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Development of a strategy for transgenic studies and monitoring of transgene expression in two closely related Moricandia species possessing a C 3 or C 3 –C 4 intermediate photosynthetic phenotype
Author(s) -
Thole Vera,
Rawsthorne Stephen
Publication year - 2003
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.2003.00135.x
Subject(s) - biology , transformation (genetics) , transgene , cauliflower mosaic virus , gene , population , reporter gene , botany , genetically modified crops , genetics , microbiology and biotechnology , gene expression , demography , sociology
In order to establish a model system for comparative studies of C 3 and C 3 –C 4 intermediate photosynthesis, the development of efficient transformation systems and the monitoring of transgene behaviour and stability were carried out in two closely related Moricandia species ( Brassicaceae ): the C 3 –C 4 photosynthetic intermediate species M. arvensis and the C 3 species M. moricandioides . In this study the green fluorescent protein ( gfp ) reporter gene was used as a vital marker gene while the use of the β ‐glucuronidase ( gusA ) gene was based on the highly sensitive detection of its activity. For Agrobacterium ‐mediated transformation of leaf explants, a cauliflower mosaic virus 35S promoter‐driven, modified version of gfp , the mgfp5‐ER gene and the gusA gene, respectively, were introduced into the new dual binary transformation vector system pGreen/pSoup (Hellens et al. 2000, Plant Mol Bio 42: 819–832). GFP5 produced bright‐green fluorescence in transformed tissues that was distinctly detected 5–12 days following transformation in developing calli of the two species. Visual screening, combined with antibiotic selection, enabled early and easy identification of transformation events and contributed to improvements in the transformation strategies. Transgene integration studies demonstrated that mgfp5‐ER was inserted with low copy number in the M. arvensis plant lines and the transgene was transmitted in a Mendelian fashion to T 1 and T 2 progenies. GFP5 expression levels in a population of 100 independent primary transformed M. arvensis plant lines (T 0 ) showed great variation between transformation events (coefficient of variation of 108%). The mgfp5‐ER or gusA reporter genes were expressed in 90–95% of the kanamycin‐resistant M. arvensis plant lines and in up to 98% of the independent M. moricandioides plant lines.