DEVELOPMENT OF AN EFFICIENT PLANT
3.2 RESULTS AND DISCUSSION .1 Multiple shoot induction
Cotyledonary node explants cultured on MSB medium produced an average of 1.9 shoots in 98% of the cultures. The frequency of shoot multiplication and shoot number were significantly higher on BAP-supplemented media followed by kinetin and TDZ (Table 2).
BAP at 5.0 µM induced maximum number of shoots (6.9) in 94% of the explants within 2 weeks of culture whereas the response was minimal at 7.5 µM TDZ with regeneration being restricted to stunted bud formation (Table 2). BAP alone or in combination with other plant growth regulators has been reported to favorably induce multiple shoot induction from diverse explants of cowpea (Muthukumar et al. 1995; Pellegrineschi, 1997; Brar et al. 1999; Popelka et al. 2006; Mao et al. 2006). However, the mean shoot length declined with increase in BAP concentrations (Table 2) and furthermore, elongation was very slow even after 4 weeks of culture. Lowering the concentration of BAP during subsequent subculture was not favorable for shoot elongation. The BAP have often been reported to stimulate shoot proliferation while inhibiting shoot elongation (Brassard et al. 1996; Figueiredo et al. 2001).
Like BAP, the effects of other cytokinins (TDZ and kinetin) on frequency of shoot regeneration were also examined. Addition of TDZ to the basal medium also induced a variable amount of callus at the base of the explants followed by induction of adventitious shoot buds from the explant. One interesting observation was made in the case of explants cultured on TDZ-supplemented media; with the increase in TDZ concentrations, there was an apparent decrease in frequency of shoot regeneration with drastic reduction in emergence of shoots in the responding explant with regeneration response restricted to stunted bud formation. TDZ at 1.0 µM induced a maximum of 0.9 shoots per explant in 74% of the cultures. Albeit TDZ found to have a higher cytokinin activity or a different action than other cytokinins during differentiation in different legumes, including pea, chickpea, lentil (Malik and Saxena, 1992c Malik and Saxena, 1992c), peanut (Gill and Saxena, 1992), white clover (Beattie and Garrett, 1995), tepary bean (Dillen et al. 1995) and goat’s rue (Collen and Jarl, 1999), no such induction was observed in cowpea. This may be attributed to non-responsive genetic makeup or internal hormone concentration of the cotyledonary node tissue of cowpea towards TDZ.
Both BAP and Kinetin favored multiple shoot development; however, frequency of shoot multiplication and average number of shoots were less on kinetin containing medium (Table
2) clearly demonstrating that the type and concentration of the cytokinin used had a profound effect on frequency of shoot multiplication and number of shoots induced. However, the mean shoot length on kinetin was significantly higher than BAP, signifying the role of kinetin in shoot elongation.
The developmental behavior of cotyledonary node explants from seeds germinated in the presence of a high concentration of BAP is known to have definite advantages on shoot multiplication. Presumably, pre-cultivation on BAP activates and/or induces proliferation of pre-existing competent cells in the tissue (Veltcheva et al. 2005). The beneficial effect of pre- conditioning of seeds in BAP on shoot regeneration efficiency from cotyledonary explants has been reported in soybean (Thorne et al. 1995), pigeonpea (Shiv Prakash et al. 1994) and common bean species (Santalla et al. 1998). The modification of the physiological state of the initial explants is one of the approaches that may alter in vitro regenerative capacity (Malik and Saxena, 1991; Santalla et al. 1998; Cruz de Caravalho et al. 2000). This could be achieved by pre cultivation of the explants in various plant growth regulators for determination of organogenesis and embryogenesis in some of the legumes like bean.
However, the physiological and molecular mechanism underlying cytokinins pre-cultivation in regeneration is still not properly known. However, since a specific physiological state is required for cells to be competent for regeneration, preconditioning might help in achieving the competence.
3.1.2 Shoot elongation
Incorporation of 0.5 µM of kinetin to MSB media enhanced the shoot elongation by three-fold in 86% of cultures within 2 weeks while higher concentrations proved ineffective whereas GA3 containing medium induced non-uniform elongation and moreover, the elongated shoots were found thin and lanky, unsuitable for rooting (Table 3). However, the interval at which kinetin (0.5 µM) was supplemented to SIM was critical for obtaining optimal response. The maximum number of shoots with optimal shoot length was obtained when explants were cultured initially for 1 week on SIM, followed by 3 weeks on SIM supplemented 0.5 µM kinetin (SMM) (Table 4; Fig. 1b) suggesting that the time of exposure to the combination of these two cytokinins was critical for accelerated regeneration response from cotyledonary node explants of cowpea.
3.1.3 Cyclic organogenesis
The BAP-habituated cultures tended to produce shoots continuously on the subculture medium and therefore, after harvesting elongated shoots, the mother explants were repeatedly subcultured on MSB media supplemented with 5.0 µM BAP and 0.5 µM kinetin for 3 weeks for cyclic induction of shoots. These mother explants continuously produced shoots during
successive subculture without losing their shoot forming potential. An average of 36–38 shoots was produced from each explant in four consecutive harvests. Repeated transfer of meristematic explants on media containing cytokinins was found to cause rejuvenation of explants tissue, activation, conditioning of meristems and retention of their morphogenetic potential (Shekhawat et al. 1993; Naik et al. 2000; Vengadesan et al. 2002; Gopi et al. 2006).
Continuous proliferation of BAP habituated culture of cotyledonary nodes during successive subcultures indicates its persistence in cowpea tissues.
The regeneration protocol described here has the basis for both adventitious and a non- adventitious system. The ability of proliferating meristematic/organogenic tissue to be maintained in a cyclic manner confers adventitious characteristics. However, the callus-like tissue that has developed upon prolonged culture on the regeneration medium itself is differentiated and produces small shoot clumps leaving small meristems, which are most probably the source of the formation of new buds in the next multiplication cycle, giving it non-adventitious characteristics.
Among the eight commercially important cultivars compared for their shoot regeneration potential, Pusa Komal produced maximum shoots (9.1 shoots/explant) in 94% of the cultures.
3.1.4 Rooting and acclimatization
Formation of roots was observed in 100% of shoots transferred to MS medium containing 2.5 µM IBA (Fig. 1c). Popelka et al. (2006) grafted rootless shoots onto cowpea seedlings in order to overcome the difficulties in rooting. Plantlets with developed roots were successfully established in soil (95%), and were eventually grown to maturity with normal seed set in greenhouse (Fig. 1d, e). The plants were apparently normal and showed no variation with respect to their morphology and growth characteristics.
The present study established a simple, efficient, rapid and direct plant regeneration through multiple shoot proliferation from cotyledonary node explants, precultured on BAP containing medium, on simple medium containing BAP and kinetin as growth regulators, and applicable to large number of genotypes.