Optimisation of gusA gene transfer into cocoa (Theobroma cacao) via agrobacterium tumefaciens-mediated transformation

• Main Author: Anisah Savantil
• Format: Thesis
• Language: English; English
• Published: 2011
• Subjects: QK474.8-495 Spermatophyta. Phanerogams
• Online Access: https://eprints.ums.edu.my/id/eprint/41848/1/24%20PAGES.pdf; https://eprints.ums.edu.my/id/eprint/41848/2/FULLTEXT.pdf; https://eprints.ums.edu.my/id/eprint/41848/

This study describes the optimization of parameters (pH, temperature, periods of co-cultivation and age of explant) for gusA gene transfer into two cocoa clones (PBC 123 and BR 25) via Agrobacterium tumefaciens-mediated transformation by using staminode cocoa bud flower collected one day before anthesis as explants. Super avirulent A. tumefaciens strain AGL 1 harbouring the binary vector pGPTVKan/ GUS was used. The binary vector contains a CaMV 35S-driven β-glucuronidase (GUS) gene and a complete neomycin phosphotransferase II (nptII) gene for conferring plant resistance to the antibiotic paromomycin. Callus induction medium (IM) for clone PBC 123 consisted of Driver and Kuniyuki Walnut (DKW) minerals, glucose, vitamins, 2 mg l-1 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.005 mg l-1 Thidiazuron (TDZ) pH5.3. Callus IM for clone BR 25 was identical to IM for clone PBC 123 except the TDZ was replaced with 100 mg l-1 2-iP. Callus was allowed to proliferate on semi-solid callus inducing medium for 14 days, 21 days, and 28 days prior co-cultivation with A. tumefaciens on semi-solid co-cultivation media for one, two, and three days at various temperatures (190C, 210C, 230C, and 250C), and pHs (4.8, 5.3, and 5.8). Each set of parameter was design in triplicate plate. Half amount of treated calli in each tested parameter was examined for GUS expression, while another half was selected on selective medium containing 100 μg ml-1 paromomycin. GUS expression analyses were done after 18 weeks selection. Based on the GUS expression analyses, up to 86% of calli had at least one blue sector after 18 weeks on selection media containing 100 μg ml-1 paromomycin. The best transformation frequency (86.1%) for clone BR 25 was obtained when explant was 21 days old and transformation condition was with pH 5.8, 250C, and three days cocultivation period. For clone PBC 123, the best transformation frequency (83.3%) was obtained when explant was 14 days old and transformation condition was with pH 4.8, 190C, and three days co-cultivation period. A PCR-walking method (Cottage et al. 2001) with modification was used to verify the integration of the T-DNA into genome of the putatively transformed cocoa calli. Based on the sequencing results, this method worked with purified plasmid pGPTV-KAN/GUS but the sensitivity too low for putatively transformed cocoa calli.