Effects of salt stress on some biochemical and growth attributes of

castor bean plant (Ricinus communis . L) varieties.

By

Masoom Ali Ujan Fakir.

A THESIS

Submitted in the partial fulfillment of requirements for the degree of

Master of Science (M.S)

DEPARTMENT OF BOTANY

SHAH ABDUL LATIF UNIVERSITY KHAIRPUR

2015
ii
 CERTIFICATE

Certified that the contents and form of the thesis entitled (Effects of salt stress on

some biochemical and growth attributes of castor bean plant “Ricinus communis”

varities) submitted by Masoom Ali Ujan Fakir have been found satisfactory for the

requirement of the degree.

Prof. Dr. Abdul Razak Mahar

Chairman Dept. Botany SALU
Khairpur.

iii
 DEDICATED

To

My beloved

MOTHER

And affectionate

FATHER

Who taught me

The first word to speak,

The first alphabet to write

First step to take.

And

Always wish and pray

for my highest academic level

To

Those who Dwell in my Heart, mind, and Prayers

Throughout the whole span of my life

And are

Nearest and Dearest

To me

iv
 Acknowledgements

All praises belong to almighty Allah, The most merciful the most
beneficent and compassionate who is entire source of knowledge and wisdom to
Man. Who knows whatever is there in the universe, lord of all things, master of
the Day of Judgment and hath power over all things, and who bestowed upon me
the intellectual ability to complete this research work successfully.

Countless salutation be upon the Holy prophet Hazrat Muhammad (peace

be upon him), the city of knowledge, greatest social reformer and revolutioner,
who is the most prefect and forever a torch of guidance for humanity as a whole.

I would like to take this opportunity to thank Professor Dr. Abdul Razak
Mahar for providing me an opportunity to perform research under his kind
supervision to complete my M.S. I am most grateful to the Dean faculty of
Natural sciences Professor Dr Yasmeen Faiz Kazi for her kind support,
providing facilities, and stimulating atmosphere in my home Department ‘The
Department of Botany Shah Abdul Latif University Khairpur Sindh Pakistan. I
am very much thankful to Dr. Aziz Ahmed Ujjan Botany SALU and Dr. Prof
Saeed Ahmed Abro from Sindh University Jamshoro for statistical analysis of
data. Thanks are also due for Mr. Zulfiqare Ali Shaito for valuable suggestion on
research. My friends deserve thanks: Mr. Sajid Ahmed Mirani, Lecture Botany
Islamia College Sukkur. Mr. Ghulam Sarwar Chana Subject Special Botany, I
am also thankful to the staff of Botany Department for their help specially Zaffar
Ahmed Kandiro Lab: assistant. And at last a great thanks to my family for all
their love, which inspired me with the feeling that anything is possible.

Last but not least, I am grateful to all my nears and dears who
remembered me in their prayers and extended wholehearted encouragement
throughout this study. May Allah bless all of them “Ameen”.

v
 CONTENT

CHAPTER# TITLE PAGE#

1 INTRODUCTION 1

2 MATERIALS AND METHODS 10

3 REVIEW OF LITERATURE 19

4 RESULTS 37

5 RESEARCH PICTURES 55

6 DISCUSSIONS 60

7 SUMMARY 63

8 LITERATURE CITED 64

9 APPENDICES 95

vi
 LIST OF FIGURES
Fig.# TITLE PAGE#
1 Effect of salt stress on percent germination of castor oil
plant. 46
2 Effect of salt stress on germination rate of castor oil
plant. 46
3 Effect of salt stress on stem girth of castor oil plant
(mm). 47
4 Effect of salt stress on number of leaves of castor oil
plant. 47
5 Effect of salt stress on leaf area index (cm2) of castor
oil plant. 48
6 Effect of salt stress on number of nodes of castor oil
plant. 48
7 Effect of salt stress on shoot length of castor oil plant
(cm). 49
8 Effect of salt stress on root length of castor oil plant
(cm). 49
9 Effect of salt stress on shoot fresh weight of castor oil
plant (g). 50
10 Effect of salt stress on root fresh weight of castor oil
plant (g). 50
11 Effect of salt stress on shoot dry weight of castor oil
plant (g). 51
12 Effect of salt stress on root dry weight of castor oil
plant (g). 51
13 Effect of salt stress on chlorophyll a content of castor
oil plant (ppm). 52
14 Effect of salt stress on chlorophyll b content of castor
oil plant (ppm). 52
15 Effect of salt stress on sodium (Na+) content of castor
oil plant (ppm). 53
16 Effect of salt stress on chloride content (Cl-) of castor
oil plant (ppm). 53

vii
 LIST OF FIGURES
Fig.# TITLE PAGE#
17 Effect of salt stress on magnesium content of castor oil
plant (ppm). 54
18 Effect of salt stress on calcium content of castor oil
plant (ppm). 54
19 Effect of salt stress on potassium content of castor oil
plant (ppm). 55
20 Effect of salt stress on proline content of castor oil
plant (ppm). 55

viii
 LIST OF ABBREVIATIONS
mm : Millimeter

cm : Centimeter

ppm : Parts Per Million

SL : Shoot Length

RL : Root Length

SFW : Shoot Fresh Weight

RFW : Root Fresh Weight

SDW : Shoot Dry Weight

RDW : Root Dry Weight

mM : Millimolar

kg ha-1 : Kilogram Per Hector

CRD : Completely Randomized Design

CBM : Castor Bean Meal

SAR : Sodium Absorption Ratio

EC : Electrical Conductivity

SCARP : Salinity Control and Reclamation Project

FAO : Food and Agriculture Organization

WAPDA : Water and Power Development Authority

QACs : Quaternary Ammonium Compounds

PDH : Proline Dehydrogenase

SALU : Shah Abdul Latif University

RSC : Residual Sodium Carbonate

ANOVA : Analysis Of Variance

ix
 Abstract

The aim of study was to investigate the effect of salt stress on some biochemical and growth
attribute of castor bean plant (Ricinus communis. L) varieties. The experiments were conducted
in Petri dishes for 16 days and other in pot experiments for four month, using six treatments;
T1 (control), T2 (20mM), T3 (40 mM), T4 (60 mM), T5 (80mM) and T6 (100mM) to two plant
varieties with four replicates. The germination percent was decreased at 100 mM in green and
red varieties (83.8 and 87.0) as compare to control (49.7 and 54.2), germination rate and
germination rate was decreased at 100mM in green and red varieties (10.4 and 10.5) as compare
to control (9.3and9.8), stem girth was decreased at 100mM in green and red varieties (9.0 and
10.3 mm) as compare to control (7.0 and 7.0 mm), No. of leaves was decreased at 100mM in
green and red varieties (10.3 and 9.3) as compare to control (7.3 and 7.0), leaf area index was
decreased at 100mM in green and red varieties (341.3 and 319.2 cm) as compare to control
(108.9 and 110.4 cm), No. of nodes was decreased at 100mM in green and red varieties
(14.3and 14.8) as compare to control (10.5 and 10.0), shoot length was decreased at 100mM in
green and red varieties (69.2 and 81.9 cm) as compare to control (45.1 and 48.6 cm), root
length was decreased at 100mM in green and red varieties (22.2 and 18.7 cm) as compare to
control (13.7 and 12.7 cm), shoot fresh weight was decreased at 100mM in green and red
varieties (79.4 and 88.3 g) as compare to control (25.7 and 29.1 g), root fresh weight was
decreased at 100mM in green and red varieties (9.0 and 8.6 g) as compare to control (2.3 and
2.9 g), shoot dry weight was decreased at 100mM in green and red varieties (14.4 and 13.6 g) as
compare to control (3.2 and 4.0 g), root dry weight was decreased at 100mM in green and red
varieties (1.5 and 1.5 g) as compare to control (0.3 and 0.5 g), chlorophyll a was decreased at
100mM in green and red varieties (11.9 and 12.2 ppm) as compare to control (1.3 and 3.2 ppm),
chlorophyll b was decreased at 100mM in green and red varieties (4.8 and 7.0 ppm) as compare
to control (1.9 and 3.0 ppm), magessium was decreased at 100mM in green and red varieties
(78.7 and 71.0 ppm) as compare to control (21.9 and 33.2 ppm), calcium was decreased at
100mM in green and red varieties (77.3 and 106.0 ppm) as compare to control (28.7 and 30.0
ppm), potassium also was decreased at 100mM in green and red varieties (36.4 and 38.1 ppm)
as compare to control (8.9 and 21.9 ppm), while Sodium was increased at 100mM in green and
red varieties (10.9 and 11.0 ppm) as compare to control (30.1 and 23.5 ppm), chloride was
increased at 100mM in green and red varieties (10.7 and 9.7 ppm) as compare to control (29.0
and 23.6 ppm), and proline was laso increased at 100mM in green and red varieties (1.1 and 1.8
ppm) as compare to control (10.8 and 14.7 ppm).Salinity inhibits the growth and nutrients
content. Although, the growth, chlorophyll, K+, Ca+, and Mg2+ content of both varieties
decreased under saline treatments but Red variety produced a significantly (P ≤0.05) higher as
compared green variety. The results confirmed that salt stress decrease growth, and ions content

x
in both varieties of castor oil plant but the red variety perform better than green variety. On the
basis of germination, growth, biomass, chlorophyll, proline, K +, Ca2+, Mg2+, Na+, and Cl-
accumulation ratio. Both varieties emerged as salt tolerant.

xi
Chapter No. 1
INTRODUCTION

Castor oil plant (Ricinus communis L.) is most useful specie of family
Euphorbiaceae, it consist of 245 genera and 6,300 species (Vasconcelos et al., 2010),
and according to (Chan et al., 2010) Castor oil plant concern to monotypic genus
Ricinus, subtribe Ricinae, within family Euphorbiaceae. Other important plants of this
family consist of starchy ornamental poinsettias (Euphorbia pulcherrrima), the invasive
leafy weed spruge (Euphorbia esula), tuber cassava (Manihot esculenta), and a
potential bio-fuel crop, Jatropha curcas (Rivarola et al., 2011). Though, the genus
Ricinus is well thought-out as monotypic, castor plant shows greatly variety in its oil
content, seed size, color of foliage, stems size, and growth habit (Weiss, 2000, and Li,
et al., 2008). Castor oil plant is native to Africa mostly Ethiopia, (Anjani, 2012) and
now found various environmental conditions of world (Vasconcelos et al., 2010). The
castor is latin word which means tick, it is so named because of its seed marking and a
dump at the end (Weiss, 2000), some other common names are castor bean plant,
wonder boom, castor oil plant castor plant, Palma Christi (Armstrong, 1982). In Greek
and Roman legend, literature castor was one of the twin sons of Jupiter and Leda
(Weiss, 1971). According to Trease and Evans (1989), the plant is a native of India
with about 17 species that have been grouped into two: as shrubs and trees that produce
large seeds or as annual herbs that produce smaller seeds. In temperate regions it grows
as an annual, while in tropical climates it is a perennial plant (Litzenberger, 1974).
Castor oil is the primary product of the plant and it is cultivated widely throughout the
world for its economic significance (Rivarola et al., 2011). In 43 countries of world
castor oil plant is grown including Pakistan, and more than 30 countries on commercial
based.

The name “castor bean” is controversial; in many research articles it is named

as castor bean, but it is not included in legume, so the be stuck-up to use the name
“castor bean”. Dwarf cultivars grown in the Texas High Plains and Trans- Pecos region
are 1 to 2 m in height (Brigham 1993) as compared to the normal internode varieties,
which range from 1.8 to 3.7 m tall (Brigham, 1970). The cotyledon leaves are oval

1
shaped and directly opposite on the stem, but true leaves are alternately arranged and
born on long and sturdy petioles (Weiss, 1971).

India, China, Brazil and Mozambique currently lead the world in castor
production, with Paraguay, Thailand, Ethiopia, Angola, Vietnam, and South Africa
contributing relatively minor amounts (FAOSTAT, 2013). According to FAOSTAT
(2012) world 97% castor oil producing countries are India, China, Brazil, Mozambique,
Ethiopia, Paraguay, Vietnam and Thailand. Leading producing areas are India (with
over 60% of the global yield), China, Brazil, Eastern Africa and Ethiopia.
Manufacturers and companies in the US almost exclusively purchase castor from India
because India contributes approximately 90% of the total international export trade
market (Kumar, 2012). Castor has been historically cultivated as an agronomically
important crop for the oil derived from its seeds (Anjani, 2012).

In Pakistan, the plant is distributed in Sub-Himalayan track and also in plain

areas. In Pakistan castor bean production is very low; it was 3204 ha with an annual
production of “2089” tonnes and average seed yield of “652” kg ha-1 (Anonymous
2006).

MEDICINAL PLANT

Castor bean plant is significant because of its oil yield, which is used in most
popular medicinal for example effective laxative (Kamal et al., 2006). Castor bean is
effective medicinal plant because it contains phytochemical compound such as
flavonoids, alkaloids and tannin (Ilavarasan et al., 2006). Abiotic factors light,
temperature, rainfall, latitude, soil characters, altitude and nutrition affect the quality
and quanitity of castor oil (Omidbaigi, 2009). The environmental factories soil type,
weather conditions and seed varieties affect the physicochemical properties of oil such
as by combination of fatty acids, triacyl glycerol and oil content (Ogunniyi, 2006). The
root, leaf and oil of the castor bean plant used in the treatment of inflammation, liver
disorders, hypoglycemic and laxative (Kensa et al., 2011). The stem of castor bean
plant showed the anticancer, antidiabetic aand antiprotozoal activity (Kumar Singh et
al., 2010), Inhibition of the mitochondrial respiratory chain reactions (Ferraz et al.,
2
1999), the oil of castor bean plant having moderate antioxidant activity (Kadri, et al.,
2011). Pharmacological activities of Ricinus communis are also remarkable such as
antiviral (Wang et al., 2001), antioxidant (Ilavarasan et al., 2006), antifertility ( Isichei
et al., 2000, Sandhyakumary et al., 2003), antipsychotic, convulsant (Ferra et al.,
1999), hepatoprotective, insecticidal (Upasani et al., 2003), haemagglutination, and
anti-inflammatory Ilavarasan et al., (2006), Phytochemical analysis revealed that it
contain steroids, saponins, alkaloids, and glycosides (Kang, et al., 1985). Leaves
content tannins and phenol (Yadav & Agarwala, 2011; Ilavarasan et al., 2006). Castor
oil plant also used for the treatment of hepatitis, skin and breast cancer in initial stage
(Rana et al., 2012).

