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Standardization of Protocol for Pretreatment, Surface Sterilization,

Regeneration, Elongation and
Acclimatization of Chrysanthemum
morifolium Ramat
Arvind Kumar Verma , K.V. Prasad , T.J anakiram , S. Kumar
Division of Floriculture and Landscaping, Indian Agricultural Research Institute, New
Delhi, 110 012, India
Author
Correspondence author
International Journal of Horticulture, 2012, Vol. 2, No. 3 doi: 10.5376/ijh.2012.02.0003
Received: 10 Dec., 2012 Accepted: 17 Dec., 2012 Published: 26 Dec., 2012
2012 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Preferred citation for this article:
Arvind K.V. et al., 2012, Standardization of Protocol for Pre-treatment, Surface Sterilization, Regeneration, Elongation
and Acclimatization of Chrysanthemum morifolium Ramat, 2012, Vol.2, No.3 7-12 (doi: 10.5376/ijh.2012.02.0003)

Abstract
In the present study we have developed proficient in vitro regeneration protocol from the
ray florets of chrysanthemum cv. Thai Chen Queen to isolate the novel mutants. The
maximum survival percentage (89.44) of cultures was found when the ray florets were
per-treated with mancozeb-45 (0.1%)+carbendazim (0.1%)+8-HQC (200 mg/L) for 3
hours. Culture establishment was found best (91.04%) when the ray florets surface
sterilized with HgCl2 (0.1%) for duration of four minutes. Murashige and Skoog (MS)
media supplemented with 4 mg/L BAP and 0.1 mg/L NAA was found to the best for
regeneration percentage (93.33), number of microshoots per explants (5.67) and
minimum days required for shoot regeneration (30.67). The regenerated plantlets were
transferred on the MS media supplemented with GA3 0.50 mg/L was found to the best
for shoot elongation after 30 days of elongation. The successful acclimatization of
elongated plantlets was achieved by transferring them into glass jar with polypropylene

cap filled with peat+soilrite (1:1). The hardening of plantlets was completed after 25 days
of acclimatization and then plants were transferred to greenhouse for flowering.
Keywords
Chrysanthemum; Chrysanthemum morifolium; Growth regulators; In vitro culture; Shoot
regeneration; Acclimatization
Chrysanthemum (Chrysanthemum morifolium Ramat.) is an important ornamental plant for cut flower and pot plant in the
international flower market. After rose it is the second cut flower and dominates the ornamental plant trade in the world
market (Kumar et al., 2006).Traditional breeding and more recently, together with genetic, molecular techniques, has
focused on the enhancement of the plants ornamental value through the improvement of flower colour, size and form,
vegetative height, growth form and sensitivity to lightquality/quantity (Rout and Das, 1997). The improvement of
chrysanthemum has been done by several breeding methods but the chrysanthemum consider as the good plant for
induced mutation. The purpose of induced mutation is to enhance mutation rate in a short duration fir the developing
new plant varieties. Novelty is always in demand in the modern and industrialized floriculture in which there is need
for the new varieties to routinely meet the consumers demand and satisfaction (Misra et al., 2003).
The main bottleneck in vegetative propagated plants is when the mutation appears as partial chimeras after treatment
with physical and/or chemical mutagens. Although it is possible to isolate a portion of a branch or an entire branch if it
is completely mutated, but it is difficult to isolate such mutants/chimeras, which are often limited in extent and may
only be expressed as strip of colour in a single floret (Mandal and Datta, 2005). Mutation breeding coupled with in
vitro regeneration would be a useful approach to establish novel mutants in pure form and facilitate production of a
wide range of novel coloured cultivars in chrysanthemum (Verma et al., 2012). Indirect regeneration via callus was
reported from stem, petal and shoot tips, but adventitious shoot regeneration derived from an initial callus phase may
result in somaclonal variation and in chimerism while direct shoot regeneration from leaf or stem explants may
eliminate such undesirables. Tissue derived from the culture of ray florets of C. Morifolium could be isolated solid
mutants, despite of somaclonal variation occurring in the vegetative tissues (Pillai and Zulkifli, 2000).The direct
regeneration protocol from the ray florets has been successfully used not only for the isolation of chimeric mutant
tissues developed through sports, but also to develop a series of new flower color/shape mutants through induced
mutagenesis (Datta et al., 2005). The efficiency of recovery of solid colour mutants in chrysanthemum was different
due to type of explants used. The plants regenerated from the ray florets gave the maximum recovery of solid colour
mutants in chrysanthemum (Mandal et al., 2000). Therefore, it is prerequisite to establish the in vitro regeneration
protocol from the ray florets of chrysanthemum to utilise the induced mutation and to isolate the novel mutants in pure.
1 Results and Analysis
1.1 Standardization of pre-treatment
Subjecting the ray florets to different pre-treatments resulted in a marked influence on culture establishment. Data
presented in the Table 1 indicated that the pre-treatment of explants with different concentrations of fungicides namely
carbendazim, mancozeb and biocide, i.e. 8-HQC significantly reduced the microbial load and improved the survival
percentage as compared to control (distilled water dip). The pre-treatment of ray florets with mancozeb-45 (0.1%) +
carbendazim (0.1%) + 8-HQC (200 mg/L) for 3 hours was found to be the best with regards to minimum contamination
(15.56%) with highest survival (89.44%) of explants when compared to the other treatments and control. Pre-treatment
with mancozeb-45 (0.2%)+carbendazim (0.2%) + 8-HQC (200 mg/L) for 3 h recorded the 8.89% microbial
contamination with 75.56%survival which were statistical lower than the mancozeb-45 (0.1%)+ carbendazim (0.1%)
+8-HQC (200 mg/L) for 3 hours. The present findings are in conformity with the Bala et al (2010) in rose who found
the carbendazim (0.2%)+diathane M-45 Indofil (0.2%)+ 8-hydroxy quinnoline citrate (200 mg/L) for 3 h agitation gave
the highest explant survival (62.47%) and bud sprouting (56.63%). Kadam et al (2010) find the best response by pretreated thepetals and immature flower of tuberose with carbendazim 0.1%+ mancozeb 0.1% and 8-HQC (200 mg/L) for 2
h.

