Transgene integration, expression and inheritance in genetically engineered oats.

摘要

Integration, expression and inheritance of two marker transgenes, bar and uidA, delivered by microprojectile bombardment were studied in transgenic oat tissue cultures and regenerated plants. Presence of at least one intact copy of one or both of the transgenes was detected in 100 of 102 transgenic tissue cultures and 22 of 23 transgenic plant lines that were examined by Southern blot analyses. Intact transgene copies were often accompanied by multiple rearranged and/or truncated transgene fragments. The number of integrated bar copies was often different from the number of uidA copies present in the same line suggesting that the transforming plasmid carrying the two transgenes underwent extensive fragmentation. All fragments of the transgenic DNA always cosegregated in the progeny of transgenic plants indicating that they were integrated at just one genetic locus. Transgenic loci in the majority of analyzed lines showed fragments of transgenic DNA interspersed with host plant DNA. We hypothesize that integration of transgenes during the DNA replication process could account for the observed discontinuity of transgenic loci.; Analysis of inheritance of transgenic phenotype through up to four generations of progeny of transgenic plants showed Mendelian single locus segregation in 11 of 23 tested transgenic lines. Segregation distortions, indicated by a lower than expected number of individuals showing expression of the transgenic phenotype, were detected in the remaining lines. Coexpression of the phenotypes encoded by the uidA and bar transgenes was found in 12 of 24 examined lines while plants that expressed one of the transgenes, but failed to express the other, were detected in the remaining lines. Transgene silencing was found in 20 transgenic lines and was the primary cause of the segregation distortions and lack of transgene coexpression.; Analysis of four transgenic lines, which produced only transgene-positive plants in their progenies, suggested that the primary transgenic plants in these lines were homozygous for the transgenic locus. While it is not entirely clear how homozygosity could arise in transgenic tissue cultures or plants, we suggest that a genetic recombination event such as gene conversion or mitotic crossing-over was the most likely cause for creating homozygous transgene loci.