OIL PRODUCING PLANT

Castor oil plant is so economical important because of its oil. Castor bean oil is
used more than 700 products (Cosmetic Ingredient Review Expert Panel et al., 2007,
Weiss. et al., 2000). India, China, Brazil and Mozambique currently lead the world in
castor production, with Paraguay, Thailand, Ethiopia, Angola, Vietnam, and South
Africa contributing relatively minor amounts (FAOSTAT, 2013). Castor bean plant at
the maturity gives fruit which are globular having at least three beans, each bean
containing different percentages of oil and protein (Ibrahim et al., 2005). According to
Salihu, et al., (2013) the weight of seeds increase as the decrease in seed number plant-
1
. The whole seed contained 2.9-3.28kcal/kg ME (Nsa, and Ukachukwu, 2007). The
seeds of castor oil plant contain 40% to 60% oil, which mainly consist of triglycerides
type ricinolein. The oil of castor is free from ricin a poison, which left into residue
during extracting process (Sims and Frey, 2005). The oil is major constitute of seeds it
contain about 35 to 55 percent weight of castor oil seeds (Oplinger et al., 1990), and
Bafor et al.,(1991) estimated that the castor oil contain 85 to 90 percent of ricinoleic
acid. Ricinus communis Lin., is a very important plant both ecologically and can be
assigned the status of biological resource. The crude oil is processed and used in
multiple forms, for example: hydrogenated oil, dehydrogenated oil, blown oil, cold
pressed oil (HardestyA, 2012), and as a potential feedstock for biodiesel production
(Scholz and Nogueira da Silva, 2008). Its industrial applications starting from aviation
lubricant, biofuel and medicinal properties, it contains ricin a poisonous protein which
3
was used in the terrorism and suicide attacks (Coopman et al., 2009). Castor bean plant
is one of the oil producing plant which has capability as oil feedstocks because of very
high oil content (48-60%) of seed and (Brigham et al., 993). Bahia the state of Brazil
which situated in semi-arid northeast, produce 80% of oil of world (Carvalho et al.,
2005, Conab, et al., 2013). India is second number in production castor oil, which is
about 59 lakh tone (FAO, 2002).

Castor bean plant at the maturity gives fruit which are globular having at least
three beans, each bean containing different percentages of oil and protein (Ibrahim et
al., 2005). This oil used in lubricant, lamp fuel, as a component of cosmetics, in the
manfacture of soaps, printer’s ink, plastics, fibres, hydraulic fluid, brake fluid,
varnishes, paints embalming fluid, textile dyes, leather finishes, adhesives, waxes,
fungicides (Rehman et al., 2007), protective coatings, grease, linoleum, oil cloth, raw
material in manufacturing of various chemicals sebacic acid, undecylenic acid, udes in
production of plasticizer and Nylon (Dole et al., 1950). Castor bean oil is used more
than 700 things (Cosmetic Ingredient Review Expert Panel et al., 2007, Weiss, et al.,
2000). The whole seed contained 2.9-3.28kcal/kg ME (Nsa, E.E. and S.N. Ukachukwu,
2007), the cake contained about 32-48 % crude protein (Rao, 2004).

ECOLOGICAL IMPORTANT PLANT

Castor oil plant treated as secondary, it cultivated marginal soil where salinity is
present (Litzenberger, 1974), it is environmental significant because it is excellent
invader of waste places (Roger & Rix, 1999, Anonymus, 2006, James & Harden,
1990). Farmer cultivated the Castor oil plant in those soils which are not unsuitable for
food crop plant (Canoira, et al., 2010, Hincapie, et al., 2011). It can be developed as
weed even along the road sides in the tropical warm regions (Ivan, 1998).

FERTILIZER AND FOODER

The castor cake, high in nitrogen and proteins, could be used as livestock feed
(Diniz et al., 2010) or as an organic fertilizer (Lima et al., 2011) to enrich soil. Its
4
residue of castor oil seed may be utilized in many ways for insteant as organic fertilizer
in field, for making paper, (Khaleq-Uezzaman, M. 1990, Sannappa, B., M. Jayaramaiah
and D. Chandrappa. 2002). The seed cake of castor oil used as manure in the soil and
their shell are use as termite control agent (Moshkin, 1986, Maiti et al., 1988). In India
castor bean meal (CBM) is byproduct used as source of protein for live stock feeding
(Dookia, 1980), but it contains antinutritional factors ricin, ricinine, allergen and
chlorgenic acid, so it need to detoxified before used in livestock (Lade et al., 2013), it
not only contain protein but has great amount of nitrogen (Diniz et al., 2010), which is
used as organic fertilizer (Lima et al., 2011). No of processes were introduce to
improve nutritive value of castor bean meal (CBM) successfully such as water soaking,
steam cooking, roasting, use of various chemicals alone or in combination (Anandan et
al., 2005), plate agguutination technique (Rao et al., 1986). Heat treatment will
denature ricin; thus castor meal (the residue left after the oil has been extracted from
the seeds) may be used as a feed protein source for livestock. The cake contained about
32-48 % crude protein (Rao, 2004).

TOXIC PLANT

Castor oil plant in nature produces three types’ toxins: ricin (RCA60), Ricinus
communis agglutinin (RCA120), and ricinine (Severino et al., 2012). Seed maturation
about 44 days and after 28 days of pollination, ricin starts develop (Barnes et al., 2009).
Ricin is present in the endosperm of the seed until approximately 6 days after the
radicle emerges (Barnes et al., 2009). The seed oil of castor contains 85 to 90%
ricinoleic acid (Bafor et al., 1991). The most notorious uses of ricin have been as an
assassination weapon by the secret intelligence services (Carus, 1998). One milligram
of ricin can kill an adult (Knight, 1979). It contains ricin a poisonous protein which was
used in the terrorism and suicide attacks (Coopman et al., 2009). If the castor seed is
swallowed without chewing and there is no damage to the seed husk, it passes
harmlessly through the digestive tract. However, if it is chewed and then swallowed,
the intestines absorb the ricin toxin (Wesche et al., 1999).

DEFENCE POINT OF VIEW

5
 In defence point of view America classified the castor as a strategic material.
Although the US is not currently invested in commercial production of castor, there has
been a history of US production during World War (I) and World War (II) with
preliminary research on castor in Florida as early as 1917 (Rolfs, 1917). In 1984,
congress passed Agricultural Materials Act P.L. 98-284 which classified castor oil as a
strategic material and Public Law 81-774 requires that strategic materials be acquired
and stored in the US for national defense purposes (Roetheli et al. 1991). The US is
required to keep 5 million pounds of castor oil with stocks managed by the Department
of Defense (Congress, 1991).

DIFFERENT USAGE OF CASTOR OIL PLANT

The seed oil of castor oil plant not edible, but 700 used, ranging from medicine,
cosmetics, plastics, lubricants and most important bio-diesel (Anonymous. 2003,
Cheema, 2011). This oil used in lubricant, lamp fuel, as a component of cosmetics, in
the manufacture of soaps, printer’s ink, plastics, fibers, hydraulic fluid, brake fluid,
varnishes, paints embalming fluid, textile dyes, leather finishes, adhesives, waxes,
fungicides (Rehman, et al., 2007), protective coatings, grease, linoleum, oil cloth, raw
material in manufacturing of various chemicals sebacic acid, undecylenic acid, udes in
production of plasticizer and Nylon (Dole, et al., 1950). The oil and its derivatives also
have numerous industrial uses, being used in a wide variety of products, such as the
basic ingredient in racing motor oil for high-performance engines, a fuel additive for
two-cycle engines, a primary raw material in the production of nylons and other resins
and fibers, and a component in paint and varnish, insulation, fabric coatins, soap, ink,
plastics, brake fluids, guns, insecticidal oils, and so forth (Sims, et al., 2005). Leaves
have been reported to have flavones and tannins (Khogali, et al., 1992), whereas roots
of this plant have been found to contain alkaloids also along with flavonoids and
tannins (Ilavarasan, R. et al., 2006). It is considered to be purgative, counter-irritant in
scorpion-sting and fish poison (Duke, and Wain, 1981). The oil of castor have unique
properties because it can be chemically broken down into exceptional subunits which
are used in a different money-making goods such as coatings, inks, paint, lubricants and
other products (Ogunniyi, 2006). The bigest reported concentration of castor oil was
6
used in a cosmetic product lipstick which has contained about 81% castor oil (Elmore,
2007).

Castor oil was the preferred lubricant for rotary engines, such as the Gnome
engine after the widespread adoption of engine for aviation in Europe in 1909. Castor
oil, like currently less expensive vegetable oils, can be used as feedstock in the
production of biodiesel. The resulting fuel is superior for cold winters (Oduvaldo,
2007). In recent days castor oil plant is most attractive crop because its seed oil can be
used as biodiesel, it great energy values and has admitting properties of fuel (Drown, et
al., 2001), (Refaat, 2009, Berman, et al., 2011). In ancient India, castor oil has been
reportedly used in traditional Ayurvadic medicine as a mild laxative (Morris, and
Morse, 2011). Castor oil was historically used as a purgative, skin ointment, and lamp
oil (Weiss, 2000). Economically it is important because it yields oil, with a wide range
of uses including its most popular medicinal use as an effective laxative (Kamal et al.,
2006).

SEED YIELD OF CASTOR OIL PLANT

The castor oil plant yield depends upon the planting dates repoted by Baldwin et al.,
(2009), with six planting dates and different location, its range was between 89 to 1954
kg ha–1. Planting dates not only influenced but it effect on plant diseases and pests
observed by (Zuchi et al., 2010a). Fanan et al., (2009), and Zuchi et al., (2010b) state
that the increase in number seeds per plant, does not mean increase the yield of plant,
but it affected the oil contain of seeds as the number of seed increased The yield of
plant also affected by the position of racemes as first, second, third racemes, it due to
the abiotic factores and genotype of plant (Vallejos et al., 2011; Neto et al., 2009;
Fanan et al., 2009; Zuchi et al., 2010c). The yield plant depend upon the high number
of female and male flowers, Severino et al., (2006e), and Bertizzo et al., (2011) state
that if the number of female flower increase than the yield will be higher. The number
of female flower is very critical to temperature, when temperature above 30oC female
flowers reduced, the imbalance in nutrients, and the age of plant also affect the number
of female flowers (Lavanya, 2002; Lakshmamma et al., 2002; Neeraja et al., 2010).

7
According to Severino, (2012) the yield of castor oil is not sink limited but source-
limited.

IRRIGATION OF CASTOR OIL PLANT

Castor oil plant showed grate tolerance against salinity and drought, but it was
observed that under saline and drought conditions seed yiled reduced. Castor oil plant is
non-edible oil, annual producing plant, it tolerance to diverse environmental conditions.
Castor bean plant has showed drought tolerance well developed root system, which
helps increase aeration, water retention and distribution in soil (Embrapa et al., 2006).
In arid conditions plant survive only on rainfall, in these plant aerial parts of plant
growth become very slow, the root growth is more than aerial parts (Weiss, E.A, 1983).
The castor oil plant survived in drought conditions up to 300mm of precipitation during
vegetative conditions. The prolong drought reduced the seed yield of plant, it yield ca
be increased for little extand of Seed yields of rain-fed castor fields can be increased by
small amounts of water (Sharma et al., 2010). A castor oil plant field yield “1774” kg
ha–1 of seeds drought conditions, 2199 kg ha–1 with irrigation conditions at the end
season, and 4252 kg ha–1 with irrigation before and after the rainy season (Souza et al.,
2007). The castor plant is a thermophilic plant, that is, it suffers from frosts. Shoots
perish at temperatures below -1°C; adult plants die at temperatures below -3 to -4°C.
Temperatures required for castor plant should not below 20-25°C to endorse normal
growth and development. Castor oil plant needs flourishing soil, produces high yield on
suitable soil, well-aerated soils having pH of 6-7.3. The water content of plant essential
for development of leaves, reduction of water content affect the leaf development
(Weiss, 2000).The castor oil plant considered as day long plant, it need 12 to 18 hour
day light for growth and flower development, less than 12 hour day light adversely
affect the plant growth and development (Weiss, 1983).

SALINITY TOLERANCE OF CASTOR OIL PLANT

8
 Castor bean plant (Ricinus communis L.) is non-edible oil, annual producing
plant, it tolerance to diverse environmental conditions. Castor bean plant has showed
drought tolerance well developed root system, which helps increase aeration, water
retention and distribution in soil (Embrapa et al., 2006). Castor bean plant grown in
those areas particularly in semiarid regions salinity stress may affect germination and
plant growth (Pinheiro et al., 2008).

The objectives of present work are:

1. To check the behavior and adaptations of castor bean in salinity.
2. The tolerance of Ricinus communis will be assesses and limits will be
quantified with respect to salinity.

3. The effects of growing this plant on long basis on the soil characteristics
especially salinity parameters will be evaluated. Similarly, long-term effect of
salt-affected soil on plant growth will also be investigated.
4. To compute effect salt stress on growth (agronomical parameters) such as
number of leaves, number of nodes, plant height, root leght, biomass.
5. To check the association of various growth characters and biochemical
attributes to salt stress of castor bean plant.

9
Chapter No. 2

MATERIALS AND METHODS

The research work presented in this manuscript was conducted to evaluate the
performance of Castor bean plant under different concentration of saline water in the
wire house at Department of Botany Shah Abdul Latif University of Khairpur Sindh
Pakistan.

