Plant Tissue Cult. 7(2) : 71-80, 1997 (December)

Transformation of an Indica Rice Cultivar Binnatoa with Agrobacterium tumefaciens

Noorain M. Rasul, K. Mohammad Ali, M. Rafiqul Islam and Zeba I. Seraj¹

Department of Biochemistry, University of Dhaka, Dhaka-1000, Bangladesh

Key words: Transformation, Indica rice, Binnatoa, Agrobacterium

Abastract

Transformation of 7-14-days-old scutellum-derived calluses from mature embryos of a highly regenerating indica rice cultivar, Binnatoa, using Agrobacterium tumefaciens LBA4404 harbouring the super virulent vector pTOK233 is reported. The plasmid pTOK233 contains the hygromycin resistant gene (hygR) as selectable marker and ?-glucuronidase gene (GUS A containing as intron) as a screenable marker. Callus induction and regeneration media supporting high regeneration frequency was used for transformation and selection with the addition of 30 mg/1 hygromycin. Although selection of transformation callus appeared to take place within two weeks after Agrobacterium infection, micro-calluses from the sides of brown calluses were subcultured at least once to ensure proper screening of transformants. After 4-6 weeks in selection, 25% infected calluses continued to grow in hygromycin. Embryogenic calluses were transferred to regeneration medium and subsequently to rooting medium containing hygromycin. Only 4% of regenerated plantlets survived in selective rooting medium, indicating a high per cent of transformation escapes in initially selected calluses. Integration of T-DNA was confirmed by the presence of a hygR-specific band only in Hind III-digested transformed plant DNA following Southern hybridization.

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