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

Reproducible, efficient Agrobacterium-mediated transformation has been established for cultivars of Indica, Japonica and Javanica rice. Embryogenic calli derived from mature seed scutella were cocultivated 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, P-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, I-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.