CALLUS CULTURE OPTIMATION AND GENETICS TRANSFORMATION ON JARAK PAGAR (Jatropha curcas L.) USING Agrobacterium tumefaciens STRAIN LBA4404 WITH BINARY VECTOR pCambia 1304

• Main Author: Budiharto, Ari
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/34894
• Institution: Institut Teknologi Bandung

An optimal in vitro culture and suitable transformation technique are needed in obtaining transgenic Jatropha curcas plants. However, few report is available concerning transformation as well as in vitro culture of J. curcas. In order to obtain transgenic J. curcas plants, optimalization J. curcas callus culture and genetics transformation had been conducted on this research. The objectives of this research were to optimize callus culture, to genetically transform J. curcas tissue using Agrobacterium tumefaciens strain LBA4404 harboring binary vector pCambia 1304, to evaluate and analyze J. curcas genetics transformation. J. curcas leaf explants were placed on MS (Murashige & Skoog’s) medium supplemented with NAA (1-Naphthaleneacetic acid) and BAP (6- Benzylaminopurine) in variety of concentrations to induce callus formation. J. curcas genetics transformation was conducted by cocultivation of J. curcas leaf explants with A. tumefaciens strain LBA4404-pCambia 1304 for three days with the addition of 100 ?M acetosyringone. J. curcas genetics transformation was evaluated based on GUS gene transient expression and explants resistance to hygromycin. Stable transgene integration was analyzed by electrophoresis. J. curcas DNA was isolated by CTAB (cetyl trimethyl ammonium bromide) method and then amplificated with DNA Thermal Cycler (GeneAmp PCR System 2400) by touchdown method. Afterward, J. curcas DNA which had been amplificated was run on electrophoresis 1.5% (b/v) agarose gel with constant voltage on 110 volt for 30 minutes. The results indicated that J. curcas callus showed induction and optimum growth on medium supplemented with 0.1 ?M NAA + 5 ?M BAP. Callus culture reached to maximum growth on the 27-th day. Callus remain survived and grew well over several time subcultured on the same fresh medium. Evaluation on GUS transient expression indicated that on both transformed leaf explant and callus of J. curcas were emerged blue color dichlorodibromoindigo (ClBr-indigo) product. Both transformed leaf explant and callus on the selection medium which containing 10 mg/L hygromycin showed their resistance. Electrophoregram of J. curcas DNA which had been amplificated showed the fragment with length of 123 bp which correspond to CAMV DNA. Based on GUS transient expression on both transformed leaf explant and callus, hygromycin resistance of callus, and the presence of fragment with length 123 bp which correspond to CAMV DNA, are proved that the GUS and hygromycin genes from pCambia 1304 of A. tumefaciens LBA4404 were integrated into the genome of J. curcas.