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Rapid and proven production of transplastomic tobaccoplants by restoration of pigmentation and photosynthesis Sebastian M. J. Klaus, Fong-Chin Huang, Christian Eibl, Hans-Ulrich Koopà and Timothy J. GoldsICON Genetics AG, Research Centre Freising, Lise-Meitner-Str. 30, 85354 Freising, Germany Received 9 May 2003; revised 5 June 2003; accepted 13 June 2003.
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Tobacco chloroplast transformation is typically achieved using dominant, selectable antibiotic resistancegenes such as aadA, nptII and aphA-6. An improvement would be the combination of such a marker with avisual screening system for the early and conclusive detection of plastid transformants. As such, we inves-tigated the use of three photosynthesis-deficient plastid mutants, DpetA, Dycf3 and DrpoA, for thedevelopment of a phenotypic selection system. Mutant plants were used as an alternative to the wild-type as source tissue for transformation, re-introducing deleted plastid sequences and using the aphA-6gene as a selection marker. The reconstitution of the deleted genes in transformed regenerants resulted inshoots with a visually distinct phenotype comparable to the wild-type. This transformation/selectionsystem overcomes the common problems associated with plastid transformation, e.g. the recovery ofspontaneous mutants or nuclear insertions. In addition to the benefits offered by phenotypic selection,phenotype reconstitution leads to restoration of photosynthesis, which we assume drives reconstitutedplants rapidly towards homoplasmy. As such, repeated cycles of regeneration in the presence of anantibiotic selection agent are no longer required.
Keywords: tobacco plastid transformation, chloroplast mutants, antibiotic selection, petA, ycf3, rpoA.
Compared to conventional nuclear transformation, the advantages of expressing genes in plastids are numerous (Kavanagh et al., 1994). In tobacco, vectors containing seg- and have been extensively reviewed (Bock and Hagemann, ments of tobacco plastid DNA carrying specific point muta- 2000; Daniell et al., 2002; Maliga, 2002). To date, four domi- tions in the 16S rRNA gene (giving resistance to nant selectable markers have been described for plastid spectinomycin and streptomycin) were initially used to transformation in higher plants: the aadA gene (aminogly- demonstrate the principle of plastid transformation in coside adenyltransferase) detoxifying spectinomycin and tobacco (Svab et al., 1990). However, in this pioneering streptomycin (Svab and Maliga, 1993), the nptII and aphA-6 work, the transformation frequencies were extremely genes (aminoglycoside phosphotransferases) detoxifying low; from a total of 62 spectinomycin-resistant cell lines, kanamycin (Carrer et al., 1993; Huang et al., 2002) and the only three were shown to be plastid transformants, the rest badh gene (betaine aldehyde dehydrogenase) in combina- were spontaneous mutants. The recovery of spectinomy- tion with betaine aldehyde as a selection agent (Daniell cin-resistant mutants also remains an issue where the aadA et al., 2001). A major disadvantage of such selection sys- gene is used as a plastid marker. During the last decade, the tems is that they can produce ‘false positives’ (a term used aadA gene has been used for the production of plastid throughout this paper to describe all selected regenerants, transformants in tobacco (Svab and Maliga, 1993) and other which are not true plastid transformants). Random point species like Arabidopsis (Sikdar et al., 1998), potato (Sidorov mutations within plastid-encoded genes can result in the et al., 1999) and tomato (Ruf et al., 2001). All of these insensitivity to some antibiotics. Such a phenomenon reports describe the recovery of false positives because has been documented for the antibiotics spectinomycin, of the occurrence of spontaneous spectinomycin-resistant

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