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Subject: Plant Biochemistry × Author: KAU × Author: Karthika, Karunakaran × End date: 2011 × Type: Thesis × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    Agrobactrium tumefaciens mediated genetic transformation in dendrobium variety sonia 17 with 1- aminocyclopropane- 1 carboxylic acid (acc) synthase antisense gene
    (Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Karthika, Karunakaran; KAU; Rajmohan, K
    A study on “Agrobacterium tumefaciens mediated genetic transformation in Dendrobium sonia 17 using ACC synthase antisense gene.” was conducted at the Department of Plant Biotechnology, College of Agriculture, and Vellayani during 2004-2006. Orchid is an important cut flower crop. Dendrobium sonia 17 is the most popular commercial orchid grown in Kerala. It has sufficiently higher vase life. However, increasing vase life can reduce the per day cost of flower. The present study was undertaken with the main objective of evolving a protocol for Agrobacterium tumefaciens mediated genetic transformation in Dendrobium sonia 17 using ACC synthase antisense gene. PLBs were initiated from the meristematic shoot tip on half strength MS medium supplemented with growth regulators. MS medium supplemented with BA 0.2 mg l-1 was proved to be the best in terms of induction of PLBs (92.5 %). The Agrobacterium tumefaciens strain GV3101, harbouring the plasmid pA4A2AB was used for genetic transformation. As the plasmid harbour nptII and ACS2 genes, the sensitivity of Agrobacterium strain and PLBs to different concentrations of kanamycin was evaluated. The lethal doses of kanamycin to Agrobacterium and PLBs were 300 and 150 mg l-1, respectively. The effective dose of cefotaxime for the elimination of bacterial strain GV3101 was 50 mg l-1 and the lethal dose of cefotaxime to PLBs was 300 mg l-1. Genetic transformation was achieved by co-cultivating PLBs with bacterial suspension. Conditions like infection and co-cultivation time, selection agent were optimized. The most effective infection time was 20 min, followed by a co-cultivation period of four days. The survival of tissues transformed on the selection media was 76.47 per cent. The transformation efficiency was increased when acetosyringone 200 µM was added to infection and co-cultivation media. Transformation was confirmed by PCR and southern hybridisation of putative transformants. This study provides a protocol for genetic transformation in Dendrobium sonia 17 using ACC synthase antisense gene.