PhD

Production and productivity of rice, the staple food for over half of the world's population, is insufficient to meet the demand of the increasing population in the rice consuming nations. One of the readily available and economically viable technological options for meeting the projected global demand of rice is exploitation of heterosis through large-scale cultivation of rice hybrids. The role of hybrids in enhancing the productivity is widely acknowledged. The commercial rice hybrids are currently based on cytoplasmic genic male sterility (CGMS) system. The availability of stable cytoplasmic male sterility and fertility restoring system is vital for commercial exploitation of heterosis in rice. The cytoplasmic male sterile (CMS) lines introduced from China were found unsuitable to use as such in developing hybrid rice in India. Limited resources of CMS and low variation of CMS lines causes genetic vulnerability. Therefore, to diversify CMS lines, breeders can transfer this character to the existing putative maintainer lines through repeated backcrosses. So the present study entitled “Development of cytoplasmic male sterile line in an identified rice variety of Kerala through marker assisted back crossing” was undertaken as an initial step for the development of hybrid rice for Kerala with the objective of transferring the cytoplasmic male sterility from the CMS line to the Kerala rice variety, identified as maintainer for CGMS system through marker assisted back crossing. This study was conducted from December 2016 to August 2019.
In the present study, four Kerala rice varieties were used, which were reported as potential maintainers of CRMS31A line in the research work conducted by Das (2017). For identification of the best maintainer among the four potential maintainers under study, these lines were crossed with CMS line CRMS31A. The pollen fertility per cent and spikelet fertility percent of hybrids was lowest in the cross CRMS 31A x Jyothi and highest in the cross CRMS 31A x Aruna. The highest pollen and spikelet fertility percent was observed in cross with Aruna with 60% seed set. Kanchana and Bharathy on crossing with CRMS31A showed 91% and 57% pollen sterility which lead to 99.11% and
74.83% of spikelet sterility respectively. Among 30 F1’s obtained from the cross CRMS31A x Jyothi, two F1 plants showed 99.99% pollen sterility and 100% spikelet sterility. Pollen and spikelet sterility of the hybrids indicated that Kanchana, Bharathy and Aruna were partial maintainers for CMS line. Rice variety Jyothi, alone was identified as maintainer for cytoplasmic male sterile line CRMS31A.
The transfer of ms gene was confirmed at molecular level using mitochondrial WA-CMS-specific marker “drrcms”. Foreground selection was carried out in 30 F1 plants and it showed homozygous band similar to male sterile line. F1 plants with 99.99% pollen sterility and 100% spikelet sterility, were backcrossed with maintainer Jyothi to obtain BC1F1 seeds.. These BC1F1plants were screened with cytoplasmic male sterility specific marker and all the plants were identified with gene of interest i.e. cms gene.
Selection of polymorphic markers was done for background selection in the identified plants with 100% spikelet sterility. 170 genome wide SSR primers were used for screening of the maintainer and CMS line to locate 31 SSR markers specific to the maintainer. The background selection was carried out on 17 BC1F1 plants using 31 loci polymorphic in the parents. The BC1F1 plants with highest percentage of recurrent parent genome recovery were selected based on the background markers. The maximum percentage of recovery of recurrent parent genome was found to be 68.13% in BC1F1 generation.
Pollen sterility in these plants was also studied by screening for fertility restoration gene (Rf). Out of 10 Rf markers used for screening only four markers for Rf4 Rf6 and Rf7 gene were present in the maintainer line Jyothi. The identified markers were then used for screening of BC1F1 plants. It was observed that the BC1F1plants had only Rf7 gene. Pollen and spikelet sterility percent of these plants could not be recorded due to absence of flowering even on the 240th day after sowing. The plants were under vegetative phase upto 260-270 days, after which they started wilting.
Due to absence of flowering, these plants were screened with marker specific to flowering time gene Hd3a. On gel documentation the band was obtained revealing the presence of Hd3a gene at 179bp. To confirm the possible reason for absence of flowering even in the presence of Hd3a gene, the expression of Hd3a gene was studied. Expression was studied at different stages of plant growth i.e. 15, 30, 45, 60, 75 and 110 days after sowing in Jyothi variety and 240 days old BC1F1 plants.
It was observed that the expression of target gene in the variety Jyothi was reduced at 30 days after sowing. But in later stages, gradual increase in the expression level was observed. A drastic increase in expression was recorded at flowering stage (75DAS) of the Jyothi variety and then the expression was gradually reduced towards maturity stage. Whereas the Hd3a gene was not expressed in BC1F1 plants i.e. the gene was silenced. The reason for non expression of Hd3a gene in the background of sterile cytoplasm and ms gene requires further investigation.
Advanced generation of the cross between another CMS line, UPRI95-17A and maintainer line Kanchana, which was developed earlier under a project for transferring male sterility to Kanchana variety was further studied. Sterility was studied by pollen test and spikelet test. Three of the plants showed 99.09% - 100% pollen sterility and 100% spikelet sterility. All the plants were screened with 28 SSR markers. Foreground selection was carried out using mitochondrial WA-CMS-specific marker “drrcms”. Band obtained in the advanced generation plants were similar to the band of male sterile parent. Recovery of the recurrent parent genome was analysed using 28 SSR markers. One plant of this advanced generation, recovered 92.86% of recurrent parent genome. Observations were taken on eight morphological traits viz. plant length, leaf length, leaf breadth, total number of tillers, panicle length, days for flowering, number of spikelets per panicle and pollen fertility percentage. It was observed that out of eight quantitative traits, four traits i.e. plant length, leaf length, total number of tillers
and panicle length were almost similar to the maintainer line Kanchana. The number of spikelets per panicle and leaf breadth was less than Kanchana.
In the present study, Kerala rice varieties, Jyothi and Kanchana showed 100% pollen and spikelet sterility when crossed with CMS line CRMS 31A and UPRI95-17A respectively. On the basis of pollen and spikelet sterilirty Jyothi and Kancjana were identified as maintainers for WA-CMS line CRMS 31A and UPRI95-17A. The reason for non expression of Hd3a gene in the background of sterile cytoplasm i.e. backcrossed plants of Jyothi variety needs to be further investigated. The identified plants from the backcross population of UPRI95-17A and Kanchana, with higher recurrent parent genome (RPG) recovery percentage can be backcrossed for another generation to get RPG of 99%. Later it can be used for commercial hybrid seed production with compatible restorers for UPRI95-17A. This will be the first CMS line in the background of Kerala rice variety