In vitro regeneration of two varieties of pigeon pea (cajanus cajan) grown in Kenya
Pigeon pea (Cajanus cajan (L) Millsp.) is an important multipurpose grain legume that is a good source of protein for populations living in the semi-arid tropics. Being a crop that is cultivated under rain-fed agricultural system, its production is threatened by several biotic and abiotic stresses. Attempts to address these problems through conventional breeding have achieved partial success due to narrow genetic variability among the cultivated species. In addition, breeding incompatibility problems associated with wild species warrant exploration of alternative approaches like gene transfer to introduce desirable traits. Development of in vitro regeneration protocols amenable to genetic transformation offer an attractive opportunity for improvement of pigeon pea. Therefore the aim of this study was to develop a protocol for in vitro regeneration of two pigeon pea (Cajanus cajan) varieties, KAT 60/8 and ICEAP 00557 grown in Kenya. Murashige and Skoog (1962) (MS) basal media supplemented with various auxin and cytokinin concentrations alone or in combinations and different explant types (embryos or leaves) were tested for callus initiation, induction of somatic embryos, shoot and root regeneration. For callus induction, MS medium supplemented with 0.5 – 4.0 mg/l 2, 4- dichlorophenoxyacetic acid (2, 4-D) and thidiazuron (TDZ) were tested. For shoot and root regeneration, 0.1 mg/l and 0.5 mg/l 6- benzyl amino purine (BAP) and 0.1- 1.0 mg/l Indole- 3- butyric acid (IBA) were tested respectively. Embryogenic calli were obtained on MS medium with 2 mg/l TDZ and 1 mg/l 2, 4- D. Six week old embryogenic calli were transferred to MS medium supplemented with 0.1 mg/l or 0.5 mg/l benzyl amino purine (BAP) or MS medium without hormones for shoot regeneration. Regenerated shoots (>3 cm) were excised after approximately ten weeks and transferred to MS medium with indole-3-butyric acid (IBA), for regeneration of roots. Shoot regeneration (6.7%) was achieved with KAT 60/8 variety from leaf callus induced on 1 mg/l 2, 4- D for 4 weeks and sub cultured on regeneration medium with 0.5 mg/l BAP for five weeks. No regenerants were obtained from ICEAP 00557 embryo and leaf callus induced on 2, 4- D and sub cultured on regeneration medium with BAP. The highest frequency of regeneration from ICEAP 00557 was achieved with leaf explant on 0.5 mg/l TDZ giving 20.0%. On the other hand, a regeneration frequency of 16.67% was obtained with KAT 60/8 leaf explants on 2 mg/l TDZ. IBA at 0.5 mg/l gave the most profuse rooting. Rooted shoots were hardened in a mixture of soil and vermiculite (1:1) for 21 days after which they withered. From the results reported in this study, the best explants for in vitro regeneration of pigeon pea varieties KAT 60/8 and ICEAP 00557 are leaf discs. The results obtained show that genotype, plant growth regulator and explant type have a great influence on the success of an in vitro regeneration system. Moreover, optimization of the protocols generated in this study will step up the prospects for mass production of pigeon pea and genetic manipulation.