Regeneration and plastid transformation approaches in Arabidopsis thaliana and Rapid-Cycling Brassica rapa

5 2 Summary Plastid transformation is a well-established tool for biotechnology as it allows high expression levels of proteins and as it provides biological transgene containment because of maternal inheritance of cytoplasmic genes in most crops. In basic research the function and regulation of plastid encoded genes can be further studied with the help of plastid transformation. This study is focused on the three most critical steps to achieve plastid transformation in Arabidopsis thaliana and Rapid-Cycling Brassica rapa (RCBr), the establishment of a regeneration protocol, the construction of species specific vectors and the use of different transformation protocols. First, to allow the generation of fertile plants out of transformed cells, a reliable regeneration protocol was established. Fertile plants of Arabidopsis thaliana were obtained with high regeneration efficiencies from cotyledons, seed derived calli and protoplasts. In RCBr, fertile plants could be regenerated from tissue culture of hypocotyls. For introduction of genes into plastids, appropriate vectors with different selection and visual markers were constructed. These ensure site specific integration of desired sequences and enable selection of transformed cell lines. In this study, species specific vectors for Nicotiana tabacum, Arabidopsis thaliana and RCBr were designed and cloned, using aminoglycoside resistance markers such as the aadA gene (confers resistance to spectinomycin and streptinomycin), the nptII or the aphA6 gene (conferring resistance to kanamycin). To allow the selection through the herbicides phosphinothricin or bialaphos, the bar gene was employed. In addition, fluorescence markers such as GFP, DsRed and AmCyan were taken as visual markers. After the establishment of a regeneration protocol and the construction of specific vectors, two different protocols for plastid transformation, the particle gun-or PEG (Polyethylene glycol)-mediated transformation method, were applied. However, no transformed plant in Arabidopsis thaliana and RCBr could be Summary 6 recovered so far. That the transformation protocol does work in principal could be shown by the use of Nicotiana tabacum. Plastids of Nicotiana tabacum were successfully transformed using both transformation methods and AmCyan as new visual marker. Out of these experiments, fertile homoplasmic tobacco plants could be obtained as was shown by Southern blot analysis and reciprocal crosses. Thus, the functionality of the used transgene expression cassettes was clearly proven.