In Vitro Regeneration of ICP 8863 Pigeon Pea ( Cajanus cajan (L.) Millsp.) Variety using Leaf Petiole and Cotyledonary Node Explants and Assessment of their Genetic Stability by RAPD Analysis

摘要

Objectives: A reliable in vitro regeneration protocol by direct organogenesis was developed in ICP 8863 variety of pigeon pea using leaf petiole and cotyledonary node explants. Methods: For direct shoot bud induction, leaf petiole explants from seven-day-old in vitro grown seedlings and cotyledonary node explants from twelve-day-old were cultured on MS medium supplemented with various combinations and concentrations of BAP, NAA and Kinetin. Induced shoot buds of both the explants were elongated on MS medium fortified with different concentrations of BAP, NAA and GA3. The wellelongated shoots of both the explants were transferred to MS medium supplemented with various concentrations of IBA. Finally, the regenerated plants were transferred to soil and vermiculate mixture in 1:1 ratio for acclimatization. Further, molecular characterization of the in vitro regenerated plants was carried out using eight OPP and OPAZ RAPD primer series. Findings: High frequency of shoot bud induction (92 %) was observed in leaf petiole explants with 2.0 mg/L 6-BAP concentration compared to cotyledonary node explants. The induced shoots were kept for elongation and maximum percentage of elongation (93 %) was noticed in leaf petiole explants with 1.0 BAP + 0.1 NAA + 2.0 GA3 mg/L concentrations compared to cotyledonary node explants. The well-developed shoots of both the explants showed profuse rooting, where high percentage of rooting (95 %) was observed in leaf petiole explants with 0.5 mg/L IBA concentration. The pattern of amplification resulted through RAPD analysis confirmed the genetic stability of in vitro regenerated plants. Improvement: The regeneration protocol standardized in this study is suitable and reliable to develop transgenic pigeon pea plants by agrobacterium mediated genetic transformation.