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        Agrobacterium-Mediated Cry1A(b) Gene Transfer in Punica granatum L. cv. Kandhari Kabuli Using Different In Vitro Regeneration Pathways

        Verma, Vipasha,Kanwar, Kamlesh,Tufchi, Mahak,Kashyap, Monika 한국작물학회 2014 Journal of crop science and biotechnology Vol.17 No.1

        Three different regeneration systems, viz. regeneration through callus cultures using embryonic explant, direct regeneration using shoot bud/nodal segments as explant and regeneration through cell suspension culture using cotyledonary explant (for the induction of transgenic callus for suspension culture) were evaluated to see their effect on transfer of Cry1A(b) gene to Punica granatum L. cv. Kandhari Kabuli through Agrobacterium mediated transformation. Pre-conditioning and co-cultivation durations had a marked effect on transformation frequency of different explants. Out of different explants used (embryo, shoot bud, and cotyledon) for different regeneration systems cotyledonary explant showed highest putative transformation frequency (13.54%) inducing callus on selective medium for carrying out cell suspension culture to regenerate transgenic shoots. Despite of the highest transformation frequency obtained from the cotyledon explant, the plating efficiency of the transgenic cells generated through the transgenic callus (callus formed from the cotyledonary explant) during cell suspension culture was found to be very low (0.7%). Thus the plating efficiency has also played worth mentioning role in the regeneration of transformants following cell suspension culture. Among the three regeneration systems, regeneration through callus cultures using embryonic explant was found to be best for regeneration of transformants. The highest per cent regeneration of 23.33 was obtained from the putative transgenic embrogenic calli. Successful genetic transformation in the transformed plantlets was confirmed by PCR analysis. The transformation system thus developed is valuable and may be used to produce insect resistant trees.

      • KCI등재

        Agrobacterium-Mediated Cry1A(b) Gene Transfer in Punica granatum L. cv. Kandhari Kabuli Using Different In Vitro Regeneration Pathways

        Vipasha Verma,Kamlesh Kanwar,Mahak Tufchi,Monika Kashyap 한국작물학회 2014 Journal of crop science and biotechnology Vol.17 No.1

        Three different regeneration systems, viz. regeneration through callus cultures using embryonic explant, direct regeneration usingshoot bud/nodal segments as explant and regeneration through cell suspension culture using cotyledonary explant (for the inductionof transgenic callus for suspension culture) were evaluated to see their effect on transfer of Cry1A(b) gene to Punica granatum L. cv. Kandhari Kabuli through Agrobacterium mediated transformation. Pre-conditioning and co-cultivation durations had a marked effecton transformation frequency of different explants. Out of different explants used (embryo, shoot bud, and cotyledon) for differentregeneration systems cotyledonary explant showed highest putative transformation frequency (13.54%) inducing callus on selectivemedium for carrying out cell suspension culture to regenerate transgenic shoots. Despite of the highest transformation frequencyobtained from the cotyledon explant, the plating efficiency of the transgenic cells generated through the transgenic callus (callusformed from the cotyledonary explant) during cell suspension culture was found to be very low (0.7%). Thus the plating efficiencyhas also played worth mentioning role in the regeneration of transformants following cell suspension culture. Among the three regenerationsystems, regeneration through callus cultures using embryonic explant was found to be best for regeneration of transformants. The highest per cent regeneration of 23.33 was obtained from the putative transgenic embrogenic calli. Successful genetic transformationin the transformed plantlets was confirmed by PCR analysis. The transformation system thus developed is valuable and maybe used to produce insect resistant trees

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