In vitro regeneration from ray florets: An efficient protocol to establish flower mutants in chrysanthemum (Chrysanthemum morifolium)

Introduction

Increase in urbanization emphasizes the interest in various coloured flowers in urban and semi urban areas where the consumers are preferring for novel types especially in flower crops. Ornamental and flower crops with added advantage of vegetative propagation offers to develop new and novel forms and colours. Mutation breeding is one of the tools for ornamental and flower crops which provides ease in development of novel types especially in chrysanthemum. So far, several mutants have been developed in chrysanthemum as solid and chimeric forms as reported by Mahure et al., 2010 [9] . Though the isolation of these mutants is the bottleneck in chrysanthemum if the variation occurs as chimera, it is difficult to isolate and establish (Mandal and Datta, 2005) [10] . Keeping this in view the present study was taken up to standardize a protocol for rapid multiplication of plants through florets in chrysanthemum.

Material and Methods

The present experiment was conducted at Bidhan Chandra Krishi Viswavidyalaya during 2014-15. The cultivar BC-8-05 which is spray type with dwarf nature good for pots, beds, borders. The flowers are decorative type with and yellowish-brown colour florets. Terminal cuttings collected from AICRP on Floriculture, BCKV and treated with different doses of gamma rays i.e. 10 Gy, 15 Gy, 20 Gy, 25 Gy 30 Gy and planted in field. Variations were observed in M 1 V 1 generation in terms of flower colour such as chimeric flowers as it appeared as brown stripes on yellow ray floret at 10 Gy. These ray florets were taken as explant for standardization of in vitro regeneration. At first the ray florets washed thoroughly in running tap water for 15 minutes and treated with 0.01% of antifungal (Bavistin) solution for 10 minutes and for another 5 minutes with 0.1% aqueous solution of teepol and then again washed in tap water for 5 minutes. Then, the explants were brought under laminar air flow. Later, the explants were disinfected with 70 % ethanol for 30 seconds and then rinsed with sterile water for 3-4 times. The individual explants were subjected to surface sterilization with different concentrations of mercuric chloride for 2-6 minutes followed by three washings with sterilized distilled water. The individual ray floret was pinched with help of a sterile needle and then inoculated in MS medium (Murashige and Skoog 1962) supplemented with 7% gelling agent (Agar-Agar type-1), 30% sucrose and different concentrations of 6-benzylaminopurine, α-naphthalene-acetic acid and gibberellic acid for different micropropagation stages like callus induction, shoot regeneration, shoot proliferation, elongation and rooting. The pH of the medium was adjusted to 5.7-5.8 before autoclaving at 121 °C and 20 psi for 20 min. The cultures were incubated under controlled atmosphere in air conditioned culture room provided with a constant photoperiod of 16/8 h light/dark regimes at 24 ± 1 °C temperature and light intensity of 3,000 lux at plant level provided by cool white fluorescent tubes. The experiment was laid out in completely randomized design (CRD). Each treatment had 10 units with five replications. All the percentage values were subjected to data transformation before calculating ANOVA.

Results and Discussion:

Growth and survival of uncontaminated cultures varied with the concentration and duration of the treatments (Table 1). The explants which were exposed to 0.1% mercuric chloride for 4 minutes gave the maximum survival percentage (93.97%) with less contamination (6.03%). Present findings were in the line of earlier work done by Misra et al. (2003) [11] ; Ilahi et al. (2007) [5] . The lower concentration resulted in contamination due to microbial load in the explants and higher concentration coupled with longer duration caused phytotoxicity and drying of explants leading to death of tissues. Similar results were also reported in chrysanthemum by many workers (Verma et al., 2012) [16] .

Callus induction and callus establishment

MS medium supplemented with different combinations of cytokinin (BAP) and auxin (NAA) for callus induction and shoot regeneration. Various concentrations of BAP and NAA showed significant difference for callus induction as well as callus establishment (Table 2). High percentage of callus induction was observed on MS medium supplemented with different concentrations of BAP and NAA combination as compared to control (MS medium devoid of hormones). Cent percent callus was attained with the ray florets on MS medium supplemented with 1 mg/l BAP + 0.1 mg/l NAA (T 11 ) with minimum number of days to callus induction (12.93). Present findings are in the line with the earlier work done by Datta et al. (2001) [3] . It was suggested by Nahid et al. (2007) [13] that BAP is more crucial for callus induction in Chrysanthemum. The callus initiation was first observed on wounded parts of the ray florets turning to green, which later spread over the whole ray florets. Similar findings were reported by Arvind et al. (2012) who reported that the wounded parts of ray florets showed faster callusing as compared to those inoculated without wounding. It was direct exposure of tissue to the culture medium containing higher levels of cytokinin in combination with auxin (NAA), that resulted in rapid cell division and callus formation (Dash et al. 2000) [2] .

