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20131
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Subject: Biotechnology × Author: Aradhna Sonik × Start date: 2012 × Type: Thesis × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    INTRODUCTION AND EXPRESSION OF ANTIFUNGAL CHITINASE AND β-1, 3-GLUCANASE GENES IN ARBOREUM COTTON TO INDUCE RESISTANCE AGAINST Fusarium oxysporum
    (PAU Ludhiana, 2013) Aradhna Sonik; Jagdeep Singh, Sandhu
    In the present study, immature embryos of diploid cotton (Gossypium arboreum L. cv. RG8) were transformed with three recombinant plasmids viz., H1Z, H2Z and pBI121 containing -1, 3-glucanase, chitinase and GUS gene cassettes respectively, under the control of CaMV 35S promoter and NOS terminator, with an objective to induce resistance against Fusarium oxysporum. The immature embryos were excised from 18-day old cotton bolls obtained during early, mid and late flowering seasons of the diploid cotton plants, cultured on germination medium (GM) [basal MS medium containing proline (560mg/l), BAP (2mg/l), GA3 (40mg/l), activated charcoal (0.20%), omnatax (500ppm) and casein hydrolysate (500mg/l)] followed by incubation under dark at 28±2oC. After five days of culture establishment, the germinating embryos were transferred under 16 hr(s) photoperiod during which they elongated into shoots and developed true leaves. Root multiplication was induced by placing elongated shoots on root induction medium (1/2 strength MS medium fortified with 0.20% activated charcoal). The plantlet formation efficiency of the embryos cultured during the mid flowering season was found to be significantly higher (37.67%) compared to the embryos excised during late (32.80%) flowering season and at par with the efficiency of embryos excised during the early season (36.28%). The three recombinant plasmids–H1Z, H2Z and pBI121, after being confirmed for the presence of gene cassettes by PCR and restriction digestion were introduced in the immature embryos using particle bombardment. The integration of gene cassettes was analysed in transient histochemical GUS assay and PCR, 48 hr(s) post bombardment. The results revealed 91.6% bombarded embryos expressing GUS gene, whereas 66.6% embryos had the co-integration of H1Z and H2Z plasmids containing -1, 3-glucanase and chitinase coding region respectively. The bombarded embryos were cultured on media GM supplemented with 210mg/l ampicillin for five selection cycles of 20 days each. After five selection cycles, 12 plantlets survived and the plantlet formation efficiency of 0.65% was obtained, which was significantly higher than the efficiency of non-bombarded embryos (0%). The PCR analysis, 100 days post bombardment, showed co-integration of -1, 3-glucanase and chitinase genes in eight plantlets with a transformation efficiency of 0.43%, while the remaining 4 plantlets showed integration of either of the genes. These putative transgenic plantlets are currently growing in the glass-house.