Genetic Transformation and Hybridization

Plant Cell Reports

, Volume 26, Issue 10, pp 1733-1744

Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment

  • Cheng-Wei LiuAffiliated withDepartment of Post-Modern Agriculture, Ming Dao University Email author 
  • , Chin-Chung Lin Affiliated withInstitutes of Biomedical Sciences and Molecular Biology, National Chung-Hsing University
  • , Jeremy J. W. ChenAffiliated withInstitutes of Biomedical Sciences and Molecular Biology, National Chung-Hsing University
  • , Menq-Jiau TsengAffiliated withDepartment of Horticulture, National Chung-Hsing University Email author 

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Abstract

The objectives of this research were first to isolate plastid gene sequences from cabbage (Brassica oleracea L. var. capitata L.), and to establish the chloroplast transformation technology of Brassica. A universal transformation vector (pASCC201) for Brassica chloroplast was constructed with trnVrrn16S (left) and trnItrnArrn23S (right) of the IRA region as a recombination site for the transformed gene. In transforming plasmid pASCC201, a chimeric aadA gene was cloned between the rrn16S and rrn23S plastid gene borders. Expression of aadA confers resistance to spectinomycin and streptomycin antibiotics. The uidA gene was also inserted into the pASCC201 and transferred into the leaf cells of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by 200 mg/l spectinomycin and streptomycin. After antibiotic selection, the regeneration percentage of the two cabbage cultivars was about 2.7–3.3%. The results of PCR testing and Southern blot analysis confirmed that the uidA and aadA genes were present in the chloroplast genome via homologously recombined. Northern blot hybridizations, immunoblotting and GUS histochemical assays indicated that the uidA gene were stable integrated into the chloroplast genome. Foreign protein was accumulated at 3.2–5.2% of the total soluble protein in transgenic mature leaves. These results suggest that the expression of a variety of foreign genes in the chloroplast genome will be a powerful tool for use in future studies.

Keywords

aadA gene Bombardment Cabbage Chloroplast transformation uidA gene