increased to 0.054 mg/g tissue at 100 mM NaCl level after which its

134 value remained unchanged at 120 mM NaCl level and approached zero at still higher concentrations. At day 150, all the NaCl-treated callus cultures had less values of peroxidase activity as compared to the peroxidase activity in control callus culture at 0 mM salt concentration (0.022 mg/g tissue).
Fig. 6.3: Peroxidase activity in callus cultures of cv. SPF 234 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C
0.09

Peroxidase activity (mg/g tissue)

0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0 20 40 60 80 100 120 140 160

90 days 120 days 150 days

Salt concentration (mM)

Fig. 6.4: Peroxidae activity in callus cultures of cv. HSF 240

maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C
0.07

Peroxidase activity (mg/g tissue)

0.06 0.05 0.04 0.03 0.02 0.01 0 0 20 40 60 80 100 120 140 160 Salt concentration (mM)

90 days 120 days 150 days

135

Table 6.2: Peroxidase activities of sugarcane callus cultures treated with different NaCl concentrations (0-160 mM) at day 90, 120 and 150 Peroxidase activity A
(mg/g tissue) Days 120 Cultivars SPF 234 MS + 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 20 mM NaCl MS + 13.5 M 2,4-D + 40 mM NaCl MS + 13.5 M 2,4-D + 60 mM NaCl MS + 13.5 M 2,4-D + 80 mM NaCl MS + 13.5 M 2,4-D +100 mM NaCl MS + 13.5 M 2,4-D +120 mM NaCl MS + 13.5 M 2,4-D +140 mM NaCl MS +13.5 M 2,4-D +160 mM NaCl Significance df 0.023 e 0.023 e 0.032 de 0.044 cd 0.040 c 0.049 b 0.084 a 0.042 cd 0f * 8 and 45 HSF 240 0.018 c 0.018 c 0.064 a 0.054 ab 0.039 b 0.054 ab 0.054 ab 0c 0c * 8 and 45 SPF 234 0.017 c 0.044 b 0.052 b 0.072 a 0.075 a 0.071 a 0.045 b 0.024 c ND * 6 and 35 HSF 240 0.022 c 0.027 c 0.022 c 0.018 c 0.044 b 0.059 a 0.065 a ND B ND * 7 and 40 SPF 234 0.012 cd 0.029 ab 0.017 c 0.037 a 0.030 ab 0.026 b 0.014 c 0.008 d ND * 6and 35 HSF 240 0.022 a 0.009 bc 0.015
abc

90 Medium composition

150

0.014 abc 0.017 ab 0.017 ab 0.011 bc ND B ND * 7 and 40

Data presented here are the means of 6 values per NaCl treatment. Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. Data were transformed using log (y) (where y is the value of peroxidase activity) to normalize the data. Non-transformed mean values are presented. B ND represents that a particular value could not be determined due to complete callus necrosis. *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

136

6.3 Catalase Activity (units/ml enzyme) of Sugarcane Callus Cultures Maintained on 9 Different Salt Stress Treatments Supplemented to MS Medium at Day 90, 120 or 150 Under Dark Conditions (27 2C)
The catalase activities of the callus cultures of cv. SPF 234 and cv. HSF 240 are given in Table 6.3. During the study, it was observed that the values of catalase activity of sugarcane cultivar SPF 234 have generally shown an increasing trend from 5.75 units/ml enzyme at 0 mM NaCl level to 20.98 units/ml enzyme at 140 mM NaCl concentration (Fig. 6.5). Almost the same general increasing trend was observed in callus cultures at day 120 as depicted in Fig. 6.5. However, deviation from this general trend in this cultivar was observed at 80 mM NaCl level. At this level, the value of catalase activity was slightly lower (9.31 units/ml enzyme) as compared to catalase activity at 60 mM NaCl (11.37 units/ml enzyme). At day 150, the values of catalase activity were generally less as compared to the catalase activities both at day 90 and 120. For cv. HSF 240, Table 6.3 and Fig. 6.6 indicate an increasing trend in the catalase activity at day 90 up to 100 mM NaCl level in the medium that remained almost the same at the next tested NaCl level, i.e., 18.03 units/ml enzyme before approaching zero at still higher concentrations. It was also observed that callus cultures of cv. HSF 240 at 0 mM had almost similar catalase activities at day 90 and 120 (5.46 and 5.86 units/ml enzyme, respectively). At day 120, a statistically significant effect of different salt concentrations on catalase activity was recorded for callus cultures of cv. HSF 240. The maximum value of catalase activity (11.14 units/ml enzyme) at day 120 was observed at 60 mM salt level. At day 150, no significant effect of different salt concentrations on catalase activities of cv. HSF 240 was observed.

137

Fig. 6.5: Catalase activity in callus cultures of cv. SPF 234 maintained on different NaCl levels at day 90, 120, or 150 under dark conditions at 27 2 C
25 20 15 10 5 0 0 20 40 60 80 100 120 140 160

Catalase activity (units/ml enzyme)

90 days 120 days 150 days

Salt concentration (mM)

Fig. 6.6: Catalase activity in callus cultures of cv. HSF 240 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C
20 18 Catalase activity (units/ml enzyme) 16 14 12 10 8 6 4 2 0 0 20 40 60 80 100 120 140 160 Salt concentration (mM) 90 days 120 days 150 days

138

Table 6.3: Catalase activity of sugarcane callus cultures treated with different NaCl concentrations (0-160 mM) at day 90, 120 and 150
Catalase activity A (units/ml enzyme) Days 120 Cultivars HSF 240 5.46 d 6.25 d 7.72 cd 9.67 bc 11.3 b 18.82 a 18.03 a 0e 0e * 8 and 45 SPF 234 4.63 e 6.57 de 7.55 cde 11.37 abc 9.31bcd 11.75 ab 13.21 ab 14.56 a ND * 7 and 40 HSF 240 5.86 c 4.79 c 6.98 bc 11.32 a 10.58 ab 11.14 a 9.53 ab NDB ND * 6 and 35

Medium composition

90

150

SPF 234 MS + 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 20 mM NaCl MS + 13.5 M 2,4-D + 40 mM NaCl MS + 13.5 M 2,4-D + 60 mM NaCl MS + 13.5 M 2,4-D + 80 mM NaCl MS + 13.5 M 2,4-D +100 mM NaCl MS + 13.5 M 2,4-D +120 mM NaCl MS + 13.5 M 2,4-D +140 mM NaCl MS +13.5 M 2,4-D +160 mM NaCl Significance df 5.75c 6.81c 7.29 c 11.18 b 11.74 b 18.32 a 20.25 a 20.98 a 0d * 8 and 45

SPF 234 2.55 bc 2.48 bc 3.81 ab 2.37 bc 4.10 a 2.25 c 2.85 abc 1.53 c ND * 7 and 40

HSF 240 3.53 a 3.50 a 3.13 a 2.89 a 2.95 a 2.99 a 2.43 a ND ND NS 6and 35

Data presented here are the means of 6 values per NaCl treatment. Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. Data were transformed using log (y) (where y is the value of catalase activity) to normalize the data. Non-transformed mean values are presented. B ND represents that a particular value could not be determined due to complete callus necrosis *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

139

6.4 Superoxide Dismutase Activity (units/mg protein) of Sugarcane Callus Cultures Maintained on 9 Different Salt Stress Treatments Supplemented to MS Medium at Day 90, 120 or 150 Under Dark Conditions (27 2C)
The values of superoxide dismutase activities of sugarcane callus cultures (cvs. SPF 234 and HSF 240) maintained on MS medium supplemented with various salt levels are given in Table 6.4. It is evident from the table that the values of superoxide dismutase activities of both the cultivars were significantly influenced by the addition of salt in MS medium. Fig. 6.7 also depicts the observed changing profile of SOD activities with increasing salt concentration during different subcultures at day 90, 120 or 150 for cv. SPF 234. The SOD activities of the salt-treated callus cultures at day 90 or 120 were generally greater as compared to control callus cultures. At day 90, there was a general tendency towards an increase in superoxide dismutase activity that reached its peak at 120 mM NaCl level (58.02 units/mg protein). Then a decrease (42.69 units/mg protein) in SOD activity was observed at 140 mM NaCl level. Superoxide dismutase contents approached zero at still higher NaCl level, i.e., 160 mM NaCl treatment. With some minor variations, the SOD activity generally increased in cv. SPF 234 under increasing salt levels at day 120. However, at day 150, the differences in SOD activities in NaCl-treated callus cultures were not as pronounced as at day 90 or 120. Consequently, the differences in SOD activities at various NaCl levels were also statistically non-significant at day 150. The SOD activity of the callus cultures of cv. HSF 240 maintained on the medium without salt (0 mM salt level) was found to be less (2.63 units/mg protein) as compared to control callus cultures of cv. SPF 234. The maximum value (25.45 units/mg protein) was observed at 120 mM NaCl, which was again less than superoxide dismutase activity of callus cultures of cv. SPF 234 at the same salt levels. At day 120, superoxide dismutase activity at all

140 the salt levels was statistically different as compared to the control. An increase was recorded in SOD activity of the control callus culture (0 mM NaCl) at day 120 as compared to the control callus culture at day 90 (Fig. 6.8). It is also evident from Fig 6.8 that generally an increase was recorded in SOD activities at day 120 with increasing salt level with the maximum value at 120 mM NaCl level (17.45 units/mg protein). At day 150, once again a significant effect of various NaCl levels was recorded on the values of SOD activities.

141

Fig. 6.7: Superoxide dismutase activity in callus cultures of cv. SPF 234 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2C 60 Superoxide dismutase activity (units/mg protein) 50 40 30 20 10 0 0 20 40 60 80 100 120 140 160 Salt concentration (mM) 90 days 120 days 150 days

Fig. 6.8: Superoxide dismutase activity in callus cultures of cv. HSF 240 maintained on different NaCl levels at day 90, 120 and 150 under dark conditions at 27 2 C
30
Superoxide dismutase axtivity (units/mg protein)

25 20 15 10 5 0 0 20 40 60 80 100 120 140 160 Salt concentration (mM) 90 days 120 days 150 days

142 Table 6.4: Superoxide dismutase activity of sugarcane callus cultures treated with different NaCl concentrations (0-160 mM) at day 90, 120 and 150
Superoxide dismutase activity A (units / mg protein) Days 90 120 Cultivars SPF 234 MS + 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 20 mM NaCl MS + 13.5 M 2,4-D + 40 mM NaCl MS + 13.5 M 2,4-D + 60 mM NaCl MS + 13.5 M 2,4-D + 80 mM NaCl MS + 13.5M 2,4-D +100 mM NaCl MS + 13.5M 2,4-D +120 mM NaCl MS + 13.5M 2,4-D +140 mM NaCl MS +13.5 M 2,4-D +160 mM NaCl Significance df 5.79 de 5.73 de 7.46 de 9.7 d 12.2 d 33.68 c 58.02 a 42.69 b 0 * 8 and 45 HSF 240 2.63 de 5.08 cd 7.74 bc 6.77 bcd 11.08 b 21.92 a 25.45 a 0 0 * 8 and 45 SPF 234 6.20 d 12.97 cd 16.28 c 14.83 c 16.76 c 41.49 b 53.91 a 53.69 a ND * 7 and 40 HSF 240 3.85 d 4.1 cd 5.44 cd 7.46 c 12.64 b 13.15 b 17.45 a NDB ND * 6 and 35 SPF 234 3.96 a 3.11 ab 3.49 a 3.80 a 3.78 a 2.41 b 3.85 a 3.65 a ND NS 7 and 40 HSF 240 3.55 c 6.22 bc 4.01 ab 6.55 a 8.09 a 9.97 ab 4.10 a ND ND * 6and 35 150

Medium composition

Data presented here are the means of 6 values per NaCl treatment. Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. Data were transformed using log (y) (where y is the value of superoxide dismutase activity) to normalize the data. Nontransformed mean values are presented. B ND represents that a particular value could not be determined due to complete callus necrosis. *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

143

6.5 Soluble Protein Contents (mg/g tissue) of the Regenerated Plants of Sugarcane (cvs. SPF 234 and HSF 240)
Table 6.5 shows the soluble protein contents of the regenerated sugarcane plants from callus cultures subjected to various levels of salt stress. Regenerated plants from callus cultures of cv. SPF 234 and cv. HSF 240 grown on 0 mM NaCl level (control) had 2.56 and 2.58 mg/g tissue protein contents, respectively. The maximum value of soluble protein contents for cv. SPF 234 was observed in the plants obtained from callus cultures treated with 20 mM NaCl, whereas, the minimum value of soluble protein contents (1.59 mg/g tissue) was observed in the regenerated plants from callus cultures treated with 60 mM salt concentration. For cv. HSF 240, the maximum value of soluble protein contents (3.04 mg/g tissue) was recorded in the plants regenerated from 100 mM NaCl-treated callus culture.

144 Table 6.5: Soluble protein contents of regenerated plants from 0-160 mM NaCl-treated sugarcane callus cultures Soluble protein contents A (mg/g tissue) Cultivars SPF 234 B 0 20 40 60 80 100 120 140 160 Significance df 2.56a 3.23a 2.73a 1.59 b 2.7a 1.66 b 2.51a ND ND * 6 and 35 HSF 240 C 2.58 ab 2.14 bc 1.38 c 3.22 a 2.65 ab 3.04 ab ND D ND ND * 5 and 30

NaCl treatment (mM)

Data presented here are the means of 6 values per NaCl treatment. Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. B Data of protein content for cv. SPF 234 were transformed using log (y) (where y is the value of protein content) to normalize the data. Non-transformed mean values are presented. C Data of protein contents for cv. HSF 240 were transformed using y (where y is the value of protein content) to normalize the data. Non-transformed mean values are presented. D ND represents that a particular value could not be determined due to complete callus necrosis. *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

145

6.6 Peroxidase, Catalase and Superoxide Dismutase Activities of the Regenerated Plants of Sugarcane (cvs. SPF 234 and HSF 240)
The values of peroxidase, catalase and superoxide dismutase activities of cv. SPF 234 and cv. HSF 240 are given in Table 6.6 and 6.7, respectively. For cv. SPF 234, regeneration of the callus cultures was recorded up till 120 mM NaCl. Plants regenerated from callus cultures of cv. SPF 234 maintained on medium supplemented with 0 or 20mM NaCl had the same peroxidase activity (0.016 mg/g tissue). Plants regenerated from callus cultures treated with 40 mM NaCl showed a slight increase (though non-significant) in peroxidase activity to 0.017mg/g tissue. The maximum value for peroxidase activity (0.04 mg/g tissue) was observed in the plants regenerated from 80 mM NaCl-treated callus cultures. For cv. SPF 234, the control plants had catalase activity of 2.16 units /ml enzyme. The maximum value of catalase activity for this cultivar (4.38 units /ml enzyme), like peroxidase, was also observed in plants regenerated from calluses treated with 80 mM NaCl. The overall effect of various NaCl levels on catalase activity of regenerated plants (derived from 0-160 mM NaCl-treated callus cultures), however was non-significant. It is evident from Table 6.6 that the values of superoxide dismutase activity of the plants regenerated from 0 mM was 3.19 units/mg protein which increased to 7.44 units/mg protein at 40mM NaCl level and afterwards remained almost the same at 60 mM NaCl (7.38 units/mg protein). The maximum value of superoxide dismutase activity (8.74 units/mg protein), was however, observed in the plants regenerated from callus cultures treated with 120 mM NaCl. For cv. HSF 240, the maximum level of salt treatment still allowing some regeneration was 100 mM. For this cultivar, the peroxidase activities of plants regenerated from callus cultures were found to be significantly affected with various salt concentrations. The plants regenerated from callus cultures of cv. HSF 240 treated with different NaCl concentrations,

146 however, did not show significant difference in catalase as well as superoxide dismutase activities (Table 6.7). Table 6.6: Peroxidase, catalase and superoxide dismutase activities of regenerated plants from 0-160 mM NaCl-treated callus cultures of sugarcane (cv. SPF 234) A NaCl treatment (mM) 0 20 40 60 80 100 120 140 160 Significance Peroxidase activity B (mg/g tissue) 0.016 c 0.016c 0.017 c 0.035 ab 0.04 a 0.028 bc 0.015 c ND E ND * Catalase activity C (units/ml enzyme) 2.16 b 2.32b 1.97 b 3.48 ab 4.38 a 3.18 ab 2.75 ab ND ND NS Superoxide dismutase activity D (units/mg protein) 3.19
c

4.98 bc 7.44 ab 7.38

ab

5.05 bc 5.07 bc 8.74 a ND ND **

A

Data presented here are the means of 6 values per NaCl treatment. Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. B Data for peroxidase activity were transformed using y (where y is the value of peroxidase activity) to normalize the data. Non-transformed mean values are presented. C Data for catalase content were transformed using y+1/2 (where y is the value of catalase activity) to normalize the data. Non-transformed mean values are presented. D Data for superoxide dismutase activty were transformed using log (y) (where y is the value of superoxide dismutase activity) to normalize the data. Non-transformed mean values are presented. E ND represents that a particular value could not be determined due to complete callus necrosis. *, NS Significant at 1 % level (*), 5 % level (**) or non-significant (NS) according to F test with 6 and 35 df.