Table 1 Effect of different pre-treatments on contamination and explants survival

1.2 Standardization of surface sterilization for explants
Perusal data presented in Table 2 revealed that surface sterilization of explants with 0.1 % HgCl 2 for 4 min resulted in
lower contamination percentage (15.56) along with maximum survival (91.04%) over control (10.00%) and other
treatment. Surface sterilization of explants with HgCl 2 (0.1%) for a duration of 6 minutes significantly reduced
microbial contamination (7.78%) but also reduced the survival percentage (51.77%) and caused drying of explants
within 10 days of inoculation which may be due to phytotoxicity caused by long exposure of explants with HgCl 2. All
the treatment was statistically significantly different to each other. Verma et al (2011) also reported that surface
sterilization with 0.1% HgCl2 gave the highest survival percentage and lower the microbialcontamination in stevia
which were quite close to our findings. In another report nodal explants washed with household detergent to
remove the impurities and then washed thoroughly under tap water and then sterilization was done with 70%
alcohol for 2 minutes followed by 1% HgCl 2 for 2~3 minutes (Ilahi et al., 2007).

Table 2 Effect of surface sterilization on contamination and explants survival

1.3 Effect of BAP and NAA on shoot regeneration
The wounded ray florets culture on MS medium fortified with different concentration of BAP and NAA for the
regeneration. The data presented in Table 3 showed that MS medium supplemented with BAP and NAA significantly
enhanced shoot regeneration percentage from the ray florets over the control (MS medium devoid of hormones).
Significantly higher shoot regeneration (93.33%) was observed on MS medium supplemented with BAP (4 mg/L) +
NAA (0.1 mg/L) than those on MS + BAP (4 mg/L) + NAA (0.5 mg/L) (71.11%) followed by MS + BAP (3 mg/L) +
NAA (1.0 mg/L) (66.67%) and MS + BAP (3 mg/L) + NAA (0.5 mg/L) (51.11%). The highest (5.67) number of
microshoots in minimum days (30.67) was observed on MS medium supplemented with BAP (4 mg/L) + NAA (0.1
mg/L) which were statistically significant with other treatment. These observations are quite close to the
recommendations of other workers in chrysanth- emum (Kumar et al., 2004; Latado et al., 2004) who regenerates
microshoots from the chimeric ray florets of chrysanthemum. Mechanical wounding in leaf explants by brushing the
leaf surfaces was shown to increase the shoot regeneration capacity in D. grandiflora (de Jong et al., 1993) therefore
we also cultured the wounded ray florets and find the earliest regeneration from the wounded parts of the ray florets.
Medium supplemented with higher concentration of BAP in combination with low concentration of NAA initiates
differentiation process following adventitious bud and shoot initial formation. It is well known that cytokinins
accelerate the cell division process in the first step of their application and, then, large amount of callus is formed
followed by differentiation of shoot initials, organogenesis and shoot regeneration.

