Intron hairpin and transitive RNAi mediated silencing of orfH522 transcripts restores male fertility in transgenic male sterile tobacco plants expressing orfH522

The present work was aimed at developing vector construct(s) suitable for restoring fertility in transgenic male sterile tobacco plants expressing male-sterility-inducing ORFH522 in tapetal cell layer (Nizampatnam et al. Planta 229:987–1001, 2009 ). PTGS vectors that could produce either intron spliced hairpin RNA against the orf H522 or induce silencing of orf H522 by heterologous 3′UTR region were developed using the selected 316 bp ( orf 316) fragment of orf H522. The constructs were independently mobilized into Agrobacterium and used for transforming tobacco. The T 1 generation plants carrying the restorer gene cassettes in homozygous condition were identified and crossed with the male sterile transgenic tobacco plants to obtain the hybrid seeds. PCR analysis of hybrid plants indicated segregation for the sterility inducing cassette while all the plants carried the restorer cassette. Hybrid plants produced fertile pollen grains and formed normal capsules upon selfing. Further molecular analyses of these hybrid plants with RT–PCR, Northern blotting and siRNA detection, revealed that intron interrupted hairpin RNA (ihp-RNA) mediated gene silencing was more effective compared to silencing by heterologous 3′UTR (SHUTR) as indicated by the complete degradation of orf H522 transcripts and formation of higher levels of orf 316 specific siRNA molecules in plants carrying ihp-RNA restorer construct. Segregation analyses of F 2 (selfed hybrid) plants confirmed the co-segregation of gene cassettes and the traits in Mendelian di-hybrid ratio (9:3:3:1). Taken together, the results established that intron hairpin and transitive RNAi mediated silencing of orf H522 transcripts restored fertility in transgenic male sterile tobacco plants expressing orf H522 and ihp-RNA was more efficient in silencing orf H522 transcripts.