Research Report

Standardization of Protocol for Pre-treatment, 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.
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Arvind K.V.,  Prasad  K.V., Anakiram T.J., and Kumar S., 2012, Standardization of Protocol for Pre-treatment, Surface Sterilization, Regeneration, Elongation and Acclimatization of Chrysanthemum morifolium Ramat, International Journal of Horticulture, 2(3): 7-12 (doi: 10.5376/ijh.2012.02.0003)


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.

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 plant’s 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 pre-treated thepetals and immature flower of tuberose with carbendazim 0.1%+ mancozeb 0.1% and 8-HQC (200 mg/L) for 2½ 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 % HgCl2 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 HgCl2 (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 HgCl2. 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% HgCl2 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 GA3 which was considerably higher than those cultured on MS medium supplemented with 0.25 mg/L GA3 (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 GA3 on shoot elongation of in vitro grown stevia and found that medium containing 0.5 mg/L GA3 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 M1 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 µm) 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 6-Benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA) and GA3. The pH of the medium was adjusted to 5.8±0.1 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, (25±1)℃ 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 GA3 (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.
Senior author is highly thankful to the financial help provided by ICAR, New Delhi in the form of Junior Research Fellowship during the study.
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