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.