Towards plastid transformation in rapeseed (Brassica napus L.) and sugarbeet (Beta vulgaris L.)

In the current study tissue cultures of rapeseed (cv. “Drakkar”, cv. “Westar”) and sugarbeet (cv. ”Viktoria”, cv. “VRB”, cv. ”31-188”, cv. ”7T1308” and 47 other breeding lines, Appendix 1) have been investigated for the establishment of conditions that make possible plastid transformation in both species. Tobacco leaf protoplasts (cv. ”petite Havana”, cv. ”Wisconsin 38”) were used to develop a novel technique – the TAL (thin-alginate-layers) technique. The TAL technique in combination with new culture media resulted in very rapid protoplast development and fast shoot regeneration (in less than two weeks). This method was also successfully applied to improve protoplast culture of rapeseed and of the extremely recalcitrant species sugarbeet. Factors, which included protoplast source, mineral and organic composition of isolation and culture media, influence of growth regulators etc. were investigated and conditions for protoplast culture and regeneration were established for both species. According to reports in the literature, only protoplasts from guard cells could be regenerated into plants. Thus, an alternative and reproducible method of shoot regeneration from protoplasts isolated from hypocotyl derived callus was successfully developed. While no shoot regeneration was observed from guard cell protoplasts in our experiments, plant regeneration (efficiencies up to 30%) from callus protoplasts could be achieved for the first time in this study. The influence of different parameters on the efficiency of callus formation from etiolated hypocotyl explants was investigated. Protoplasts from callus and hypocotyl derived callus were used for the experiments on nuclear transformation in sugarbeet. Both, the PEG method and the biolistic method were successfully applied to obtain nuclear transformants as confirmed by molecular methods (PCR analysis and Southern blot hybridisation). The biolistic method was applied for plastid transformation experiments in sugarbeet. Species specific vectors containing the aadA cassette were constructed for plastid transformation in rapeseed and sugarbeet. However, difficulties to select plastid transformants were observed due to a high natural resistance to spectinomycin and streptomycin in rapeseed. In sugarbeet spectinomycin at a concentration of 100 mg/l was found efficient for selection and spectinomycin and streptomycin resistant colonies were obtained after callus bombardment. The presence of the aadA gene in antibiotic-resistant lines was proven by PCR analysis, but an integration of DNA into the plastome could not be verified so far. Efficient regeneration systems and methods of DNA transfer were established for rapeseed and sugarbeet and straightened the way for successful plastid transformation in either species