147 Table 6.7: Peroxidase, catalase and superoxide dismutase activities of regenerated plants from 0-160mM NaCl-treated callus cultures of sugarcane (cv. HSF 240) A

NaCl treatment (mM)

Peroxidase activity B (mg/g tissue)

Catalase activity C (units/ml enzyme)

Superoxide dismutase activity D (units/mg protein)

0 20 40 60 80 100 120 140 160 Significance

0.014 b 0.013 b 0.024 ab 0.033 a 0.029 a 0.028 a NDE ND ND *

1.57a 2.32 a 1.60 a 2.08

a

4.55 b 7.71 5.51 5.80 5.05 4.61 ND ND ND NS

A

a ab

ab

1.90 a 2.32 a ND ND ND NS

Data presented here are the means of 6 values per NaCl treatment. Different letters within a specific column represent significant difference at P= 0.05 according to Duncans Multiple Range Test. B Data were transformed using y (where y is the value of peroxidase activity) to normalize the data. Nontransformed mean values are presented. C Data were transformed using y+1/2 (where y is the value of catalase activity) to normalize the data. Non-transformed mean values are presented. D Data were transformed using log (y) (where y is the value of superoxide dismutase activity) to normalize the data. Non-transformed mean values are presented. E ND represents that a particular value could not be determined due to complete callus necrosis. *, NS Significant at 1 % level (*), 5 % level (**) or non-significant (NS) according to F test with 5 and 30 df.

148

DISCUSSION
The present investigation highlights some of the biochemical aspects (soluble protein contents and antioxidant enzyme activities) associated with salt (NaCl) stress in sugarcane callus cultures. It has earlier been suggested that protein contents may be used as a good biochemical marker for analyzing salt stress (Pareek et al., 1997). Consequently, the changes associated with NaCl stress within the affected plant tissues have been reported in the literature (Fadzilla et al., 1997; Fahmy et al., 1998). Evidence exists for both the positive as well as negative correlation between the protein contents and NaCl stress (Lutts et al., 1999; Muthukumarasamy et al., 2000, Niknam et al., 2006). Streb and Feierabend, (1996) and Fidalgo et al., (2004) while working on rye and potato respectively reported that salt stress interferes negatively with protein synthesis both under in vitro and in vivo conditions. Quantitative analysis of the soluble protein contents during the present work indicated that soluble protein contents in the callus cultures of both the sugarcane cultivars (cv. SPF 234 and cv. HSF 240) significantly decreased in response to various NaCl treatments. A somewhat similar trend of decrease in protein contents as a result of salt stress was also observed in rice (Reddy and Vaidyanath, 1986) and chick pea (Ashraf and Waheed, 1993). Salt stress has been reported to stimulate protein hydrolysis and this hydrolysis is considered a primary effect of the salt resulting in decreased protein level (Uprety and Sarin, 1976). Apart from these reports, the available literature also reveals an increase in protein contents in the plant tissues under salt stress (Munns et al., 1979; Skriver and Munday, 1990). Cano et al., (1998) observed that protein contents increased with salinity in different tomato genotypes. Ashraf and OLeary (1999) also reported that total soluble proteins increased in spring wheat due to salt stress in all the tested cultivars. The reason for the increased protein levels according to these workers was

149 thought to be due to some proteins that could have been synthesized in response to salt stress. The introduction of such proteins (generally referred to as stress proteins) could have resulted in an increase in the overall quantity of the soluble proteins in the plant tissues exposed to salt stress. Apart from many other factors, it has also been reported that the phenomenon of salt tolerance is dependent upon the nature of the plant species or cultivars (Ashraf and Harris, 2004). During the present study, it was thus observed that the soluble protein contents of cv. HSF 240 were generally greater as compared to cv. SPF 234 at day 90 but after second and third subcultures on salt-containing medium (i.e., at day 120 and 150), the salt-treated callus cultures of cv. SPF 234 had greater soluble protein contents as compared to cv. HSF 240. It may consequently be suggested that the callus cultures of cv. SPF 234 perhaps acquired greater resistance to salt stress during the salt-adaptation process. Higher soluble protein contents have also been observed in salt-tolerant than in salt-sensitive cultivars of barley (Ramagopal, 1987; Hurkman et al., 1989), sunflower (Ashraf and Tufail, 1995), pea (Olmos and Hellin, 1996) and rice (Lutts et al., 1996 b; Pareek et al., 1997). In sugarcane callus cultures, a general decreasing trend in soluble protein contents was recorded with increase in NaCl concentration in the medium. This decreased protein synthesis in sugarcane callus cultures, especially at higher NaCl levels, might be due to damaged cell growth as a result of salt stress. Considering all the above-mentioned aspects, it can be suggested that not only different plants but different cultivars of the same plant species show variation in their soluble protein contents in response to salt stress. There is now conclusive evidence that production of reactive oxygen species (ROS) is enhanced in plants in response to different environmental stresses including salinity (Ashraf

150 and Harris, 2004). ROS scavenging machineries of the cell are thus vital to overcome salinityinduced oxidative stress. Antioxidant enzymes (peroxidase, catalase and superoxide dismutase) are the main scavengers of ROS. It has been reported by many workers that plants with higher levels of antioxidants, either constitutive or induced, have a greater resistance to oxidative damage (Harper and Harvey, 1978; Dhindsa and Matowe, 1981; Wise and Naylor, 1987; Monk and Davies, 1989; Spychalla and Desborough, 1990; Manda- manchi and Alscher, 1991; Polle and Rennenberg, 1994). It has also been reported in the literature that a relationship exists between total peroxidase activity and changes in cell wall and membrane integrity under salt stress thus indicating an important role of peroxidase in response to salt stress (Bradley et al., 1992; Chen et al., 1993). In plants, it has also been reported that peroxidases are involved in the mechanisms reducing oxidative stress, which enable the plants to withstand salt stress. Peroxidase enzyme also decomposes H2O2 by oxidation of co-substrates such as phenolic compounds and/or antioxidants (Gaspar et al., 1991). Results of the present study indicated that when salt was supplied to the growth media, generally there was a significant increase in peroxidase activity of the callus cultures as compared to the control in both the cultivars. This high activity of peroxidase may, therefore, be correlated with the capability of the cells to quash oxygen-free radicals, which can damage the cell compartment. The increased total peroxidase activities in response to salinity have also been reported by Sancho et al., (1996) in tomato. Dionisio-Sese and Tobita (1998) reported similar results in rice. Sreenivasulu et al., (2000) also reported an increase in peroxidase activity under NaCl stress in two cultivars of millet. The activity of antioxidant enzymes such as peroxidase has also been reported to have

151 increased under salt stress in maize (Lewis et al., 1989), cucumber (Lechno et al., 1997), rice (Lee et al., 2001) and wheat (Sairam et al., 2005). The data for the peroxidase contents of both the sugarcane cultivars at day 90 indicate 3-3.6 times maximum increase in peroxidase activity in 120 mM NaCl-treated callus cultures as compared to the non-treated controls. At day 120, the callus cultures of cv. SPF 234 showed an even more (4 times) increase at 80 mM NaCl level as compared to the control while in cv. HSF 240, relatively less (3 times) increase was recorded at 100 mM NaCl level. Interestingly, almost a similar increasing pattern of peroxidase activity was observed in chickpea by Sheokand et al., (1995). They observed similar peroxidase activity and reported a maximum of 2.5 or 3.0 times increase in peroxidase activity at 50 or 100 mM NaCl concentrations, respectivly. It has been reported earlier that peroxidase activities of the callus cultures depend upon the salt tolerance levels of different cotton cultivars (Gossett et al., 1994 a, b). By comparing the mechanisms of antioxidant production in salt tolerant and sensitive plants, Sreenivasulu et al., (1999) also reported that tolerant wheat cultivar had more peroxidase activity. Likewise, during the present study, the callus cultures of cv. SPF 234, which could tolerate NaCl up to 120mM, showed greater peroxidase activity as compared to cv. HSF 240 whose sub-lethal salt tolerance level was 100mM. On the basis of the results from this study, it may be suggested that the cells subjected to NaCl stress might have developed a better protection against ROS by an increased peroxidase activity. Catalase enzyme is localized in the microbodies, mitochondria and cytosol. Even though catalase primarily is not essential for some cell types under normal conditions, nonetheless, it plays an important role in the acquisition of tolerance to oxidative stress in the

152 adaptive response of cells (Hunt et al., 1998). Catalase is reported to be quite important for the maintenance of normal cellular processes under stress conditions (Guan and Scandalios, 1995). It has been reported that catalase plays an important role in the removal of electrons that can lead to the production of O 2 free radical (Abassi et al., 1998). Some workers have reported decreased catalase activity in response to salinity (Garratt et al., 2002; Muscolo et al., 2003; Neto et al., 2006). These workers have suggested that the protective action of catalase in response to salinity is limited because of its relatively poor affinity towards its substrate and its sensitivity to light (De Gara and Tommasi, 1999). The present investigation, however, indicated that catalase activity of both the sugarcane cultivars under study showed an increasing trend with a correspondingly increasing salt level in the medium. Since it has been reported that catalase dismutates H2O2 (which is toxic to the cells) into non-toxic H2O and O2, therefore, this enhanced level of catalase may be described as a key enzyme involved in detoxification processes linked to H2O2 accumulation as a result of salt stress. The results of our experiments are thus consistent with many previous observations indicating an enhanced catalase activity under different environmental stresses including salt (Gossett et al., 1994 b), heat (El-Shintinawy, 2004) or cold (Scandalios et al., 1984). Catalase has also been found to be an important antioxidant enzyme in cotton (Gossett et al., 1994 a) and barley (Liang et al., 2003). Gossett et al., (1994 b) reported that the activity of catalase under salt stress increased in the callus tissue of the salt-tolerant cultivars of cotton and decreased or remained unchanged in non-tolerant cultivars. The protective role of catalase is also evident from the observation that catalase when injected into the intercellular space of the leaf, can protect the plant against H2O2 (Willekens et al., 1997). Similarly, Bellaire et al., (2000) have also observed increase in catalase activity with the addition of salt in the medium in callus cultures of cotton. Increase in

153 catalase activity was also reported by Vaidyanathan et al., (2003) under salt stress in a salttolerant rice cultivar. El- Baz et al., (2003) have also reported that salt treatment enhanced the catalase activity in cucumber plants. Similar increase in catalase activities as a result of salt stress was also observed in the seedlings of four potato cultivars by Rahnama and Ebrahimzadeh (2005). Our results with the two sugarcane cultivars have indicated that cv. HSF 240 had comparatively more elevation in catalase activity at different salt levels at each subculture as compared to cv. SPF 234. Rhanama et al., (2003) also found varietal differences in catalase activity in different potato cultivars according to their salt tolerance level. Increased catalase activity in response to salt stress as observed during the present study might suggest the synthesis of new enzyme. Therefore, greater catalase activities in salt-treated callus cultures as compared to the control can be correlated to the ability of this enzyme to detoxify the damaging effects of hydrogen peroxide produced under stressed conditions. Superoxide dismutase enzyme also plays a key role in the antioxidative defense mechanism. During electron-transport chain of mitochondria, molecular oxygen gets four electrons to form water accompanied by occasional leaking of a single electron. Superoxide reduces Fe (III) to Fe (II), releasing the iron from storage sites so that it can react with hydrogen peroxide and produce hydroxyl radicals. SOD enzyme converts superoxide to hydrogen peroxide and molecular oxygen. Therefore, SOD activity has been reported to be negatively correlated to the concentration of O2 and H2O2. Another function of superoxide dismutase is to protect dehydratases (dihydroxy acid dehydratase, aconitase, 6phosphogluconate dehydratase and fumarase A and B) against inactivation by the free radical superoxide (Benov and Fridovich, 1998). An increase in superoxide dismutase activity in

154 response to salt stress was observed during the present study. So our results support previous reports indicating increased SOD activity in plants exposed to different environmental stresses, including salinity (Hernandez et al., 1999; Santos et al., 1999; Savoure et al., 1999; Benavides et al., 2000). It has been demonstrated that salt-tolerant cotton (Gosset et al., 1996), barley (Acar et al., 2001), tomato (Shalata and Tal, 1998) and wild beet (Bor et al., 2003) exhibit higher constitutive and induced levels of SOD as compared to their salt-sensitive counterparts. Lee et al., (2001) also reported that in the leaves of the rice plant, salt stress preferentially enhanced the content of H2O2 as well as the activities of SOD enzyme. Rajguru et al., (1999) have also observed that SOD activity is enhanced as a result of salt treatment to cotton ovules. Increase in total SOD activity was also observed in mulberry by Harinasut et al., (2003) who found that SOD activity increase with increasing salinity level up to150 mM NaCl. Bueno et al., (1998) have also found an up-regulation of SOD activity in response to salt stress in tobacco. On the basis of our results, we may also suggest that superoxide dismutases are important enzymes within the antioxidant defense system of sugarcane to detoxify ROS and confer salt tolerance. A comparison of the SOD activities of both the sugarcane cultivars (SPF 234 and HSF 240) indicated an increased SOD activity as compared to the respective control. Sreenivasulu et al., (2000) while comparing the response of antioxidant compounds to salinity stress in salttolerant and salt-sensitive seedlings of foxtail millet observed that under conditions of salt stress, the salt-tolerant cultivar exhibited increased total SOD activity whereas it decreased in acutely salt-stressed seedlings of the sensitive cultivar. Sairam et al., (2002) have observed increase in SOD activity in two wheat genotypes subjected to salinity stress. Vaidyanathan et al., (2003), however, reported decrease in superoxide dismutase activity in seedlings of rice

155 cultivars subjected to salt stress. As mentioned, in our study, both sugarcane cultivars expressed elevated levels of superoxide dismutase activity under NaCl stress, so it may be suggested on this basis as well that none of the two cultivars was extremely salt sensitive. It was interesting to note during the present study that the plants regenerated from salttreated callus cultures had generally more antioxidant enzyme activities as compared to the control plants (regenerated from non-treated callus cultures). Thus it can be suggested that NaCl treatment at the callus level probably triggered the synthesis of antioxidant enzymes which was retained in the regenerated plants from these callus cultures. Although there is no prior study regarding the antioxidant enzymes in sugarcane plants regenerated from NaCltreated calluses but it is well documented that plants with high levels of antioxidant enzymes have more salinity tolerance because of resistance to oxidative damage (Wise and Naylor, 1987, Sehmer et al., 1995). Therefore, it can be suggested that higher antioxidant enzyme activities of the regenerated plants from NaCl-treated callus cultures could result in the increased ability of these plants to withstand NaCl stress. As mentioned before, there might be the production of reactive oxygen species in sugarcane callus cultures such as superoxide (O2-) and hydrogen peroxide (H2O2) in response to NaCl stress as confirmed by previous studies on other plants (Halliwell and Gutteridge, 1985; Fadzilla et al., 1997; Uchida et al., 2002). In order to quench these ROS, elevation was observed in the activities of antioxidant enzymes peroxidase, catalase and superoxide dismutase in NaCl-treated sugarcane callus cultures as compared to the control. Evidently, this increase in antioxidant enzyme activity, however, did not contribute towards the total soluble protein contents of the callus cultures and a decline was hence observed in the soluble protein contents of the callus cultures after NaCl treatment. In conclusion, this investigation has shown

156 that salt tolerance is correlated with higher levels of antioxidant enzyme activities and these enzymes can act as a defense team in protecting the cells from oxidative damage imposed by salt stress. Hence, soluble protein, peroxidase, catalase and superoxide dismutase contents of callus cultures seem quite useful as biochemical parameters of salt tolerance in plants including sugarcane. This study has also shown that healthy and vigorously growing sugarcane were successfully regenerated from calluses maintained under various NaCl levels. The morphological features of the regenerated sugarcane plants as observed in this study have shown promise. Because such higher NaCl levels generally prove lethal for most sugarcane tissues maintained under in vitro conditions, the successful regeneration of sugarcane plants from callus cultures maintained at higher NaCl levels (120 mM in cv. SPF 234 and 100 mM in cv. HSF 240) seems quite a significant step forward. Though not tested till the compilation of present work, it is optimistically assumed that regenerated salt-tolerant plants retain salttolerance characteristics under greenhouse or field conditions. Further studies on salt tolerance characteristics of regenerated plants from this study under either in vitro or greenhouse/field conditions are highly recommended.

