Optimized Agrobacterium tumefaciens - mediated transformation parameters for cocoa callus
2006
Helda Souki | Anisah Savantil, Malaysia Sabah Univ. (UMS), Locked Bag 2073, 88999 Kota Kinabalu, Sabah (Malaysia). School of Science and Technology. Centre for Postgraduate Study | Douglas B. Furtek (eds.), Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Sabah (Malaysia)
We describe optimized parameters for Agrobacterium tumefaciens-mediated genetic transformation of Theobroma cacao L. using staminodes from cocoa buds. The A. tumefaciens strain was the super avirulent AGLI with the binary vector pGPTV-Kan/Gus which confers aminoglycoside resistance to transformed cells through the neomycin phosphotransferace II (nptII) gene and contains the beta-glucuronidase (GUS) reporter gene. Callusing media contained DKW minerals, glucose, vitamins, 2 mg L-1 2,4D and 0.0005muM TDZ, pH 5.3. Co-cultivation media excluding pH was identical to callusing media but contained 100 muM acetosyringone. Experiments were conducted with five clones: KKM19, P22, PBC123, UP6, and BR25. Staminodes were cultured on soilid callusing media for 14 days, 21 days, and 28 days in the dark before the transformation process. Staminodes were co-cultivated with A. tumefaciens on solid 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). One set of treated calli was selected on callusing media containing 100 mug ml-1 paromomycin. Another set was cultured without paromomycin but tested for GUS activity. The best transformation frequencies (86%) as revealed by GUS staining at 20 weeks after A. tumefaciens treatment were obtained with three week-old calli from clone BR25 and co-cultivation at pH 5.8, 25°C, and for two days. Second best (85%) was obtained with two week-old callus from clone P22and co-cultivation at pH 5.8, 25°C, and for one day. Third best (83%) was from two week-old PBC 123 callus and pH 4.8, 19°C, and for three days. Fourth best (80%) was from three week-old KKM19 callus and pH 4.8, 19°C and for three days. The least best transformation frequency (48%) was obtained with two week-old UP6 callus and pH 5.3, 23°C, and for three days.
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