TRANSFORMATION AND REGENERATION OF Jatropha curcas L. Agrobacterium tumefaciens STRAIN GV3101-MEDIATED WITH POLIHYDROXYALKANOATS GENE (phaC)

• Main Author: Novatiano, Chesara
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34232
• Institution: Institut Teknologi Bandung

Polyhidroxybutyrate is a component of bioplastics synthesized by the enzyme encoded by multigene pha (phaA, phaB, and phaC). These three genes are naturally present in bacteria Rastolnia eutropha, but not found in the plant. Plant has abundant source of biomass, so that plant can be a good candidate of PHB production. Therefore, objectives of this study were to insert phaC gene (one encoding gene of PHB synthesis) in tissue of Jatropha curcas which can be indirectly mediated by Agrobacterium tumefaciens strain GV3101 which contained pARTC expression vector, to regenerate transformation plant, and to confirm the presence of inserted genes by PCR technique. In this study, phaC gene with nptII gene as marker gene, were constructed in pARTC in previous study. Genetic transformation of J. curcas was conducted via A. tumefaciens- mediated transformation of cotyledon using acetosyringone 100 ?M for 30 minutes, and cocultivated for 3 days. Explants of J. curcas were planted in selection medium MS in addition of 13,3 ?M BAP and 0,05 ?M IBA with kanamycin 20 mg/L and Augmentin 300 mg/L. Results showed putative transformant explants of J. Curcas were able to regenerate forming shoot in selection medium for 1 – 2 months and rooting in liquid half – strength MS with 4,9 ?M IBA generating putative transformant planlet. Molecular analysis of J. curcas putative transformant was performed using two primers, phaC and nptII. PCR products obtained were 659 base pair using nptII primer and 700 base pair with phaC primer, indicating integrated incomplete T – DNA in pARTC in J. curcas genome presumably because deletion of phaC gene in T – DNA.