CHAPTER 7 ROLE OF POLYETHYLENE GLYCOL (PEG) IN IMPROVING SALT (NaCl) TOLERANCE OF SUGARCANE

157

CHAPTER 7 ROLE OF POLYETHYLENE GLYCOL (PEG) IN IMPROVING SALT (NaCl) TOLERANCE OF SUGARCANE RESULTS 7.1 Effect of Polyethylene Glycol (PEG) Pretreatment on Fresh Weights, Browning and Necrosis in Sugarcane Callus Cultures

In order to study the possible effect of PEG in improving salt tolerance, sugarcane calluses were treated with four different salt concentrations including one as control (0 mM NaCl) after giving 1 % PEG pretreatment. To proceed, experimental design as described in Materials and Methods (section 3.6.4) was followed. The three other salt concentrations tested included one sublethal NaCl level, one above and one below the sublethal NaCl concentration as previously determined for each cultivar during the present investigation. The effect of PEG pretreatment on fresh weights of callus cultures of both the sugarcane cultivars (SPF 234 and HSF 240) is given in Table 7.1. It was observed that although fresh weights of sugarcane callus cultures of cv. SPF 234 were significantly affected by different media tested for this cultivar but no significant change in fresh weights was recorded as a result of PEG pretreatment. At 0 mM NaCl concentration, the fresh weights of PEG-pretreated and nonpretreated callus cultures at day 90 were 1.22 and 1.17 g, respectively. However, at the other salt concentrations tested for this cultivar, the PEG-pretreated callus cultures had somewhat greater fresh weights as compared to non-pretreated callus cultures.

158 Similarly, for cv. HSF 240, although the medium was found to have a significant effect on fresh weights of calluses but PEG pretreatment was found to have an overall nonsignificant effect on fresh weights at day 90. Generally, at all the salt concentrations tested for cv. HSF 240, PEG-pretreated callus cultures had almost the same values for the fresh weights in comparison with the non-pretreated callus cultures.

159 Table 7.1: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on fresh weights of sugarcane callus cultures at day 90 and 120 A Fresh weights of callus cultures C (g) Days 90 SPF 234
MS + 13.5M 2,4-D + 0mMNaCl MS + 13.5M 2,4-D + 80 mM NaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl

Medium composition

PEG Pretreatment B (1%)

120

90 HSF 240

120

_ + _ + _ + _ + _ +

1.22 1.17 Not tested D Not tested D 1.06 1.08 0.97 1.10 0.99 1.01 ** NS NS

1.21 1.23 Not tested D Not tested D 1.04 1.09 1.06 1.04 1.08 1.08 * NS NS

1.20 1.19 1.14 1.14 1.05 1.04 1.04 1.05 Not tested D Not tested D * NS NS

1.10 1.17 0.97 0.95 1.05 1.02 0.90 1.01 Not tested D Not tested D * NS NS

MS + 13.5M 2,4-D + 140 mM NaCl

Effect of medium (with 3 and 152 df) Effect of pretreatment (with 1 and 152 df) Effect of medium x pretreatment (with 3 and 152 df)
A

The above results are based on 20 replicates for each NaCl treatment. B The + or - signs represent respective media supplemented with (+) or without (-) PEG. C Data were transformed using log10 (y) (where y is the value of fresh weight) to normalize the data. Nontransformed mean values are presented. D Not tested because according to the experimental plan only three salt concentrations (sublethal NaCl level, one above and one below the sublethal NaCl concentration) in addition to control (0 mM NaCl) were tested for each sugarcane cultivar. *,**, NS Significant at 1% level (*), 5% level (**) or non-significant (NS) according to F test with df mentioned against each.

160 Table 7.2 depicts the effect of salt stress on callus browning and necrosis of two sugarcane cultivars according to callus necrosis scale A-E. It was observed that callus cultures became necrotic and brown in color when maintained on the medium containing salt. In callus cultures of both the sugarcane cultivars maintained at 0 mM NaCl, most of the callus cultures were without browning and necrosis while some had very little browning and necrosis (category E). No change in callus necrosis was recorded at this salt level after PEG pretreatment and category E necrosis was observed in pretreated as well as non- pretreated callus cultures at 0 mM NaCl. Figures 7.1 and 7.2 show PEG-pretreated and non-pretreated callus cultures at 0 mM NaCl level, respectively.

Fig. 7.1-7.2: Morphology of PEG-pretreated or non-pretreated sugarcane callus (cv. SPF 234) at 0 mM NaCl level at day 120 under 16 h photoperiod at 27 2 C

Fig. 7.1

Fig. 7.2

Fig. 7.1: Off-White non-pretreated callus culture showing category E necrosis at 0 mM NaCl level (1.2x) Fig. 7.2: Callus culture maintained at 0 mM salt level after PEG pretreatment at day 120 (1.4x) When callus cultures of cv. SPF 234 were transferred to the medium containing different salt concentration, it was observed that callus cultures after PEG pretreatment showed less necrosis when subcultured on salt medium as compared to the non-pretreated

161 cultures maintained at the same salt level. Category B necrosis was recorded in nonpretreated callus cultures maintained at 120 mM NaCl level, which showed reduction in callus necrosis (Category C and D) after PEG pretreatment at day 90 and 120, respectively. A comparison of callus necrosis at 100 and 120 mM NaCl levels is depicted in Figures 7.3-7.6. Fig. 7.3-7.4: Morphology of PEG-pretreated sugarcane callus (cv. SPF 234) at 100 mM NaCl level at day 120 under 16 h photoperiod at 27 2 C Fig. 7.5-7.6: Morphology of PEG-pretreated sugarcane callus (cv. SPF 234) at 120 mM NaCl level at day 120 under 16 h photoperiod at 27 2 C

Fig. 7.3

Fig. 7.4

Fig. 7.5

Fig. 7.6

Fig. 7.3: A callus culture without PEG pretreatment at day 120 (1x) Fig. 7.4: Category D necrosis in callus maintained at 100 mM NaCl level at day 120 after PEG pretreatment (1.4x) Fig. 7.5: Brownish-yellow, non-pretreated callus culture maintained at 120 mM NaCl level (1.2x) Fig. 7.6: A callus culture maintained at 120 mM NaCl level after PEG pretreatment (1.1x) PEG pretreatment also resulted in less necrosis at 140 mM NaCl level. A transition from category A to category B necrosis was recorded at this salt concentration both at day 90 and 120. Fig. 7.7 and 7.8 show necrosis in callus culture of cv. SPF treated with 140 or 160 mM NaCl level at day 90.

162 Fig. 7.7-7.8: Morphology of PEG-pretreated sugarcane callus (cv. SPF 234) at 140 mM NaCl level at day 90 under 16 h photoperiod at 27 2 C

Fig. 7.7

Fig. 7.8

Fig. 7.7: A reddish-brown (category A necrosis) non-pretreated necrotic callus culture maintained at 140 mM NaCl level (1.1x) Fig. 7.8: PEG-pretreated callus with some off-white portion (category B necrosis) maintained at 140 mM NaCl at day 90 (1x) Most of the callus cultures of cv. HSF 240 (like the other cultivar) did not show any necrosis regardless of PEG pretreatment at 0 mM NaCl level. However, some of the callus cultures showed signs of necrosis. Generally category E necrosis was recorded in all the pretreated as well as non-pretreated callus cultures at 0 mM NaCl level (Fig. 7.9 and 7.10) Fig. 7.9-7.10: Morphology of PEG-pretreated sugarcane callus (cv. HSF 240) at 0 mM NaCl level at day 120 under 16 h photoperiod at 27 2 C

Fig. 7.9

Fig. 7.10

Fig. 7.9: A non-pretreated callus culture transferred to saltfree medium (1.2x) Fig. 7.10: A PEG-pretreated greenish-yellow callus (category E necrosis) on 0 mM NaCl level at day 120 (1x)

163 No change was observed in callus necrosis percentage at 80 mM where category C necrosis was recorded in pretreated as well as non-pretreated callus cultures both at day 90 as well as 120. Fig. 7.11 and 7.12 indicate the callus morphology of non-pretreated and PEGpretreated callus cultures respectively at 80 mM NaCl level. Fig. 7.11-7.12: Morphology of PEG-pretreated sugarcane callus (cv. HSF 240) at 80 mM NaCl level at day 120 under 16 h photoperiod at 27 2 C

Fig. 7.11

Fig. 7.12

Fig. 7.11: A callus culture maintained at 80 mM NaCl level without PEG pretreatment (1.2x) Fig. 7.12: PEG-pretreated callus at 80 mM salt concentration at day 120 (2x) At 100 mM NaCl level, a transition from scale C to D was recorded after PEG pretreatment both at day 90 as well as 120. A non-pretreated callus culture at day 120 is shown in Fig. 7.13. The brownish off-white callus culture after PEG treatment with less necrosis is shown in Fig. 7.14. Similarly, at 120 mM NaCl concentration, less necrosis (category B) was recorded after PEG pretreatment at day 90 while at day 120, no change in callus necrosis category was recorded. Fig. 7.15 and 7.16 show the non-pretreated and pretreated callus cultures of cv. HSF 240 maintained at 120 mM NaCl concentration at day 90.

164 Fig. 7.13-7.14: Morphology of PEG-pretreated sugarcane callus (cv. HSF 240) at 100 mM NaCl level at day 120 under 16 h photoperiod at 27 2 C

Fig. 7.13

Fig. 7.14

Fig. 7.13: A 120-days-old non-pretreated callus culture transferred to 100 mM NaCl (1.2x) Fig. 7.14: Brownish off-white callus culture maintained at 100 mM NaCl level after PEG pretreatment (1.4x) Fig. 7.15-7.16: Morphology of PEG-pretreated sugarcane callus (cv. HSF 240) at 120 mM NaCl level at day 90 under 16 h photoperiod at 27 2 C

Fig. 7.15

Fig. 7.16

Fig. 7.15: Yellowish-brown non-pretreated callus culture maintained at 120 mM NaCl level (1.1x) Fig. 7.16: PEG-pretreated 90-days-old off-white callus culture showing category B necrosis with browning in the lower portion at 120 mM NaCl concentration (1.2x)

165 Table 7.2: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on callus browning and necrosis in sugarcane (cvs. SPF 234 and HSF 240) callus cultures at day 90 and 120 a Callus necrosis (Scales A-E) c Medium composition PEG Pretreatment b (1%) 90
MS+ 13.5M 2,4-D + 0mMNaCl MS + 13.5M 2,4-D + 80 mM NaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl MS + 13.5M 2,4-D + 140 mM NaCl
a

Cultivars cv. SPF 234 Days 120 E E Not tested d Not tested d C D B D A B 90 E E C C C D A B Not tested d Not tested d 120 E E C C C D B B Not tested d Not tested d cv. HSF 240

_ + _ + _ + _ + _ +

E E Not tested d Not tested d B D B C A B

Data on callus browning and necrosis were based on 20 replicates for each NaCl treatment. The + or - signs represent respective media supplemented with (+) or without (-) PEG. c Callus necrosis ( Scales A-E); A: 81-100 %, B: 61-80 %, C: 41-60 % D: 21-40 %, E: 0-20 % callus necrosis. d Not tested because according to the experimental plan only three salt concentrations (sublethal NaCl level, one above and one below the sublethal NaCl concentration) in addition to control (0mM NaCl) were tested for each sugarcane cultivar.
b

166

7.2 Effect of Polyethylene Glycol (PEG) Pretreatment on Soluble Protein Contents of Sugarcane Callus Cultures
The soluble protein contents of the callus cultures of the two sugarcane cultivars at day 90 were found to be significantly affected both by the medium as well as PEG pretreatment (Table 7.3 and 7.4). It was observed that PEG pretreatment enhanced the biosynthesis of soluble protein contents in the callus cultures of both the sugarcane cultivars as compared to the control callus cultures maintained at the same salt level without PEG pretreatment at day 90 (Fig. 7.17- 7.18). Table 7.3 indicates that at 0 mM salt concentration, the value of soluble protein contents in the PEG-pretreated callus cultures of cv. SPF 234 was 3.71 mg/g tissue, which was little greater than the soluble protein contents of the nonpretreated callus cultures (3.54 mg/g tissue). This increase in the protein content was more pronounced at 140 mM NaCl concentration at which pretreatment resulted in an increase in soluble protein contents from 0.71 mg/g tissue to 1.30 mg/g tissue. At day 120, similar differences were observed in PEG-pretreated and non-pretreated callus cultures of cv. SPF 234 at 0, 100 or 120 mM NaCl concentration except at 140 mM NaCl level. At this NaCl level, in contrast to the other cultivar, a decrease in soluble protein contents from 0.63 mg/g tissue (in pretreated) to 0.53 mg/g tissue (in non-pretreated callus cultures) was recorded. It is evident from Table 7.4 that for cv. HSF 240, different media as well as PEG pretreatment had a significant effect on the soluble protein contents. At day 90, the observed value of soluble protein contents in the control callus cultures (without PEG or salt treatment) was 3.85 mg/g tissue, which showed an increase up to 4.08 mg/g tissue in the PEG-pretreated callus cultures maintained at 0 mM NaCl level. Comparison of the soluble protein contents indicated that at all the salt concentrations tested for cv. HSF 240, the soluble protein

167 contents significantly changed with pretreatment of polyethylene glycol (PEG). Even at 120 mM NaCl concentration, a fairly good amount of soluble protein contents (1.46 mg/g tissue) was recorded in the callus cultures after PEG pretreatment as compared to the non-pretreated callus cultures (0.42 mg/g tissue). However, at day 120, although different media had a significant effect on soluble protein contents but the soluble protein contents were not significantly affected with PEG pretreatment.

168 Table 7.3: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on soluble protein contents of sugarcane (cv. SPF 234) callus cultures at day 90 and 120 PEG Pretreatment A (1%) _ + _ + _ + _ Soluble protein contents B (mg/g tissue) N At day 90 6 6 6 6 6 6 6 3.54 3.71 2.67 3.62 1.56 2.00 0.71 1.30 * ** NS At day 120 2.68 3.28 1.43 1.95 0.47 1.75 0.63 0.53 * * **

Medium composition
MS+ 13.5M 2,4-D + 0 mM NaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl MS + 13.5M 2,4-D + 140 mM NaCl

6 + Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)

A B

The + or - signs represent respective media supplemented with (+) or without (-) PEG. Data were transformed using log (y) (where y is the value of soluble protein contents) to normalize the Significant at 1% level (*), 5% level (**) or non-significant (NS) according to F test with df

data. Non-transformed mean values are presented.

*, **,NS

mentioned against each.

