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b'In the current study tissue cultures of rapeseed (cv. \\u201cDrakkar\\u201d, cv. \\u201cWestar\\u201d) and sugarbeet (cv.\\n\\u201dViktoria\\u201d, cv. \\u201cVRB\\u201d, cv. \\u201d31-188\\u201d, cv. \\u201d7T1308\\u201d and 47 other breeding lines, Appendix 1)\\nhave been investigated for the establishment of conditions that make possible plastid\\ntransformation in both species. Tobacco leaf protoplasts (cv. \\u201dpetite Havana\\u201d, cv. \\u201dWisconsin\\n38\\u201d) were used to develop a novel technique \\u2013 the TAL (thin-alginate-layers) technique. The\\nTAL technique in combination with new culture media resulted in very rapid protoplast\\ndevelopment and fast shoot regeneration (in less than two weeks). This method was also\\nsuccessfully applied to improve protoplast culture of rapeseed and of the extremely recalcitrant\\nspecies sugarbeet. Factors, which included protoplast source, mineral and organic composition of\\nisolation and culture media, influence of growth regulators etc. were investigated and conditions\\nfor protoplast culture and regeneration were established for both species.\\nAccording to reports in the literature, only protoplasts from guard cells could be regenerated into\\nplants. Thus, an alternative and reproducible method of shoot regeneration from protoplasts\\nisolated from hypocotyl derived callus was successfully developed. While no shoot regeneration\\nwas observed from guard cell protoplasts in our experiments, plant regeneration (efficiencies up\\nto 30%) from callus protoplasts could be achieved for the first time in this study.\\nThe influence of different parameters on the efficiency of callus formation from etiolated\\nhypocotyl explants was investigated. Protoplasts from callus and hypocotyl derived callus were\\nused for the experiments on nuclear transformation in sugarbeet. Both, the PEG method and the\\nbiolistic method were successfully applied to obtain nuclear transformants as confirmed by\\nmolecular methods (PCR analysis and Southern blot hybridisation). The biolistic method was\\napplied for plastid transformation experiments in sugarbeet.\\nSpecies specific vectors containing the aadA cassette were constructed for plastid transformation\\nin rapeseed and sugarbeet. However, difficulties to select plastid transformants were observed\\ndue to a high natural resistance to spectinomycin and streptomycin in rapeseed. In sugarbeet\\nspectinomycin at a concentration of 100 mg/l was found efficient for selection and\\nspectinomycin and streptomycin resistant colonies were obtained after callus bombardment. The\\npresence of the aadA gene in antibiotic-resistant lines was proven by PCR analysis, but an\\nintegration of DNA into the plastome could not be verified so far. Efficient regeneration systems\\nand methods of DNA transfer were established for rapeseed and sugarbeet and straightened the\\nway for successful plastid transformation in either species.'