SITES OF EXPERIMENTS

Exp1-The first experiment on seed germination of castor bean plant was

conducted at department of Botany laboratory, Shah Abdul Latif University Khairpur
Sindh Pakistan, under room temperature and no control over temperature, During
experiment period day and night temperature in laboratory 20±5 oC and 30±5 oC
respectively.

Exp2- The second pot experiment effect of salt stress on some biochemical and
growth attribute of castor bean plant varieties was conducted in department of Botany
Shah Abdul Latif Khairpur Sindh Pakistan; during year 2014-2015 in wired house of,
normal conditions as there was no control on light, temperature and humidity. The day
and night temperature of wired house ranged between 30±1 oC and 40±2 oC, and
humidity was in between 30±7%.

SEED SOURCE

10
 Castor oil plant seeds were collected from local market, locally called Shao and
Gharo hiran, green and red castor oil respectively. The two varieties are morphological
different from each other as they are named.

EXPERIMETAL DETAILS

To investigate the effect of salt stress on Castor bean plant (L. R.communus )
two studies were conducted, First effect salt stress on seed germination of castor bean
plant, and Second effect salt stress on growth and biochemical characters of Castor
bean plant in pot experiment. On the first experiment was conducted in Petri dishes, the
base of petri dishes two layers of filter paper whatman No1 were placed than in each
petri dishes 10 seed were kept, after this process aggregation of salt concentration given
to the each petri dished of 5ml every day. The concentration are 20, 40, 60, 80, and
100mM respectively, and Second experiment the castor bean plant were cultivated in
pot which filled soil about 12 Kg soil and aggregated with tap water regularly, after
reaching to the trifoliate stage than 20, 40, 60, 80, and 100mM salt concentration were
applied for four month. The objective of second study was to evaluate relative salt
tolerance of two varieties of castor bean plant under salt stress. Subsequently, two
varieties (Red and Green castor bean plant varieties) were selected for this study. The
following parameters were investigate the growth attribute specially plant height,
diameter, No of leaves, No of nodes, fresh and dry weight of shoot, root, biochemical
character such as chlorophyll, proline, Ca+, Na+, K+, Mg2+, and chlorides under salt
stress.

Methodology for saline water

Treatments

T1 = control

T2 = 20mM Nacl

T3 = 40mM Nacl

T4 = 60mM Nacl

11
T5 = 80mM Nacl

T6 = 100mM Nacl

SEED GERIMENATION EXPERIMENTS

Petri dishes washed with distilled water and then autoclaved for sterilization tag
them according to treatment and replica, in the base petri dishes filter paper whatnam
No1 is placed for moisture, The seed coat of seed was remove with scissor than socked
with water for one hour, the seeds are dried and sterilized with sodium hypochloride,
the seed were placed in sterilized petridishes, lined with 2 layers of tissue roll followed
by 2 layers of filter paper. Experiment has six treatments and four replicas and it was
completely randomized design (CRD). The salt treatment are applied which are 20, 40,
60, 80, and 100mM. The germination was monitored after every 24 hours for 2 weeks.

The first study was conducted in laboratory under room temperature in petri
dishes, In the base of petri dishes two layers of filter papers were placed. Distilled
water used for different salt concentration, the concentration are 20, 40, 60, 80, and
100mM were applied every day of 5 ml to each petri dishes. Experiment has six
treatments and four replica, Petri dishes were arranged according to completely
randomized design (CRD). After 24 hours germination of seed recorded, the
experiment runs for 16 days. The pH and EC were regularly recorded day wise.

Germination Parameteres

The germination was monitored after every 24 hours for 2 weeks. The data was
recorded as follows:

 No of seeds germinated per 24 hours.

 Seed germination rate.
 % germination.

12
Growth: The length of radical and plumule will be monitored after each 24 hours.

Formula germination rate is given below.

GR=100(A1+A2+ ------ +Ax)/(A1T1+2T2+ ------- +AxTx).

 A1, A2,-------Ax: are no of germinate seed counted on the 1st day, 2nd day, and
so on until the last day (x) and T1.
 T2------ Tx: are no of days between 1st, 2nd, 3ird germination day and so on (x).

Formula for percent germination is given below.

No of germinated seed after 16 days

Germination percent = --------------------------------------------- X 100
Total No of seeds

POT CULTURE EXPERIMENTS

experiment was conducted in wire house uncontrol conditions, Surface soil (0-
15 cm) in bulk was collected from surrounding of Shah Abdul Latif University and
analyzed for textural class, EC, pH, the size pots was 12 inches height and 12 inches
dia. In the base of pots hole was made for leaching than these pot were fill with soil
about 10kg.

Healthy seeds were sown in pot (12 inches × 12 inches) having 10 Kg of soil
filled in pot. Tap water was used for irrigation to maintain optimum moisture before
two days of sowning. In each pot five seeds of two genotypes were sown and after
germination plants were thinned and one plants for further observations. All pots were

13
irrigated with tap water until they reached the trifoliate stage, than seedlings were
subjected to five levels of salinity 20, 40, 60, 80, and 100mM were applied every day
for four months. The experiment was (CRD) having six treatment and four replica. The
pH and EC were regularly recorded day wise. After four month plants were harvest
data was recorded.

THE SOIL PROPERTIES

Sand (%) Silt ~ Clay ~ ST Soil pH ECe Organic

matter %
50.25 25.01 24.74 Sandy. 9.32 1.83 0.81
Clay.
Loam.

GROWTH PARAMETERS

 Number of leaves per plant

 Leaf area (cm2)
 Number of nodes per plant
 Shoot length (cm)
 Root length (cm)
 Shoot fresh weight (g)
 Root fresh weight (g)
 Shoot dry weight (g)
 Root dry weight (g)

SHOOT LENGTH (SL)

Shoot length (SL) was measured through inches tape in centimeters (cm).

14
ROOT LENGTH (RL)

Root length (RL) was measured through inches tape in centimeters (cm).

SHOOT FRESH WEIGHT (SFW)

Shoot fresh weight was measured by electric balance in grams (g).

ROOT FRESH WEIGHT (RFW)

Root fresh weight was measured by electric balance in grams (g).

SHOOT DRY WEIGHT (SDW)

After taking fresh weight of shoot, was kept in an oven at 70oC for 7 days and
then their shoot dry weight was measured.

ROOT DRY WEIGHT (RDW)

After taking fresh weight root, was kept in an oven at 70oC for 7 days and then
their root dry weight was measured.

NUMBER OF LEAVES

Number of leaves count per plant.

NUMBER OF NODES

Number of nodes count per plant.

STEM GIRTH

Stem girth of each plant was measured with vernier caliper in millmeter (mm).

15
LEAF AREA

Leaf area of each plant was measured in both length of maximum length and
maximum width with following formula.

LAM .N
LAI= -----------------
A
“N” is the no of leaves in the plant.
“A” is area (cm2) of one plant.
The results were analysed by means of slope, and coefficient of determination (r2)

BIOCHEMICAL ATTRIBUTES.

Cholorphyll content

The photo-synthetic pigments were analysied as Arnon (1949). First 1g fresh

leaves were washed with distilled water carfully, than were ground in a porcelain
mortar adding 2 ml (80%) acetone, filtered through a filter paper (Whatman No. 1).
Collect the extract and add 10 mL of (80%) acetone and made it up to 10 mL, than mix
with a vortex mixer. Finaly absorbance of the samples was determined at 645, 652 and
663 nm. The formulae for calculation of chlorophyll content are given below

Chl. “a” ( mg g-1 f.wt. ) = {12. 7 (OD 663)-2.69(OD 645) ×V/1000 x W}

Chl. “b” ( mg g-1 f.wt. ) = {22. 9 (OD 645)- 4.68(OD 663) ×V/1000×W}

Where V = volume of the leaf extract (mL)

W = weight of fresh leaf tissue (g)

Proline content

For estimation of proline standard method of (Bates et al 1973) was used. Fresh
leaf tissue of (0.5 g) was ground and extracted with 10 mL of (3%) sulfo-salicylic acid.

16
The extract was filtered through a filter with paper (Whatman No. 2), add 2mL acid
ninhydrin solution, and 2 mL of glacial acetic acid was added to 2mL of the filtrate and
homogenized. The heated the homogenate at 75oC for 60 min. in water bath than placed
in ice to terminate the reaction, add 4 mL of toluene. Finally chromophore phase
containing toluene was separated from mixture and the absorbance of the chromophore
phase was done through spectrophotometer reading at 520 nm. The blank sample was
also obtained through same steps. Formula for calculation of proline:

μg proline mL-1 × mL toluene/115.5

μmole proline 1g fresh weight= ----------------------------------------------------

g of sample

Chloride content

The chloride content was determind through (Richard 1954). The 0.1g of air
dried ground sample of plant material were placed in test tube and add 10ml of distilled
water, then heated it at 80oC as the volum become half to original. The volum of extract
maintained to 10ml by adding deionized water. The reading was get through chloride
analyzer.

Na+, And K + Content

The sodium (Na+) and potassium (K+) content determind through Wolf (1982)
method. The 1g of dried ground material into digestion tube, 2.5mL of H2SO4 added,
then incubated at room temperature for a night. After this in each digestion tube 1mL of
H2O2 added, and then heated the digestion tube up to 350oC for 20min. until the fumes
are produced. Remove the digestion block and cool it, then again heated them for
20min. until the fumes are produced. The digestion became colorless. To maintained
voulum of digested material to 50 mL by adding de-ionized distilled water and filter it
through filter paper (Whatman No. 1). Flame photometer was used, and series of

17
standards (from 5 to 25 mg per litter) for each cation were prepared. In last the standard
curves were drawn after measuring takeing the reading.

Ca2+ and Mg2 + Content

For determination of calcium (Ca2+) and magessium (Mg2+) content titration with
EDTA (Richards, 1954).

Formula for Calcium (Ca2+) or Magnesium (Mg2+) content.

(V - B) × N × R × 1000
Ca or Ca + Mg (meq/L) = - ___________________________
Wt

Mg (meq/L) ={ Ca + Mg (meq/L) - Ca (meq/L)}

Where: V = is volume of EDTA (Ethylene Diaminetetraacetic Acid) titrated for the

sample (mL)
B = is blank titration volume (mL)
R = is ratio between total volume of the extract and extract volume used (for titration).
N =is normality of EDTA (Ethylene Diaminetetraacetic Acid) solution.
Wt= is weight of air-dried (g)

STATISTICS OF WORK

The data obtained from present work were tablized by performing “ ONE
WAY ANOVA” using SPSS IBM 19.0 statistical software.

Chapter No. 3
18
 REVIEW OF LITERATURE

Throughout the world majority of the crop yield reduced due to the abiotic
factores, Salinity is one of them; it is most agitating forms of land erosion which
severely affects crop products remarkably in arid and semiarid regions of worldwide
(Shomeili et al., 2011). According to Tuteja (2007) 20% of irrigated land crop yield,
severely affected by high salinity stress in the world. Those plants grown in salinity
render in growth was observed in terms of biomass, root and shoot growth as compare
to control plants (Rodriguez et al., 2006).

The 25% soil facing the problem of salinity throughout of world (Taffouo, et
al., 2010) , different techniques are applied to reduce the salinity damage in the world.
In coming 25 years in arable land salinity become 30% and up to 2050 it would be
50%, it very alarming issue of the world (Choudhury, et al., 2012). Stochle, et al.,
(2013) recently state that salinity in irrigated areas increase country wise the Iraq at top
of the countries with (50%), followed by (30%) of Egypt, (28%) of Pakistan, (27%) of
India, and (20%) of Australia.

Our county Pakistan is located in arid and semiarid regions of the world, Alam
et al., (2000) reported that each year 40,000 ha irrigated land becoming saline with 6.30
Mha totally in Pakistan. According to Anonymous, 2003 salinity causes damage to the
productivity of Agriculture crops about Rs 29 million each year. There are many reason
of salinity, particularly in Pakistan are water logging, increase in water table in the
Panjab, Sindh, Balochistan, and KPK, Pakistan stand at six number according to
population, so it increase pressure to utilized the those land which are salinity affected.

The term “salinity” refers accumulation excessive salts on surface of soil for
long periods of time. Salt affected soil is classified into three kinds on the basis of
sodium absorption ratio (SAR), and electrical conductivity (EC) According to Richard
(1954), one saline soil, second sodic soil and saline sodic soils Richard (1954), but the
majority of soils are saline or sodic. Soil having electric conductivity (EC) more than
4dSm-1 and sodium absorption ratio (SAR) smaller than 13 is saline soil, while electric

19
conductivity (EC) less than 4 dSm-1 and sodium absorption ratio over 13 are sodic soil
(Richard, 1954)., in sodic saline has more sodium as exchangeable cation. According to
Brady, Weil (2010), and Flowers, Rozema (2008) 50% of soil salinity is sodic soils, in
which more than 15% sodium is as exchangeable cation.

According to the Rasool et al., (2013) the main reasons of salinity are bad
practices of agriculture for example excessive use of inorganic fertilizers, unbefitting
irrigation, reduction of native trees, introduction of exotic species, (Azevedo Neto et
al., 2006) uses of saline water or poor water quality in agriculture fields, high
temperature, low rainfall, high soil, The other reasons are importation of sea water
(saline water) to land, the rain and wind also deposit the salt from sea to land (Rasool et
al., 2013). In recent days large number of deep rooted trees are cutting down which
bring the water table up ride (Anon., 2001a), the vegetation with shallow roots absorb
less water as compare to deep rooted trees, due to this water table increase and in this
regard the salt from lower surface of soil comes on the surface (Sadava et al., 2009).
The main causes of salinity is parental material of soil formation which have already
salt, it form the salt bearing sediments, and it is more effective in the river and low
lying areas (Hillel, 2005).

Salinity causing cautions are sodium (Na+), calcium (Ca2+), and magnesium
(Mg2+) and chloride (Cl-), carbonate, and sulphate (SO4 2-) are anions of total soluble
salt, in the majority of the saline soils the most common caution is sodium (Na+) and
chloride (Cl-) is anion, According to the Rengasamy (2010) sodium chloride (NaCl)
50% to 80% of total soluble salt of saline soil. So the sodium chloride (NaCl) got
attraction of the researchers in recent days (Kronzucker et al., 2013).

