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Subject: Agricultural Biotechnology × Author: Karminderbir Kaur × Start date: 2018 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Development of an in vivo haploid induction system in rice through distant hybridization and manipulation of CenH3 gene
    (Punjab Agricultural University, Ludhiana, 2020) Karminderbir Kaur; Kumari Neelam
    In rice, an in vivo haploid induction system, that can produce both maternal and paternal haploids at promising rates is not available. In the present study, four different experiments were carried out to explore various possibilities of developing an in vivo haploid induction system in rice. Rice X maize and rice X pearl millet crosses were analyzed for pollen tube penetration to study fertilization barriers. A total of 38,468 spikelets were pollinated, but no true caryopsis was observed. Fluorescent microscopy revealed that callose depositions hinder the release of generative nuclei to the ovule. 73 wild species accessions of genus Oryza were re-sequenced for mining alleles of the OsCENH3 gene. Based on the sequence data, 16 haplotypes for OsCENH3 gene were identified. The most consistent variation obtained with respect to Nipponbare reference was SNP A/G at position 69 (codon 23). Phylogenetic analysis of 24 accessions revealed that the O. rufipogon accessions carrying amino acid changes in the N-terminal tail domain region to be closer to O. nivara accessions than they were to OsCENH3 reference. In addition to the wild species, two HFD-region mutants were also identified from a Nipponbare TILLING population. Ros 5783 TILLING mutant showed 50% incidence of seed death upon crossing; indicating it to be a promising haploid inducer candidate. Genome editing was carried out using an M4 specific guide RNA, complemented by three different mutant CENH3 versions, in separate events. The plantlets obtained after transformation with the guide and the complements were genotyped. Cas9 and complementtransgenes were found to be present in four and ten T0 plants respectively. The transformation was successful, but edits could not be detected. Both TILLING mutants and transformed plants are available at PAU for the further crossing to check for haploid induction capabilities.