169 Table 7.4: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on soluble protein contents of sugarcane (cv. HSF 240) callus cultures at day 90 and 120 PEG Pretreatment A (1%) _ + _ + _ + _ Soluble protein contents B (mg/g tissue) N At day 90 6 6 6 6 6 6 6 3.85 4.08 2.47 3.38 1.46 2.1 0.42 1.46 * * NS At day 120 2.62 3.05 1.27 1.85 0.96 0.95 0.16 0.42 * NS NS

Medium composition
MS+ 13.5M 2,4-D + 0mMNaCl MS + 13.5M 2,4-D + 80 mM NaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl

6 + Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)

A B

The + or - signs represent respective media supplemented with (+) or without (-) PEG. Data were transformed using y (where y is the value of soluble protein contents) to normalize the data. Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

Non-transformed mean values are presented.

*, NS

170

Fig. 7.17: Effect of Polyethylene glycol pretreatment on soluble protein contents in callus cultures of sugarcane (cv. SPF 234) at day 90 and 120
4 Soluble Protein Contents (mg/g tissue) 3.5 3 2.5 2 1.5 1 0.5 0 0 100 120 NaCl concentration (mM) 140
NP 90 P 90 NP 120 P 120

Fig. 7.18: Effect of Polyethylene glycol pretreatment on soluble protein contents in callus cultures of sugarcane (cv. HSF 240) at day 90 and 120
4.5 Soluble Protein contents (mg/g tissue) 4 3.5 3 2.5 2 1.5 1 0.5 0 0 80 100 120 NaCl concentration (mM)
NP 90 P 90 NP 120 P120

NP 90: Non-pretreated callus cultures at day 90 P 90: Pretreated callus cultures at day 90 NP 120: Non-pretreated callus cultures at day 120 P 120: Pretreated callus cultures at day 120
Note: The same abbreviations as given for above Figures have been used throughout this chapter.

171

7.3 Effect of Polyethylene glycol (PEG) Pretreatment on Peroxidase Activity of Sugarcane Callus Cultures
Peroxidase activities of cv. SPF 234 and cv. HSF 240 are given in Table 7.5 and 7.6, respectively. It is evident from Table 7.5 that both media as well as PEG pretreatment had a statistically significant effect on peroxidase activities of the callus cultures of cv. SPF 234 at day 90. A comparison of the PEG-pretreated and non-pretreated callus cultures at day 90 indicated that generally at each salt concentration, PEG-pretreated callus cultures had relatively greater peroxidase activity as compared to non-pretreated callus cultures maintained at the same salt level (Fig. 7.19). The peroxidase activity in the callus cultures of cv. SPF 234 maintained at 0 mM NaCl level after PEG treatment was 0.020 mg/g tissue which was also somewhat greater than 0.016 mg/g tissue, the peroxidase activity, in nonpretreated callus cultures. The difference in the peroxidase activities was, however, more pronounced in the callus cultures maintained at higher levels of salt concentrations. It may thus be suggested that PEG pretreatment followed by salinity stress generally stimulated the synthesis of peroxidase enzyme in this sugarcane cultivar at day 90. No statistically

significant effect of PEG pretreatment was recorded on the peroxidase activities at day 120 in cv. SPF 234. It is evident from the data depicted in Table 7.6 that the peroxidase activities of callus cultures of cv. HSF 240 showed significant difference with respect to different media both at day 90 and 120. No significant effect of PEG pretreatment on peroxidase activity was recorded for this cultivar at day 90. However, at day 120, a statistically significant difference in peroxidase activities was observed as a result of PEG pretreatment as well as NaCl treatment. Furthermore, a significant combined effect of pretreatment and the medium was

172 also observed in this cultivar at day 120. Maximum value of peroxidase activity was observed at 100mM NaCl level (0.081 and 0.090 mg/g tissue for non-pretreated and PEGpretreated callus cultures, respectively). An increase in peroxidase activity from 0.024 mg/g tissue to 0.037 mg/g tissue was also recorded at 120 mM NaCl level after PEG treatment.

173 Table 7.5: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on peroxidase activity of sugarcane (cv. SPF 234) callus cultures at day 90 and 120 PEG Pretreatment A (1 %) _ + _ + _ + _ Peroxidase activity B (mg/g tissue) N At day 90 6 6 6 6 6 6 6 6 0.016 0.020 0.067 0.087 0.075 0.088 0.058 0.068 * * NS At day 120 0.023 0.020 0.061 0.061 0.075 0.079 0.033 0.042 * NS NS

Medium composition

MS + 13.5M 2,4-D + 0 mM NaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl MS + 13.5M 2,4-D + 140 mM NaCl

+ Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)

A B

The + or - signs represent respective media supplemented with (+) or without (-) PEG. Data were transformed using log (y) (where y is the value of peroxidase content) to normalize the data. Non-transformed mean values are presented. Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against

*, NS

each.

174 Table 7.6: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on peroxidase activity of sugarcane (cv. HSF 240) callus cultures at day 90 and 120 PEG Pretreatment A (1%) _ + _ + _ + _ Peroxidase activity B (mg/g tissue) N At day 90 6 6 6 6 6 6 6 0.020 0 .015 0.084 0.083 0 .085 0 .084 0.054 0.061 * NS NS At day 120 0.022 0.025 0.073 0.082 0.081 0.090 0.024 0.037 * ** *

Medium composition
MS + 13.5M 2,4-D + 0 mM NaCl MS + 13.5M 2,4-D + 80 mM NaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl

6 + Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)

A B

The + or - signs represent respective media supplemented with (+) or without (-) PEG. Data were transformed using y+1/2 (where y is the value of protein content) to normalize the data. Significant at 1% level (*), 5% level (**) or non-significant (NS) according to F test with df

Non-transformed mean values are presented.

*,**, NS

mentioned against each.

175

Fig. 7.19: Effect of Polyethylene glycol on peroxidase activity in callus cultures of sugarcane (cv. SPF 234) at day 90 and 120
0.1 0.09 Peroxidase activity (mg/g tissue) 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0 100 120 140 NaCl concentration (mM)
NP90 P90 N120 P120

Fig. 7.20: Effect of Polyethylene glycol on peroxidase activity in callus cultures of sugarcane (cv. HSF 240) at day 90 and 120
0.1

Peroxidase activity (mg/g tissue)

0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0 80 100 120
NP90 P90 NP120 P120

NaCl concentration (mM)

176

7.4 Effect of Polyethylene glycol (PEG) Pretreatment on Catalase Activity of Sugarcane Callus Cultures
The data for catalase activities at day 90 and 120 for cv. SPF 234 and cv. HSF 240 are given in Table 7.7 and 7.8, respectively. Significant effect of medium as well as PEG pretreatment was observed on catalase activity of the callus cultures of cv. SPF 234 at day 90. Fig. 7.21 indicates increasing trend in catalase activities of callus cultures of cv. SF 234 with increasing salt level as well as with PEG pretreatment. It was observed that catalase activity of the callus cultures maintained at 0 mM NaCl significantly increased from 3.23 units/ml enzyme to 13.54 units/ml enzyme after PEG treatment. A somewhat similar increasing trend (but far less pronounced) in catalase activity was observed as a result of PEG pretreatment at other NaCl levels except for 140 mM NaCl level where catalase activities of both PEG-pretreated as well as non-pretreated callus cultures were almost the same (16.15 units/ml enzyme and 16.97 units/ml enzyme, respectively). At 120 mM NaCl level, the catalase activity of PEG-pretreated callus cultures was 18.18 units/ml enzyme which was greater as compared to the catalase activity of the non-pretreated callus cultures (17.23 units/ml enzyme). Only the medium had a statistically significant effect on catalase activity in sugarcane callus cultures of cv. SPF 234 at day 120. The rest of the parameters, i.e., pretreatment alone or in combination with medium had no influence on catalase activity. The catalase activities of PEG-pretreated and non-pretreated callus cultures of sugarcane cv. HSF 240 are given in Table 7.8. It was observed that at day 90, the catalase activity was significantly affected by the medium as well as PEG pretreatment. Fig. 7.22 indicates the changing profile of catalase activities of PEG-pretreated and non-pretreated callus cultures of cv. HSF 240. An increase was recorded in catalase activity after PEG

177 pretreatment at all the salt concentrations except at 100 mM NaCl level where the catalase activity of PEG-pretreated and non-pretreated callus cultures were almost the same (17.37 and 17.16 units/ml enzyme, respectively). The highest catalase activity (25.31 units/ml enzyme) was recorded for the callus cultures maintained at 120 mM salt concentration after PEG treatment. At day 120, significant difference was also observed in the catalase activities of callus cultures as a result of PEG pretreatment. Catalase activities increased with the increasing NaCl concentration both in pretreated as well as in non-pretreated callus cultures. Non-PEG pretreated callus culture on MS medium without any salt (0 mM NaCl) had 5.86 units/ml enzyme while catalase activity of the PEG-pretreated callus culture at the same salt level was 9.12 units/ml enzyme. Both PEG-pretreated as well as non-pretreated callus cultures at higher NaCl levels had greater catalase activities. The highest catalase activity at day 120 was recorded in callus cultures maintained at 120 mM NaCl level after PEG pretreatment (19.07 units/ml enzyme). .

178 Table 7.7: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on catalase activity of sugarcane (cv. SPF 234) callus cultures at day 90 and 120 Catalase activity B (units /ml enzyme) N At day 90 6 6 6 6 6 6 6 6 3.23 13.54 11.11 15.52 17.23 18.18 16.97 16.15 * * ** At day 120 6.01 9.62 11.96 11.19 7.98 7.99 15.42 16.37 * NS NS

Medium composition
MS+ 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 100 mM NaCl MS + 13.5 M 2,4-D + 120 mM NaCl MS + 13.5 M 2,4-D + 140 mM NaCl

PEG Pretreatment A (1%) _ + _ + _ + _ +

Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)
A B

The + or - signs represent respective media supplemented with (+) or without (-) PEG. Data were transformed using y (where y is the value of catalase activity) to normalize the data. Significant at 1% level (*), 5% level (**) or non-significant (NS) according to F test with df

Non-transformed mean values are presented.

*,**, NS

mentioned against each.

179 Table 7.8: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on catalase activity of sugarcane (cv. HSF 240) callus cultures at day 90 and 120 Catalase activity B (units/ml enzyme) N At day 90 6 6 6 6 6 6 6 6 5.46 12.33 10.81 15.93 17.16 17.37 15.93 25.31 * * At day 120 5.86 9.12 9.67 10.92 8.13 13.17 12.14 19.07 * * NS

Medium composition
MS + 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 80 mM NaCl MS + 13.5 M 2,4-D + 100 mM NaCl MS + 13.5 M 2,4-D + 120 mM NaCl

PEG Pretreatment A (1%) _ + _ + _ + _ +

Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)
A B

The + or - signs represent respective media supplemented with (+) or without (-) PEG. Data were transformed using y (where y is the value of catalase activity) to normalize the data. Nontransformed mean values are presented. Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against

*, NS

each.

180

Fig. 7.21:Effect of Polyethylene glycol on catalase activity in callus cultures of sugarcane (cv. SPF 234) at day 90 and 120
20 18 16 14 12 10 8 6 4 2 0 0 100 120 NaCl concentration (mM) 140 Catalase activity (units/ml enzyme)

NP90 P90 NP120 P120

Fig. 7.22: Effect of Polyethylene glycol on catalase activity in callus cultures of sugarcane (cv. HSF 240) at day 90 and 120
30 Catalase activity (units/ml enzyme) 25 20 15 10 5 0 0 80 100 NaCl concentration (mM) 120
NP90 P90 NP120 P120

181

7.5 Effect of Polyethylene Glycol (PEG) Pretreatment on Superoxide Dismutase Activity of Sugarcane Callus Cultures
The data for changes in superoxide dismutase activities of callus cultures of cv. SPF 234 are given in Table 7.9. It was observed that different levels of NaCl significantly affected superoxide dismutase activity of callus cultures both at day 90 as well as 120. A comparison of superoxide dismutase activities of pretreated and non-pretreated callus cultures maintained at various NaCl concentrations at day 90 revealed interesting data. An increase was recorded in superoxide dismutase activity after PEG pretreatment at each salt level (except for 140 mM NaCl level) though not statistically significant. Fig. 7.23 indicates generally greater peroxidase activity in the pretreated callus cultures of cv. SPF 234 as compared to their nonpretreated controls at day 90 as well as 120. For cv. SPF 234, superoxide dismutase activity of PEG-pretreated callus cultures maintained at 0 mM salt concentration was 6.51mg/g protein, which was quite greater than SOD activity of the non-pretreated callus cultures (1.79 mg/g protein). At day 90, the pretreated callus cultures maintained at 100 and 120 mM NaCl level had somewhat higher values of SOD activity as compared to their non-pretreated controls. However, at 140 mM NaCl level, the value of SOD activity in the non-pretreated callus cultures was comparatively little greater (32.52 mg/g protein) as compared to the PEGpretreated callus culture (31.98 mg/g protein). At day 120, a significant increase in SOD activity was observed after PEG pretreatment at all the salt concentrations tested for cv. SPF 234 (0, 100, 120 or 140 mM NaCl). Non-pretreated callus cultures had maximum SOD activity at 140 mM NaCl level (17.80 mg/g protein) while the highest SOD activity in PEGpretreated callus cultures was at 100 mM NaCl level (20.89 mg/g protein).

182 Table 7.10 (Fig. 7.24) shows the SOD activity in PEG-pretreated and non-pretreated callus cultures of cv. HSF 240 at day 90 and 120. Significant effect of the pretreatment with polyethylene glycol (PEG) on superoxide dismutase activities was observed in the callus cultures of this cultivar at day 90. PEG-pretreated callus cultures had greater superoxide dismutase activities as compared to their non-pretreated controls maintained at the same NaCl level. In non-pretreated callus cultures at 0 mM NaCl concentration, the value of superoxide dismutase activity was 0.79 mg/g protein, which rose to 4.07 mg/g protein in the PEG-pretreated callus cultures. In the callus cultures maintained at 120 mM NaCl, maximum SOD activity was observed in pretreated callus cultures (63.88 mg/g protein). At day 120, however, no significant effect of PEG pretreatment was recorded on the activity of SOD enzyme.

183 Table 7.9: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on superoxide dismutase activity of sugarcane (cv. SPF 234) callus cultures at day 90 and 120 Superoxide dismutase activity B (mg/g protein) N At day 90 6 6 6 6 6 6 6 6 1.79 6.51 15.47 21.52 23.95 25.84 32.51 31.98 * NS NS At day 120 2.17 3.96 11.29 20.89 13.28 18.23 17.80 20.46 * ** NS

Media composition
MS + 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 100 mM NaCl MS + 13.5 M 2,4-D + 120 mM NaCl MS + 13.5 M 2,4-D + 140 mM NaCl

PEG Pretreatment A (1%) _ + _ + _ + _ +

Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)

A B

The + or - signs represent respective media supplemented with (+) or without (-) PEG. Data were transformed using 4y (where y is the value of superoxide dismutase activity) to normalize the Significant at 1% level (*), 5% level (**) or non-significant (NS) according to F test with df

data. Non-transformed mean values are presented.

*,**, NS

mentioned against each.

184

Table 7.10: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on superoxide dismutase activity of sugarcane (cv. HSF 240) callus cultures at day 90 and 120 Media composition
MS + 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 80 mM NaCl MS + 13.5 M 2,4-D + 100 mM NaCl MS + 13.5 M 2,4-D + 120 mM NaCl

PEG Pretreatment A (1%) _ + _ + _ + _ +

Superoxide dismutase activity B (units/mg protein) N At day 90 6 6 6 6 6 6 6 6 0.79 4.07 10.64 10.84 25.79 32.48 45.34 63.88 * At day 120 4.51 4.54 24.05 16.21 30.91 28.93 113.60 121.86 * NS NS

Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)
A B

* NS

The + or - signs represent respective media supplemented with (+) or without (-) PEG.

Data were transformed using log10 (y) (where y is the value of superoxide dismutase activity) to normalize Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

the data. Non-transformed mean values are presented.