There are two main groups of plant on the basis of salinity one is halophytes
and other is glycophytes, the halophytes are those plant can growth and developed into
salinity up to large scale, and the glycophytes are those plant which cannot grow in
salinity or cannot survive in salinity finally die.

Salinity affects the plant in different ways, it cause reduction in metabolism,

water potential, ions toxicity, imbalance in ions, finally crop yield (Joseph et al. 2011),
Jenks et al., (2007) it causes osmotic stress, addition of salt to the corrosive levels in

20
the cell, According to Manneh (2004) the primary of salt injury are nutrient imbalance
through osmotic stress, with these salt stress reduce root mass and length so the root
become thicker and thinner (Munns and Termaat, 1986). The most of the crops are
highly sensitive to high salinity and shows reduction in agricultural productions (Zorb
et al., 2004).

ASSESSMENT OF WATER QUALITY

In the term of soil plant management analysis of water quality has great importance
and it is consider as the best parameter, the water given to plant must be fit for plant, if
it contain certain amount of salt, which influence the plant growth, development, it not
only affect the plant but also the soil physical and chemical properties. For the
successful agriculture production, good quality of water must be given to the crop. The
classification of irrigated water is based on salinity, sodium hazard, pH, alkalinity and
presence of ions.

SALINE WATER

In many parts of Sindh and Pakistan earth crust water are saline, so it is not fit
for irrigation. It salt concentration is measured through electric conductivity (EC)
which describes the quantity of dissolved salt or ions present in it. In the beginning it
was only used in SCARP (Hameed et al., 1966). Usually the water has electric
conductivity up to 1.5 dSm-1 regard as marginal and greater than 3 dSm-1 not fit for
plant irrigation. Sheikh (1989) give an idea about the analysis of quality water for
irrigation, EC when EC ≥ 3 dS m-1 and it the effect on plant growth and development,
during 1954 the value of EC from 075 to 2.25 dSm-1 recognized as highly saline. In
recent days the classification of saline water was based on FAO, the high saline water
has EC between 10 to 25 dSm-1, and it was studied that the high EC water effect plant
productivity, the plant was unable to get water because the high saline water cause
physiological drought.

SODIUM TOXICITY

21
 The EC of water provide better approach to applicability of irrigation water,
sodium effect are different in term of EC, it has specific impact on soil properties,
particularly in soil physical properties. In the reference with sodium two relationships
were described one sodium absorption ratio and carbonate/ bicarbonate levels. Increase
in sodium concentration is correlated increase in bicarbonates and decrease in calcium
(Ca2+) and magnesium (Mg2+) levels, this enhance the hazards of sodium in plant as
well as in soil. There is an standard through which we can measured the sodium
concentration is sodium absorption ratio (SAR), it shows the ratio of sodium (Na+) to
calcium (Ca+2) and magnesium (Mg2+) ions in given sample. The role of Ca2+ ions in
soil maintains the soil structure and the Mg+2 maintain the secondary properties of soil.
The soil profile affected by high concentration of sodium (Na+), it banishing the
calcium (Ca2+) ions which finally disturb the clay fraction. The sodium ions breakdown
the soil physical and chemical properties, it promote weaker force or having charge
which holds together the clay particles. Sodium concentration and other salt in
irrigating water negative impact on the soil aeration and it create the binding effect in
the soil and soil becomes impermeable (Oster, 2004). The water having SAR less than
7 and RSC 1.25 having no negative impact on crop development and soil properties
Chaudhry and Rana (1975), the Bauder et al., (2006) reported that the water having
SAR less than 9 sodium hazards are minimum, if the SAR is in between 18-25 the
effect of sodium hazard higher. Sodium concentration effect the soil physical factores
like soil texture, soil pH, soil nutrients content, soil drainage, and water holding
capacity.

pH AND ALKALINITY

The pH is another standard method to calculate the concentration of ions in

given sample, mostly pH is the primary method to analysis the soil ions concentration,
the pH values from 6.5 to 8.4 is advised normal for many crops of the world, from these
values low or high pH is recommend as hazard for the crops. In the salinity sodium
considered as the major salt which cause salinity, it increase when high carbonates
occur in the soil, the carbonates are main causes of sodium ions in solution by creating
insoluble minerals from calcium and magnesium ions. Irrigation of such type of water
22
increases the sodic soil conditions. The pH of water has very short term effect on soil
pH, because the many soil has its buffering effect, for significant change it needs many
year,

CHLORIDE (Cl-) HAZARDS

Many other ions also have significant effect on plant like chloride, chloride also
required for plant growth but in low quantity. The high chloride (Cl-) concentration
range from (70 ppm) has deleterious effect on plant growth particularly in sensitive
plants (Bauder et al., 2006).

UNDERGROUND WATER QUALITY

The quantity and quality of water for crop production has great important,
because the effect of lower quality of water on reduce the yield of crop, and it causes
the economic loss of the country. Pakistan facing same kind of problem, however the
Pakistan has great canal system, canal system has many advantage but it main causes of
salinity, water logging, change the underground water properties. Unless in many
regions of Pakistan underground water quality not fit for irrigation of crop. According
to Ibrahim et al., (1988) river water the concentration of different solute ranging from
1035-345 mgL-1, RSC and SAR 0-4.37mM/L and 0-1.2mM/L respectively. The
Ahmad and Chaudhry (1987) reported that the ground water qualities in the canal
irrigation areas 60% have salt less than 1000mg/l, 15% have up to 2000mg/L, and 25%
have above 2000mg/L, this depend upon the depth soil. In some cases underground
water quality has more salt concentration as the depth increase (Gupta et al., 1999).

In Pakistan has well developed tube well system namely Salinity Control and
Reclamation Project (SCARP) having tube well 565,000, but the quality of these are
from useable to hazardous. GOP (2004) state that 70% to 80% of water pumped by tube
well is not suitable for irrigation of crops, it have high EC, SAR/RSC, same thing
reported by Azhar et al., (2003).

23
Nasim et al., (1994) reported that majority of the sample take from the underground
water has salinity to hazard levels with increase in depth of soil profile. The ground
water 23.8% is fit for crops, 7.6% is to some extent fit, and 68.6% atrocious for the
crops (Shah et al. 1992). In Punjab the underground water has 1252mg/L, and
18.39mM/L, 3.42mM/L SAR and RSC respectively. In Sindh underground water
quality from upper to lower was too bad (Chaudhry, 1977), but in the Indus plain
WAPDA claimed that 63% of tube well water was fit for irrigation (Younus, 1977). In
Punjab 97.5% salt developed due to tube wells, 11.3% by canal system, and 9.2%
because of dry conditions (Awan et al., 1992). The number of research were conducted
in term of underground water these suggest that 70% of tube wells water contain salt.
The shortage of river water especially during 1999 to 2000, to meet this shortage,
utilization of underground saline water is essential. So it important to understand the
genetic traits, mechanisms determining salt tolerance is required to increase agriculture
production (Paranychianakis and Chartzoulakis, 2005).

SALINITY EFFECTS ON GROWTH

Among the abiotic stress salinity is major stress which renders the plant growth
and yield. Effect of salinity on plant growth and development is different in different
stages, but it corrosive effect was observed in early stage. Salinity inhibit agronomical
characters for example plant height, number of nodes, number of leaves, yield such as
number of pods per plant, number of seed per pod, seed weight (Hossain, et al.,2008),
fresh weight, dry weight, (Zhani, et al., 2012, Munns, 2003), shoot and leaves dry
weight (Kaouther, et al., 2013, Aguilar, et., 2009, Zapryanova and Atanassova. 2009).
Salinity/ salt increase in root zone produce physical drought (plant unable to get water
from the soil by lowering the water potentail) (Lloyd et al, 1989). Kandil et al., (2012),
Saberi et al., (2011), and Bashir et al., (2011) reported that salinity increased reduction
the root length and plant height was observed. When plant was subjected to salinity
plant photosynthesis rate also affected, it leads to reduction in productivity (Netondo et
al. 2004). The canola cultivars show noticeable taming of growth and seed germination
at various stages of growth under saline conditions (Mer et al., 2000; Tunuturk et al.,
2011). Another reports when plant grown in 200mM concentration of salt, such as

24
sugarbeet decrease 20% in dry weight, in cotton 60% and soybean a salt sensitive
specie die (Munns, 2002). Salinity stress not affect the plant growth but also on physio-
chemical performs of plant for instant chlorophyll content in tomato Hajer et al.,
(2006), cotton Basal (2010) and Catharanthus roseus (Jaleet et al., 2008) other
parameters of plant performs studies in different species in chickpea (Maliro et al.,
2008), rice (Zeng, 2005), seashore paspalum (Lee et al., 2008), maize (Neto et al.,
2004), and wheat (El-Hendawy et al., 2005),

EFFECT ON NUMBER OF LEAVES

The leaf is a very important organ of plant in respect to photosynthesis,

transpiration, gaseous exchange; definitely reduction in number of leaves per plant can
affect all these process. It is common observation that under salinity stress number of
leaves reduced in many species (Zhu et al., 2001). According to Iqbal and Ashraf,
(2005), actually reduction of leaves per plant because of decrease in turgor potential, it
so essential for cell elongation which increase number of leaves and leaf area, for
example in Zea mays (Hassine and Lutts, 2010) and Solanum tubersum by Albacete et
al., (2008). The other researcher Romero-Aranda, et al.,(1998), Dong and Dong.
(2007), get similar results. (Akram, et al., 2010), also observed decrease in number of
leaves due to salinity.

EFFECT ON SHOOT, ROOT FRESH WEIGHT

Salinity has negative impact on the plant shoot and root fresh weight. As the
mention early reduction of photosynthesis rate, leaf are, number of leaves, finally
compact the shoot and root fresh by developing the shrinking effect on the content of
protoplasm, which influence the differentiation of tissues, cell elongation, and root,
shoot development Kent and Lauchli (1985) and water uptake, decreased osmotic
potential by Terry and Waldron, (1984). According to Levitt, (1980) this is because of
toxicity and nutrition imbalance. Qureshi et al., (1991), Akhtar et al., (2000) reported
that increase in salinity levels decrease in root fresh weight in rice and wheat.

25
EFFECT ON SHOOT, ROOT DRY WEIGHT

When plants expose to the salinity levels in shoot and root dry weight depletion
with increase in salinity revealed by Shannon et al. (1998). The reason of depression in
shoot dry weight is because absorption (Brugnoli and Lauteri, 1991) and sodium (Na+)
toxicity in plant (Shannon et al. 1998). When plant take excess toxic ions such as
sodium (Na+) and chloride (Cl-) subtraction growth, biomass, (root and shoot dry
weight), the availability of nutrients to root (Qadir and Shams, 1997) and finally death
of plant (Munus and Termat, 1986; Sharma, 1989).

EFFECTION ROOT AND SHOOT LENGTH

Salinity not only reduce fresh weight, dry and number of leaves but it also
inhibit the root and shoot length, salinity stress considerable reduce the root and shoot
length studied by (Netondo G.W., 2004). Similar results were obtained by (Keutgen
and Pawelzik, 2008; Kasukabe et al., 2006; Ibrahim et al., 2007). According to West G,
D Inze and GTS Beemster (2004) reported salinity reduce the root growth of
Arabidopsis. In castor oil plant root length and shoot length decrease under the salinity
studies by Pinheiro et al., (2008). With the reduction of root length the capacity of
water and minerals absorption from soil influenced, which in return decrease shoot
length (Neumann PM. 1995), growth and development.

EFFECT ON GERMINATION OF CASTOR OIL PLANT

SEED OF CASTOR OIL PLANT:

For successful crop yield seed germination has great importance among the
crops (Almansouri et al., 2001). Mostly the castor oil plant seeds were classified into
five types according to seed coat, seed coat colour, seed pattern. The seeds are have
different colours from immature to mature, yellow to black and having different oil
contain. In nature seed coat of castor oil is impermeable to different chemical
26
compounds as compare to other seed for example soybean, switchgrass (Salanenka and
Taylor, 2009). The caruncle is common in many species of family of Euphorbiaceae.
According to Bianchini et al., (1996) stated that the caruncle in castor seed enhance the
germination. The removal of caruncle has no affect on germination, and in some causes
it promotes germination (Martins et al., 2006; Lagoa et al., 1987; Sousa et al., 2009),
another report removal of caruncle under different levels of salinity and moisture show
no effect on germination (Severino et al., 2012).

STORAGE AND DRY EFFECT ON SEED GERMINATION

It was observed that storage of seed for longer time has not influence the
germination, the seed of castor oil plant stored for one year no affect was observed in
germination, and after five years storage 65 to 70% seed survival was obtained (Pandey
and Radhamani, 2006). The position of racemes does not affect the germination
(Machado et al., 2010). The water and dry weight occurrence in seed was equal in the
different positions of racemes.

REQUIREMENTS OF GERMINATION

The conditions required for seed germination for example temperature,

moisture, were different in different species. Minimum and maximum temperature
required for germination of castor oil plant is 14 to 15°C, and 36°C respectively, and
the optimum 31°C, Moshkin (1986) reported that low temperature render the
germination. Requirement of moisture for castor oil seed germination minimum to
maximum is 60to80% in field (Severino et al., 2012).

SALINITY STRESS ON GERMINATION

The process of seed germination is a complex, seed germination influence by

number of factores such as hormonal and environmental (Finch-Savage and Leubner-
Metzger, 2006). One of them is salinity, salinity effects the seed germination by
27
developing external osmotic potential, which inhibit water intake, and this is because of
toxic effect of sodium and chloride ions (Khajeh-Hosseni, 2003; Kandil et al., 2012).
There are many reports shows that different salinity levels reduce the germination
percent (Mauromicale, et al., 2001). The both glycophytes (Zhu, 2003) and halophytes
(Khan and Huang, 1988; Gul and Weber, 1998; Li et al., 2005) germination was
affected by salinity. The species which showed reduction in germination under salinity
bread wheat (Ashraf and McNeilly, 1988), durum wheat (Almansouri et al., 2001),
barley (Norlyn and Epstein, 1982), tomato (Kurth et al., 1986) and lucerne (Rogers et
al., 1995).

