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GrainGenes Reference Report: MBR-11-203

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Reference	MBR-11-203
Title	Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.)
Journal	Molecular Breeding
Year	2003
Volume	11
Pages	203-211
Author	Popelka JC Altpeter F
Abstract	Summary: A system for the genetic transformation of rye by co-cultivation with Agrobacterium tumefaciens is described. A total of 45 independent transgenic plants were regenerated with a transformation efficiency of 1 to 4% of the inoculated explants. The co-cultivation of Agrobacterium-strain AGL(0), harboring plasmid pJFnptII and rye immature embryos in liquid medium allowed a high throughput and facilitated washing of the cultures to avoid Agrobacterium overgrowth. Transgenic plants were phenotypically normal and fully fertile, which might be a consequence of the short time in tissue culture. The selection with paromomycin exclusively during the regeneration allowed the efficient recovery of transgenic events without interfering with somatic embryogenesis. Southern blot analysis confirmed the independent nature of the analyzed plants and indicated single copy inserts in more than 50% of them. Segregation analysis confirmed single locus integration and stable transgene expression in most of the lines, while one line with multiple locus integration was also observed. The analysis of T-DNA::plant DNA boundary sequences revealed examples of exclusion of vector sequences, deletion of a few bases of the T-DNA or insertion of up to 29 bases of the vector backbone. This stresses the importance of detailed analysis of the inserted transgenes in order to identify events with the desired integration profile
Keyword	[ Hide all but 1 of 52 ] agrobacterium agrobacterium tumefaciens agrobacterium-mediated gene transfer base cereale l cocultivation culture dna efficient efficient transformation embryo embryogenesis explants fertile genetic transformation genetic-transformation high-throughput immature embryo inbred line insertion integration locus media molecular-weight dna multiple nptii oryza sativa plant transformation plasmid rapid production regeneration rice rye rye (secale cereale l.) secale secale cereale segregation selection sequence sequence analysis single-copy somatic embryogenesis stress system t-dna boundaries tissue transformation transgene transgene expression transgenes transgenic vector

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