Genetic engineering for bacterial blight resistance in maintainer lines for hybrid rice breeding

The optimal response of six maintainer lines (IR58025B, IR62829B, PR3B, IR69627B and 913B) to tissue culture was determined by using dehulled manure seeds, three callus induction, and three plant regeneration media. The test genotypes exhibited differential response to the media used. MNK and MSCI media showed similar callus inducing ability, which is better than RS medium. Unlike in callus induction, the test genotypes exhibited very strong differential response to plant regeneration media. IR58025B and PR3B were identified for transformation with the Xa21 gene for bacterial blight resistance based on good tissue culture response and current status in hybrid rice breeding. Plasmid pB822 containing Xa21 gene, and plasmid pB1222 containing the hpt selectable gene for hygromycin resistance were co-bombarded to these B lines using the particle gun. A total of 185 regenerants IR58025B and 134 PR3B that putatively contain the Xa21 gene was produced. PCR analysis revealed the presence of the 1.4 kb xa21 specific amplicon together with the 1.3 kb non-specific amplicon in many of the regenerants. Twenty six out of an initial sample of 36 IR58025B regenerants contained the Xa21 target gene,giving a transformation efficiency of 72.2%. For PR3B, 21 out of 26 regenerants tested for PCR produced the 1.4 kb band and gave a transformation efficiency of 80.7%. Regenerants were evaluated by inoculation with Xoo race 3 through the clipping method. Preliminary result indicated that the non-transformed control plants of the two genotypes showed high susceptibility to the pathogen. Susceptibility was comparable to the susceptible check, IR24. The primary transformation of the two genotypes showed varying reactions to BB inoculation, ranging from moderate resistance to susceptibility. One plant was identified to be resistant out of the 26 randomly selected PR3B primary transformants. In contrast, three out of the 36 randomly selected IR58025B primary transformants had intermediate reaction to bacterial blight. Some of the transgenic plants showed greater resistance to the pathogen as compared to IRBB21. However, some PCR-positive transgenic plants showed high percentage of DLA, suggesting that the Xa21 transgene was not expressed or it mutated. Interestingly, some PCR-negative plants had relative low percentage of DLA. Further evaluation on the transformation is recommended. This should be done in terms of inheritance of the transgene, reaction to BB, and their ability to restore the corresponding A lines.