MATERIALS AND METHODS

Seeds of pigeon pea cultivars viz., IPA-2013, IPA-3088, Pusa-9, IPA-34, IPA-204, IPA-242, T-7, IPA-61, IPA-337, IPA-341 and IPA-98-3 were procured from Indian Institute of Pulses Research, Kanpur.

Seeds were surface sterilized with 1% cetrimide for 10 min followed by 0.2% HgCl₂ for 5 min and thoroughly rinsed 4-5 times with sterile distilled water and germinated aseptically on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) with 0.3% sucrose under cool white fluorescent light at 25±2°C. The morphogenic potential of leaf explants of 10, 15 and 20 days old seedlings were analyzed on different concentration of growth hormones viz., BAP, Kinetin and TDZ. For multiple shoot bud induction about 0.5 cm² section from petiolar end of the leaf was cultured on MS media supplemented with variable concentration of growth hormones. The pH of all media was adjusted to 5.8 before adding agar and sterilized by autoclaving at 1.08 kg cm² for 20 min. Cultures were incubated under light intensity of 1500l ux with 16 h light and 8 hs dark period at 25±20°C. For each experimental set up 10 explants were used with each concentration and experiment was repeated twice. After each successive subculture within 15 days, the well developed shoots were rooted on MS media augmented with three different concentration i.e., 0.1, 0.2 and 0.3 mg L^-1 of three growth regulators namely NAA, IAA and IBA. Finally few plants of each cultivar were successfully acclimatized to soil.

RESULTS AND DISCUSSION

In vitro regeneration via organogenesis has been extensively attempted as compared to somatic embryogenesis in pigeon pea using explants like apical meristem, undifferentiated callus, leaves, hypocotyls, cotyledons, cotyledonary nodes, epicotyls and embryonal axes for more than 51 diverse genotypes/cultivars (Krishna et al., 2010). Efforts have been made for developing in vitro regeneration protocol via organogenesis using leaf and leaf petiole as explants for more than 17 cultivars of pigeon pea (Eapen and George 1993; Eapen et al., 1998; Geetha et al., 1998; Singh et al., 2002; Dayal et al., 2003; De Villiers et al., 2008).

In the present study eleven cultivars of pigeon pea not attempted for regeneration studies earlier (Krishna et al., 2010) were assessed for multiple shoot bud induction and regeneration using leaf explants under the influence of different cytokinins namely 6-Benzylaminopurine (BAP), Kinetin (KIN) and Thidiazuron (TDZ) cultured on Murashige and Skoog (MS) media. The leaf explants obtained from 10 days old in vitro germinated seedlings were found to be better for multiple shoot bud induction on MS media supplemented with different concentration of BAP, KIN and TDZ. In vitro regeneration using leaf lamina as explants with leaves from 5-12 -day-old seedlings has been reported (Eapen and George, 1993; Geetha et al., 1998).

The response of eleven cultivars for multiple shoot bud induction on MS media supplemented with variable concentration of BAP ranging from 0.5 to 4.0 mg L^-1 revealed callus of diverse nature and morphology for most of the cultivars (Table 1). Only four cultivars namely IPA-2013, Pusa-9, T-7 and IPA- 98-3 showed appreciable multiple shoot bud induction (Fig. 1a-d). In case of cultivars IPA-2013 and T-7 higher concentration of BAP i.e., 3.0 mg L^-1 was found to be effective for multiple shoot bud induction resulting with 6 and 5 buds per explants, respectively. In case of Pusa-9 and IPA- 98-3 a total of 6 buds per explants were obtained in 2.0 mg L^-1 of BAP. Other cultivars did not responded for multiple shoot bud induction in variable concentration of BAP. Among different hormones used BAP has been reported to be most effective for in vitro regeneration of pigeon pea (Geetha et al., 1998; George and Eapen 1994). BAP alone has been used for regeneration via organogenesis using leaf explants of cultivars ICPL 93115 (Yadav and Padmaja, 2003) and one for unknown cultivar (Kumar et al., 1983). The response of these cultivars for multiple shoot bud induction in the presence of variable concentration of KIN ranging from 0.5 to 4.0 mg L^-1 was very poor. In vitro regeneration via organogenesis of leaf explants is not reported in the presence of KIN alone though combination of BAP and KIN has been preferred by several workers (Tyagi et al., 2001; De Villiers et al., 2008).

