Molecular analysis of transgenic rice var. Chinsurah Boro 2
1996
Oliva, N. | Vasquez, A. | Datta, K. | Muthu-Krisnan, S. | Datta, S. (International Rice Research Inst., P.O. Box 933, 1099 Manila (Philippines). Plant Breeding, Genetics and Biochemistry Div.)
First generation (T1) progenies of Chinsurah boro 2 transformed with plasmid containing both hpt and chitinase genes (pGL2-CH2) were analyzed using enzyme assays, Southern and Western blotting. Using hygromycin phosphotransferase (HPT) assay, seven T1 families were found to fit the 3 positive:1 negative Mendelian ratio suggesting that the parental (To) plants were heterozygous and the expression of the hpt gene is controlled by a single dominant gene. The presence of the hpt gene was also determined using Southern blot analysis. In the five T1 families analyzed, all plants found to have hygromycin phosphotransferase activity by enzyme assays were confirmed to have the gene integrated into the plant genome by blot analysis. It was also observed that the gene integrated into one to three locations into the genome. Western blot analysis of extracts of uninfected transgenic plants with a chitinase antibody revealed the presence of one immunologically-related with an apparent molecular weight of 35 kDa in plants from two families, SKD 354 and SKD 211. This suggests that both the chitinase and hpt genes are present and expressed in the T1 generation. Biological assay from T1 and T2 generations showed encouraging data indicating at least the two transgenic lines are moderately resistant to sheath blight. Transformation was also undertaken using plasmid containing either bt toxin, rice ragged stunt virus coat protein, and chitinase gene/s with hpt as selectable marker gene. Initial HPT assay result showed that 48 out of 51 plants were positive for the expression of the hpt gene
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