The introduction of multiple genes into the genome at preferably a single locus/chromosome is very important in transgenic breeding especially while engineering for a complete metabolic pathway. Agrobacterium tumefaciens-mediated transformation is of wide preference in the recent days for the simple pattern of integration of the one to very few copies of the transgene(s) in the same chromosomal locus as compared to the complex, multiple and rearranged copies of transgene(s) integration in more than one locus in biolistic method (Baisakh et al. 1999; Tinland et al. 1996). The unavailability of a suitable virulent strain of Agrobacterium with a suitable binary vector is considered a limitation. Herewith, we report on engineering a supervirulent Agrobacterium strain EHA101 that harbors a reconstructed binary vector carrying three genes in the T-DNA region.

Agro-vector pCAMBIA1301 carrying gus (reporter gene) and hph (selectable marker gene) in the T-DNA, and pC822 carrying Xa21 gene for resistance to bacterial blight disease of rice were digested overnight with Kpn I that linearises the former at multiple cloning site (MCS), and releases a 9.6-kb fragment spanning Xa21 from pC822. The linearised pCAMBIA1301 (- 11.837-kb) and Xa21 (9.6-kb) were ligated overnight in a proportion of (1:3). The ligated product i.e., the reconstructed plasmid vector (herein after designated as pTCL5, Fig. 1) was mobilized into competent cells of a supervirulent strain of Agrobacterium (EHA101) by a simple heat-shock treatment. pTCL5 was recovered from the transformed Agrobacterium (EHA101-pTCL5) cells growing under kanamycin (Km) and rifampicin. The integrity and orientation of Xa21 was confirmed by restriction digestion and PCR (data not shown). The functional integrity of pTCL5 was evident from the transient expression of gus gene by the calli bombarded with the plasmid, and the calli of Basmati 370 cocultivated with EHA101-pTCL5.

This engineered Agrobacterium (EHA101-pTCL5) was used for transformation of one- or one and half-month-old calli of an elite indica rice IRRI-bred maintainer line IR68899B used in hybrid rice breeding program. The methods for preparation of Agrobacterium suspension, cocultivation, selection and regeneration of putative transformants were essentially the same as described earlier (Datta et al. 2000). A duplicate set of the putative transgenic calli after 3-4 cycles of selection in hygromycin B was assayed for GUS expression before putting for regeneration.

A number of putative transformants from independently selected GUS-positive calli were obtained. These primary regenerants were screened for T-DNA integration by PCR for the presence of three genes (gus, hph, and Xa21) with three gene-specific set of designed primers. Since a small amount (- 50-100 ng) of DNA is sufficient for the reaction, DNA isolated from 4-5 cm-long leaf excised from the regenerated shoots (while transferring

to growth medium or to the Yoshida's nutrient solution) was used as template. Amplification products of 1.2-kb corresponding to gus gene (Fig. 2A), 0.78- kb for hph (Fig. 2B), and 1.4-kb for Xa21 (Fig. 2C) indicated the stable integration of the

at least one copy of the intact T-DNA in the rice genome. The follow-up molecular analysis for copy number, integration site(s), inheritance pattern and bioassay of the transgenic rice against bacterial blight pathogen will be published elsewhere. We have been successful in transforming other indica genotypes like IR64, IR72, and Basmati 370 with this engineered strain (our unpublished results) and should be able to make the homozygous transgenic rice with agronomically important genes within a year from the start of the transformation deploying anther culture technique (Baisakh et al. 2000).

Acknowledgement:

The work was generously supported by BMZ-GTZ, Germany, and the Rockefeller Foundation, USA. The authors are thankful to Dr. R. Jefferson for pCAMBIA1301, Dr. P. C. Ronald for pC822, and Dr. E. E. Hood for EHA101.

References:

Baisakh, N., K. Datta, N. Oliva and S.K. Datta, 1999. Comparative molecular and phenotypic characterization of transgenic rice with chitinase gene developed through biolistic and Agrobacterium-mediated transformation. Rice Genet. Newslett. 16: 149-152.

Baisakh, N., K. Datta, N. Oliva, G.J.N. Rao and S.K. Datta, 2000. Rapid development of homozygous transgenic rice using anther culture harboring rice chitinase gene for enhanced sheath blight resistance. Plant Biotechnol. (in press).

Datta, K., Z. Koukolikova-Nicola, N. Baisakh, N. Oliva and S.K. Datta, 2000. Agrobacterium-mediated engineering for sheath blight resistance of indica rice cultivars from different ecosystems. Theor. Appl. Genet. 100(6): 832-839.

Tinland, B., 1996. The integration of T-DNA into plant genomes. Trends in Plant Sci. 1(6): 178-184.