Development of protocol for Agrobacterium Tumefaciens mediated transformation of cocoa (Theobroma cacao)

Helda Souki, (2009) Development of protocol for Agrobacterium Tumefaciens mediated transformation of cocoa (Theobroma cacao). Masters thesis, Universiti Malaysia Sabah.

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Abstract

This research describes the optimization of parameters (including pH, temperature, period of co-cultivation and age of callus) for Agrobacterium tumefaciens-mediated genetic transformation of Theobroma cacao L. using staminodes from cocoa buds as explants. The A. tumefaciens strain used was the super avirulent AGLl with the binary vector pGPTV-Kan/Gus. The strain confers aminoglycoside resistance to transformed cells through the neomycin phosphotransferase II (nptII) gene. Callus induction medium contained DKW minerals, glucose, vitamins, 2 mg/L 2,4D and 0.005 mg/L TDZ (0.5nM) pH 5.3. Co-cultivation medium was identical to callus induction medium but contained 0.02mg/L acetosyringone. Experiments were conducted using two clones of cocoa: KKM19 and P22. Staminodes were cultured on callus induction medium in the dark before the transformation process. After 14 days and 21 days on callus induction medium, callus-derived staminodes were co-cultivated with A. tumefaciens on semi-solid co-cultivation media for one, two, or three days with various temperatures (19°C, 21°C, 23°C, and 25°C) and pHs (4.8, 5.3, and 5.8). Half amount of treated calli in each parameters used was selected on selective medium containing 100µg/ml paromomycin. Another half amount was cultured without paromomycin but tested for GUS activity. Analysis for GUS activities were done 20 weeks after A. tumefaciens treatment. The result revealed that the best transformation frequency (86%) was obtained with two week-old calli from clone P22 and co-cultivation at pH 5.8, 25°C, and for one day. For clone KKM19, the best transformation frequency (80%) was obtained with three week-old calli, co-cultivation at pH 4.8, 19°C and three days. Putatively transgenic cells of cocoa (after 20 weeks of A. tumefaciens treatment) were first extracted and then digested using restriction enzyme (Sau3A) which cut at T-DNA right border (GATC). Base on modified Cottage method, both ends of digested putative DNA samples were ligated with special designed adaptors and primers for PCR to detect the integration of T-DNA into the cocoa genome. Based on the DNA sequencing results, adaptors and primers from both ends of digested DNA were successfully sequenced but not for the expected sequence for cocoa/ T-DNA. It might occur when no DNA transformation happened. However, for control sample (Binary Plasmid DNA), all bases, adaptors and primers were successfully sequenced. Cottage modified method shows that it works and more efforts should be done in optimizing Cottage method specifically for identification of cocoa DNA Sequences.

Item Type:Thesis (Masters)
Uncontrolled Keywords:pH, temperature, age of callus, period of co-cultivation, agrobacterium tumefaciens-mediated, cocoa, theobroma cacao, T-DNA
Subjects:T Technology > TP Chemical technology
Divisions:SCHOOL > School of Science and Technology
ID Code:6373
Deposited By:IR Admin
Deposited On:21 Jun 2013 16:44
Last Modified:21 Jun 2013 16:44

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