The response of these eleven cultivars for multiple shoot bud induction in the presence of variable concentration of TDZ ranging from 0.05-0.4 mg L^-1 is shown in Table 1. Only one cultivar IPA-2013 showed multiple shoot bud induction resulting in 6 buds per explants in the presence of TDZ at 0.05 mg L^-1 while other cultivars revealed callus formation with variable morphology and nature (Table 1). TDZ-induced shoot regeneration in pigeon pea cultivars ICPL 161, ICPL 88039, UPS 120 has been reported (Eapen et al., 1998).

Multiple shoot buds obtained from leaf explants were subjected to rooting in full strength MS basal medium supplemented with three different hormones viz., NAA, IAA and IBA at three different concentrations namely 0.1, 0.2 and 0.3 mg L^-1. NAA was found to be best for inducing rooting in multiple shootlets derived from leaf explants.

Table 1:	Multiple shoot bud induction, callus morphology from leaf explants of 10 days invitro germinated seedlings cultured on MS media supplemented with different concentration of BAP and TDZ after 8 weeks of culture
NR: No response, YWCC: Yellowish white compact callus, WGCC: Whitish green compact callus, GCC: Green compact callus, BCC: Brown compact callus, WCC: White compact callus, YWFC: Yellowish white friable callus, WGFC: Whitish green friable callus, YBCC: Yellowish brown compact callus, YGCC : Yellowish green compact callus, YGFC: Yellowish green friable callus

Fig. 1(a-d):	Multiple shoot bud induction from leaf explants of different cultivars of Cajanus cajan on MS media supplemented with different concentration of BAP (a) IPA2013 (3.0 mg L-1), (b) Pusa-9 (2.0 mg L-1) and (c) T-7 in (3.0 mg L-1) and (d) IPA-98-3 (2.0 mg L-1)

The rooting response at different concentration of NAA for the cultivars IPA-2013, Pusa-9, T-7 and IPA- 98-3 showing efficient multiple shoot bud induction on MS media supplemented with BAP and TDZ is shown in Fig. 2a-d, respectively. Cultivar IPA-2013 showing efficient multiple shoot bud induction in the presence of BAP and TDZ revealed efficient rooting with 0.1 mg L^-1 NAA (Table 2). Efficient rooting of multiple shoot bud induction from leaf explants in the presence of IBA, NAA and IAA has been reported (Srinivasan et al., 2004).

The rooted plantlets were hardened in small pots and transferred to the soil for acclimatization.

Table 2:	Rooting responses of in- vitro regenerated multiple shoot buds from leaf explants of four cultivars of pigeonpea at different concentration of NAA recorded after 4 weeks of culture

Fig. 2(a-d):	Rooting response for multiple shoot buds from leaf explants of different cultivars of Cajanus cajan on MS media supplemented with different concentration of NAA (a) IPA-2013 (0.1 mg L-1), (b) Pusa-9 (0.1 mg L-1), (c) T-7 (0.2 mg L-1) and (d) IPA-98-3 (0.1 mg L-1)

Fig. 3(a-d):	Acclimatized plants of the cultivars (a) IPA 2013, (b) Pusa-a, (c) T-7 and (d) IPA-98-3

The percentage acclimatization ranged from 25 to 50% with cultivar IPA-2013 showing best response. The acclimatized plants of cultivars IPA2013, Pusa-9, T-7 and IPA-98-3 are shown in Fig. 3a-d, respectively.

CONCLUSION

The response of eleven cultivars not attempted earlier for regeneration via organogenesis using leaf explants has been assessed for multiple shoot bud induction. The age of the explants donor seedling greatly influenced the differentiation of shoot buds with leaves from 10 days old seedling gave better response as compared to the leaves of 15, 20 and 25 days seedling explants. Further higher concentration of BAP (3.0 mg L^-1) and lower concentration of TDZ (0.05 mg L^-1) responded best for multiple shoot induction. NAA was found to be best for rooting of multiple shootlets derived from leaf explants as compared to IAA and IBA. Among the eleven cultivars studied the response of IPA-2013 for multiple shoot bud induction was best which could be further standardized for developing efficient in vitro regeneration protocol via organogenesis.

ACKNOWLEDGMENT

The authors wish to acknowledge Dr. B.B. Singh and Dr. S.D. Dubey, Indian Institute of Pulses Research, Kanpur, Dr. Harpal Singh, Punjab Agricultural University, Gurdaspur, Director Experiment Station, G.B. Pant University of Agriculture and Technology, Pantnagar for providing seeds of the pigeon pea cultivars used in the present study.