Apomixis in plants consists of asexual reproduction by seeds. Here we characterized at structural and functional levels an apomixis-linked sequence of Paspalum simplex homologous to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3). ORC is a multiprotein complex which controls DNA replication and cell differentiation in eukaryotes. Three PsORC3 copies were identified, each one characterized by a specific expression profile. Of these, PsORC3a, specific for apomictic genotypes, is a pseudogene that was poorly and constitutively expressed in all developmental stages of apomictic flowers, whereas PsORC3b, the putative functional gene in sexual flowers, showed a precise time-related regulation. Sense transcripts of PsORC3 were expressed in the female cell lineage of both apomictic and sexual reproductive phenotypes, and in aposporous initials. Although strong expression was detected in sexual early endosperm, no expression was present in the apomictic endosperm. Antisense PsORC3 transcripts were revealed exclusively in apomictic germ cell lineages. Defective orc3 mutants of rice and Arabidopsis showed normal female gametophytes although the embryo and endosperm were arrested at early phases of development. We hypothesize that PsORC3a is associated with the down-regulation of its functional homolog and with the development of apomictic endosperm which deviates from the canonical 2(maternal):1(paternal) genome ratio.

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One hundred and sixty accessions representing global germplasm of guinea grass (Panicum maximum Jacq.), an important apomictic (aposporous) fodder crop, were subjected to study on reproductive diversity in apomictic seed development utilizing ovule clearing and Xow cytometric seed screen (FCSS). Single seed FCSS of selected 14 tetraploid and Wve hexaploid lines demonstrated uncoupling between the three apomixis components, viz. apomeiosis, parthenogenesis and functional endosperm development, in natural as well as experimental populations, though it diVered across ploidy levels and genotypes. Reconstruction of reproductive pathways yielded a total of eight diVerent pathways of seed development, arising by uncoupling/recombination between apomixis components. Amongst these, two pathways involving modiWcations in embryo-sac (ES) (presence of two polar nuclei in aposporous ES that fuse prior to fertilization) and fertilization process (fusion of only one polar nucleus in a sexual ES) have been reported for the Wrst time. Some of the combinations, such as M I (haploids arising from parthenogenetic development of reduced egg cell), were found viable only in hexaploid background.Germplasm lines with higher expression of individual components were also identiWed. These components (including autonomous endosperm development) were also experimentally partitioned in hexaploid progenies (derived from a tetraploid parent viz. accession IG 04-164) that showed segregation in their reproductive capacities, and are reported for the Wrst time. Occurrence of several apomixis recombinants (phenotypic) in guinea grass lines suggested their hybrid origin, favors a multigene model for apomixis, with their penetrance aVected by modiWers and epigenetic mechanisms, in contrast to earlier reports of single locus control. Implications of partitioning components on apomixis research are discussed.

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The embryo rescue technique was successfully used to raise hybrids between Trifolium alexandrinum and T. constantinopolitanum. As a result of its narrow genetic base, genetic improvement in Egyptian clover (syn. Berseem; T. alexandrinum), an important fodder crop in tropical and subtropical countries, is hampered, thereby making it imperative to introduce alien genes from related species. In a conventional interspecific hybridization program, hybrids could not be raised due to post-fertilization barriers. Of the several combinations tried, pollination 2 days after emasculation was found to be the best. Globular embryos were observed 5-7 days after pollination (DAP), followed by heart-shaped embryos 10-12 DAP. Embryos excised at the heart-shaped stage responded well to EC3 culture medium. Of 612 crosses, 33 healthy embryos could be excised and cultured on EC3 medium. The plumule emerged 8-12 days following inoculation. The embryo-rescued plants were hardened, inoculated with Rhizobium and transferred to the field. The hybrids showed intermediate morphological features with reduced pollen fertility (55-65%) and a chromosomal complement of 2n = 16. Biochemical characterization using isozymes confirmed hybridity.