*, NS

185

Fig. 7.23: Effect of Polyethylene glycol on superoxide dismutase activity in callus cultures of sugarcane (cv. SPF 234) at day 90 and 120
35 Superoxide dismutase activity (mg/g protein) 30 25 20 15 10 5 0 0 100 120 NaCl concentration (mM) 140
NP90 P90 NP120 P120

Fig. 7.24: Effect of Polyethylene glycol on superoxide dismutase activity in callus cultures of sugarcane (cv. HSF 240) at day 90 and 120
140 Suoeroxide dismutase activity (units/ mg protein) 120 100 80 60 40 20 0 0 80 100 NaCl concentration (mM) 120
NP90 P90 NP120 P120

186

7.6 Effect of Polyethylene Glycol (PEG) Pretreatment on Regeneration Potential of Sugarcane (cv. SPF 234 and cv. HSF 240) Callus Cultures
PEG-pretreated and non-pretreated callus cultures of sugarcane maintained at different salt concentrations (0, 100, 120 or 140 mM for cv. SPF 234 and 0, 80, 100 or 120 mM NaCl for cv. HSF 240) were shifted to an already standardized regeneration medium after 120 days. The effect of PEG pretreatment on regeneration potential of the two sugarcane cultivars (cv. SPF 234 and cv. HSF 240) is depicted in Table 7.11 and 7.12 respectively. It is evident from the data given in Table 7.11 that PEG pretreatment to the callus cultures of cv. SPF 234 had no effect on the regeneration frequency. Shoot regeneration from both PEG- pretreated and non-pretreated callus cultures (though at a much reduced regeneration frequency) was observed at 120 mM NaCl level for cv. SPF 234. Regeneration frequency of the non-pretreated callus cultures at 0 mM NaCl level was 85 % while 82 % regeneration frequency was recorded in PEG-pretreated callus cultures. Similarly, reduction (though more than the one observed for the previous NaCl level) was observed in the regeneration frequency of callus cultures at 100 mM NaCl when pretreated with 1 % PEG. Fig. 7.25 and 7.26 shows shoot initiation from the callus culture treated with 100 mM NaCl. PEG pretreatment also increased the regeneration frequency of the callus cultures from 10 % (the regeneration frequency in non-treated controls) to 13 % at 120 mM NaCl level. Initiation of shoots from pretreated callus cultures and their further development is shown in Fig. 7.27 and 7.28. However, no significant effect of PEG pretreatment was observed on number of regenerated shoots per culture vessel or shoot length.

187 Fig. 7.25-7.26: Shoot regeneration from NaCl-treated callus cultures of sugarcane (cv. SPF 234) after PEG pretreatment on MS basal medium supplemented with 8.87 M BAP + 0.5 M TDZ at day 30 under 16 h photoperiod at 27 2 C

Fig. 7.25

Fig. 7.26

Fig. 7.25: Pretreated callus culture with a high number of regenerating shoots at day 30 upon transfer to regeneration medium after treatment with 100 mM NaCl level (1.5x) Fig. 7.26: Well-developed shoots on MS medium regenerated from PEG-pretreated callus culture maintained at 100 mM NaCl (1.2x)

Fig. 7.27

Fig. 7.28

Fig.7.27: Shoot initiation from callus culture of cv. SPF 234 maintained at 120 mM NaCl level after PEG pretreatment (1.4x) Fig. 7.28: Rooting of the regenerated shoots treated with 120 mM NaCl after PEG pretreatment (1.2x)

188 Table 7.11: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on regeneration potential of 120-days-old NaCl-treated callus cultures of sugarcane A (cv. SPF 234) Number of shoots /culture vessel C 42 43 54 62 65 64 0 0 * NS NS

Medium composition
MS + 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 100 mM NaCl MS + 13.5 M 2,4-D + 120 mM NaCl MS + 13.5M 2,4-D + 140 mM NaCl

PEG Pretreatment B (1%) _ + _ + _ + _ +

N 20 20 20 20 20 20 20 20

Regeneration Frequency (%) 85 82 82 73 10 13 0 0

Shoot length D (cm) 0.96 1 0.89 0.95 0.24 0.28 0 0 *

Effect of medium (with 3 and 136 df) Effect of pretreatment (with 1 and 136 df) Effect of medium x pretreatment (with 3 and 136 df)
A B

NS NS

Data were recorded at day 30 upon transfer to the regeneration medium. The + or - signs represent respective media supplemented with (+) or without (-) PEG. C Data for shoot number were transformed using y (where y is the value of mean shoot number) to normalize the data. Non-transformed mean values are presented. D Data for shoot length were transformed using arcsiny (where y is the value of mean shoot length) to normalize the data. Non-transformed mean values are presented. *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

189 Table 7.12 indicates that the salt-treated callus cultures of cv. HSF 240 had regeneration potential up to 100mM NaCl level with 82 % and 75 % regeneration in callus cultures with or without PEG pretreatment, respectively. PEG pretreatment increased the regeneration frequency from the callus cultures treated with 0 mM NaCl from 76 % to 83 %. In callus cultures maintained at 80 mM salt level, PEG pretreated callus cultures had 83 % regeneration frequency as compared to 70 % regeneration frequency in callus cultures to which no PEG treatment was given. Fig. 7.29-7.31 shows the different stages of regeneration from pretreated callus cultures treated with different NaCl concentrations. Although variation in number of shoots per culture vessel and shoot lengths was recorded for this cultivar after PEG pretreatment but like cv. SPF 234 these differences were not statistically significant. 7.29-7.31: Shoot regeneration from NaCl-treated callus cultures of sugarcane (cv. HSF 240) on MS basal medium supplemented with 8.87 M BAP + 0.5 M TDZ at day 30 under 16 h photoperiod at 27 2 C

Fig. 7.29

Fig. 7.30

Fig. 7.31

Fig. 7.29: Shoot initiation from callus culture of cv. SPF 234 maintained at 80 mM NaCl level after PEG pretreatment (1x) Fig. 7.30: Bunch of shoots developed from callus culture maintained at 100 mM NaCl level after PEG pretreatment (1.5x) Fig. 7.31: Well-developed in vitro-grown plants from callus cultures treated with 100 mM NaCl concentration after PEG pretreatment (1.2x)

190 Table 7.12: Effect of medium and/or Polyethylene glycol (PEG) pretreatment on regeneration potential of 120-days-old NaCl-treated callus cultures of sugarcane A (cv. HF 240) PEG Pretreatment B (1%) _ + _ + _ + _ + Regeneration Frequency (%) 76 83 70 83 75 82 0 0 Number of shoots /culture vessel C 44 60 47 72 54 67 0 0 * Shoot length D (cm) 0.95 0.94 0.93 0.94 0.88 0.89 0 0 * NS NS

Medium composition
MS + 13.5 M 2,4-D + 0 mM NaCl MS + 13.5 M 2,4-D + 80 mM NaCl MS + 13.5 M 2,4-D + 100 mM NaCl MS + 13.5 M 2,4-D + 120 mM NaCl

N 20 20 20 20 20 20 20 20

Effect of medium (with 3 and 136 df) Effect of pretreatment (with 1 and 136 df) Effect of medium x pretreatment (with 3 and 136 df)
A

NS NS

Data were recorded at day 30 upon transfer to the regeneration medium. The + or - signs represent respective media supplemented with (+) or without (-) PEG. C Data for shoot number were transformed using y (where y is the value of mean shoot number) to normalize the data. Non-transformed mean values are presented. D Data for shoot length were transformed using 1/y (where y is the value of mean shoot length) to normalize the data. Non-transformed mean values are presented. *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.
B

191

DISCUSSION
There are many reports which indicate that plants exposed to one kind of stress often show tolerance to other stresses (cross-tolerance). For example, salt stress has been reported to stimulate cold hardiness in potato (Solanum tuberosum L.) and spinach (Spinacia oleracea L.) seedlings (Ryu et al., 1995); water stress induced chilling resistance in rice (Oryza sativa L.) (Takahashi et al., 1994); heat stress has also been reported to increase tolerance to several abiotic stresses in plants (Sabehat et al., 1998). On the basis of these observations, this experiment was planned to observe the effect of osmotic stress induced by Polyethylene glycol on NaCl tolerance of callus cultures of sugarcane. During the present experiment, a single molecular form of Polyethylene glycol (PEG 4000) was used to simulate the osmotic effects. PEG is a polymeric osmoticum, which, unlike NaCl, does not penetrate plant cells and is relatively non-toxic (Chazen and Neumann, 1994). Plant responses to salt and water stress have much in common. Several halophytic plants respond similarly when subjected to seawater or PEG, indicating that most of the effects can be ascribed to low osmotic potentials and not due to salinity (Jefries, et al., 1979). Salinity reduces the ability of plants to take up water, and this quickly causes reduction in growth rate, along with a number of metabolic changes identical to those caused by water stress (Munns, 2002). Therefore, it can be suggested that exposure to salt or drought stress triggers many common reactions in the plant cell. Both stresses lead to cellular dehydration, which causes osmotic stress and removal of water from the cytoplasm into extra cellular space resulting in the reduction of cytosolic and vacuolar volumes. However, it has also been reported that salt stress at equivalent osmotic potentials is lethal as compared to osmotic stress. Hampson and Simpson (1990) observed that wheat seeds remain viable for a

192 considerable period under water stress but not under salt stress. Alam et al., (2002) have also observed that in rice cultivars, the onset of germination, germination rate and seedling growth, all declined with increasing concentrations of both NaCl and PEG, but the NaCl stress was more inhibitory. Results of the present study indicated that regardless of the PEG pretreatment, calluses subjected to salt stress showed less fresh weight as compared to control. It has been shown that both stresses cause inhibition of growth of various calluses (Ben-Hayyim and Kochba, 1882, 1883; Ben-Hayyim et al., 1985). The reason is that the cells grown under any kind of stress may have to spend more metabolic energy than those grown in the absence of stress (Croughan et al., 1981). This extra energy most probably is used up in regulating osmotic adjustment thus decreasing the growth of the cells. In plants, rapid (essentially instantaneous) changes in growth rates have been observed with a sudden change in salinity. Rapid and transient reductions in leaf expansion rates after a sudden increase in salinity have been recorded in maize (Cramer and Bowman, 1991; Neumann, 1993), rice (Yeo et al., 1991) and wheat and barley (Passioura and Munns, 2000). The same changes occur in plants when polyethylene glycol (PEG) is applied (Yeo et al., 1991; Chazen et al., 1995). So our results for reduction in fresh weights of callus cultures after PEG and NaCl treatment are inline with these earlier findings in other plants. It is evident from the results that PEG pretreatment increases the tolerance of sugarcane callus cultures to NaCl. The callus cultures after PEG pretreatment showed less necrosis as compared to NaCl-treated callus cultures. So it can be suggested that the damage caused by PEG is relatively less as compared to NaCl. Drought pretreatment induced by PEG has been reported previously to increase the tolerance to the osmotic effect, the main effect

193 induced by salinity in a number of crops e.g., parsley (Pill, 1986; Pill and Kilian, 2000), sunflower (Chojnowski et al., 1997), pea seeds (Sivritepe and Eris, 2000), carrot, lettuce and onion (Yeon et al., 2000). In tomato and carrot cell lines, the relationship between the degree of resistance to water stress and that of tolerance to salt has been described by Bressen et al., (1981) and Harms and Oertli (1985). It has been reported that increased tolerance to NaCl conferred increased tolerance to osmotic stress in tobacco cells (Heyser and Nabros., 1981; Binzel et al., 1985;). It was observed by these workers that tomato and carrot cells which have been selected for better growth in increasing PEG concentration also showed enhanced ability to grow in the presence of NaCl. Gonzalez-Fernandez (1996) also reported that tomato plants, which had previously been subjected to a drought stress pretreatment, were able to grow better than non-pretreated plants after 21 days of salt treatment. Similarly, Balibrea et al., (1999) found a positive effect on salt tolerance of tomato plants proceeding from pretreated seedlings with PEG. Ashraf et al., (2003) studied the effect of PEG and NaCl on improvement of salt tolerance of pearl millet and observed that soaking of seeds in PEG solution increased the final germination percentage of seeds under salinized conditions but the rate of germination (days to 50 % germination) was unaffected. PEG pretreatment was also able to promote salt tolerance of transgenic tobacco plants (Chen et al., 2005). It has also been reported that increased tolerance to NaCl conferred increased tolerance to osmotic stress in tobacco cells (Heyser and Nabros., 1981; Binzel et al., 1985). In all the above-mentioned reports, a strong correlation was observed between salt and PEG- induced water stress. On the other hand, some workers were of the view point that plant cells might show variable response in their performance under salt or water stress. No correlation, hence, was observed between salt and PEG-induced water stress in tomato (Cayuela et al., 1996), Atriplex

194
(Katemb et al., 1998) and soybean (Khajeh-Hosseini et al., 2003). Tal and Katz (1980) have

also reported that a cell line of tomato, which exhibited enhanced tolerance to NaCl, was less resistant to PEG than a cultivated NaCl-sensitive cell line. The actual mechanism of PEG-induced salt tolerance is not well understood. The reason is that adaptation to any kind of stress is a complex process and involves numerous physiological and biochemical changes (Guy, 2003). However, some workers suggest that specific proteins are induced by one kind of stress that is involved in the protection against other kinds of stresses (Pareek et al., 1995; Sabehat et al., 1998.) An important consequence of NaCl stress is the production of reactive oxygen species, which effects cellular metabolism negatively. This aspect has already been discussed at length in chapter 6 of this manuscript. Prevention of oxidative damage to cells during stress has been suggested as one of the mechanisms of stress tolerance, which is attributed to enhanced antioxidant enzyme activity (Senaratna et al., 1988; Kraus and Fletcher, 1994). Many workers have reported increase in the activity of antioxidant enzymes in response to high salinity (Bowler et al. 1992, Hernandez et al. 1993, 1994; Gossett et al., 1994, a,b; Olmos et al., 1994). Water stress has also been reported to cause an enhancement in the generation of ROS and antioxidant defenses (Smirnoff, 1993; Bartoli et al., 1999; Loggini et al., 1999). It was observed during the experiments of this study that pretreatment with PEG enhanced the levels of antioxidant enzymes (peroxidase, catalase and SOD) in the callus cultures subjected to NaCl treatment. Keeping this background information in view, it may be suggested that non-toxic PEG stress may trigger a few (or perhaps more) biochemical changes. In this study, it enhanced the antioxidant enzyme activities that could have resulted in better growth parameters and resistance of callus cultures to elevated salt levels.

195 On the basis of above discussion, it is quite evident that such an effect of PEG on the biochemical aspects using calluses/plants is a new observation in sugarcane in general and the cultivars tested in particular. The PEG pretreatment to the callus cultures maintained on various NaCl concentrations resulted in decreased callus necrosis as well as increased activities of antioxidant enzymes (peroxidase, catalase and superoxide dismutase) in sugarcane. PEG pretreatment also improved the regeneration frequency of the salt-treated callus cultures. Therefore, it can be suggested that PEG pretreatment to callus cultures can be helpful in alleviation of salt stress in both sugarcane cultivars (cv. SPF 234 and cv. HSF 240).

