GrainGenes Reference Report: MBR-11-203
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
agrobacterium
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GrainGenes Reference Report: MBR-11-203
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GrainGenes is a product of the Agricultural Research Service of the US Department of Agriculture. | |||