Fig. 1. Southern blot analysis of transgenic IR58 plant.
1) Tissue Culture Laboratory, Plant Breeding, Genetics and Biochemistry Division, International Rice Research Institute, P. O. Box 933, Manila, Philippines.
2) Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, U.S.A.
Transgenic rice plants have been successfully produced using protoplasts as the initial recipient host for DNA transformation (Cao et al., 1991). Since indica rices are widely grown, establishment of an efficient reproducible transformation system for indica protoplasts is an important research objective. To date, protocols for the DNA transformation and regeneration have been developed for only a limited number of indica varieties (Hodges et al., 1991). In this report, we describe our procedures for the efficient production of transgenic plants of indica variety,IR58.
Protoplasts were isolated from established suspension cultures of IR58, which were subcultured weekly in R2 medium supplemented with 2mg/l 2, 4-D. After removing the enzyme solution, protoplasts were mixed with 50 Ug plasmids (in 100ul distilled water) for 10 min, followed by dropwise addition of appropriate volume of 40% PEG 8000 MaMg solution to 20% final concentration. After 2O- 30 min of incubation, PEG was removed by centrifugation. Protoplast culture and plant regeneration was obtained following the procedures described by Wu and Zapata (1992).
Three gene constructs were used in our experiments: pCallGc (courtesy of V. Walbot through R. Nelson, Plant Pathology, IRRI) carrying a GUS reporter gene, pTRA132 carrying a hygromycin (HPH) resistant gene (courtesy of N. Mural, Louisiana State University), and PKAN (courtesy of T. Hodges, Purdue University) containing a G418-resistant gene NPTII. These three gene constructs were driven by the CaMV35S promoter. Protoplasts were cotransformed with GUS and HPH or GUS and NPTII. Selection was performed with 12-14 day-old pro-
Fig. 1. Southern blot analysis of transgenic IR58 plant.
toplast colonies at 100 mg/l hygromycin B or 20 mg/l G418 for 25 days. In order to determine whether the employed transformation protocol was successful, colonies were stained with X-gluc before selection. Histochemical analysis of transgenic plants was conducted on various tissues such as roots, leaves, stems, leaf sheaths and spikelets. For Southern blot analysis, DNA was isolated from leaves of one transformed plant of IR58.
The results of several experiments showed 3-21% of the unselected colonies stained blue. Hygromycin- or kanamycin-resistant colonies were also stained and 38-58% co-transformation efficiencies were obtained. Blue color was also observed in root tips and in other organs of the transgenic plants after incubation in X-gluc solution.
Using the 1.8Kb GUS fragment of pCallGc as a probe, Southern blot analysis also confirmed the presence of GUS gene in the high molecular weight region, and in the 1.8Kb band corresponding to the intact GUS gene coding sequence (Figure 1). These results show that our protocol for obtaining transgenic plants of IR58 is very efficient.
References
Cao J., W. Zhang, D. McElroy and R. Wu. 1991. Assessment of rice genetic transformation techniques. Pages 175-198 in Rice Biotechnology, G.S. Khush and G.H. Toenniessen, eds. Int. Rice Res. Inst. and C.A.B. International.
Hodges, T.K., J. Peng, L.A. Lyznik and D.S. Koetje. 1991. Transformation and regeneration of rice protoplasts. Pages 157-174 in Rice Biotechnology, G.S. Khush and G.H. Toenniessen, eds. Int. Rice Res. Inst. and C.A.B. International.
Wu, C. and F.J. Zapata. 1992. Plant regeneration from protoplasts isolated from primary callus of four japonica rice (Oryza sativa L.) varieties. Plant Science 86: 83-87.