CHAPTER 8 ROLE OF ASCORBIC ACID IN IMPROVING SALT (NaCl) TOLERANCE OF SUGARCANE

196

CHAPTER 8 ROLE OF ASCORBIC ACID IN IMPROVING SALT (NaCl) TOLERANCE OF SUGARCANE RESULTS
To investigate the effect of ascorbic acid on salt tolerance of sugarcane, the experiments were performed both on callus cultures as well as in vitro plants of sugarcane (cvs. SPF 234 and HSF 240). To study the effect of ascorbic acid pretreatment on callus cultures, 60-days-old calluses were shifted to the medium containing 0.5 mM Ascorbic acid. The duration of ascorbic acid pretreatment was either 24 or 48 hours. No callus culture of cv. SPF 234 survived whereas only 1 % culture survival was observed for cv. HSF 240 after ascorbic acid pretreatment for 48 hours. Consequently, 48 h pretreatment of callus cultures with 0.5 mM ascorbic acid was no longer continued. To analyze the effect of ascorbic acid pretreatment, in vitro-grown plants were pretreated with ascorbic acid for 24 hours and then shifted to MS medium supplemented with NaCl. The data recorded for various parameters of study after 24 hours of ascorbic acid pretreatment of the callus cultures and in vitro plants of sugarcane and further maintained at various salt levels is given below:

8.1 Effect of Ascorbic Acid Pretreatment on Fresh weight, Browning and Necrosis in Sugarcane Callus Cultures
The data for fresh weights of callus cultures after ascorbic acid pretreatment and their non-pretreated controls at day 90 is given in Table 8.1. It is clear from the data that both medium as well as ascorbic acid pretreatment had a significant effect on fresh weights of

197 callus cultures of both the sugarcane cultivars (cvs. SPF 234 and HSF 240). The control callus cultures of cv. SPF 234 (without ascorbic acid or salt treatment) had 1.24 g fresh weight at day 90 while the callus cultures subjected to ascorbic acid pretreatment and then shifted to callus maintenance medium without salt had 1.01 g fresh weight. The fresh weights of callus cultures maintained at 100 mM salt concentration were 0.94 g and 1.06 g for ascorbic acid pretreated and non-pretreated callus cultures, respectively. Likewise ascorbic acid pretreatment also caused callus necrosis and some decrease in fresh weight at 120 or 140 mM NaCl. Callus cultures of cv. HSF 240 also showed decreased fresh weights after ascorbic acid pretreatment. The pretreated callus cultures maintained at 80 mM NaCl level had a decrease in fresh weight from 1.09 to 0.88 g. At the highest salt concentration tested for this cultivar (120 mM NaCl), 0.84 g fresh weight of ascorbic acid-pretreated callus cultures was recorded. These results suggest that ascorbic acid pretreatment causes decrease in fresh weights of sugarcane callus cultures. The callus necrosis after ascorbic acid pretreatment in terms of callus necrosis scales A-E was also recorded for both the sugarcane cultivars (Table 8.2). All the callus cultures of cv. SPF 234 maintained at 0 mM NaCl level showed category E necrosis as depicted in Fig. 8.1. The callus cultures showed browning and necrosis with the addition of NaCl in the medium (Fig. 8.2-8.4). Category C necrosis was observed in the ascorbic acid pretreated callus cultures at 0 mM (Fig. 8.5). Increased necrosis was recorded in the callus cultures after ascorbic acid pretreatment and category A necrosis was observed at 100, 120 or 140 mM NaCl level.

198 Fig. 8.1-8.4: Morphology of Ascorbic acid non-pretreated sugarcane callus cultures (cv. SPF 234) at 0, 100, 120 or 140 mM NaCl level at day 90 under 16 h photoperiod at 27 2 C

Fig. 8.1

Fig. 8.2

Fig. 8.3

Fig. 8.4

Fig. 8.1: A control callus culture maintained at 0 mM NaCl level (1.2x) Fig. 8.2: Non-pretreated callus culture maintained at 100 mM NaCl level (1.4x) Fig. 8.3: A callus culture maintained at 120 mM NaCl level (1x) Fig. 8.4: Callus morphology at 140 mM NaCl level (1.4x) Fig. 8.5-8.8: Morphology of Ascorbic acid-pretreated sugarcane callus cultures (cv. SPF 234) at 0, 100, 120 or 140 mM NaCl level at day 90 under 16 h photoperiod at 27 2 C

Fig. 8.5 Fig. 8.6 Fig. 8.7 Fig. 8.5: Some browning initiating (arrow) at 0 mM NaCl (1.2x) showing necrosis (1.2 x)

Fig. 8.8

Fig. 8.6: Ascorbic acid-pretreated callus culture at 100 mM NaCl concentration Fig. 8.7: A 90-days-old callus maintained at 120 mM NaCl level after ascorbic acid pretreatment (0.9x) Fig. 8.8: Necrotic callus culture after Ascorbic acid pretreatment at day 90 (1x)

199 No necrosis was recorded in the control non-pretreated callus culture of cv. HSF 240 maintained at 0 mM NaCl level (Fig. 8.9) but ascorbic acid pretreatment resulted in some browning of the callus culture (Fig. 8.13). Similarly, Fig. 8.14 indicates more callus browning and necrosis at 80 mM NaCl concentration after ascorbic acid pretreatment as compared to the non-pretreated control (Fig. 8.10). Even more necrosis (category A) was recorded after ascorbic acid pretreatment in callus cultures at 100 mM NaCl (Fig. 8.11, 8.15). It is also evident from Table 8.2 that both ascorbic acid pretreated and non-pretreated callus cultures had category A necrosis at 120 mM NaCl concentration (Fig. 8.12 and 8.16).

Fig. 8.9-8.12: Morphology of Ascorbic acid non-pretreated sugarcane callus cultures (cv. HSF 240) at 0, 80, 100, or 120 mM NaCl level at day 90 under 16 h photoperiod at 27 2 C

Fig. 8.9

Fig. 8.10

Fig. 8.11

Fig. 8.12

Fig. 8.9: Callus morphology at 0 mM NaCl level (1 x) Fig. 8.10: A 90-days-old callus at 80 mM NaCl level without ascorbic acid pretreatment (1.2 x) Fig. 8.11: A callus culture at 100 mM NaCl level showing category B necrosis (1x) Fig. 8.12: Non-pretreated callus culture maintained at 120 mM NaCl level (1.1 x)

200 Fig. 8.13-8.16: Morphology of Ascorbic acid-pretreated sugarcane callus cultures (cv. HSF 240) at 0, 80, 100, or 120 mM NaCl level at day 90 under 16 h photoperiod at 27 2

Fig. 8.13

Fig. 8.14

Fig. 8.13: Off-white callus with some black portion at 0 mM NaCl showing necrosis after ascorbic acid pretreatment (1.2x) Fig. 8.14: Callus browning after ascorbic acid pretreatment at 80 mM NaCl concentration (0.9x)

Fig. 8.15

Fig. 8.16

Fig. 8.15: Callus maintained at 100 mM NaCl level after ascorbic acid pretreatment (1x) Fig. 8.16: Ascorbic acid-pretreated callus at 120 mM NaCl level (1.2x)

201

Table 8.1: Effect of medium and/or Ascorbic acid pretreatment on fresh weights of sugarcane callus cultures maintained on MS medium supplemented with NaCl at day 90 Fresh weight of callus cultures at day 90(g) B Ascorbic acid Medium pretreatment A Cultivars composition (0.5 mM) SPF 234 HSF 240
MS + 13.5M 2,4-D + 0 mM NaCl MS + 13.5M 2,4-D + 80 mM NaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl MS + 13.5M 2,4-D + 140 mM NaCl

_ + _ + _ + _ + _ +

1.24 1.01 Not tested Not tested 1.06 0.94 1.01 0.90 0.99 0.90 * * NS

1.26 1.01 1.09 0.88 1.05 0.91 0.95 0.84 Not tested Not tested * * NS

Effect of medium (with 3 and 152 df) Effect of pretreatment (with 1 and 152 df) Effect of medium x pretreatment (with 3 and 152 df)

The + or - signs represent respective media supplemented with (+) or without (-) Ascorbic acid. B The results are based on 20 replicates for each treatment. Data were transformed using arcsin y (where y is the value of fresh weight) to normalize the data. Non-transformed mean values are presented. *, NS Significant at 1% level (*) or non-significant (NS) according to F test with df mentioned against each.

202 Table 8.2: Effect of medium and/or Ascorbic acid pretreatment on callus browning and necrosis in sugarcane callus cultures maintained on MS medium supplemented with NaCl at day 90 a Callus necrosis (Scales A-E) c Cultivars SPF 234
MS + 13.5M 2,4-D + 0 mMNaCl MS + 13.5M 2,4-D + 80 mM NaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl MS + 13.5M 2,4-D + 140 mM NaCl
a

Medium composition

Ascorbic acid pretreatment b (0.5 mM) _ + _ + _ + _ + _ +

HSF 240 E C C A B A A A Not tested Not tested

E C Not tested Not tested B A C A C A

Data on callus browning and necrosis were based on 20 replicates for each NaCl treatment. b The + or - signs represent respective media supplemented with (+) or without (-) Ascorbic acid. c Callus necrosis ( Scales A-E); A: 81-100 %, B: 61-80 %, C: 41-60 % D: 21-40 %, E: 0-20 % callus necrosis.

203

8.2 Effect of Ascorbic Acid Pretreatment on Soluble Protein Contents and Antioxidant Enzyme Activities in Callus Cultures of Sugarcane (cv. SPF 234) 8.2.1 Soluble Protein Contents
The effect of ascorbic acid pretreatment on soluble protein contents of sugarcane callus cultures (cv. SPF 234) is depicted in Table 8.3. It was observed that soluble protein contents of the callus cultures were significantly affected by ascorbic acid pretreatment. A gradual decreasing trend was observed both with increasing salt level as well as ascorbic acid pretreatment (Fig. 8.17). Thus it can be suggested that ascorbic acid pretreatment had resulted in a decrease in soluble protein contents in this sugarcane cultivar. This decrease was significant even at 0 mM NaCl level where a decrease in soluble protein contents from 3.54 mg/g tissue to 1.43 mg/g tissue was recorded after ascorbic acid pretreatment. Similar reduction in the soluble protein contents was observed at 100 mM NaCl level where ascorbic acid pretreated callus cultures had less value of soluble protein contents (1.24 mg/g tissue) as compared to non-pretreated controls (2.67 mg/g tissue). However, the pretreated callus cultures at 120 mM NaCl level had somewhat greater soluble protein contents (1.67 mg/g tissue) than non-treated callus cultures (1.56 mg/g tissue) although this was not statistically significant. At 140 mM salt concentration, once again ascorbic acid pretreatment reduced the soluble protein contents of the callus tissue.

8.2.2 Peroxidase Activity

Table 8.3 also shows the changes in peroxidase activities of ascorbic acid pretreated and non-pretreated callus cultures of cv. SPF 234 at day 90. Ascorbic acid pretreatment to callus cultures of this cultivar had a significant effect on peroxidase activity at day 90. An overall significant effect of medium as well as pretreatment was observed in callus cultures

204 of cv. SPF 234. Fig. 8.18 indicates a decreased peroxidase activity in the callus cultures after ascorbic acid pretreatment as compared to non-pretreated callus cultures. Pretreated callus cultures showed decrease in peroxidase activity at all the salt concentrations tested for this cultivar except for 140 mM NaCl level. At 140 mM NaCl, the peroxidase activity of the nonpretreated callus cultures was 0.06 mg/g tissue which increased to 0.07 mg/g tissue in the ascorbic acid pretreated callus cultures.

8.2.3 Catalase Activity

The activity of catalase enzyme in callus cultures of cv. SPF 234 at day 90 was found to be significantly affected by both the medium as well as ascorbic acid pretreatment either alone or in combination. At 0 mM salt level, the catalase activity in the ascorbic acid pretreated callus cultures was greater (5.33 units/ml enzyme) as compared to the nonpretreated controls (3.23 units/ml enzyme). A slight increase (from 11.10 to 12 units/ml enzyme) was recorded in the callus cultures after ascorbic acid pretreatment at 100 mM NaCl level. However, non-pretreated callus cultures at 120 mM salt level had higher value of catalase activity (17.23 units/ml enzyme) as compared to pretreated callus cultures (12.67 units/ml enzyme). Similarly, decrease in catalase activity was also recorded after ascorbic acid pretreatment of the callus cultures from 16.97 units/ml enzyme to 8.33 units/ml enzyme at 140 mM NaCl level. Fig. 8.19 also indicates the graphical representation of catalase activities in ascorbic acid-pretreated and non-pretreated callus cultures.

205

8.2.4 Superoxide Dismutase Activity

Although superoxide dismutase activity of the callus cultures of cv. SPF 234 was significantly affected by various salt levels, no significant effect of ascorbic acid pretreatment was observed on superoxide dismutase activity in the callus cultures (Table 8.3). It is evident from Fig. 8.20 that the ascorbic acid-pretreated callus cultures had greater peroxidase enzyme activity as compared to their non-pretreated control except at 120 mM NaCl level. At 0 and 100 mM NaCl level, an increase in superoxide dismutase activity (5.2 units/mg and 24.52 units/mg protein) in ascorbic acid pretreated callus cultures was recorded as compared to 1.80 and 15.47 units/mg protein in non-pretreated controls at the same salt levels. Afterwards, at 120 mM salt concentration, decrease was observed in superoxide dismutase activity (19.33 units/mg protein) of the callus cultures after ascorbic acid pretreatment as compared to non-treated callus cultures (23.96 units/mg protein). At 140 mM salt level, the non-treated callus cultures had 32.41 units/mg protein superoxide dismutase activity which increased relatively considerably to 51.67 units/mg protein in ascorbic acid pretreated callus cultures.

206 Table 8.3: Effect of medium and/or Ascorbic acid pretreatment on soluble protein contents, peroxidase, catalase and superoxide dismutase activities of sugarcane (cv. SPF 234) callus cultures maintained on MS medium supplemented with NaCl at day 90 Ascorbic acid pretreatment A (0.5 mM) _ + _ + _ + _ Soluble Protein contents (mg/g tissue) 3.54 1.43 2.67 1.24 1.56 1.67 0.71 0.37 * * * Peroxidase activity (mg/g tissue) 0.02 0.01 0.07 0.01 0.07 0.02 0.06 0.07 * * * Catalase activity (units/ml enzyme) 3.23 5.33 11.10 12 17.23 12.67 16.97 8.33 * ** ** Superoxide dismutase activity (units/mg protein) 1.80 5.2 15.47 24.52 23.96 19.33 32.41 51.67 * NS NS

Medium composition

N 6 6 6 6 6 6 6 6

MS + 13.5M 2,4-D + 0mMNaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl MS + 13.5M 2,4-D + 140 mM NaCl

+ Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)

The + or - signs represent respective media supplemented with (+) or without (-) Ascorbic acid Significant at 1% level (*), 5% level (**) or non-significant (NS) according to F test

*, NS

207

Fig. 8.17
Soluble Protein contents (mg/g tissue) 4 3.5 3 2.5 2 1.5 1 0.5 0 0 100 120 140 NaCl concentration (mM)
NP P

Fig. 8.18
0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0 100 120 140 NaCl concentration (mM) Peroxidase activity (mg/g tissue)

NP P

Fig. 8.19
20 18 16 14 12 10 8 6 4 2 0 0
NaCl concentration (mM)
60 Superoxide dismutase activity (units/mg protein) 50 40 30 20 10 0

Fig. 8.20

Catalase activity (units/ml enzyme)

NP P

NP P

100

120

140

0 100 120 140 NaCl concentration (mM)

NP: Non-pretreated P: Ascorbic acid pretreated

Note: The same abbreviations as given for above Figures have been used throughout this chapter

Fig. 8.17: Effect of Ascorbic acid pretreatment on soluble protein contents of callus cultures of sugarcane (cv. SPF 234) at day 90 Fig. 8.18: Effect of Ascorbic acid pretreatment on peroxidase activity of callus cultures of sugarcane (cv. SPF 234) at day 90 Fig. 8.19: Effect of Ascorbic acid pretreatment on catalase activity of callus cultures of sugarcane (cv. SPF 234) at day 90 Fig. 8.20: Effect of Ascorbic acid pretreatment on superoxide dismutase activity of callus cultures of sugarcane (cv. SPF 234) at day 90

208

8.3: Effect of Ascorbic Acid Pretreatment on Soluble Protein Contents and Antioxidant Enzyme Activities in Callus Cultures of Sugarcane (cv. HSF 240) 8.3.1 Soluble Protein Contents
Table 8.4 indicates effects of media and/or ascorbic acid pretreatment on soluble protein contents, peroxidase, catalase and SOD activities in callus cultures of cultivar HSF 240. There was a significant effect of medium on soluble protein contents. It was interesting to note that there was no significant effect of ascorbic acid pretreatment on soluble protein contents of the callus cultures. However, a combined effect of medium as well as pretreatment had a statistically significant effect on soluble protein contents. Ascorbic acid pretreated callus cultures had less soluble protein contents as compared to non-pretreated callus cultures except at 100 mM NaCl level (Fig. 8.21). At 0 and at 80 mM NaCl concentration, the ascorbic acid pretreated callus cultures had less soluble protein contents (2.29 and 0.77 mg/g tissue, respectively) as compared to 3.85 and 2.47 mg/g tissue on the respective media without ascorbic acid pretreatment. In callus cultures maintained at 100 mM NaCl level, it was observed that the value of soluble protein contents after ascorbic acid pretreatment increased to 2.2 mg/g tissue as compared to 1.5 mg/g tissue in non-treated cultures. Similar results were observed for 120 mM salt level at which the value of ascorbic acid pretreated callus cultures had comparatively greater value (0.47 mg/g tissue) as compared to the control (0.42 mg/g tissue).

