Chloroplast transformation

Until very recently, high-efficiency chloroplast transformations have been limited to Chlamydomonas and the higher plant Tobaccum. Arabidopsis chloroplasts have been successfully transformed, but none of the regenerated transformed plants were fertile. Chloroplasts from potato leaf cells and rice suspension cells have also been transformed but with poor efficiency as well, and no fertile plants were recovered (Khan et al., 1999 Hibbard et al., 1998). Therefore, a major hurdle is the development of fertile, transgenic, economically important plants. 

Four basic strategies for recombinant protein expression in plants are transient expression, stable nuclear transformed plants, chloroplast transformed plants, and suspension cultures derived from stable transgenic lines. 

Revertant of no-active RuBisCO tobacco mutant, Sp25, obtained by chloroplast transformation method using microprojectile bombardment... 

Boynton, J.E., et al., Chloroplast transformation in Chlamydomonas with high-velocity microprojectiles. Science, 1988 240(4858) 1534-1538. 

Sidorov, V.A., et al.. Technical Advance Stable chloroplast transformation in potato use of green fluorescent protein as a plastid marker. Plant J, 1999 19(2) 209-216. 

Reddy, V. S., et ah. Analysis of chloroplast transformed tobacco plants with cryllaS under rice psbA transcriptional elements reveal high level expression of Bt toxin without imposing yield penalty and stable inheritance of transplastome. Mol Breed, 2002 9 259-269. 

Cell culture-based methods suitable for obtaining chloroplast-encoded herbicide resistance in crops will be reviewed. These will Include mutant selection in cultured cells, transfer of chloroplasts via protoplast fusion, and selection of recombinant chloroplast genomes. Examples will be limited to resistance to triazine herbicides. Chloroplast transformation involving introduction of novel DNA into chloroplasts, will not be covered. Preliminary data indicate that chloroplast transformation may be feasible (4) but have not been confirmed. The genetics, physiology... 

Figure 16.2 Schematic of a typical Chlamy-domonas chloroplast transformation vector. The gene of interest and the selectable marker are expressed in two separate cassettes. Two sites are commonly targeted the 3HB site is a neutral site in the chloroplast genome that tolerates transgene insertion,...

Electroporation is generally the first technique tried in most algae for nuclear transformation, while chloroplast transformation often requires a more aggressive... 

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