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20131
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Subject: Agricultural Biotechnology × Author: Atul Dev × End date: 2020 × Start date: 2000 ×
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    ESTABLISHING EFFICIENT IN VITRO REGENERATION PROTOCOL AND GENETIC TRANSFORMATION OF PIGEONPEA (Cajanus cajan L. Millsp.) THROUGH BIOLISTIC GUN USING cry1Ac GENE
    (Punjab Agricultural University, Ludhiana, 2013) Atul Dev; Ajinder Kaur
    The present investigation entitled “Establishing efficient in vitro regeneration protocol and genetic transformation of pigeonpea (Cajanus cajan L. Millsp.) through biolistic gun using cry1Ac gene” was carried using two varieties of pigeonpea ICPL87 and ICPL 88039. Regeneration from callus and direct organogenesis, both approaches were used to establish a tissue culture baseline to carry out transformation. Different explants viz., leaves, cotyledons, epicotyls, embryonic axes, mature and immature embryos were used for callus induction. None of the calli regenerated into plants on any of the growth hormone combinations and concentrations used. Organogenesis approach worked well with the induction of multiple shoots from cotyledonary nodes. Both the varieties exhibited a high frequency of shoot buds (60-65%) on MS + 5mgL-1 BAP medium. Elongation of the shoot buds was carried out on MS + 30 mgL-1 adenine sulphate medium and an average of 5-6 shoots per explant was obtained. Rooting and hardening were done ex vitro with a success rate of 75-94%, using a rooting mixture ROOTEXTM. For genetic transformation, biolistic approach was used to introduce cry1Ac gene. Mature seeds after 12 days of culturing produced axillary meristems in cotyledonary nodes that were used as target tissue for bombardments. Bombarded explants were kept on shoot induction medium for further induction of shoot buds for 10-12 days that were later transferred to shoot elongation medium. In total, 8 transformation experiments were conducted and a total of 326 axillary meristem explants (169 of ICPL 87 and 157 of ICPL 88039) were bombarded, from which 416 shoots in ICPL 87 and 384 shoots in ICPL 88039 were regenerated. Transformation results were promising in both the varieties with transformation efficiencies of 1.18% in ICPL 87 and 3.18% in ICPL 88039