Table 3 Effect of BAP and NAA on percentage of shoot regeneration from ray florets of chrysanthemum
1.4 Effect of GA3 on elongation
The regenerated microshoots individually transferred to elongation media consisting of basal MS medium
supplemented with various concentrations of GA3 significantly affected the growth of the shoots and made them

elongated, with thick, sturdy and strong stem and well developed dark green expended leaves. In this process, the micro
shoots attained an optimum height so that they could be transferred for rhizogenesis. Maximum (5.55 cm) shoot length
was recorded after 30 days of culturing on MS medium supplemented with 0.50 mg/L GA 3 which was considerably
higher than those cultured on MS medium supplemented with 0.25 mg/L GA 3 (3.88 cm) and 1.0 mg/L GA3 (3.48 cm)
(Figure 1). All these treatments were statistically significant with each other. The MS medium devoid of hormones
(control) showed minimum (2.28 cm) length of microshoots. Shruti et al (2011) study the effect of GA 3 on shoot
elongation of in vitro grown stevia and found that medium containing 0.5 mg/L GA 3 showed maximum elongation
(3.5). Elongated microshoots reduce the chance of mortality during the rooting and hardening processes. Gibberellins
are known for inducing stem elongation in a number of crops. The elongation of the stems is not due to increased
formation of nodes and internodes but results from rapid elongation of internodes, which is due to both cell division
and cell elongation. Gibberellic acid is involved in several important biochemical and morphogenetic responses which
include the promotion of elongation in axial organs, such as stems and flower pedicels, along with the stimulation of
root growth (Srivastava, 2005).

Figure1 Effect of Gibberellic acid (GA3) on microshoot elongation

1.5 Acclimatization of plantlets
For obtaining the high success during the plantlet acclimatization, plantlets transferred in glass jars with polypropylene
lids filled with peat + soilrite (1:1) and moistened with half-strength MS medium (devoid of growth regulators,
calcium, organics and sucrose) was found to be the best in terms of maximum survival percentage (95.00), plant height
(22.45 cm), number of leaves per plant (31.50) and minimum days taken for transfer to greenhouse (21.00) (Table 4).
After 20~25 days of acclimati- zation the plants ready to transfer in the green house. These results are in line with the
earlier workers in chrysanthemum (Mandal and Datta, 2005; Nahid et al., 2007).

Table 4 Effect of different strategies on hardening

2 Materials and Methods
2.1 Plant material
Cuttings of chrysanthemum cultivar Thai Chen Queen was planted under greenhouse after irradiation with acute
gamma rays. When the plants came in the flowering, different types of flower colour mutants were emerged in the
population.

2.2 Selection and preparation of explants

The ray florets of mutant which appear in the population of M 1 generation were taken as explants for in vitro
regeneration. The collected material was brought to the laboratory and washed thoroughly with running tap water for
30 min. The whole flower were washed with 0.1% Teepol solution for 8~10 min followed by washing under running
tap water for 10~15 min. The whole flower were then pre-treatments before inoculation with 0.1% mancozeb 45 +
0.1% carbendazim + 200 mg/L 8-hydroxyquinoline citrate (HQC) for 2 h. then individual ray florets surface sterilized
with 0.1% HgCl2 for 3 min followed by 3~4 times rinsing with sterile double distilled water.
2.3 Inoculation and incubation conditions
All aseptic operations were conducted in the laminar air flow chamber fitted with HEPA filter (0.22 m) and a burner.
The working table of laminar airflow chamber was wiped thoroughly with ethanol (100%) before use. The material