SALINITY AND BIOCHEMICAL ASPECT

The biochemical, physiological processes for example photosynthesis, nitrogen,

metabolism, ion homeostasis, are affected by salinity (Misra et al., 2006; Ashraf, 2004),
and accumulation of proline as osmolytes (Misra and Gupta, 2005). In the response to
salinity plant adopted different ways to limit the effects of salinity by changing in
morphology, anatomy, and physiology (Poljakoff- Mayber, 1988). Salinity affects the
carbon dioxide (Co2) assimilation, in photosynthesis which finally reduced the growth
of plant (Cha-Um et al., 2009; Seeman et al., 1986). Salinity causes chemical changes
such as osmotic stress, toxicity of ions which finally the plant growth (Nublat et al.,
2001).

SALINITY STRESS AND NUTRIENTS IMBALANCE

Mostly salinity stress disturb the ions status, and causing hyperosmotic stress,
change the water potential, which leading to molecular damage, and growth inhibition,
which lead to death of plant (Zhu,2001).

SALINITY STRESS AND POTASSIUM

ROLE OF POTASSIUM

28
 Plant growth, development depend up the uptake of minerals with water from
soil and photosynthetic activates, in this way plant needs nutrients, Potassium (K+) is
necessary mineral for plant development and growth (Taiz et al., 2009), and it has some
functions such as the reactions that occur in pyruvate formation, activation of essential
enzymatic reactions, it has role in agencement of osmotic stress and, therefore, cell
turgor, which provides support and hardness to non-lignified cells (Maathuis et al.,
1999), protein synthesis, animating photosynthesis (Freitas et al., 2001; Ashraf, 2004).
Besides this, Potassium (K+) shortage limited rate of photosynthesis, photosynthetic
pigments and assimiliation carbon (Zhao et al., 2001). Potassium (K+) also required
maintain the structure and functions of plasma membrane (Wenxue et al., 2003).
Maintenance of adequate amount of potassium (K+) in plant parts or tissue under
salinity influence, it was noticed, it based on selective uptake of potassium (K+) and
selective cellular potassium (K+) and (Na+) compartmentation and allocation in the
shoots (Carden et al., 2003; Munns et al., 2000).

REDUCTION OF POTASSIUM CONTENT

When NaCl was applied to the soil, the levels of K+ in plant were reduced in
accordance with the antagonism between Na+ and K+ (Alberico and Cramer, 1993;
Azevedo and Tabosa, 2000). Cramer et al. (1985) reported that surplus sodium chloride
(NaCl), has role in deduction of potassium due to damage of cell membrane by sodium
(Na+) ions. Hu et al., (2005) reported reduction in potassium, effect the calcium and
magnesium content under salt stress, due to sodium interference and ionspairing of
sodium. In this scenario sodium potassium ration is so important to understand the
salinity tolerance of plants (Apse et al., 2007). So the competition between sodium and
potassium for binding sites, for cell metabolism brought to noticed by Tester et al.,
(2007). Decreased in K+ concentration with increasing salinity was recorded in many
plants; rice Aslam et al. (1993), in wheat (Qureshi et al., 1991; Akhtar et al., 2001;
Nawaz et al., 1998). Accumulation of high concentration Na+ and Cl- ions in root zone,
which aggravate the selective uptake of potassium and caused in reduction of potassium
(Gadallah, 1999). For this reason in sodium potassium ratio, sodium increased under sal
stress (Weimberg 1987). The deficiency of potassium (K+) initially brings about
chlorosis and then necrosis (Gopal et al., 2003). The excessive of Na+ ions in plant
29
affect the in many ways for example stomatal opening and closining, inflexion of
enzyme fuction which finally disturb the plant growth and development. Salinity was
shown to increase the uptake of Na+ or decrease the uptake of Ca2+ and K+ (Neel et al.,
2002). Britto et al., 2010, and Coskun et al., (2013) reported that decrease in K+
concentrations when plants are exposed to high Na+ concentrations.

SALINITY STRESS AND CALCIUM, MAGNESSIUM

ROLE OF CALCIUM, MAGNESSIUM

Calcium is known to increase salinity tolerance and mitigate the adverse effects
of salt stress on plant growth (Jaleel et al., 2007). In plant cells, calcium has significant
role in both intercellular and extracellular stimuli, and carried the different reaction as
co-enzyme (Sneeden et al., 2001), as nutrient calcium regulate plant growth and
development (Arshi et al., 2006). According to Trofimova et al., (1999) calcium may
be involved genetical process such as in signal transduction involving new gene
expression under oxidative stress. It also controls osmoregualtion, turgity of guard-cell,
opening and closing of stomata (Bhattacharjee, 2009). Calcium (Ca2+) plays a key role
in limiting the toxic effects of Na+ on integrity of plasma membrane in root cells.
Calcium can by stabilizing membranes and protect it from stresses such as oxidative
and salt (Larkindale et al., 2002). Salinity affects plant growth and its deleterious
effects are attributed to a reduced osmotic potential of the growing medium, specific
ion toxicity and nutrient deficiency (Luo et al., 2005; Bhattacharjee, 2008). Ca2+
sustains K+ transport and K+/Na+ selectivity in Na+ challenged plants (Rengle, 1992). In
salt affected plant it was noticed that both calcium (Ca+) and magnesium (Mg2+)
protected the cell membrane and give support to perform the function of transport and
osmoregulation (Wenxue et al., 2003). The ions has much significant because they
regulate the different metabolic process of plant, magnessium (Mag2+) is one of them.
The magnesium (Mg2+) worked as co-factor of different enzymes particular in
phosphate transfer, and magnesium (Mg2+) is integral part of chlorophyll molecules,
without magnesium chlorophyll cannot perform the function of photosynthesis.

30
REDUCTION OF CALCIUM, MAGNESSIUM CONTENT

Rout et al., (2001) studies the effect of salt stress on Najas gramenia, Najas
indica, and Hydrilla verticillata found that salt induced the reduction in calcium and
magnesium in their tissues. In root zone effect of salt stress disturb the cell membrane
and their properties, in regard Ca2+/Na+ ratio decreases, so the selective up take of
sodium was increased and calcium reduced (Kinaraide, 1998). An experiment was
conducted on plotted rice plans irrigated with different concentration of Japan sea water
(Electrical conductivity 0.9, 5.7, 11.5, or 21.5 ds/m) from 75 to 100 DAT showed that
the leaf Na+ concentrations increased with increasing salinity level while Ca2+ and k+
concentrations decreased (Sultan, 2002). The sodium (Na+) ions replacing the calcium
(Ca2+) ions which disturb the cell membrane and causing the leakiness of cell
membrane (Orcutt et al., 2000). Excess Na+ displaces Ca2+ from binding sites at plasma
membrane with consequent loss of K+/Na+ selectivity (Cramer et al., 1985). Increasing
the Ca2+/Na+ ratio in the external solution has been reported to alleviate the effects of
salinity on depolarization and selectivity of plasma membrane (Rinaldelli et al., 1996).
The majority of the scholar reported that reduction of nutrients uptake for example K +,
Ca2+, and Mn2+ is because of high concentration of deleterious ionic such as sodium
(Na+) and chloride (Cl-) in tissues of plant (Karimi et al., 2005). So to check the
content, uptake, and transportation of calcium (Ca+); in reference to the salinity stress is
considered as one of the important parameter to identify the salt tolerance of different
plant species (Soussi et al., 2001; Unno et al., 2002). Higher concentrations of Na+
resulted in the disintegration of the membranes because of osmotic stress and ionic
displacement (especially Ca2+), which further resulted in the release of cytoplasmic
contents, including water and K+ (Nassery, 1975; Lynch and Lauchi, 1984; Cramer et
al., 1985; Britto et al., 2010; Coskun et al., 2013). When plant are grown in salinity
reduction of magnesium was noticed by (Rout et al., 2001; Niaz et al., 1998; Esteves et
al., 2008; Japeetong et al., 2009), another scholar Renault et al. (2001) stated that
magnesium ions concentration decrease against sodium chloride (salt stress) in Cornus
stolonifera, this may occur in different way when combined with SO42–, and the excess
of sodium (Na+) and SO42– render the uptake of magnesium (Mg2+) from the soil
(Esteves, 2009).

31
SALINITY STRESS AND CHLOROPHYLL

Salinity is a abiotic factor which influence the plant growth in numbers of ways,
one of them is reduction in the chlorophyll, It has been reported that the chlorophyll
level of trees decreases with aggravated salt stress or grow in saline soil (Rao, et al.,
1986), due to enzymatic chlorophyll degradation (Carter and Cheeseman. 1993, Xu, et
al., 2000). It also observed by (Gadallah. 1999, Agastian, et al., 2000), decrease content
of chlorophyll and carotenoids by salinity as reported in a number of glycophytes. For
screening of salt tolerance plants species, chlorophyll is considered as best parameter,
because chlorophyll is the principal agent, and responsible for photosynthesis (XinWen
et al., 2008). Salinity induced chlorophyll degradation in NaCl sensitive pea cultivar as
compared with tolerant one (Hernandez, et al., 1995), have also been reported earlier.

REASIONS FOR REDUCTION OF CHLOROPHYLL

Accumulation of toxic ions especially sodium Na+, and chloride Cl-, reduce the
chlorophyll content in three ways 1) reduction in biosynthesis of chlorophyll, 2)
degradation of chlorophyll, 3) reactive oxygen. The reduction in chlorophyll content
can be attributed to the inhibitory effects of accumulated ions such as sodium and
chloride, on the biosynthesis of the chlorophyll as reported by Mohammed (2007).
According to Arulbalachandran et al., (2009), the reduction of photosynthetic pigment
under salinity occurs due to degradation of chlorophyll by enzyme chlorophyllase and
reactive oxygen species generated during photorespiration. Some time decrease of
Chlorophyll content in the plants grown under salt stress conditions may be ascribed to
both of the increased degradation and the inhibiting effect on synthesis of that pigment
(Garsia-Sanchez et al., 2002). The leaf is chief photosynthetic oragn of plant, in leaves
aggregation of injurious ions inversely influenc Chl concentration (Meloni et al., 2003).
The salt developed the ionic effect on structure of cell and cell organelles, including
chloroplast, these changes influence the photosynthetic activity of plants (Sade et al.,
2010; Lawlor 2009). Under the salt stress conditions, deleterious ions such as sodium
(Na+) and chloride (Cl-) damaged the membrane of chloroplast, because these ions high
32
concentration aggregate into chloroplast (Wu and Zou 2009, Omoto et al., 2010). For
example, salt stress can break down chlorophyll (Chl), the effect impute to increased
level of the toxic cation, Na+ (Li et al., 2010, Pinheiro et al., 2008, Yang et al., 2011).
According to studies, Iqbal et al., (2006) reported that chlorophyll is reduced under
saline conditions.

REDUCTION OF PHOTOSYNTHESIS UNDER SALINITY

The reduction in photosynthesis under salinity can be attributed to a decrease in

chlorophyll content (Jamil et al. 2007) and activity of photo-system ΙΙ(Ganivea et al.
1998). Luorescence of chlorophyll reflected the photochemical activities of photo-
system ΙΙ(Ganivea et al. 1998). Photochemical efficiency of photo-system ΙΙ(fv/fm)
could be reduced by salinity stress (Jamil et al. 2007; Netondo et al. 2004). Also salt
stress caused change in leaf pigment content that terminal photosynthesis level,
distillation speed and stomatal transport is changing in salinity stress condition
(Doganlar et al., 2010). Salinity can affect chlorophyll content through inhibition of
chlorophyll synthesis or an acceleration of its degradation (Reddy and Vora 1986).

CHLOROPHYLL “a” LESS SENSITIVE

In many reports chl “a” is less sensitive stated by (Fang et al., 1998, Eckardt
2009), in satress condition chl b is degradate into chl “a” , as result in the increase in
Chl “a” content. Increase in pigment content was observed in salinity stressed plant
such as Rice (Doganlar et al., 2010) and Purslane (Rahdari et al., 2012). It is may be
due to increase in the number of chloroplast in the stressed plant leaves (Chaum et al.,
2009). Interestingly, Chl.a was less sensitive or better protected against salt stress
compared to Chl b. It was also established that carotenoid decreased to a lesser extent
than Chl.

IN SOME PLANTS CHLOROPHYLL DECREASE UNDER SALINITY

33
 Reduction in the photosynthetic pigments, such as Chl “a” and “b” under
salinity was reported in many studies in different plants, e.g in wheat (Arfan et al.,
2007, Perveen et al., 2010), alfalfa (Winicov et al.,1990), sunflower (Ashraf et
al.,2000, Akram et al., 2011), and castor oil plant (Pinheiro et al., 2008). A decrease in
chlorophyll content has been observed in Spinacia oleracea by Kaya et al., (2001)
under salt stress. Similarly decrease in chl content has been reported in sweet sorghum
(Almodares et al., 2008) and Vicia faba (El Sayed, 2011) under saline conditions. The
salt stress alterations in a leaf Chl content could be due to impaired anabolism or
accelerated pigment degradation.

SALINITY STRESS AND PROLINE

The most common mechanism for salt tolerance is the accumulation of

osmolytes or osmoprotectants, such as glycinebetaines, proline and sugars (de Oliveira
et al., 2013), glycerol, sucrose, trehalose, pinitol, proline and quaternary ammonium
compounds (QACs) such as glycinebetaine, alainebetaine, prolinebetaine, choline O-
sulfate, hydroxyprolinebetaine and pipecolatebetaine (Yang et al., 2003;Rhodes et al.,,
1993; Mohanty et al., 2002; Mansour, 2000; Martino et al., 2003; Abraham et al.,
2003; Ashraf and Harris, 2004; Vinocur and Altman, 2005), these are organic
metabolites of low molecular weight that are collectively known as compatible solutes
(Bohnert et al., 1995; Serraj and Sinclair, 2002; Ashraf and Harris, 2004; Vinocur and
Altman, 2005). The accumulation of these osmolytes results in a negative water
potential, compared to the external environment, thereby increasing water uptake
(Rhodes et al., 2002). These solutes tend to be uncharged at neutral pH and are highly
soluble in water (Bonhert et al., 1995). These are the metabolites which serve as
osmolytes; these osmolytes maintain osmoregulation of cells, without disturbing
normal physiological functions cells (Yancey et al., 1982).

