| his thesis work has been designed to develop an effective and efficient transformation system for higher plant chloroplast, and to explore the possibility of expressing foreign genes transiently and stably in chloroplast, using the recently developed and improved biolistic transformation process.;Using GUS as a reporter gene and using an improved biolistic device, optimal bombardment conditions were established, consistantly producing several hundred transient chloroplast transformants per petri plate. This system provides a highly effective mechanism for introducing and transiently expressing plasmid DNA within higher plant chloroplast.;Chloroplast expression vectors containing the NPTII gene under the control of the psbA promoter (psbA-NPTII) were constructed and biolistically delivered into tobacco suspension cells or leaf strips, in attempts to transform plant chloroplast stably. Analysis of the resulting kanamycin-resistant transgenic plants showed that the psbA-NPTII gene was consistently located in the nucleus in very high copy number, but not in the chloroplast. This conclusion is based on results from: (1) kanamycin screening of progenies from reciprocal crosses; (2) pulse-field gel electrophoresis; and (3) Southern blots of chloroplast and nuclear DNAs using NPTII, chloroplast-marker, and nuclear-marker probes. These results indicate that the chloroplast psbA promoter used in this study can function in the nucleus. However, its nuclear activity is very limited since an extremely high number of copies of the insert (over 100) was required to provide effective levels of kanamycin resistance.;Two reporter genes, coding for chloramphenicol acetyltransferase (cat) and... |
