STUDIES ON IN VITRO AND IN PLANTA TRANSFORMATION PIGEONPEA (Cajanus cajan L.) cv. ICPL-8863 (Maruti)
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Date
2003
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University of Agricultural Science, Dharwad
Abstract
In vitro and in planta transformation of pigeonpea was achieved using
Agrobacterium tumefaciens strain EHA105 harboring pBinBtS plasmid
having crylAc gene linked to the cauliflower mosaic virus (CaMV) 35 S
promoter and neomycin phosphotransferase (npt II) gene under the control of
nopaline synthase (nos) promoter and terminator.
Initially, protocol was developed for in vitro multiple shoot
regeneration. Using this protocol in vitro transformation were attempted.
Cotyledonary node with cotyledon were pre-cultured on MS medium with
2 mg 1-' BAP for two days, co-cultured for two days in dark, washed with
MS broth containing cefatoxime transferred to shoot induction (MS + 2 mg 1'
BAP) followed by elongation (MS + 0.1 mg BAP) and rooting medium
(MS + 0.5 mg 1-' IBA). Rooted shoots were established in greenhouse. This
protocol for pigeonpea transformation using crylAc gene was repeated 8
times through all the steps. In one batch 2 per cent of established plants
showed PCR amplification for npt 7/and crylAc primer.
In in planta approach, different methods such as simple dipping
injury followed by dipping, injection and vaccum infiltration were used at
different developmental stages like germination, seedling and at flowering
stage. Among different methods vaccum infiltration of the germinating seeds
and flower injection were found to be suitable for transformation.
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