PROLINE

Among the all osmolytes, proline is well known osmolytes; it was observed that
it accumulate in many organisms for example algae, marine invertebrates, plants, in the
34
response stress (Delauney et al,, 1993). The Kemble and McPherson were the first
scholar, studies the proline content in tissue of rye grass in 1954. Proline is synthesis
from glutamate; it is an essential amino acid and main component of protein synthesis
(Szabados et al., 2009). Proline accumulates in plant under the response to abiotic
stresses such as including low temperatures, nutrient deficiencies, and acidic
environments or where water is a limiting factor, soils contaminated with heavy metals
(Delauney et al,, 1993).

BIOSYNTHESIS OF PROLINE

According to Charest et al., (1990), and Yoshiba et al., (1997) biosynthesis of

proline in different plant occur in four ways; one an additional biosynthesis of proline,
second more consumption or degradation of proline, and third hydrolysis of protein in
stress conditions. In hyperosmotic stress (salt stress), out three reasons, first two got
more acception of researchers as compare to third (Delauney et al., 1993; Peng et al.,
1996), but in other plant proline synthesis occur in different ways. Decreased proline
catabolism may also add more proline against osmotic stress conditions. Biosynthesis
and degradation of proline are linked at glutamate, Proline degradation happen when
proline oxidase, the enzyme which carry this reaction known as proline dehydrogenase
(PDH) it convert proline into (P5C), the (P5C) then oxidized to glutamate by another
enzyme called (P5C) dehydrogenase (P5CD) (Kiyosue et al., 1994). The more
accumulation proline was observed in Solanum tuberosum (Forlani et al., 2000), rice
(Lin et al., 2002) and Arabidopsis (Kiyosue et al., 1996). Delauney et al., (1993) stated
that biosynthesis of proline occur from ornithine or glutamate, the synthesis of proline
from glutamate in stress conditions particularly salt stress reported Higher level of
proline content in stem and leaf maybe due to expression of genes encoding enzymes of
proline synthesis such as pyrroline-5-carboxylate or decrease in enzymes of proline
oxidative such as proline dehydrogenase which is controlled by osmotic and salinity
stress (Amini et al., 2005).
ROLE OF PROLINE AGAINST SALINITY

Proline is a compatible solutes prevent the plants from overextend abomination

in different ways, it provide precautions for scavenging of hydroxyl radicals, cytoplasm
35
and chloroplasts from sodium ions (Na+) effects (Smirnoff et al., 1989), maintaining
structure of cell membrane (Crowe et al., 1992) composition of proteins (Bohnert et al.,
1996), and stabilized the plant physiological conditions in different abiotic stresses
(Rhodes et al., 1993; Galinski et al., 1994). The proline in plant detoxifying the
harmful effects of ions in plants when plant exposed to salinity stress, and cell osmotic
adjustment (Kavi et al., 2005; Ashraf et al., 2007).

PROLINE ACCUMLATION AGAINST STRESSES

Proline increase in many species because of it is sensible to different stresses,

such as salinity and drought (Nanjo et al., 1999; Jain et al., 2001), Heat and Cold
Temperatures, atmospheric pollution and heavy metal toxicity (Bohnert et al., 1995;
Sleator and Hill., 2002), sodium high content in tissue (Bajji et al., 2001). According to
Lehmann et al., (2010) proline is positively correlated with developing embryo,
dividing tissues. Proline is highly found in pollen and seeds. The high content of
proline in leaves is one of plant physiological respons to various stresses.

PROLINE INCREASE IN DIFFERENT PLANTS UNDER SALINITY

Proline is accumulated under the stress conditions especially in salinity stress

for example it increased in salt stressed durum wheat (Mattioni et al., 1997), cowpea
and high in salinity tolerance tomato (Somal & Yapa, 1998). The high concentration of
proline was observed in root of Eucalyptus microtheaca which grow in 200mM
concentration of sodium chloride (NaCl) (Morabito, Jolivet, Prat & Dizengremel,
1996), root tips of maize (Raymond & Smirnoff, 2002) and many other reports proline
increase in response to salinity in Capsicum annuum (Chookhampaeng, 2011) Brassica
genotypes (Siddiqui et al., 2010) Jatropha curcas (Patel et al., 2010), and alfalfa (Mezni
et al., 2010).

Chapter No. 04

Results

36
The objective of the study was to evaluate relative salt tolerance of castor oil two
varieties under saline water application. Our study was divided into three steps.

 Assessment of stress of salt on germination of Castor bean plant (L. Ricinus

communius) in petri dishes.
 Study of effect of salt stress on growth of Castor bean plant (L. Ricinus
communis) in pots.
 Investiging effect of salt stress on biochemical attributes of Castor bean plant
(L. Ricinus communis) harvest after four months.

Study-1 conducted in petri dishes for analysis of germination response of castor oil
plant under various levels of salinity at room temperature. The data from study one
shows that castor oil germinate up to high level of salinity (0, 20, 40, 60, 80, 100) and
very little effect was observed on germination rate and percent (%) germination.

Percent germination (%):

The data related to the effect of salinity (NaCl) on germination (%) is given in
(Fig. 1 and Table 1 & 2). The results obtained from the study indicates that the salinity
reduce the % germination significantly. The highest value in red variety at control
(87.0%) followed by (83.8%) in green, and the lowest value at 100mM in green variety
(49.7%) followed by (54.2%) in red variety, the salinity render the percent germination
stating from T1(20mM), its values decreased with high numbers after T1(80mM)
concentration of salt. The overall values of this plant character were decreases with
increase in salinity at higher (20, 40, 60, 80, and 100mM) concentrations by 69.21-
81.91, 62.23-69.53, 57.47-64.45, 56.51-57.78, 52.39-52.38, and 45.08-48.57
respectively in green and red varieties.

Germination Rate:

The data concerned with germination rate under salt stress given in a (Fig. 2 and
Table 3 & 4). Looking into the data obtained from the experiment that little effect on

37
germination rate was observed, reduction start from T4 (60mM) to T6(100mM). The
highest value at control 10.39 in green, followed by 10.49 in red, the lowest at
T6(100mM) 9.29 in green, and 9.83 in red. The overall values of this character was
decreases with increase in salinity at higher (20, 40, 60, 80, and 100mM) concentrations
by 10.4-10.5, 10.4-10.5, 10.4-10.5, 10.0-10.2, 9.8-10.1 and 9.3-9.8 respectively in
green and red varieties. The results show the stepwise decrease in germination rate by
increase salinity significantly (Fig. 2 and Table 3).

Study-2 Study of tolerance to salinity on growth of Castor bean plant (L. Ricinus
communis). This experiment was conducted in net house, no control over temperature,
humidity. Growth results are given under.

Stem girth (mm):

Salinity effect not only the % germination, and germination rate of plant but
also the plant stem girth it significantly reduced (Fig. 3 and Table 5 & 6), the negative
effect of salinity start from T2(20mM) concentration of salt and up to T6(100mM)
concentration, the overall values was decreases with increase in salinity at higher (20,
40, 60, 80, and 100mM) concentrations by 9.0-10.3, 8.8-9.8, 8.5-8.5, 7.6-7.9, 7.5-7.8,
and 7.0-7.0 (mm )respectively in green and red varieties. Their little difference between
control and T2(20mM) but from T3(40mM) to T6(100mM) the stem girth reduced.

Number of leaves:

Like stem girth, salinity also decreased number of leaves per plant (Fig. 4 and
Table 7 & 8).The data brings to notice that varieties decrase significantly in number of
leaves production; the difference between two varieties against salt stress (NaCl) was
clear. The overall results decreases with increase in salinity at higher (20, 40, 60, 80,
and 100mM) concentrations by 10.3-9.3, 8.5-9.0, 8.5-83, 7.5-8.0, 8.3-7.5, and 7.3-7.0
respectively in green and red varieties. Generally, red variety produced more leaves,
followed by green variety, the maximum number of leaves at T1(control) 10.25, the
lowest number of leaves 7.00 at T6(100mM).

38
Leaf area index (cm2):

The (Fig. 5 and Table 9 & 10) shows the data pertaining to the effect of (NaCl)
salt stress on the leaf area index. The results show that significantly reduction in leaf
area index was observed. The negative impact was studied from T2(20mM) with 189.67
in green, 319.24 in red. Overall results decreases with increase in salinity at higher (20,
40, 60, 80, and 100mM) concentrations by 341.3-319.2, 189.7-231.8, 178.5-188.8,
149.3-196.4, 134.7-150.5, and 108.9-110.4 respectively in green and red varieties.

Number of nodes plant-1:

The data effect of (NaCl) salinity on the number of nodes per plant presented in
(Fig. 6 and Table 11 & 12). Number of nodes indicator the development of plant, the
results shows that number of nodes per plant significantly reduced against salinity, the
difference between varieties was distinct from the data, it reduces as the concentration
of salt increases from T1 (control) to T6 (100mM). The high number was observed at
control 14.25 in red, the lowest at 10.00 in green variety, the overall results decreases
with increase in salinity at higher (20, 40, 60, 80, and 100mM) concentrations by 14.3-
14.8, 12.5-13.0, 12.0-11.8, 11.5-11.3, 11.3-10.8, and 10.5-10.0 respectively in green
and red varieties.

Shoot length (cm):

The effect of (NaCl) salinity on the shoot length (cm), data of both varieties is
entered in (Fig. 7 and Table 13 & 14). The height result was observed at T1 (control)
81.91 in red variety and the lowest at T6(100mM) 45.08 in green variety. The overall
results slightly decreases with increase in salinity at higher (20, 40, 60, 80, and 100mM)
concentrations by 69.2-81.9, 62.2-69.5, 57.5-64.5, 56.5-57.8, 52.4-52.4, and 45.1-48.6
respectively in green and red varieties.

Root length (cm):

The (Fig. 8 and Table 15 & 16) shows the results regarding to root length under
(NaCl) salinity effect. Similarly to the shoot length root length also decreased under salt

39
stress significantly as compare to control. But here the green showed better adoption
against salinity than red variety, the height value obtained at T1(control) 22.22 in green
and the lowest at T6 (100mM) 12.70 in red. The root length decreases with increase in
salinity at higher (20, 40, 60, 80, and 100mM) concentrations by 22.2-18.7, 18.7-16.2,
16.8-16.5, 16.2-14.6, 15.6-14.9, and 13.7-12.7 respectively in green and red varieties.
The results show the stepwise decrease in root length by increase salinity significantly
(Fig. 8, Table. 16).

Shoot fresh weight (g):

The data effect of (NaCl) salinity treatments on shoot fresh weight of both
varieties presented in (Fig. 9 and Table 17 & 18). The highest value 88.29 in red at
T1(control) and the lowest value 25.69 in green variety at T6(100mM) concentration of
salt, the overall results decreases with increase in salinity at higher (20, 40, 60, 80, and
100mM) concentrations by 79.4-88.3, 56.3-66.1, 48.2-47.4, 37.4-43.7, 30.9-38.5 and
25.7-29.1 respectively in green and red varieties. The results show the stepwise
decrease in root length by increase salinity significantly (Fig. 9, Table. 18).

Root fresh weight (g):

The entered data in (Fig. 10 and Table 19 & 20).is related with effect of salinity
on root fresh weight, the data shows a significant decrease in root fresh weight of plant.
The detectable effect on this parameter started from T2(20mM) as assessed after four
months. The overall results decreases with increase in salinity at higher (20, 40, 60, 80,
and 100mM) concentrations by 9.0-8.6, 6.1-7.5, 5.3-5.7, 4.0-4.8, 2.9-4.0 and 2.3-2.9
respectively in green and red varieties. From the data it quite clear that green variety
has great root fresh weight as compare to red variety.

Shoot dry weight (g):

The (Fig. 11 and Table 21 & 22).shows the data of shoot dry weight under salt
stress. The results indicate that with increasing salinity (NaCl) concentration, shoot dry

40
weight per plant decreased. The results expressed that the interaction of salt (NaCl)
treatments was significant for this trait. The overall results slightly decreases with
increase in salinity at higher (20, 40, 60, 80, and 100mM) concentrations by 14.4-13.6,
9.1-10.9, 8.0-7.6, 6.0-7.1, 4.6-5.1, and 3.2-4.0 respectively in green and red varieties.
The highest shoot dry weight was observed at T1 control(0mM) 14.44g in green and the
lowest at T6 (100mM) 3.17g in green, the red variety perform better than green under
salt stress.

Root dry weight (g):

Root dry weight of plant was also affected inversely with increasing salinity
levels (Fig. 12 and Table 23 & 24), the effect and patter of variation of root dry weight
was similar as recorded in shoot dry weight. The decreased in root dry weight was
observed significantly and was differentiated statistically at all treatments. The great
content of root dry weight was assessed in red 1.50g, and the smallest in green variety
0.31g. The overall results slightly decreases with increase in salinity at higher (20, 40,
60, 80, and 100mM) concentrations by 1.5-1.5, 1.1-1.3, 0.9-0.9, 0.7-0.7, 0.5-0.6 and
0.3-0.5 respectively in green and red varieties.

Study-3 Investiging effect of salinity on biochemical attributes of Castor bean plant (L.
Ricinus communis). The biochemical parameters result are given below

Chlorophyll a (ppm):

With increasing salinity of water both varieties exhibited a trend of significantly

declining of chlorophyll a (Fig. 13 and Table 25 & 26).The highest chlorophyll a
content in T1 control was highest and ranged between 11.9 ppm in green, 12.2 ppm in
red variety. The lowest was observed at T6 (100mM) 1.3 ppm in green and followed by
3.2 ppm in red. The overall results decreases with increase in salinity at higher (20, 40,
60, 80, and 100mM) concentrations by 11.9-12.2, 10.7-10.3, 8.2-9.6, 2.9-7.5, 1.8-5.7
and 1.3-3.2 respectively in green and red varieties.

