T-DNA transfer, integration, expression and inheritance in rice: effects of plant genotype and Agrobacterium super-virulence

摘要

Reproducible, efficient Agrobacterium-mediated transformation has been established for cultivars of Indica, Japonica and Javanica rice. Embryogenic calli derived from mature seed scutella were co-cultivated with A. tumefaciens LBA4404 carrying (1) the binary vector pVDH65 [T-DNA encoding #beta#-glucuronidase (gus-intron) and neomycin phosphotransferase genes; strain 0065], (2) pVDH65 and the supervirulent pTOK47 (strain 1065), or (3) the super-virulent binary vector pTOK233 [T-DNA encoding the neomycin phosphotransferase, #beta#-glucuronidase (gus-intron) and hygromycin phosphotransferase genes]. GUS activity was observed in callus following co-cultivation with strains LBA4404(pTOK233) and 1065, but not with strain 0065. Regeneration of phenotypically normal transgenic plants occurred from 12-21%, 16-31%, and 10-19% of transformed tissues of the cultivars Pusa Basmati 1 (Indica rice), Taipei 309 (Japonica rice), and Tinawen (Javanica rice) respectively, following co-cultivation with LBA4404 (pTOK233) and selection on hygromycin-containing medium. Single T-DNA inserts were rare in hygromycin-resistant transformants. However, T-DNA inserts were stably inherited and expressed in T1 seed generation plants of Taipei 309, with transgenes being expressed in a 3:1 ratio in T1 progeny, indicating the presence of active T-DNA at a single locus. Comparison of T2 generation hemizygotes and homozygotes revealed a positive correlation between transgene dosage and GUS activity.