The study of rice chloroplast transformation

• Main Authors: Shih-YinChen, 陳世殷
• Other Authors: Ching-Chun Chang
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/04671031619133501874

碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 100 === Biolistic bombardment is the most common used method for plant plastid transformation. Once expression vectors were delivered into plastid, the transgene could be integrated into plastid genome via homologous recombination. Subsequently under a series of selection and regeneration process, the transplastomic plant could be obtained. Generally, the efficiency of plastid transformation by bombardment is higher than other methods, but it is costly. Although the rice plastid transformation has been demonstrated by bombardment, the homoplasmic transplastomic plant was not obtained and the efficiency is still low. In this study, several approaches were tested to improve the efficiency and reduce the cost. First, the delivery of chloroplast expression vectors carrying gfp reporter gene into plastids of rice suspension cells by electroporation was tested. Although the GFP expressing cells were transiently observed under fluorescence microscope, the transplastomic plantlet could not obtain, probably because the cells were seriously damaged after high voltage of electroporation. In addition, the delivery of chloroplast expression vector into plastids of rice mesophyll protoplasts by either PEG alone or in combination with R9 peptides or PAMAM dendrimers was also tested. The addition of R9 peptides or PAMAM dendrimers with PEG failed to increase the transformation efficiency. The GFP was transiently observed in the plastids of a few protoplasts after PEG-mediated transformation; however, subsequently the GFP expressing cells were lost after subjecting to regeneration process. Finally, the recombinant fusion proteins that the transit peptide of nuclear-encoded chloroplast RNA polymerase was fused with R9 peptide were used as a DNA carrier. It was hypothesized that the DNA-protein complex could be recognized and imported into chloroplast through translocon complex. The preliminary results suggested that it is potentially able to enhance the plastid transformation by PEG-mediated method.