Abstract: An efficient protocol was developed for direct regeneration, multiplication and rooting under in vitro conditions of Chrysanthemum. The frequency of multiple shoot regeneration response was 95 and 91%, for nodal segments and shoot tips, respectively when cultured on the medium containing MS +1.0 mg l-1 BAP. Efficient rooting was achieved on half strength of MS+0.2 mg l-1 IBA. In vitro raised plantlets were transferred to potted soil and finally transferred to the field.
Introduction
Chrysanthemum morifolium is one of the common ornamental plants of Bangladesh belonging to the family Composite. It is a perennial herb, weak and lateral branching, herbaceous angular and present wooly. It is important not only for its outstanding aesthetic beauty and a long lasting capability but also because of its good potentials for marketing as cut flowers and potted plants to many countries.
Chrysanthemum are propagated vegetatively either through root suckers or terminal cuttings. This conventional process of shoot cutting is very slow. For commercial purpose, we need large scale production. Due to the high popularity and demand for chrysanthemum it becomes one of the first commercial targets for micro propagation (Levin et al., 1988). Ben-Jaacov and Langhans (1972), described in vitro Chrysanthemum micro propagation from shoot tips and shoot initiated callus. Bhattacharya et al. (1990) reported rapid mass propagation of Chrysanthemum morifolium through callus derived from leaf and stem explants. Through the use of tissue culture, it is possible to obtain a large number of plants from one explant (Bajaj et al., 1992). It represents the optimum efficiency in terms of vegetative plant propagation and allows a large number of propagules to be produced in a relatively short period under controlled conditions, throughout the year in a relatively small space.
The present study was undertaken to establish a protocol for large-scale clonal propagation of Chrysanthemum through in vitro culture.
Materials and Methods
The experiment was conducted at Plant Tissue Culture Laboratory, Department of Botany, University of Rajshahi, Bangladesh during the period of 1997 to 1998. The shoot tips and nodes were used as experimental plant materials. The explants were collected from 4 months old grown from the stem cuttings at the Botanical garden of Rajshahi University and they were washed thoroughly under running tap and distilled water.
The material was then taken into laminar flow cabinet and surface sterilized with 0.1% HgCl2 for different durations. After sterilization, the explants were planted on the surface of the semisolid MS, MMS1 or MMS2 medium gelled with 6 gm l-1 agar, 30 gm l-1 sucrose. The pH of the medium was adjusted to 5.7 and autoclaved at 1210C for 20 minutes. All the cultures were incubated at 25±20C and culture was kept under a 14 hours photoperiod fluorescent tube light. The materials were subcultured at 3-4 weeks intervals. The shoots were separated and individual shoots were placed in the rooting medium. After hardening plantlets were transferred to soil for establishment.
Results and Discussion
Different concentrations of BAP and Kn (viz. 0.1, 0.2, 0.5, 1.0, 2.0, 5.0,10.0 mg l-1) were used in MS media for shoot regeneration from nodal and shoot tip explants of Chrysanthemum.
Proliferation of axillary shoot from the nodal and shoot tip segments of the mature plants and in vitro raised shoot origin was remarkably influenced by types and concentrations of the cytokinin used. Among different concentrations used, best response towards shoot proliferation from nodal and shoot tip explants was obtained on MS +1.0 mg l-1 BAP (Table 1). Roest and Bokelmann (1975) obtained similar results when they used 1.0 mg l-1 BAP in the medium for shoot regeneration of Chrysanthemum. Among the two explants used, nodal explant showed comparatively better response than shoot tip explants towards shoot regeneration. Similar results have been published by Hoque et al. (1995) while working on multiple shoot regeneration for carnation. For nodal explant, the highest degree of axillary shoot proliferation was found on medium containing 1.0 mg l-1 of the cytokinin (BAP) and 95% of the explants proliferation with 5.3±0.2 shoots. On the other hand, for shoot tip explants the highest degree of axillary shoot proliferation was found on medium containing 1.0 mg l-1 of the cytokinin (BAP) and 91% of the explants proliferation with 4.2±0.3 shoots (Fig. 1A, B). The effectiveness of cytokinin BAP was proved to be superior to that of Kn in regeneration of shoots from both the explants. It is in agreement with those of Hutchinson (1981) who reported that MS media supplemented with BA have been satisfactory for many species and cultivars of crop plants for their in vitro propagation. Superiority of BA over other cytokinins in producing in vitro shoots has also been confirmed in other plants like Rosmarinus officinalis (Misra and Chatruvedi, 1984), Arachis hypogaea (Mhatre et al., 1985) and Atropa beladona (Benjamin et al., 1987). The proliferation efficiency of nodal explants was significantly higher than that of shoot tip explant, when evaluated after five-six weeks of proliferation. Micro cuttings prepared from in vitro proliferated shoots with 3-5 cm length were cultured on MMS1 medium with 0.1,0.2,0.5 and 1.0 mg l-1 of IBA, NAA and IAA supplemented for root formation. Percentage of root induction and number of roots per shoots were highly influenced by concentration and type of the auxins. Among different concentrations of auxin supplemented in media lowest rooting was obtained with IAA and highest with 0.2 mg l-1 IBA (Table 2). Similar observations were found on Chrysanthemum morifolium (Hoque et al., 1995; Khan et al., 1994). NAA showed effective results in the induction of root. Using NAA at different concentrations on MS medium, several workers (Earle and Langhans, 1974a) reported root induction in Chrysanthemum. On medium with 0.2 mg l-1 concentration of the auxin the cultured shoot cuttings produced the highest number of roots per micro shoots and that were 9.1±0.2 for IBA, 8.9±0.1 for NAA 7.2±0.8 for IAA (Fig. 1C). The maximum length of the longest root was 5.3±0.1 on 0.2 mg l-1 IBA supplemented medium. Healthy and well established rooted plantlets were transferred to soil for acclimatization.
| Table 1: | Effects of cytokinin for in vitro shoot proliferation from the nodal and shoot tip explant of C. morifolium on MS medium. There were 10-15 explants in each treatment. Data were recorded after 6-8 weeks of culture |
| Fig. 1: | In vitro shoot regeneration in Chrysanthemum morifolium |
| A. | Multiple shoot regeneration from nodal explants on MS medium containing 1.0 mg l-1 BAP |
| B. | Initiation of multiple shoots from shoot tip explants on MS medium supplemented with 1.0 mg l-1 BAP |
| C. | Root formation in regenerated shoots in ½ strength MS+0.2 mg l-1 IBA |
| D. | Regenerated plantlets established in the soil |
| Table 2: | Effects of different auxins on formatting of root from the in vitro grown micro shoots cultured on MMS1 medium fortified with 6 gm l-1 agar. There were 10-15 micro shoots cultured for each treatment and data were collected after 4-6 weeks |
Among the regenerates transplanted 75% of them survived and acclimatized successfully on the soil (Fig. 1D). The results of the present study indicate that using shoot tip as well nodal explants is possible to multiply Chrysanthemum on a large scale on MS medium supplemented with BAP and Kn. This protocol can be used for commercial rapid propagation of Chrysanthemum to earn foreign exchange and also to meet the local demand.