41
Chlorophyll b (ppm):

Data presented in (Fig. 14 and Table 27 & 28) indicated that saline water
significantly decreased the chlorophyll b. Varieties also showed different responses in
term of chlorophyll b content under different salinity levels. The comparison of
treatments, the highest relative mean at T1 control in red 7.0 ppm, while the lowest at
T6(100mM) 1.9 ppm in green. The red variety performs well and compare to green
variety. The results are decreases with increase in salinity at higher (20, 40, 60, 80, and
100mM) concentrations by 4.8-7.0, 4.3-5.4, 3.7-4.6, 2.9-4.2, 2.4-3.6, and 1.9-3.0
respectively in green and red varieties.

Sodium (Na+) content (ppm):

Sodium concentration increased, with increase in salinity levels (Fig. 15 and

Table 29 & 30). The treatments significantly increased Na+ concentration with respect
to T1 control in shoot dry matter, and the great value obtained at T6 (100mM) 30.1 ppm
in green, while the smallest value in green variety at T1 control(0mM) 10.9 ppm. The
green variety has much more sodium concentration corresponding to red variety. The
overall results are increases with increase in salinity at higher (20, 40, 60, 80, and
100mM) concentrations by 10.9-11.0, 14.4-18.5, 19.4-19.9, 20.2-20.8, 22.4-21.9 and
30.1-23.5 respectively in green and red varieties. T2 (20mM) the red plant has high
value than green same results at control 0mM, and T3 (40mM).

Chloride (Cl-) content (ppm):

The effect of salinity (NaCl) cocentrations on Cl- content of both varieties is

given in (Fig. 16 and Table 31 & 32). It is express from the data that salinity increases
the chloride (Cl-) content as the concentrations increased. of shoot dry matter. The
chloride content increased significantly under the salt stress, the smallest value obtained
at control 9.7 ppm in red followed by 10.7 ppm in green, and the highest at T6
(100mM) in green 29.0 ppm, followed by red 23.6 ppm, the results indicate that the
green variety has high chloride concentration except T2 (20mM) high content of
chloride 18.1ppm in red. The overall results increases with increase in salinity at higher
42
(20, 40, 60, 80, and 100mM) concentrations by 10.7-9.7, 14.1-18.1, 19.5-18.8, 21.2-
19.6, 22.6-21.9 and 29.0-23.5 respectively in green and red varieties.

Magnesium (Mg2+) content (ppm):

The effect of increasing salinity (NaCl) treatments on Mg2+ contents of shoot of

plant is shown in (Fig. 17 and Table 33 & 34). It is observed from the data the
increasing of salinity, decreases Mg2+ content in shoot significantly. The high content
of Mg2+ was observed in green at T1 control 78.7 ppm, in green followed by red variety
71.0 ppm, the smallest at T6 (100mM) 21.9 ppm in green followed by 33.2 ppm in red
variety, except T1 control the red has great Mg2+ content than green variety. The
overall results are decreases with increase in salinity at higher (20, 40, 60, 80, and
100mM) concentrations by 78.7-71.0, 53.5-60.3, 47.7-54.8, 38.7-45.5, 29.7-39.3, and
21.9-33.2 respectively in green and red varieties.

Calcium (Ca2+) content (ppm):

The results obtained from the study are similar to Mg2+ content (Fig. 18 and
Table 35 & 36). It quite clear from the data the salinity levels increased the calcium
content in shoot dry matter significantly decreased, it is less at T6 (100mM) 28.7 ppm
in green followed by 30.0 ppm in red variety, the red variety has overall more calcium
content as compare to green. The results are decreases with increase in salinity at
higher (20, 40, 60, 80, and 100mM) concentrations by 77.3-106.0, 57.7-87.0, 51.7-64.7,
43.3-52.3, 35.7-43.0, and 28.7-30.0 respectively in green and red varieties.

Potassium (K+) content (ppm):

The effect of salinity (NaCl) on K+ content of castor oil plant in shoot dry
matter is shown in (Fig. 19 and Table 37 & 38). The results evident that it is
significantly reduced potassium content with increase in salinity levels. The
observations are similar to the Mg2+ content. The less value get from T6(100mM) in
green 8.9 ppm, followed by 21.9 ppm in red variety, the greater values obtained at T1

43
control concentration 38.1 ppm in red followed by 36.4 ppm in green variety. It can be
easily recognized that the red has more potassium content as the green variety. The
results are decreases with increase in salinity at higher (20, 40, 60, 80, and 100mM)
concentrations by 36.4-38.1, 31.8-37.1, 28.9-30.7, 25.1-25.6, 18.1-23.2, and 8.9-21.9
respectively in green and red varieties.

Proline content (ppm):

Proline one of the best indicator of the salinity tolerance in plant, it increased under the
stress conditions, so the data we get are similar to this statement (Fig. 20 and Table 39
& 40). As the salinity levels increased the concentration of proline in shoot dry matter
also increased significantly. The highest value obtained at T6 (100mM) 14.7 ppm in red
followed by 10.8 ppm in green, the similar results are at serious levels of salinity but
the 40mM concentration of salt both varieties showed same results 6.5 ppm. The lowest
value at T1 control 1.1 in green followed by red 1.8 ppm. The overall results are
increases with increase in salinity at higher (20, 40, 60, 80, and 100mM) concentrations
by 1.1-1.8, 4.5-3.5, 6.5-6.5, 9.4-8.8, 12.5-9.9 and 14.7-10.8 respectively in green and
red varieties.

44
Fig. 1. Effect of salts on percent germination (%) of castor oil verities.

Fig. 2. Effect of salt stress on germination rate of castor oil verities.

Fig. 3. Effect of salt stress on stem girth (mm) of castor oil verities.

45
Fig.4. Effect of salt stress on number of leaves per plant of castor oil verities.

Fig. 5. Effect of salt stress on leaf area index per plant of castor oil verities.

46
Fig. 6. Effect of saline waters on number of nodes per plant of castor oil verities.

Fig. 7. Effect of saline waters on shoot length of (cm) castor oil verities.

47
 Fig. 8. Effect of saline waters on root length (cm) of castor oil verities.

Fig. 9. Effect of saline waters on shoot fresh weight (g) of castor oil verities.

48
Fig. 10. Effect of salt stress on root fresh weight (g) of castor oil verities.

Fig. 11. Effect of salt stress on shoot dry weight (g) of castor oil verities.

49
Fig. 12. Effect of salt stress on root dry weight (g) of castor oil verities.

Fig. 13. Effect of salt stress on chlorophyll a (ppm) of castor oil verities.

50
Fig. 14. Effect of salt stress on chlorophyll b (ppm) of castor oil verities.

Fig. 15. Effect of salt stress on sodium Na+ (ppm) of castor oil verities.

51
 Fig. 16. Effect of salt stress on chloride Cl- (ppm) of castor oil verities.

Fig. 17. Effect of salt stress on magnesium Mg2+ (ppm) of castor oil verities.

52
Fig. 18. Effect of salt stress on calcium Ca+ (ppm) of castor oil verities.

Fig. 19. Effect of salt stress on potaasium K+ (ppm) of castor oil verities.

53
 Fig, 20. Effect of salt stress on proline content (ppm) of castor oil verities.

Seed germination pictures

54
Seed germination pictures

55
Pot experminet pictures

56
57
58
59
Chapter No. 5
DISCUSSIONS

Two separate experiments, one in petri dishes and second in pots, were
conducted at department of Botany Shah Abdul Latif University Khairpur Sindh
Pakistan to the assess salinity tolerance of two varieties of castor oil plant.

Effect of salinity/salt stress on germination of castor oil plant

The results presented in (Fig. 1, Table 1) obtained from the petri dishes
experiment showed that the influence of salinity (NaCl) was adversely affects the seed
germination of castor oil plant. It is a fact that the solute potential has pivotal role in
cell development and ultimate embryo development, which refers as seed germination.
The castor seed germination was also affected and decreased as compare to control
treatments (Fig. 1, Table 1&2) at present study. Allen et al., (1985), Machado Neto et
al., (2004) and K. Chartzoulakis and Klapaki, (2000) reported that salinity has reduced
the castor seed germination, which affirms the present study. The results show that red
varieties perform better as compare to green variety.

Effect of salinity/salt stress on growth of castor oil plant

It was resulted from the present work conducted in pots that with increasing
concentration of NaCl from 0 to 100mM concentration number of leaves decrease (Fig.
4, Table. 7&8). The results obtained from the experiment confirm the in Zea mays by
Hassine and Lutts, (2010) and in Solanum tubersum by Albacete et al., (2008), it due to
the toxic affect of sodium and chloride ions which inhibit the growth and development
of plants. From the results it was observed that both varieties show mix trend in
reduction number of leaves per plant. The salinity effect on leaf area index is presented
in (Fig. 5, Table. 9&10). Shows decrease in leaf area index per plant under salinity,
these result are similar to (Steduto et al., 2000) and Garg and Gupta, (1997). The
reduction in leaf area could be due to accumulation of high amounts of toxic salts like

60
sodium ( Na+) in the leaf apoplasm wjich leading to loss of water (dehydration) and
turgor loss, and resulted in death of leaf cells and tissues (Marschner, 1995). Shoot and
root length of some plant is sensitive to salt stress, the shoot and root length of castor
oil plant under salt stress entered in (Fig. 8, Table 15&16). These results are confirm
the by Netondo, et al., (2004); Keutgen and Pawelzik, (2008); Kasukabe et al., (2006);
Ibrahim et al., (2007) it due to the toxic affect of sodium and chloride ions which
inhibit the growth and development of plants. Also, a high concentration of salt tends to
slow down or stop root elongation (Kramer. 1983) and causes reduction in root
production (Garg and Gupta 1997). Reduction in biomass is common observation under
the salinity stress; it was resulted from the present work conducted in pots that with
increasing concentration of NaCl from 0 to 100mM concentration shoot fresh weight
decrease (Fig. 9, Table. 17&18). The results obtained from the experiment affirm the
by Kent and Lauchli (1985) and Terry and Waldron, (1984), it due to the toxic affect of
sodium and chloride ions which inhibit the growth and development of plants. The
salinity also reduces the root fresh weight (Fig. 10, Table. 19&20). The results obtained
from the experiment accord with the by Kent and Lauchli (1985), and Qureshi et al.,
(1991), Akhtar et al., (2000), the results of shoot dry decrease with increase in salinity
(Fig. 11, Table. 21&22). The results obtained from the experiment confirm the by
(Brugnoli and Lauteri, (1991) and Shannon et al. 1998), and root dry weight results
decreases from 0 to 100mM concentration are present in (Fig. 12, Table 23&24). These
results are establish by (Munus and Termat, 1986; Sharma, 1989) and Shannon et al.,
(1998).

Effect of salinity/salt stress on biochemical attributes of castor oil plant

The salinity not only affects the castor oil plant growth, it influence on the
physio-chemical activities of plant. In present study it observed with increasing
concentration of NaCl from 0 to 100mM concentration chlorophyll a (Fig. 13, Table
25&26). The results obtained from the experiment confirm the in alfalfa, Medicago
sativa by Winicov and Seemann (1990), in wheat, Triticum aestivum (Arfan et al.,
2007, Perveen et al., 2010), and castor bean, Ricinus communis (Pinheiro et al., 2008),
it due to the toxic affect of sodium and chloride ions which inhibit the development of

61
pigments or case degradation of pigments of plants. The results regarding to
chlorophyll b are given in (Fig. 14, Table 27&28), reduction in chlorophyll a was
observed from 0 to 100mM concentration of salt. The results obtained from the
experiment confirm the by (Ashraf and Sultana 2000, Akram and Ashraf 2011),
chlorophyll a is less sensitive than chlorophyll b in castor oil plant. Sodium content
increases with increases in salt concentration (Fig. 15, Table 29&30). These results are
according to Wahid, (2004), and in many species, including sunflower (Ashraf and
O’Leary, 1995), rice (Zhu et al., 2001) and wheat (Schachtman et al., 1989; Poustini
and Siosemardeh, 2004). The results obtained from experiment shows salinity
adversely affect the nutrient content of plant, creating osmotic imbalance in root zone,
the magnesium (Mg2+) content decrease with increase in salinity (Fig. 17, Table
33&34). These results are similar to (Niaz et al., 1998; Rout et al., 2001; Esteves et al.,
2008; Japeetong et al., 2009), the potassium (K+) content decrease under salt stress
(Fig. 19, Table 37&38). The results obtained from the experiment confirm the by
Alberico et al., (1993); Azevedo et al., 2000) because of antagonism between Na+ and
K+, in rice Aslam et al., (1993), Akhtar et al., 2001; Nawaz et al., (1998). Proline is
osmolyte it increases in stress conditions, the results regarding to proline are given (Fig.
20, Table. 39&40). The results obtained from the experiment confirm the by Nanjo et
al., (1999); Jain et al., 2001), in alfalfa (Mezni et al., 2010), Jatropha curcas (Patel et
al., 2010), Brassica genotypes (Siddiqui et al., 2010) and Capsicum annuum
(Chookhampaeng, 2011).

In the conculsion of this discussion, there was difference of salt tolerance both
castor oil plant varieties was observed, in the present work. Examination of
relationships of various biochemical and growth attributes of castor oil plant showed
that seed germination, agronomical parameters, sodium, chloride, calcium, magnesium,
potassium, chlorophyll and proline content were sufficient for salt tolerance. In all these
attributes difference was observed in varieties. Thus, the screening of castor oil
suggests that red variety has shows effective against salinity/salt stress.

