Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue
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A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.
KeywordsAgrobacterium tumefaciens Cryptomeria japonica Green fluorescent protein Somatic embryogenesis Transgenic plant
The authors are grateful to Dr. Y. Niwa for providing the sGFP(S65T) gene; Dr. P. Quail for permission to use the maize ubiquitin promoter; Dr. C. Koncz for providing the disarmed A. tumefaciens strain, C58/pMP90; and Ms T. Okuyama, Ms K. Isami and Ms K. Sato for their laboratory assistance.
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