A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations.
Transgenic locus composition and T-DNA linkage configuration were assessed in a population of rice plants transformed using the dual-binary vector system pGreen (T-DNA containing the bar and gus genes)/pSoup (T-DNA containing the aphIV and gfp genes). Transgene structure, expression and inheritance were analysed in 62 independently transformed plant lines and in around 4,000 progeny plants. The plant lines exhibited a wide variety of transgenic locus number and composition. The most frequent form of integration was where both T-DNAs integrated at the same locus (56% of loci). When single-type T-DNA integration occurred (44% of loci), pGreen T-DNA was preferentially integrated. In around half of the plant lines (52%), the T-DNAs integrated at two independent loci or more. In these plants, both mixed and single-type T-DNA integration often occurred concurrently at different loci during the transformation process. Non-intact T-DNAs were present in 70-78% of the plant lines causing 14-21% of the loci to contain only the mid to right border part of a T-DNA. In 53-66% of the loci, T-DNA integrated with vector backbone sequences. Comparison of transgene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgene inheritance or T-DNA linkage, as only 60-80% of the transgenic loci were detected by the expression study. Co-expression (28% of lines) and backbone transfer (53-66% of loci) were generally a greater limitation to the production of marker-free T(1) plants expressing the gene of interest than co-transformation (71% of lines) and unlinked integration (44% of loci).
Afolabi
A S
AS
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Worland
B
B
Snape
J W
JW
Vain
P
P
D015139:Blotting, Southern; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D008040:Genetic Linkage; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008962:Models, Theoretical; D012275:Oryza / Q000235:genetics*; D010641:Phenotype; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction; D019076:Transgenes / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; C036483:T-DNA; D049452:Green Fluorescent Proteins; D005966:Glucuronidase