Adventitious microshoot regeneration and proliferation:

Different combinations of cytokinin (BAP) and Auxin (NAA) supplemented with MS media was subjected for shoot proliferation and presented in Table 3. Highest shooting percentage (97.50 %) in minimum days (28.23) was found in MS + 1.0 mg/l BAP+ 0.1 mg/l NAA. For the successful shoot regeneration, a combination of auxin and cytokinin was found effective (Kumari et al., 2013) [7] .

Cytokinins accelerate the cell division process in the first step of their application and then large amount of callus was formed followed by differentiation of shoot initials, organogenesis and shoot regeneration (Verma et al. 2012) [16] . BAP is the most effective plant growth regulator in relation to shoot proliferation. These findings lend support from the various workers (Rahayu and Prayogi, 2013; Lindiro et al., 2013) [14,8] . Values in the parentheses are angular transformed data

Number of micro shoots and shoot length:

Cytokinin in combination with auxin and Gibberellin was used for production of micro shoots and was represented in table 4. Among the various treatments, MS + 1.0 mg/l BAP+ 0.1 mg/l NAA+0.1 mg/l GA 3 (T 11 ) gave maximum number of micro shoots per callus (10.15) with a shoot length of 10.53 cm. Influence of both auxin and cytokinin combination on plantlet regeneration in in vitro culture is a well-known fact and was confirmed by several researches (Jaime and da Silva, 2014) [6] . Shoot proliferation in tissue culture might be due to the role of optimum dose of BAP, which enhanced auxiliary branching and multiple shoot formation though the significant effect of GA 3 in terms of shoot differentiation and shoot elongation was clear from this study. This may be due to the significant effect of GA 3 on shoots which made them elongated, with thick, sturdy and strong stem and well developed dark green expanded leaves. These results indicate that not only the balance between auxin and cytokinin for shoot regeneration but also that GA 3 plays a role in shoot differentiation (Chakraborty et al., 2000) [1] by involving in several important biochemical and morphogenetic responses which include the promotion of elongation in axial organ, such as stem and flower pedicels. Similar findings of effect of GA 3 on organogenesis and shoot differentiation in callus culture have been demonstrated in many species (Srivastava, 2005) [15] .

Treatments Number of micro shoots per callus

Shoot length (cm) T1-Basal MS medium 0 0 T2-MS + 3.0 mg/l BAP + 0.1 mg/l NAA+0.1 mg/l GA3 7.33 b 5.83 fgh T3-MS+3.0 mg/l BAP+ 0.5 mg/l NAA+0.1 mg/l GA3 4.58 cde 7.30 cd T4-MS + 3.0 mg/l BAP+ 1.0 mg/l NAA+0.1 mg/l GA3 5.38 c 7.00 cde T5-MS + 4.0 mg/l BAP+ 0.1 mg/l NAA+0.1 mg/l GA3 4.10 def 5.20 hi T6-MS + 4.0 mg/l BAP+ 0.5 mg/l NAA+0.1 mg/l GA3 4.95 cd 5.50 gh T7-MS + 4.0 mg/l BAP+ 1.0 mg/l NAA+0.1 mg/l GA3 5.43 c 5.17 hi T8-MS + 5.0 mg/l BAP+ 0.1 mg/l NAA+0.1 mg/l GA3 3.92 efg 6.25 efg T9-MS + 5.0 mg/l BAP+ 0.5 mg/l NAA+0.1 mg/l GA3 3.03 g 4.65 i T10-MS + 5.0 mg/l BAP+ 1.0 mg/l NAA+0.1 mg/l GA3 1.03 i 3.80 j T11-MS + 1.0 mg/l BAP+ 0.1 mg/l NAA+0.1 mg/l GA3 10.15 a 10.53 a T12-MS + 2.0 mg/l BAP+ 0.1 mg/l NAA+0.1 mg/l GA3 5.53 c 7.70 bc T13-MS + 3.0 mg/l BAP+ 1.5 mg/l NAA+0.1 mg/l GA3 3.20 fg 7.03 cde T14-MS + 4.0 mg/l BAP + 1.5 mg/l NAA+ 0.1 mg/l GA3 1.07 h 3.40 jk T15-MS + 5.0 mg/l BAP + 1.5 mg/l NAA+ 0.1 mg/l GA3 1.02 h 2.65 k T16-MS + 1.5 mg/l BAP + 0.1 mg/l NAA+ 0.1 mg/l GA3 7.08 b 8.50 b T17-MS + 2.0 mg/l BAP + 0.5 mg/l NAA+ 0.1 mg/l GA3 1.65 h 3.60 j T18-MS + 2.0 mg/l BAP + 1.0 mg/l NAA+ 0.1 mg/l GA3 [4] . Furthermore, reduction in salt levels in the MS medium particularly macronutrients including potassium during in vitro rhizogenesis, utilizes and prevents the accumulation of salt complex by guard cells in the stomata which facilitates for easy rooting as opinioned by Nagatomi et al. (2003) [12] . Successful micro propagation depends on the ability to transfer in vitro plantlets to potting mixture and acclimatized them successfully to the field conditions. Primary hardening, was done with sterilized vermiculite fortified with ½ strength MS plant salt mixture (devoid of growth regulators, calcium, organics and sucrose) then the plantlets were transferred to potting mix consisting of soil + sand + FYM (2:1:1) after 3-4 weeks of acclimatization for secondary hardening. Later these plantlets were successfully transferred to filed. This protocol may be useful for isolation and establishment of chimeric variants in case of florets especially in chrysanthemum where conventional propagation is not possible without loosing the valuable mutants.

