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Subject: Plant Biochemistry × End date: 2008 × Start date: 2006 × 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.
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    ThesisItemOpen Access
    Development and analysis of ESTs (expressed sequence tags ) in black pepper (piper nigrum L )
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2008) Renu, Kushwah; KAU; Nazeem, P A
    Soil moisture is one of the major factors that influence plant growth and productivity. The stress is the second most important factor that affects crop production in black pepper. Various genotypes of black pepper are reported to vary in their response to water stress and the variety Kalluvally has been identified as a drought tolerant one among the cultivated genotypes. Plants respond to stress by adaptation of the biochemical and physiological processes. These biochemical and physiological reactions are regulated by several genes that are induced during drought conditions. Thus the gene products directly or indirectly provide tolerance to the plants so that they can survive under water stressed conditions. In the present study, an attempt was made to identify such water stress induced genes in variety Kalluvally using the molecular technique called Suppression Subtractive Hybridization (SSH) and finally to develop and analyze Expressed Sequence Tags (ESTs). Total RNA and mRNA were isolated from normal and water stressed plants and were used respectively as ‘driver’ and ‘tester’ in SSH reaction. The reactions were performed utilizing the PCR select cDNA subtraction kit provided by CLONTECH, USA. Control subtraction was carried out first using PCR select™ cDNA subtraction kit, which gave satisfactory and expected results. For experimental subtraction, double stranded cDNAs were synthesized from 2µg mRNA from normal ‘driver’ and water stressed ‘tester’. Two tester populations were created and each ligated to two different adaptors. This was followed by two hybridization reactions and finally a selective PCR amplification. Only differentially expressed cDNAs were amplified exponentially. This was confirmed by analyzing the PCR products on agarose gel, which showed a smear ranging from 0.2 to1.5kb in the subtracted sample and was different from smear pattern of unsubtracted ones. The cDNA fragments from subtracted sample were cloned in pGEMT vector and sequenced. Total twenty clones were sequenced and analysed after vector and adaptor editing. In silico analysis using bioinformatics tools revealed that some of the cloned sequences showed good homology with known sequences which play important role during water stress conditions directly or indirectly. These included Heat Shock Proteins (HSP-17 & 20), Secretory Carrier Membrane Protein (SCAMP), gamma thionins, MYB transcription factor, Ribonuclease enzyme, fatty acid desaturase, peptidylprolyl isomerase and NADH-ubiquinone oxidoreductase family protein. Also, these sequences had conserved domains for the above mentioned proteins. The rest of the clones did not show any good homology and therefore it was difficult to assign any reported role to these. In addition to this, all the sequences possessed Open Reading Frames (ORFs) many had transmembrane helices and some were found to have signal peptide. The sequences were submitted to dbEST. For further exploitation of these sequences it is necessary to clone full length cDNA. ESTs thus generated in the present study will be of great use in future for further downstream applications.