Improved protocol for efficacious in vitro androgenesis and development of doubled haploids in temperate japonica rice.

DH (Doubled haploid) is the immortal mapping population and an outcome of single meiotic cycle, contributed from male partner. An improved procedure was developed for high frequency androgenesis in japonica genotypes, K-332 and GS-88 and their F1s. A total of 207 fertile, green, di-haploid plants were generated from K-332 × GS-88 hybrids using the improved anther culture protocol. The investigation was carried out to evaluate callus induction potential and regeneration response for the genotypes and the derived F1s on N6 media and modified N6 media (N6M). Whereas, N6 failed to induce callusing, agarose solidified N6M media supplemented with 4% maltose, growth regulators; NAA (2 mg/l), 2, 4-D (0.5 mg/l), Kinetin (0.5 mg/l), and silver nitrate induced high calli percentage of 27.6% in F1s, 9.5% and 6.7% in GS-88 and K-332 respectively. Murashige and Skoog (MS) media supplemented with 3% sucrose, and the hormonal combination BAP (2 mg/l), Kinetin (1 mg/l) and NAA (1 mg/l) induced high green shoot regeneration rates (0-60.0%). The effect of cold pre-treatment at 4°C and the stage of anther collection and their interaction was studied. The effect of cold pre-treatment (CP) of collected boots at 4°C (for CP2: 2, CP4: 4, CP6: 6 and CP8: 8 days) at different stages of panicle emergence (BES4-6: 4-6, BES7-10: 7-10, BES11-13: 11-13, BES>13: more than 13 inches was worked out in relation to the effect on response of calli induction, albino regeneration, green plant regeneration and number of shoots/green calli. CP referred to the number of days for which the collected boots were incubated before they were inoculated. BES was the length (inches) between flag leaf and penultimate leaf at the time of boot collection. We concluded that CP6 and BES7-10 showed better response to callus proliferation and regeneration of plantlets across genotypes. The appropriate pre-treatment, stage of anther collection and favourable media composition resulted in high calli induction and green plant regeneration rates in recalcitrant japonica genotypes. The modified N6 media resulted into efficient callus induction and is expected to be useful for studies which aim at rapid generation of mapping populations for genetic studies

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Not AvailableThe conservation and utilization of germplasm is contingent on its proper characterization at morphological or molecular levels. The present study aimed to elucidate the population sub-structure of 470 temperate rice germplasm collections of the Kashmir Valley. Analysis was carried out using KASP (Kompetitive Allele Specific PCR) assay on 213 genomic loci. Of these, a restricted set of 114 KASP loci were selected by the elimination of redundant, i.e. tightly linked markers based on map positions. STRUCTURE grouping was carried out to reveal three distinct sub-populations, K1, K2 and K3 comprising of 209, 156 and 105 germplasm accessions, respectively. Population FST values for K1, K2 and K3 were at 0.60, 0.24, 0.69, respectively, with highest pair-wise FST obtained between K2 and K3 (0.53). Analysis using the restricted set of 114 markers gave a better inferred membership with a low average admixture of 15.1% compared with 22.6% based on the whole marker set. An improved agreement between STRUCTURE grouping and principal coordinate analysis was reached using the restricted marker set. ΦST values calculated based on nucleotide diversity also suggested three sub-populations: K2, mostly indica germplasm; K1 mostly exotic temperate japonica; and K3, local japonica varieties and landraces. Polymorphic SNPs and haplotypes were discovered which discriminated the three sub-populations. Fifteen KASP markers were most important in discriminating K2 from K1 and K3 and included SNPs associated with domestication within the Wx, Ghd7 and Ghd8 genes. KASP markers are cheaper than SSR markers. Some of the KASP markers were highly discriminatory, using both model and distance based approaches, and so can be used as a cost-effective tool for efficient maintenance and use of rice genetic resources.Innovate U.K. and Biosearch Technologies, Hoddesdon, Herts, U. K