8.3.2 Peroxidase Activity

Callus cultures of cv. HSF 240 (like the other cultivar) also showed reduction in peroxidase activity with ascorbic acid pretreatment (Fig. 8.22). It is evident from Table 8.4

209 that the peroxidase activity of the non-pretreated callus cultures at 80 or 100 mM was exactly the same (0.08 mg/g tissue) that showed a decreasing trend to 0.04 and 0.01 mg/g tissue in the corresponding pretreated callus cultures. There was no difference in the values of peroxidase activity amongst ascorbic acid pretreated and non-pretreated callus cultures for cv. HSF 240 at 120 mM.

8.3.3 Catalase Activity

Unlike cv. SPF 234, no significant effect of medium or ascorbic acid pretreatment was recorded on catalase activity of callus cultures of cv. HSF 240. The changing profile of catalase activity in ascorbic acid- pretreated and non-pretreated callus cultures is given in Fig. 8.23 which shows generally less catalase activity in callus cultures of cv. HSF 240 after ascorbic acid pretreatment.

8.3.4 Superoxide Dismutase Activity

It is evident from the data that medium had a significant effect on SOD activities in callus cultures of cv. HSF 240. Fig. 8.24 depicts increase in SOD activity with increasing NaCl concentration in the medium. However, ascorbic acid pretreatment had an overall nonsignificant effect on callus cultures (Table 8.4).

210 Table 8.4: Effect of medium and/or Ascorbic acid pretreatment on soluble protein contents, peroxidase, catalase and superoxide dismutase activities of sugarcane (cv. HSF 240) callus cultures maintained on MS medium supplemented with NaCl at day 90 Soluble Peroxidase protein activity B B contents (mg/g (mg/g tissue) tissue) 3.85 2.29 2.47 0.77 1.5 2.2 0.42 0.47 * NS ** 0.02 0.01 0.08 0.04 0.08 0.01 0.05 0.05 * * ** Catalase activity B (units/ml enzyme) 5.4 5.6 12.09 11.33 17.16 16 15.93 16 NS NS Superoxide dismutase activity B (units/mg protein) 0.79 2.80 10.64 5.33 25.79 37 31.08 36.03 * NS NS

Medium composition

Ascorbic acid pretreatment A (0.5 mM) _ + _ + _ + _ +

N 6 6 6 6 6 6 6 6

MS + 13.5M 2,4-D + 0mMNaCl MS + 13.5M 2,4-D + 80 mM NaCl MS + 13.5M 2,4-D + 100 mM NaCl MS + 13.5M 2,4-D + 120 mM NaCl

Effect of medium (with 3 and 40 df) Effect of pretreatment (with 1 and 40 df) Effect of medium x pretreatment (with 3 and 40 df)
A B

NS

The + or - signs represent respective media supplemented with (+) or without (-) Ascorbic acid Data were transformed using arcsiny (where y is the value of enzyme activity) to normalize the data. NonNS

transformed mean values are presented

*, **,

Significant at 1% level (*), 5% level (**) or non-significant (NS) according to F test with df

mentioned against each.

211

Fig. 8.21
Soluble protein contents (mg/g tissue) 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0 80 100 120 NaCl concentration (mM) NP P Peroxidase activity (mg/g tissue)

Fig. 8.22
0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
0 80 100 120 NaCl concentration (mM)

NP P

Fig. 8.23
20 Catalase activity (units/ml enzyme) 18 16 14 12 10 8 6 4 2 0 0 80 100 120 NaCl concentration (mM) NP P Superoxide dismutase activity (units/ mg protein) 40 35 30 25 20 15 10 5 0 0

Fig. 8.24

NP P

80

100

120

NaCl concentration (mM)

Fig. 8.21: Effect of Ascorbic acid pretreatment on soluble protein contents of callus cultures of sugarcane (cv. HSF 240) at day 90 Fig. 8.22: Effect of Ascorbic acid pretreatment on peroxidase activity of callus cultures of sugarcane (cv. HSF 240) at day 90 Fig. 8.23: Effect of Ascorbic acid pretreatment on catalase activity of callus cultures of sugarcane (cv. HSF 240) at day 90 Fig. 8.24: Effect of Ascorbic acid pretreatment on superoxide dismutase activity of callus cultures of sugarcane (cv. HSF 240) at day 90

212

8.4 Effect of Ascorbic Acid Pretreatment on Fresh Weight, Browning and Necrosis in in vitro-grown Plants of Sugarcane (cvs. SPF 234 and HSF 240)
To analyze the effect of ascorbic acid pretreatment on in vitro-grown plants, 60-daysold regenerated plants of cv. SPF 234 and cv. HSF 240 were pretreated with 0.5mM ascorbic acid for 24 hours and then were shifted to MS medium supplemented with various NaCl concentrations for 30 days. The data for fresh weights of plants, number of shoots per culture vessel, shoot length, numbers of roots per culture vessel and root length at day 30 are given in Table 8.5 and 8.6 for cv. SPF 234 and cv. HSF 240, respectively. It is evident from the data given in these tables that fresh weights of both the sugarcane cultivars were significantly affected by the medium as well as pretreatment of ascorbic acid. It was also observed that non-pretreated in vitro-grown plants of cv. SPF could tolerate up to 100 mM NaCl level but the pretreated plants could survive up to 160 mM NaCl concentration. At 0 mM NaCl level, an increase was observed in fresh weights from 0.50 to 0.54 g after ascorbic acid pretreatment of the plants of cv. SPF 234. Fig. 8.25 and 8.26 indicate the effect of ascorbic acid pretreatment on growth of plants of cv. SPF 234.

Fig. 8.25

Fig. 8.26

Fig. 8.25: Non-pretreated control in vitro plants of cv. SPF 234 at 0 mM NaCl level (1.8 x) Fig. 8.26: Ascorbic acid-pretreated plants at day 30 maintained at 0 mM NaCl level (0.5 x)

213 Table 8.5: Effect of medium and/or Ascorbic acid pretreatment on morphological growth parameters of in vitro-grown sugarcane plants (cv. SPF 234) maintained on MS medium supplemented with different concentrations of NaCl at day 30a MS medium + NaCl (mM) 0 20 40 60 80 100 Ascorbic acid pretreatment (0.5 mM) b + + + + + Fresh weight (g) 0.50 0.54 0.47 0.51 0.40 0.44 0.35 0.45 0.31 0.35 0.11 0.20 ND c 0.18 ND c 0.14 ND c 0.12 * * NS Number of shoots/ culture vessel 10.8 12.3 7.5 8.9 6.5 6.4 6.1 7.6 3.5 6.0 2.6 3.3 ND c 3.4 ND c 3.2 ND c 3.0 * ** NS Shoot length (cm) Number of roots/ culture vessel 6.1 5.6 5.4 6.2 5.4 6.0 6.3 5.8 6.4 5.9 3.4 3.9 ND c 4.0 ND c 3.4 ND c 3.6 * NS NS Root length (cm)

9.17 10.48 9.75 10.78 7.35 9.0 5.3 6.7 5.9 6.3 1.9 2.8 ND c 2.7 ND c 2.4 ND c 2.4 * ** NS

4.0 3.6 3.35 2.90 3.8 3.8 3.7 2.7 4.0 2.32 2.5 3 ND c 3.1 ND c 2.8 ND c 2.2 ** ** NS

+ 120 + 140 + 160 + Effect of medium (with 5 and 135 df) Effect of pretreatment (with 1 and 135 df) Effect of medium x pretreatment (with 5 and 135 df)
aa b

The results are based on 20 replicates for each treatment The + or - signs represent respective media supplemented with (+) or without (-) Ascorbic acid. c ND: Not determined *, **, NS Significant at 1 % level (*), 5 % level (**) or non-significant (NS) according to F test

214 Table 8.6: Effect of medium and/or Ascorbic acid pretreatment on morphological growth parameters of in vitro-grown sugarcane plants (cv. HSF 240) maintained on MS medium supplemented with different concentrations of NaCl at day 30a Number of shoots/ culture vessel 9.3 10.7 8.8 9 5.6 6 5.7 8.5 4.2 6.0 2.9 3.4 ND c 3.4 ND c 3.1 ND c ND c * ** NS

MS medium + NaCl (mM) 0 20 40 60 80 100 120 140

Ascorbic acid pretreatment (0.5 mM) a + + + + + + + +

Fresh weight (g) 0.45 0.51 0.43 0.45 0.35 0.38 0.22 0.33 0.23 0.26 0.10 0.16 ND c 0.14 ND c 0.11 ND c ND c * ** NS

Shoot length (cm) 8.7 10.65 8.7 9.7 6.5 6.8 6.8 8.2 6.9 8.3 2.2 3.3 ND c 3.0 ND c 2.7 ND c ND c * ** NS

Number of roots/ culture vessel 7 7.3 5.6 5.6 5.7 6.0 5.9 6.0 5.8 5.6 3.6 4.2 ND c 4.3 ND c 3.9 ND c ND c * NS NS

Root length (cm) 3.05 4.1 2.8 3.4 3.7 3.4 3.0 2.7 3.8 3.0 1.9 2.6 ND c 2.4 ND c 2.1 ND c ND c * NS NS

160 + Effect of medium (with 5 and 126 df) Effect of pretreatment (with 1 and 126 df) Effect of medium x pretreatment (with 5 and 126 df)

The results are based on 20 replicates for each treatment. The + or - signs represent respective media supplemented with (+) or without (-) Ascorbic acid c ND: Not determined *,**, NS Significant at 1% level (*), 5% level (**) or non-significant (NS) according to F test
b

215 It was observed that yellow coloration of the leaves initiated at 60 mM NaCl in nonpretreated plants of cv. SPF 234. Yellow coloration was, however, less pronounced in case of leaves of ascorbic acid-pretreated plants at the same NaCl level (Fig. 8.27). Similarly, more browning was recorded at 100 mM NaCl level in the non-pretreated plants as compared to the ascorbic acid pretreated plants at this salt level (Fig. 8.28 and 8.29).

Fig. 8.27

Fig. 8.28

Fig. 8.29

Fig. 8.27: Ascorbic acid-pretreated in vitro plants maintained at 40 mM NaCl level at day 30 (0.6x) Fig. 8.28: Initiation of browning in plants maintained at 100 mM NaCl concentration after ascorbic acid pretreatment (0.8x) Fig. 8.29: Effect of 100 mM NaCl on non-pretreated plants of cv. SPF 234 (1x)

The data given in Table 8.5 also indicates effects of ascorbic acid pretreatment on other growth parameters of the plants. A significant increase in the number of shoots per culture vessel was observed after ascorbic acid pretreatment. At 0 mM NaCl level, the number of shoots in non-pretreated plants was 10.8 that rose to 12.3 after ascorbic acid pretreatment. A somewhat similar trend was observed at other NaCl levels up to 100 mM NaCl level. An increase was observed in shoot lengths of the plants after ascorbic acid pretreatment at all the NaCl levels as compared to their non-pretreated controls. A general

216 decrease in root length was recorded after ascorbic acid pretreatment in the plants of cv. SPF 234 but no significant effect of pretreatment was recorded on number of roots per plant in this cultivar. Non-pretreated plants showed no growth at salt concentrations above 100 mM and plants became completely yellow and dry at 120, 140 or 160 mM NaCl levels while treated plants could tolerate these NaCl levels. Figures 8.30 and 8.31 show the ascorbic acid pretreated and non-pretreated plants of cv. SPF 234 at 160 mM NaCl concentration.

Fig. 8.30

Fig. 8.31

Fig. 8.30: Ascorbic acid-pretreated plants of cv. SPF 234 at 160 mM NaCl level (0.7x) Fig. 8.31: Necrosis of non-pretreated sugarcane plants (cv. SPF 234) at 160 mM NaCl level as a result of NaCl stress (1x)

It was observed that the non-pretreated plants could not be maintained on MS medium supplemented with more than 100 mM NaCl. No data, hence, could be recorded for non-pretreated plants of cv. HSF 240 for these NaCl levels. The pretreated plants of cv. HSF 240, on the other hand, survived at the above-mentioned NaCl levels except the highest tested concentration (160 mM). In cv. HSF 240, a significant reduction was observed in fresh weights with increasing salt concentrations but ascorbic acid pretreatment resulted in a significant increase in the fresh weights of the plants as compared to the non-pretreated

217 control at a particular salt level (Table 8.6). A significant effect of ascorbic acid pretreatment was recorded on number of shoots per culture vessel as well as shoot length. The highest mean number of shoots (10.7) and the highest shoot length (10.65 cm) was recorded at 0 mM NaCl level in ascorbic acid-pretreated plants (Fig. 8.32). Apart from the morphological features shown in Table 8.6, much less yellowing of leaves in ascorbic acid-pretreated plants was observed as compared to non-pretreated plants at the same salt concentration (Fig. 8.338.38). For this cultivar, it was observed that the number of roots per culture vessel as well as root lengths was significantly affected by the medium but no significant effect of pretreatment was recorded on these parameters

Fig. 8.32

Fig. 8.33

Fig. 8.34

Fig. 8.32: Ascorbic acid-pretreated plant of cv. HSF 240 maintained at 0 mM NaCl level at day 30 (0.5x) Fig. 8.33: Plant of cv. HSF 240 after ascorbic acid pretreatment at 40 mM NaCl level (0.5x) Fig. 8.34: Non-pretreated plant of cv. HSF 240 maintained at 60 mM NaCl level showing yellowing of some leaves due to NaCl stress (0.7 x)

218

Fig. 8.35

Fig. 8.36

Fig. 8.35: Necrotic plant of cv. HSF 240 at 100 mM NaCl level without ascorbic acid pretreatment (1x) Fig. 8.36: Ascorbic acid-pretreated plant at 100 mM NaCl level as compared to non-pretreated control at the same NaCl level (1.5x)

Fig. 8.37

Fig. 8.38

Fig. 8.37: Browning of non-pretreated plant at day 15 upon transfer to MS medium supplemented with 140 mM NaCl level (1.5x) Fig. 8.38: Ascorbic acid-pretreated plant upon transfer to MS medium supplemented with 140 mM NaCl at day 15 (1x)

219

8.6 Effect of Ascorbic Acid Pretreatment on Soluble Protein Contents and Antioxidant Enzyme Activities in in vitro-grown Sugarcane Plants (cvs. SPF 234 and HSF 240) 8.6.1 Soluble Protein Contents
The data in Table 8.7 indicate that the soluble protein contents of in vitro-grown plants significantly decrease with increasing NaCl concentration in the medium. A comparison of soluble protein contents of ascorbic acid-pretreated and non-pretreated plants as given in Fig. 8.31 indicated that ascorbic acid pretreatment had a positive effect on soluble protein contents. At each NaCl level, ascorbic acid pretreated plants had greater value of soluble protein contents as compared to non-pretreated controls at that salt level. The ascorbic acid pretreated or non-pretreated plants had 3.18 and 3.90 mg/g tissue soluble protein contents respectively when maintained at 0 mM NaCl level. Even at 100 mM NaCl, an increase was observed in soluble protein contents from 0.04 (in non-pretreated plants) to 0.23 mg/g tissue (in pretreated plants). It is evident from the Table 8.8 that the plants of cv. HSF 240 showed a decrease in soluble protein contents with increasing NaCl concentration in the medium. Data given in Table 8.8 also indicates that ascorbic acid pretreatment had no significant effect on soluble protein contents of this cultivar. However, it is evident from Fig. 8.35 that ascorbic acid pretreated plants maintained at NaCl levels above 40 mM had greater soluble protein contents as compared to their non-pretreated controls maintained at the same NaCl level.