required for inoculation was steam sterilized. The hands were cleaned with ethanol (70% v/v). The individual ray
florets were pinched with the help of sterile scalpel and then inoculated on culture medium (Murashige and Skoog
1962) supplemented with 3% sucrose, 0.72% (w/v) agar-agar and different concentrations and combinations of 6Benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA) and GA 3. The pH of the medium was adjusted to 5.80.1
and autoclaved at 121 for 15 min at 15 psi. The cultures were incubated in culture room after inoculation and
provided with a constant photoperiod 16/8 h light/dark with a light intensity of 3 000 Lx by white fluorescent tubes,
(251) temperature and 60%~70% relative humidity.
2.4 Media and plant growth regulators for in vitro regeneration
The Murashige and Skoog, 1962 media supplemented with different concentrations and combinations of BAP (3, 4, and 5
mg/L) and NAA (0.1, 0.5 and 1 mg/L) for regeneration. The multiplied shoots were separated and individual micro
shoot was subjected to media comprising of basal MS medium supplemented with various concentration of GA 3 (0.25,
0.5 and 1 mg/L) for shoot elongation.
2.5 Acclimatization of plantlets
For acclimatization, plantlets were transferred from in vitro to ex vitro conditions in glass jar with polypropylene cap
filled with peat + soilrite (1:1) and Plastic pot with polythene cover filled with peat + soilrite (1:1) for acclimatization.
After 20 to 25 day of hardening, plants were transferred in green house for flowering.
2.6 Experimental design and statistical analysis
The experiments were laid out in completely randomized design (CRD). Recorded data were analyzed statistically
using analysis of variance technique (ANOVA). Each treatment had 20 units with three replications. All the percentage
data was subjected to Arc Sin percentage transformation before calculating ANOVA.
Authors' contributions
Arvind Kumar Verma: Student who conducted the experiment for the dissertation of his degree; K. V. Prasad: Chairman
of the advisory commeettee; T. Janakiram: Head of the Department and provided necessory facilities to conduct the
experiment; S. Kumar: Technical assistant of the experiment.
Acknowledgements
Senior author is highly thankful to the financial help provided by ICAR, New Delhi in the form of Junior Research
Fellowship during the study.
References
Bala M., Singh K.P., and Prasad K.V., 2010, Standardization of in vitro mass multiplication protocol for hybrid tea rose
cv. Pusa Mohit, Indian J. Hort., 67(2): 225-229
Datta S.K., Misra P., and Mandal A.K.A. 2005, In vitro mutagenesis- A quick method of for establishment of solid
mutant in chrysanthemum, Curr. Sci., 88(1): 155-158
de Jong J., Rademaker W., and van Wordragen M.F., 1993, Restoring adventitious shoot formation on chrysanthemum
leaf explants following cocultivation with Agrobacterium tumefaciens, Plant Cell Tiss. Org. Cult., 32: 263-70
http://dx.doi.org/10.1007/BF00042287
Ilahi I., Jabeen M., and Sadaf S.N., 2007, Rapid clonal propagation of chrysanthemum through embroyogenic callus
formation, Pak. J. Bot., 39: 1945-1952
Kadam G.B., Singh K.P., Singh A.K., and Jyothi R., 2010, In vitro regeneration of tuberose through petals and
immature flower buds, Indian J. Hort., 67(1): 76-80
Kumar S., Kanwar J.K., and Sharma D.R., 2004, In vitro regeneration of Gerbera jamesonii Bolus from leaf and petiole
explants, J. Plant Biochem. Biotech., 13: 73-75
Kumar S., Prasad K.V., and Choudhary M.L., 2006, Detection of genetic variability among chrysanthemum
radiomutants using RAPD markers, Current Sci., 90(8): 1108-1113.
Latado R.R., Adames A.H., and Neto A.T., 2004, In vitro mutation of chrysanthemum (Dendranthema grandiflora
Tzvelev) with ethylmethanesulphonate (EMS) in immature floral pedicels, Plant Cell Tiss. Org. Cult., 77: 103-106,
doi:10.1023/B:TICU.0000016481.18358.55
Mandal A.K.A., and Datta S.K., 2005, Direct somatic embryogenesis and plant regeneration from ray florets of
chrysanthemum, Biol. Plant., 49: 29-33
http://dx.doi.org/10.1007/s10535-005-0033-6
Mandal A.K.A., Chakrabarty D., Datta S.K., 2000, Application of in vitro techniques in mutation breeding of
chrysanthemum, Plant Cell Tiss. Org. Cult.,60:33-38
http://dx.doi.org/10.1023/A:1006442316050

Misra P., Datta S.K., and Chakrabarty D., 2003, Mutation in flower colour and shape of chrysanthemum induced by
gamma radiation, Biol. Plant., 47(1): 153-156
http://dx.doi.org/10.1023/A:1027365822769
Nahid J.S., Saha S., and Hottori K., 2007, High frequency shoot regeneration from petal explants of Chrysanthemum
morifolium Ramat. in vitro, Pak. J. Biol. Sci.
http://dx.doi.org/10.3923/pjbs.2007.3356.3361
Pillai V., and Zulkifli L., 2000, Somaclonal variation in Chrysanthemum morifolium generated through petal cultures, J.
Trop. Agric. Food Sci., 28: 115-20
Rout G.R., and Das P., 1997, Recent trends in the biotechnology of chrysanthemum: A critical review, Sci. Hort., 69:
239-257
http://dx.doi.org/10.1016/S0304-4238(97)00008-3
Srivastava, L.M., 2005, Plant growth and development: Hormones and environment. Academic Press, An imprint of
Elsevier, San Diego, USA, pp:172-188
Verma A.K., Prasad K.V., Singh S.K., and Kumar S., 2012, In vitro isolation of red coloured mutant from chimeric ray
florets of chrysanthemum induced by gamma-ray, Indian J. Hort., 69(4): 562-567
Verma S., Yadav K., and Singh N., 2011, Optimization of the protocols for surface sterilization, regeneration and
acclimatization of Stevia rebaudiana Bertoni. American-Eurasian J. Agric. Environ. Sci., 11 (2): 221-227