REGENERATION OF TRANSGENIC PLANTS OF MEXICAN LIME FROM AGROBACTERIUM-RHIZOGENES TRANSFORMED TISSUES

Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vecto r pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were gene rated by two different approaches. The first approach used internodal stem segments cocultured with A. rhizogenes. These were placed onto re generation medium containing Murashige and Skoog salts and B5 organic compounds supplemented with 8g . 1(-1) agar, 7.5 mg . l(-1) 6-benzylam inopurine, 1.0 mg . l(-1) -naphthaleneacetic acid, 300 mg . l(-1) cefo taxime and 80 mg . l(-1) kanamycin as a selective agent, and incubated under continuous light at 25 degrees C. Under these conditions, 76% o f the explants produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were geneti cally transformed as determined by beta-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtai ned from internodal stem segments cocultured with A. rhizogenes were c ultured on the above regeneration medium under similar conditions. For ty-one percent of these transformed root segments produced adventitiou s shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident in the transformed roots and in all parts of both transformed shoots and regenerated plants. The pres ence of the npt II and rolB genes in the regenerated plants was confir med by PCR analysis. The pres ence of the npt II gene in the regenerat ed plants was also confirmed by Southern blot. Using these transformat ion systems, more than 300 Mexican lime transgenic plants were obtaine d, 60 of which were adapted to growing in soil.