High efficiency transformation ofBrassica napus usingAgrobacterium vectors
2011
maurice m. moloney | janis m. walker | kiran k. sharma
Maurice M. Moloney, Janis M. Walker, Kiran K. Sharma, 'High efficiency transformation ofBrassica napus usingAgrobacterium vectors', Plant Cell Reports, vol. 8(4), pp.238-242, Springer Nature, 2011
显示更多 [+] 显示较少 [-]An efficient procedure for obtaining transgenic B. napus rape plants using Agrobacterium binary vectors is described. The target tissue for the transformation is the cut end of cotyledonary petioles. These tissues, when cultured with their lamina intact, show a regeneration frequency of more than 80%. The cells of this cut surface, which undergo organogenesis, are very susceptible to topical infection by Agrobacterium. The cocultivation method used does not require feeder layers or use of exogenously applied promoters of virulence. After 72 h of infection with Agrobacterium containing a binary plasmid encoding NPT II (neomycin phosphotransferase) the explants were transferred to selective regeneration medium. Using kanamycin (15 µg/cm³) for selection, transgenic plantlets emerged within 3 weeks. These plantlets, which appeared on over half the explants, were excised and rooted for a further 7-10 days. When the plants were large enough, leaves were taken for assay of NPT II activity using dot blots. Most of the plants surviving the selection showed substantial NPT II activity. The frequency of transformation and yield of transgenic plants was higher than in previously reported methods with this species. Southern blotting revealed that integration of the T-DNA frequently occurred in multiple copies and at multiple loci in the genome. The transgenic B. napus plants all grew normally and developed fertile flowers. They were self-pollinated and their progeny studied a single embryo NPT II assay performed on developing seeds and a leaf bleaching assay performed by selection of germinating seedlings of the selfed progeny. Both assays yielded segregation ratios consistent with the number of integration events indicated by Southern blots
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