Full potential of any hybrid can be exploited by ensuring the supply of genetically pure seeds. Conventionally, hybrid seed purity assessment is done through grow out test (GOT) which is based on the morphological and floral characters of plants grown to maturity. Being land and labor intensive, time consuming and influenced by the environment, there is an immense need to replace GOT with a simple, rapid, unbiased and cost-effective DNA based assay for hybrid purity assessment. With this objective, the parental lines of three commercial safflower hybrids of India (NH-1, NH-15 and DSH-129) were screened using 74 safflower EST-SSR markers and five markers were found to be polymorphic. A PCRbased assay with these markers showed both alleles of the parental lines in pure hybrids proving the heterozygosity, while the off-types were identified by the presence of either of the parental alleles. This assay could accurately determine the genetic purity in a predetermined sample of hybrids constituted by deliberately mixing seeds of parental lines. This is the first report demonstrating the utility of EST-SSR markers for the assessment of genetic purity of hybrids in crop plants.

Nuclear-mitochondrial gene interactions governing cytoplasmic male sterility (CMS) in angiosperms have been found to be unique to each system. Fertility restoration of three diverse alloplasmic CMS lines of Brassica juncea by a line carrying the fertility-restorer gene introgressed from Moricandia arvensis prompted this investigation to examine the molecular basis of CMS in these lines. Since previous studies had found altered atpA transcription associated with CMS in these lines, the atpA genes and transcripts of CMS, fertility-restored, and euplasmic lines were cloned and compared. atpA coding and downstream sequences were conserved among CMS and euplasmic lines but major differences were found in the 5' flanking sequences of atpA. A unique open reading frame (ORF), orf108, co-transcribed with atpA, was found in male sterile flowers of CMS lines carrying mitochondrial genomes of Diplotaxis berthautii, D. catholica, or D. erucoides. In presence of the restorer gene, the bicistronic orf108-atpA transcript was cleaved within orf108 to yield a monocistronic atpA transcript. Transgenic expression of orf108 with anther-specific Atprx18 promoter in Arabidopsis thaliana gave 50% pollen sterility, indicating that Orf108 is lethal at the gametophytic stage. Further, lack of transmission of orf108 to the progeny showed for the first time that mitochondrial ORFs could also cause female sterility. orf108 was found to be widely distributed among wild relatives of Brassica, indicating its ancient origin. This is the first report that shows that CMS lines of different origin and morphology could share common molecular basis. The gametic lethality of Orf108 offers a novel opportunity for transgene containment.

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Oil content is an important yield trait in Brassica juncea (L.) Coss. Improvements to yield levels of mustard by conventional breeding methods have reached a plateau. The application of transgenic technology is an area that has not yet been explored for improving the oil content of B. juncea. In this study, the effect of overexpression of AtDGAT1 (a key gene involved in oil biosynthesis) on the seed oil content of B. juncea was investigated. For seed-specific overexpression, the gene was linked to Arabidopsis thaliana oleosin promoter and mobilized into mustard through Agrobacterium-mediated transformation. Transformants were selected on MS medium containing 50 mg/L kanamycin, and a transformation frequency of 10.5% was obtained. A total of 10 transgenic events were generated. Analyses of seed weight, oil content, and other yield traits in T1 transgenics showed that seed-specific overexpression of AtDGAT1 significantly improved the oil content and seed weight. The maximum oil content increase observed in the transgenic seeds was 8.3% compared with the wild-type plants. Total fatty acid content was increased from 4% to 14% in six of the seven events. However, the content of oleic and linoleic acid was reduced and, of these two, oleic acid content showed drastic reduction.