220

8.6.2 Peroxidase Activity

Table 8.7 indicates that the peroxidase activities of ascorbic acid pretreated and nonpretreated plants of cv. SPF 234 at 0 mM NaCl level were almost the same (0.017 and 0.018 mg/g tissue respectively). A general increase in peroxidase activities was recorded both by the medium composition as well as pretreatment at the other salt concentrations tested during the experiment (Fig. 8.32). The highest peroxidase content (0.061 mg/g tissue) was observed in salinized plants at 80 mM NaCl level after ascorbic acid pretreatment. Table 8.8 indicates a significant effect of medium as well as ascorbic acidpretreatment on peroxidase activities of in vitro-grown plants. A considerable increase in peroxidase activities was recorded in the plants of cv. HSF 240 at all the salt levels after ascorbic acid pretreatment (Fig. 8.36). The pretreated plants maintained at 0mM NaCl level had 0.021 mg/g tissue peroxidase activity which was greater than 0.013 mg/g tissue (the peroxidase activity in non-pretreated control). Even at 100 mM NaCl level, the plants showed considerable peroxidase activity with the values of 0.029 and 0.018 mg/g tissue in ascorbic acid pretreated and non-pretreated plants, respectively.

8.6.3 Catalase activity

Data given in Table 8.7 indicate that there was a significant effect of medium as well as pretreatment on the catalase activities in plants of cv. SPF 234. The highest catalase contents (16.56 /ml enzyme) were recorded in plants maintained at 60 mM NaCl level after ascorbic acid pre-treatment, which was followed by 15.94 units/ml enzyme in non-pretreated salinized plants at 60 mM NaCl. Fig. 8.33 indicates that the ascorbic acid-pretreated plants had greater catalase activities as compared to their non-pretreated controls maintained at the same NaCl level.

221 Table 8.8 indicates a significant effect of medium as well as ascorbic acid pretreatment on catalase activities of the plants of HSF 240 maintained at various NaCl concentrations. A comparison of catalase activities of in vitro-grown plants indicated that ascorbic acid pretreatment enhanced the catalase activity (Fig. 8.37). At 0 mM, catalase activity of the ascorbic acid non-pretreated plants was 7.58 units/ml enzyme which increased to 11.55 units /ml enzyme after ascorbic acid pretreatment of the plants maintained at the same NaCl concentration. The highest catalase activities were recorded at 60 mM NaCl level with the values of 16.84 and 15.61 units/ml enzyme in pretreated and non-pretreated plants, respectively.

8.6.4 Superoxide dismutase activity

Ascorbic acid pretreatment was found to have a positive effect on SOD activities of the in vitro-grown plants of cv. SPF 234 (Fig. 8.34). It is depicted in Table 8.7 that the superoxide dismutase activity of the plants maintained at 0 mM NaCl level was 3.64 units/mg protein which increased to 12.79 units/g protein in the ascorbic acid-pretreated plants maintained on the same medium without salt. At all the other salt levels, a significant increase was recorded in SOD activities after ascorbic acid pretreatment except at 20 mM NaCl level where the SOD activities of pretreated and non-pretreated plants were almost the same (15.90 and 15.26 mg/g protein). Although medium had a significant effect on SOD activities but no significant effect of ascorbic acid pretreatment was recorded on SOD activities of the plants of cv. HSF 240 (Table 8.8). However, the enzyme profile as given in Fig. 8.38 indicates that the values of SOD activity in the pretreated plants were generally greater as compared to non-pretreated controls.

222 Table 8.7: Effect of medium and/or Ascorbic acid pretreatment on soluble protein contents, peroxidase, catalase and superoxide dismutase activities of in vitro-grown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl at day 30 a MS medium + NaCl (mM) 0 20 40 60 80 100 120 140 160 Soluble protein contents (mg/g tissue) 3.18 3.90 3.07 3.32 1.70 2.32 1.73 2.46 2.01 2.30 0.04 0.23 ND c 0.20 ND c 0.16 ND c 0.12 * ** NS Catalase activity (units/ml enzyme) 8.39 11.25 13.32 14.67 8.38 13.31 15.94 16.56 9.97 14.23 5.35 10.50 ND c 8.7 ND c 8.0 ND c 7.2 * * NS Superoxide dismutase activity (units/mg of protein) 3.64 12.79 15.26 15.90 12.60 17.24 16.56 17.39 13.22 18.24 9.44 15.05 ND c 14.27 ND c 14.02 ND c 13.35 * * NS

Ascorbic acid pretreatment (0.5 mM) b + + + + + + + +

Peroxidase activity (mg/g tissue) 0.018 0.017 0.031 0.054 0.038 0.054 0.032 0.023 0.037 0.061 0.027 0.041 ND c 0.035 ND c 0.034 ND c 0.034 * ** NS

+ Effect of medium (with 5 and 75 df) Effect of pretreatment (with 1 and 75 df) Effect of medium x pretreatment (with 5 and 75 df)
a b

The results are based on 6 replicates for each treatment. The + or - signs represent respective media supplemented with (+) or without (-) Ascorbic acid c ND: Not determined *, **, NS Significant at 1% level (*), 5% level (**) or non-significant (NS) according to F test

223

Fig. 8.39
4.5 4 3.5 3 2.5 2 1.5 1 0.5 0
0 40 80 120 160 NaCl concentration (mM)
0.07 Peroxidase activity (mg/g tissue) 0.06 0.05 0.04 0.03 0.02 0.01 0

Fig. 8.40

Soluble protein contents (mg/g tissue)

N P P

NP P

0 40 80 120 160 NaCl concentration (mM)

Fig. 8.41
Superoxide dismutase activity (units/mg protein)
18 Catalase activity (units/ml enzyme) 16 14 12 10 8 6 4 2 0 0 40 80 120 160 NaCl concentration (mM) NP P

Fig. 8.42
20 18 16 14 12 10 8 6 4 2 0 0 40 80 120 160 NaCl concentration (mM)

NP P

Fig. 8.39: Effect of Ascorbic acid pretreatment on soluble protein contents of in vitro-grown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Fig. 8.40: Effect of Ascorbic acid pretreatment on peroxidase activity of in vitro-grown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Fig. 8.41: Effect of Ascorbic acid pretreatment on catalase activity of in vitro-grown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Fig. 8.42: Effect of Ascorbic acid pretreatment on superoxide dismutase activity of in vitrogrown plants of sugarcane (cv. SPF 234) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30

224 Table 8.8: Effect of medium and/or Ascorbic acid pretreatment on soluble protein contents, peroxidase, catalase and superoxide dismutase activities of in vitro-grown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl at day 30 a MS medium + NaCl (mM) 0 20 40 60 80 100 120 140 Ascorbic acid pretreatment (0.5mM) b + + + + + +

Soluble protein contents (mg/g tissue) 3.65 3.35 2.60 2.10 2.49 1.86 1.76 1.94 1.61 2.27 0.09 0.39 ND c 0.24 ND c 0.21 ND c ND c * NS NS

Peroxidase activity (mg/g tissue) 0.013 0.021 0.016 0.048 0.031 0.036 0.045 0.040 0.018 0.030 0.018 0.029 ND c 0.027 ND c 0.022 ND c ND c * * NS

Catalase activity (units/ml enzyme) 7.58 11.55 14.86 16.56 10.23 13.68 15.61 16.84 8.85 13.74 3.47 7.93 ND c 7.12 ND c 6.62 ND c ND c * * NS

Superoxide dismutase activity (units/mg of protein) 6.20 7.95 15.68 18.72 18.27 21.33 16.25 16.20 21.33 23.18 10.58 15.62 ND c 14.47 ND c 13.96 ND c ND c * NS NS

+ +

160 + Effect of medium (with 5 and 70 df) Effect of pretreatment (with 1 and 70 df) Effect of medium x pretreatment (with 5 and 70 df)
a b

The results are based on 6 replicates for each treatment. The + or - signs represent respective media supplemented with (+) or without (-) Ascorbic acid c ND: Not determined *, NS Significant at 1% level (*) or non-significant (NS) according to F test

225

Fig. 8.43
4 Soluble protein contents (mg/g tissue) 3.5 3 2.5 2 1.5 1 0.5 0 0 40 80 120 160 NaCl concentration (mM) NP P

Fig. 8.44
0.06 Peroxidase activity (mg/g tissue) 0.05 0.04 0.03 0.02 0.01 0 0 40 80 120 160
NaCl concentrations (mM) NP P

Fig. 8.45
18 16 14 12 10 8 6 4 2 0 0 40 80 120 160 NaCl concentration (mM)

Fig. 8.46
25 20 15 10 5 0 0 40 80 120 160 NaCl concentration (mM)
NP P

NT P

Fig. 8.43: Effect of Ascorbic acid pretreatment on soluble protein contents of in vitrogrown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Fig. 8.44: Effect of Ascorbic acid pretreatment on peroxidase activity of in vitro-grown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Fig. 8.45: Effect of Ascorbic acid pretreatment on catalase activity of in vitro-grown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30 Fig. 8.46: Effect of Ascorbic acid pretreatment on superoxide dismutase activity of in vitro-grown plants of sugarcane (cv. HSF 240) maintained on MS medium supplemented with NaCl concentrations (0-160 mM) at day 30

Superoxide dismutase activity (mg/g protein)

Catalase activity (units/ml enzyme)

226

DISCUSSION
The ability of plants to evolve mechanisms to detoxify toxic chemicals produced inside the cytoplasm allows them to successfully grow in adverse environmental conditions (Weigel et al., 1990). Apart from many other aspects, it has also been suggested that an important consequence of salt stress is the generation of reactive oxygen species (Foyer et al., 1994; Mittler, 2002). It has further been reported earlier that salinity affects important metabolic processes located in chloroplasts and mitochondria (Cheeseman, 1988) but little is known about its effect on activated oxygen metabolism of these organelles. In response to ROS, plants are reported to produce high amount of enzymatic and non-enzymatic antioxidants, i.e., superoxide dismutase, catalase, peroxidase, ascorbic acid and glutathione (Neto et al., 2006). Ascorbic acid is widely utilized in cell metabolism (Loewus and Helsper, 1982) and is needed to synthesize hydroxyproline-containing protein (Arrigoni et al., 1977), to activate biological defense mechanisms (Arrigoni et al., 1979), to facilitate cell division and cell expansion (Liso et al., 1984; Arrigoni et al., 1992) and/or to delay senescence of tissues (Borraccino et al., 1994). As an antioxidant, ascorbic acid has been found not only to react with hydrogen peroxide but also with O2 -, OH and lipid hydroperoxides and thus enhances plant growth (Yu, 1994; Noctor and Foyer, 1998). Ascorbic acid has an additional role on thylakoid surface in protecting and regenerating oxidized carotenes and tocopherols, which are also helpful in protecting the plants against oxidative damage (Tappel, 1977). Considering these metabolic roles of ascorbic acid in view, many workers estimated the capability of this compound in ameliorating and modifying the salt stress- induced changes in plants (Kefeli, 1981; Neubauer and Yamamota, 1992; Choudhry et al., 1993; Hamada, 1998,

227 Janda et al., 1999). There are some indications that externally applied ascorbic acid could be metabolized in tissues (Sapers et al., 1991). During the present study, ascorbic acid pretreatment was not proven to improve salt tolerance of callus cultures rather it caused more browning and necrosis of the callus tissues. However, it was also observed during this study that the exogenous supply of ascorbic acid to in vitro-grown sugarcane plants improved their salt tolerance as indicated by the studied morphological parameters. There is no prior report of ascorbic acid pretreatment given specifically to callus cultures or in vitro plants of sugarcane. Ascorbic acid pretreatment on other plant tissues/organs such as leaves, however, has been reported in some plants. For instance, application of ascorbic acid to leaves was reported to increase the concentration of ascorbic acid in plants (Hagene and Trichet, 1964; Mozafar and Oertli, 1993). This increased supply of ascorbic acid was shown to enhance the tolerance level of sunflower plants to acid mist that was attributed to a greater accumulation of compatible solutes (Gadallah, 2000). Shalata and Neumann (2001) reported that the addition of 0.5 mM ascorbic acid to the root medium, prior to salt-treatment for 9 h, facilitated the subsequent recovery and long-term survival of 50 % of the wilted tomato seedlings. Similarly, Al-Hakimi and Hamada (2001) observed that soaking of wheat grains in ascorbic acid also counteract the adverse effects of NaCl salinity on the wheat seedlings. Thus it was suggested by these workers that additional supply of ascorbic acid to seedlings might decrease the build-up of active oxygen species and thereby increase resistance to salt-stress. Results of our study for in vitro-grown sugarcane plants are thus in line with these previous studies. However, the callus cultures did not show any improvement in salinity tolerance as indicated by the studied parameters of this study. This indicates a difference of response amongst the plant tissues to the ascorbic acid

228 pretreatment. Not always does the ascorbic acid pretreatment result in improved salinity tolerance. Quite recently, Afzal et al., (2005) have also reported that ascorbic acid pretreatment did not improve the seedling growth of wheat under normal (4 dS cm-1) or saline (15 dS cm-1) conditions. As already emphasized, plants subjected to various stresses have shown decline in growth because some quantum of energy from growth and metabolism is utilized as a maintenance cost of growing cells under the stress condition (Croughan et al., 1981). Results of the present investigation indicated that ascorbic acid pretreatment did not improve the cell survival rate under salt stress, rather it caused more browning and necrosis of callus cultures. A decrease in fresh weight was also observed in both ascorbic acid pretreated as well as nonpretreated callus cultures maintained on various NaCl levels. The concentration of ascorbic acid used during the present experiment (selected on the basis of available literature) might have been quite toxic to sugarcane callus cultures. Furthermore, this concentration of ascorbic acid could have caused a level of oxidative stress callus cultures were unable to overcome and result in cell death. In rice plants, it has been reported by Yang et al., (2004) that salicylic acid results in death of plants at higher concentrations but at low concentrations is quite useful and only increases the level of ROS thus enabling the plants to survive stress conditions. It was also observed during this study that 48 hours treatment of ascorbic acid had resulted in complete browning and necrosis of the callus tissue as compared to 24 hours treatment. Potentially, the duration of ascorbic acid pretreatment given to callus cultures could have been yet another important factor. It was observed that sugarcane plants at the same concentration as given to callus cultures (0.05 mM) resulted in less yellowing of plants. Although there was reduction in

229 fresh weights of the plants subjected to various NaCl stress levels but ascorbic acid pretreatment prior to the NaCl treatment significantly increased the fresh weights of the plants at the corresponding NaCl levels. It was also observed during this investigation that ascorbic acid pretreatment resulted in decreased soluble protein contents as well as antioxidant enzyme activities in callus cultures. In contrast, ascorbic acid pretreatment to the in vitro-grown plants resulted in greater soluble protein contents as well as increased activities of antioxidant enzymes, i.e., peroxidase, catalase and SOD. It seems that the concentration of ascorbic acid used during the present study (0.5 mM) increased ROS in the plant cells which enhanced the oxidative capacity resulting in protection of plants from oxidative stress. Plants with high levels of antioxidant enzymes have been reported to be more salinity tolerant due to a betterdeveloped resistance to oxidative damage (Harper and Harvey, 1978; Wise and Naylor, 1987, Sehmer et al., 1995). So, it can also be concluded that ascorbic acid pretreatment in this study improved the growth of the sugarcane plants as indicated by morphological as well as biochemical characteristics. Thus on the basis of the antioxidant enzyme activities, it may be concluded that though ascorbic acid pretreatment did not improve the salt tolerance of the callus cultures of sugarcane but it certainly did improve the stress tolerance of in vitro sugarcane plants. This aspect is encouraging and justifies further work on sugarcane plants under both in vitro and green house conditions. Why does ascorbic acid pretreatment improve salinity tolerance in sugarcane plants grown in vitro? What are the reasons that the same treatments have resulted in quite contrasting results in sugarcane callus cultures? These are amongst several questions that perhaps are partially unanswered till this point in time.

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230

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