References

1. Chakrabarty D, Mandal AKA, Datta SK. Retrieval of new coloured chrysanthemum through organogenesis from sectorial chimera. Curr Sci. 2000;78:1060-1061.
2. Dash P, Singh RP, Voss F. Retrieval of new coloured chrysanthemum through organogenesis from sectorial chimera. Curr Sci. 2000;78:1060-1070.
3. Datta SK, Mandal AKA, Saxena M. Direct organogenesis from ray and disc florets of a newly evolved chlorophyll variegated chrysanthemum (Chrysanthemum morifolium). Indian J Agric Sci. 2001;71(10):655-657.
4. Fukaki H, Tameda S, Masuda H, Tasaka M. Lateral root formation is blocked by a gain-of-function mutation in the solitary root/IAA14 gene of Arabidopsis. Plant J. 2002;29:153-168.
5. Ilahi I, Jabeen M, Sadaf SN. Rapid clonal propagation of chrysanthemum through embryogenic callus formation. Pak J Bot. 2007;39(6):1945-1952.
6. Jaime A, da Silva TD. Ornamental chrysanthemums: Improvement by biotechnology. Plant Cell Tissue Organ Cult. 2014;79:1-8.
7. Kiran Kumari K, Dhatt KK, Kapoor M. Induced mutagenesis in Chrysanthemum morifolium variety 'Otome Pink' through gamma irradiation. Bioscan. 2013;8(4):1489-1492.
8. Lindiro C, Kahia J, Asiimwe T, Mushimiyimana I, Waweru B, Kouassi M, et al. in vitro regeneration of pyrethrum (Chrysanthemum cinerariaefolium) plantlets from nodal explants of in vitro raised plantlets. Int J Appl Innov Eng Manag. 2013;2(7):201-203.
9. Mahure HR, Choudhry ML, Prasad KV, Singh SK. Mutation in chrysanthemum through gamma irradiation. Indian J Hortic. 2010;67:356-358.
10. Mandal AKA, Datta SK. Direct somatic embryogenesis and plant regeneration from ray florets of chrysanthemum. Biol Plant. 2005;49(1):29-33.
11. Misra P, Datta SK, Chakrabarty D. Mutation in flower colour and shape of Chrysanthemum morifolium induced by γ-radiation. Biol Plant. 2003;47(1):153-156.
12. Nagatomi S, Degi K, Yamaguchi H, Morishita T, Yui M. Four mutant varieties induced by gamma rays and in vitro culture in chrysanthemum. Institute of Radiation Breeding, Technical News No. 64. 2003. pp. 1-12.
13. Nahid JS, Shyamali S, Kazumi H. High frequency shoot regeneration from petal explants of Chrysanthemum morifolium Ramat. In vitro. Pak J Biol Sci. 2007;10(9):3356-3361.
14. Rahayu SM, Prayogi HE. Addition of organic substances in media for in vitro growth of chrysanthemum (Dendranthema grandiflora Tzvelev). Bull Agrihorti. 2013;1(4):94-100.
15. Srivastava LM. Plant growth and development: Hormones and environment. San Diego (CA): Academic Press (Elsevier); 2005. pp. 172-188.
16. Verma AK, Prasad KV, Singh SK, Kumar S. in vitro isolation of red coloured mutant from chimeric ray florets of chrysanthemum induced by gamma rays. Indian J Hortic. 2012;69(4):562-567.