62
 SUMMARY

The study comprised of two separate experiments (1) Seed germination in petri dishes
(2) Pot experiment was conducted at the department of Botany SALU Khairpur
(Meirus), Sindh Pakistan, to evaluate the salinity tolerance oil plant (Ricinus communis
L.).

 General was observed that salinity effect the seed germination of castor oil
plant, very little effect was observed on germination rate, but from germination
percent it is clear that red variety performs better than green variety.
 At all salinity levels plant show decrease in plant height, biomass, number of
leaves, number of nodes, leaf are index, the red variety shows great tolerance as
compare to green variety.
 Under salinity stress reduction in calcium, magnesium, and potassium content
was observed in both varieties the red variety has more content than green
variety.
 The chlorophyll a and b content decrease with increase salinity levels in both
varieties, it was observed that chlorophyll a is less sensitive to salinity. The red
variety performs better than green variety.
 It was observed from the present study that with increasing salt concentrations,
absorption of ions decreased significantly except Na+ and Cl- concentration.
 Red variety has less accumulation Na+, Cl-.
 Antagonism effect between sodium and potassium was also observed.
 It was clear from the present study that salinity stress inversely affected the
plant growth and development of castor oil plant varieties at different salinity
levels.
 The results get from the two experiments show that the castor oil plant, green
variety grown 0 to 100mM showed deprive performance, while red variety
showed better adaption against salt stress.
 The proline content increase in both varieties in response to salinity from 0 to
100mM salt concentration, in red variety more proline content is observed than
green variety.

63
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 APPENDICES
Appendix 1. Effect of salinity stress on percent germination (%).

Green Seed Plant Red Seed Plant

NaCl treatment
Mean SEM Mean SEM
0mM 83.75 1.88 87.03 0.30
20mM 79.69 6.15 84.84 2.26
40mM 79.38 3.92 86.56 0.54
60mM 77.34 2.00 83.44 1.36
80mM 66.41 5.08 66.41 3.83
100mM 49.69 2.05 54.22 3.36

Appendix 2. Effect of salinity stress on percent germination ANOVA.

Sum of Mean
Squares df Square F Sig.
%germination Between 6866.081 5 1373.216 32.383 0.000
Groups
Within 1781.055 42 42.406
Groups
Total 8647.135 47

Appendix 3. Effect of salinity stress on germination rate.

Green Seed Germination Red Seed Germination

NaCl treatment
Mean SEM Mean SEM
0mM 10.40 0.02 10.49 0.02
20mM 10.42 0.04 10.49 0.03
40mM 10.39 0.02 10.45 0.04
60mM 10.00 0.06 10.23 0.10
80mM 9.80 0.07 10.10 0.07
100mM 9.29 0.12 9.83 0.13

Appendix 4.Effect of salinity stress on germination rate ANOVA.

Sum of Mean
Squares df Square F Sig.
Germination Between 5.115 5 1.023 26.018 .000
Rate Groups
Within 1.651 42 .039
Groups
Total 6.767 47

95
 Appendix 5.Effect of salinity stress on stem girth (mm).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 9.00 0.00 10.25 0.25
20mM 8.75 0.25 9.75 0.48
40mM 8.50 0.29 8.50 0.29
60mM 7.63 0.24 7.88 0.13
80mM 7.50 0.29 7.75 0.25
100mM 7.00 0.00 7.00 0.20

Appendix 6.Effect of salinity stress on stem girth ANOVA.

Sum of Mean
Squares df Square F Sig.
Stem Between 41.167 5 8.233 23.444 .000
girth Groups
(mm)
Within 14.750 42 .351
Groups
Total 55.917 47

Appendix 7.Effect of salinity stress on number of leaves per plant.

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 10.25 0.63 9.25 0.25
20mM 8.50 0.29 9.00 0.00
40mM 8.50 0.29 8.25 0.25
60mM 7.50 0.50 8.00 0.00
80mM 8.25 0.25 7.50 0.29
100mM 7.25 0.25 7.00 0.00

Appendix 8.Effect of salinity stress on number of leaves ANOVA.

Sum of Mean
Squares df Square F Sig.
No Between 33.354 5 6.671 15.458 .000
Leaves Groups
Within 18.125 42 .432
Groups
Total 51.479 47

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 Appendix 9.Effect of salinity stress on leaf area index (cm2).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 341.33 58.95 319.24 33.59
20mM 189.67 4.31 231.76 29.40
40mM 178.47 9.93 188.78 6.36
60mM 149.26 6.35 196.45 19.51
80mM 134.67 12.40 150.50 18.56
100mM 108.90 12.67 110.42 10.74

Appendix 10.Effect of salinity stress on leaf area index ANOVA.

Sum of Mean
Squares Df Square F Sig.
LAI Between 233042.446 5 46608.489 21.384 .000
(cm) Groups
Within 91542.389 42 2179.581
Groups
Total 324584.835 47

Appendix 11. Effect of salinity stress on number of nodes.

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 14.25 0.25 14.75 0.25
20mM 12.50 0.29 13.00 0.71
40mM 12.00 0.41 11.75 0.48
60mM 11.50 0.29 11.25 0.25
80mM 11.25 0.25 10.75 0.25
100mM 10.50 0.29 10.00 0.00

Appendix 12.Effect of salinity stress on number of nodes ANOVA.

Sum of Mean
Squares df Square F Sig.
No of Between 90.167 5 18.033 38.349 .000
Nodes Groups
Within 19.750 42 .470
Groups
Total 109.917 47

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 Appendix 13.Effect of salinity stress on shoot length (cm)

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 69.22 2.82 81.92 2.17
20mM 62.23 0.73 69.53 1.59
40mM 57.47 0.61 64.45 1.59
60mM 56.52 0.64 57.79 0.82
80mM 52.39 0.95 52.39 0.61
100mM 45.09 1.22 48.58 0.80

Appendix 14.Effect of salinity stress on shoot length ANOVA.

Sum of Mean
Squares df Square F Sig.
SL cm Between 4136.171 5 827.234 41.810 .000
Groups
Within 830.996 42 19.786
Groups
Total 4967.167 47

Appendix 15.Effect of salinity stress on root length (cm).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 22.23 1.22 18.73 0.61
20mM 18.73 0.61 16.19 1.20
40mM 16.83 0.61 16.51 1.27
60mM 16.19 0.61 14.61 0.64
80mM 15.56 0.32 14.92 0.32
100mM 13.65 0.61 12.70 0.52

Appendix 16. Effect of salinity stress on root length ANOVA.

Sum of Mean
Squares df Square F Sig.
RL cm Between 244.623 5 48.925 15.537 .000
Groups
Within 132.258 42 3.149
Groups
Total 376.881 47

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 Appendix 17.Effect of salinity stress on shoot fresh weight (g)

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 79.43 6.93 88.29 6.65
20mM 56.34 3.04 66.12 10.27
40mM 48.16 3.07 47.39 1.34
60mM 37.43 2.67 43.69 1.05
80mM 30.89 0.70 38.52 3.57
100mM 25.69 1.63 29.06 2.19

Appendix 18.Effect of salinity stress on shoot fresh weight ANOVA.

Sum of Mean
Squares df Square F Sig.
SFW g Between 16873.794 5 3374.759 40.138 .000
Groups
Within 3531.326 42 84.079
Groups
Total 20405.120 47

Appendix 19.Effect of salinity stress on root fresh weight (g).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 9.00 0.96 8.63 0.48
20mM 6.10 0.13 7.48 0.95
40mM 5.34 0.23 5.66 0.72
60mM 4.05 0.21 4.79 0.31
80mM 2.91 0.10 3.97 0.21
100mM 2.28 0.05 2.92 0.17

Appendix 20.Effect of salinity stress on root fresh weight ANOVA.

Sum of Mean
Squares df Square F Sig.
RFW g Between 208.909 5 41.782 40.612 .000
Groups
Within 43.210 42 1.029
Groups
Total 252.119 47

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 Appendix 21.Effect of salinity stress on shoot dry weight (g).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 14.45 1.57 13.62 0.99
20mM 9.05 0.63 10.88 1.31
40mM 8.02 0.36 7.61 0.22
60mM 6.02 0.43 7.07 0.26
80mM 4.59 0.14 5.14 0.32
100mM 3.17 0.10 4.01 0.22

Appendix 22. Effect of salinity stress on shoot dry weight ANOVA.

Sum of Mean
Squares df Square F Sig.
SDW g Between 571.512 5 114.302 55.509 .000
Groups
Within 86.486 42 2.059
Groups
Total 657.997 47

Appendix 23. Effect of salinity stress on root dry weight (g)

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 1.46 0.16 1.50 0.11
20mM 1.13 0.03 1.27 0.15
40mM 0.93 0.06 0.88 0.05
60mM 0.72 0.04 0.69 0.04
80mM 0.51 0.05 0.57 0.08
100mM 0.32 0.02 0.52 0.04

Appendix 24. Effect of salinity stress on root dry weight ANOVA.

Sum of Mean
Squares df Square F Sig.
RDW g Between 6.587 5 1.317 50.542 .000
Groups
Within 1.095 42 .026
Groups
Total 7.682 47

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 Appendix 25. Effect of salinity stress on chlorophyll “a” (ppm).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 11.95 0.13 12.24 1.07
20mM 10.73 0.43 10.26 0.10
40mM 8.24 0.22 9.58 0.12
60mM 2.87 0.43 7.47 0.66
80mM 1.82 0.18 5.71 0.24
100mM 1.32 0.04 3.18 1.04

Appendix 26. Effect of salinity stress on chloride content ANOVA.

Sum of Mean
Squares df Square F Sig.
Chl a Between 625.003 5 125.001 42.997 .000
(ppm) Groups
Within 122.102 42 2.907
Groups
Total 747.105 47

Appendix 27. Effect of salinity stress on chlorophyll “b” (ppm).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 4.75 0.06 7.00 0.52
20mM 4.31 0.03 5.43 0.20
40mM 3.69 0.15 4.63 0.10
60mM 2.88 0.07 4.19 0.05
80mM 2.37 0.09 3.64 0.12
100mM 1.94 0.05 2.98 0.12

Appendix 28. Effect of salinity stress on chlorophyll b ANOVA.

Sum of Mean
Squares df Square F Sig.
Chl b Between 63.127 5 12.625 19.028 .000
(ppm) Groups
Within 27.868 42 .664
Groups
Total 90.995 47

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 Appendix 29. Effect of salinity stress on Sodium (ppm).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 10.92 0.28 11.02 0.28
20mM 14.41 0.53 18.45 1.11
40mM 19.40 0.28 19.95 0.00
60mM 20.22 0.28 20.77 0.00
80mM 22.43 1.26 21.88 0.28
100mM 30.14 4.41 23.53 0.73

Appendix 30. Effect of salinity stress on sodium content ANOVA.

Sum of Mean
Squares df Square F Sig.
Na Between 864.131 5 172.826 22.340 .000
(ppm) Groups
Within 232.089 30 7.736
Groups
Total 1096.220 35

Appendix 31. Effect of salinity stress on chloride content (ppm).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 10.74 0.34 9.73 0.31
20mM 14.13 0.58 18.11 2.52
40mM 19.51 0.67 18.84 0.33
60mM 21.24 0.58 19.56 0.34
80mM 22.64 1.86 21.92 0.34
100mM 29.02 2.34 23.60 0.88

Appendix 32. Effect of salinity stress on chloride content ANOVA.

Sum of Mean
Squares df Square F Sig.
Cl (ppm) Between 907.436 5 181.487 30.003 .000
Groups
Within 181.467 30 6.049
Groups
Total 1088.903 35

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 Appendix 33. Effect of salinity stress on magnesium content (ppm).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 78.70 10.14 70.96 4.34
20mM 53.54 0.32 60.31 1.16
40mM 47.73 2.33 54.83 0.65
60mM 38.70 1.12 45.15 2.76
80mM 29.67 2.33 39.35 0.32
100mM 21.93 1.71 33.22 1.41

Appendix 34. Effect of salinity stress on magnesium content ANOVA.

Sum of Mean
Squares df Square F Sig.
Mg Between 8675.810 5 1735.162 34.314 .000
(ppm) Groups
Within 1517.037 30 50.568
Groups
Total 10192.847 35

Appendix 35. Effect of salinity stress on calcium content (ppm).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 77.33 9.28 106.00 7.57
20mM 57.67 0.33 87.00 2.65
40mM 51.67 2.03 64.67 4.67
60mM 43.33 0.67 52.33 2.85
80mM 35.67 2.03 43.00 2.52
100mM 28.67 2.40 30.00 4.16

Appendix 36. Effect of salinity stress on calcium content ANOVA.

Sum of Mean
Squares df Square F Sig.
Ca Between 15587.889 5 3117.578 21.786 .000
(ppm) Groups
Within 4293.000 30 143.100
Groups
Total 19880.889 35

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 Appendix 37. Effect of salinity stress on potassium content (ppm)

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 36.41 0.63 38.06 0.00
20mM 31.83 0.18 37.14 0.66
40mM 28.90 1.47 30.73 0.97
60mM 25.06 0.92 25.60 0.18
80mM 18.10 1.20 23.22 0.32
100mM 8.95 0.15 21.94 0.18

Appendix 38. Effect of salinity stress on potassium content ANOVA.

Sum of Mean
Squares df Square F Sig.
K (ppm) Between 2070.575 5 414.115 32.425 .000
Groups
Within 383.139 30 12.771
Groups
Total 2453.715 35

Appendix 39. Effect of salinity stress on proline content (ppm).

Green Red
NaCl treatment
Mean SEM Mean SEM
0mM 1.76 0.53 1.10 0.91
20mM 4.53 0.31 3.48 0.20
40mM 6.51 0.14 6.46 0.53
60mM 9.44 0.45 8.78 0.48
80mM 12.48 0.20 9.94 0.22
100mM 14.74 0.24 10.82 0.24

Appendix 40. Effect of salinity stress on proline ANOVA.

Sum of Mean
Squares df Square F Sig.
Proline Between 565.787 5 113.157 69.596 .000
(ppm) Groups
Within 48.778 30 1.626
Groups
Total 614.565 35

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