• Rgn13

Agrobacterium-mediated gene transfer to higher plants is a standard technique for genetic engineering of dicots. However, this technique has been employed for transformation of monocots such as rice (Hiei et al. 1994, Rashid et al. 1996) and maize (Ishida et al. 1996) only recently. Transformation efficiency is dependent on the tissue culture regenerability of rice cultivars (unpublished data). Considering the in vitro response, this technique was initially employed to transform a tissue culture responsive

Agrobacterium strain LBA4404 (pTOK233) which contains virB, virC and virG from Tl-plasmid pTiB0542, intron-gus, a hygromycin-resistance gene (hph) and a gene for kanamycin resistance, was used for rice transformation (Hiei et al. 1994). Immature embryos, scutella derived primary embryogenic calli (EC) and embryogenic cell suspension (ECS) were used as explants. Protocols for preparation of EC and ECS were the same as described by Datta et al. (1992). However, the medium was supplemented with Acetosyringone (200M) and the cultures after co-cultivation were maintained at 26-28°C. Co-cultivation was done following the protocol of Hiei et al. (1994). The OD2 of bacterial density was found suitable for efficient transformation.

Actively dividing primary EC were found most suitable for transient and stable gene expression followed by ECS and immature embryos. Hg^r calli and plants were tested for GUS expression. Many tissues showed blue staining to variable degrees. However, some chimeric expression was observed. Several HFP (hygromycin phos-photrasferase) plants did not show GUS expression and three GUS* plants did not express HPT (Table 1, Fig. 1). Such chimerism in Agrobacterium mediated gene transfer has also been reported earlier (Schmulling and Schell 1993). The data on HPT assay (based on Datta et al. 1990) clearly demonstrated that hph gene not only integrated but was also functioning (Fig. 1). Enzyme activity was absent in control and lanes 3,4. Besides the other advantages like transfer of larger segments of DNA with little rearrangement, this technique also allows integration of low numbers of gene copies into plant chromosomes, is cheaper than other methods. Additionally the recovery of transgenic plants is faster. Our first batch of transgenic rice plants are now growing in

IE = immature embryo. ND = not done.

Fig. 1. Expression of HPT activity in extracts of transgenic rice plants mediated by Agrobacterium tumefaciens: lanes:l-2 transgenic IR51500; 3-4 gus* Basmati 122, 4-7 transgenic T309; C, 8 non transformed control and transformed positve control respectively.

It is clear that genetic transformation of indica rice is possible by Agrobacterium mediated gene transfer,

We gratefully acknowledge the financial support from Deutsche Gesellschaft fur Technische Zusammenarbeit, Germany, under the Asian Rice Biotechnology Network and the Rockefeller Foundation, New York. We also thank Dr. T. Komari and Dr. T. Kumashiro of Japan Tobacco Co., Japan for providing the Agro-strain used in this experiment.

References 

Datta, K., 1. Potrykus and S.K. Datta, 1992. Efficient plant regeneration from protoplasts of the Indica rice

Rashid, H., S. Yokoi, K. Toriyama and K. Hinata, 1996. Transgenic plant production meditaed by

• Cite Gramene
• Feedback
• About
• Site Map
• Home

 

SPECIES: