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Genetic Transformation of Capsicum and Cloning of Genes for Value Addition

Vaitheeswaran, V. (2010) Genetic Transformation of Capsicum and Cloning of Genes for Value Addition. [Student Project Report]

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Abstract

This Dissertation / Report is the outcome of investigation carried out by the creator(s) / author(s) at the department/division of Central Food Technological Research Institute (CFTRI), Mysore mentioned below in this page.

Item Type: Student Project Report
Additional Information: The primary aim and scope of this work for dissertation was to learn the techniques of biotechnology and molecular biology along with specific objectives. Several techniques such as preparation of tissue culture media, explant preparation, inoculation and sub culturing, techniques on Agrobacterium mediated genetic transformation maintaining and handling Agrobacterium cultures, handling in vitro cultures infected with Agrobacterium were learnt. Molecular biology techniques such as handling of E.coli cultures, preparation of competent cells of E.coli and Agrobacterium, isolation of plasmids, transformation techniques, preparation of constructs with gene of interest for cloning and transformation studies were made. Analysis of transformants via biochemical methodology was attemped as a preliminary confirmation. The major highlights of the outcomes during the dissertation period are mentioned below. The first objective of the experiment was to make binary vector constructs for transformation studies. To achieve this we had selected an aminotransferase gene from Capsicum sp. involved in vanillylamine biosynthesis, which is also a precursor for Capsaicinoids. Previously the gene was cloned in T-tailed vector and was confirmed by various methodologies in the Institute. Further directional cloning methodology was adopted to clone the gene from T-tailed vector to a cloning vector named pRT100. This vector was selected in order to get a gene construct along with a CaMV/35S promoter. Using suitable restriction sites from T-tailed vector and pRT 100 Multiple Cloning Site (MCS) it was possible to make sense and antisense constructs of aminotransferase gene in pRT100 after digesting the plasmids, ligating the required fragments and transforming in E.coli. After confirmation of positive clones the recombinant plasmids were taken up for preparing binary vector constructs. pCAMBIA 2301 was selected as candidate binary plasmid form making the constructs. The plasmid construct pRT100 harboring aminotransferase gene in sense and antisense orientation was digested with the suitable restriction enzyme, similarly for the binary vector pCAMBIA 2301 with the same enzyme. Upon ligation of the fragments and transformation in E.coli, we were able to achieve to get binary vector constructs with aminotransferase gene. After analyzing the binary plasmid the same was mobilized to Agrobacterium tumefaciens for plant transformation experiments. The second objective of the experiment was to do genetic transformation studies in Capsicum sp. During the course of study it was decided to check the functionality of the prepared constructs on Nicotiana tabaccum also as it is a model system for any transformation experiments. This type of study has an added advantage that Vanillylamine production in Nicotiana could be demonstrated using sense binary vector construct as the metabolite is new to the plant and reduction in Capsaicinoid/Vanillyllamine production could be shown using antisense binary vector construct in Capsicum callus cultures. Initially the germplasm of both Capsicum sp. and N.tabaccum were established in vitro. Further callus induction was achieved upon subjecting the in vitro seedlings on respective media discussed in this thesis. Genetic transformation studies were initiated by infecting the fully grown callus tissues with Agrobacteriun tumefaciens strain GV 3101. After initial co-cultivation the calli were grown on Kanamycin selection media since pCAMBIA 2301 has Kanamycin as plant selection antibiotic. The confirmation of transformants was done using GUS-Histochemical staining. Most of the callus tissues selected for staining showed GUS positive nature. Further confirmatory studies needs to be taken up in future and is beyond the scope of this dissertation period.
Uncontrolled Keywords: Capsicum; genetic transformation; binary vector constructs
Subjects: 600 Technology > 08 Food technology > 23 Vegetables
600 Technology > 05 Chemical engineering > 01 Biotechnology and Bioengineering
Divisions: Plant Cell Biotechnology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 08 Jul 2010 07:01
Last Modified: 28 Dec 2011 10:15
URI: http://ir.cftri.com/id/eprint/9471

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