Molecular marker-assisted cell engineering and citrus improvement

Cell engineering is a valuable 'no GMO concern' technique for citrus cultivar improvement that has efficiently circumvented the reproductive barriers encountered in conventional citrus breeding, such as nucellar polyembryony and male/female sterility. Application of molecular markers has greatly enhanced the efficiency of cell engineering-based targeted citrus breeding. In our program, somatic hybrids were produced from over 50 fusion combinations that are either interspecific or intergeneric, and were characterized by molecular markers. Some of the somatic hybrids have already served as pollen parents for interploidy sexual crossing. Consequently, by embryo rescue, flow cytometry and simple sequence repeat (SSR) marker analysis, thousands of triploid plants were recovered. Based on molecular marker analysis of numerous citrus somatic hybrids and cybrids, we put forward a strategy of creating male sterile cybrids by symmetrically fusing the protoplasts of embryogenic callus induced from 'Satsuma' mandarin (CMS type with sterile cytoplasm) with mesophyll protoplasts of elite seedy cultivars. As a result, the regenerated diploids, which proved to be cybrids, contain sterile cytoplasm from 'Satsuma'. To date, some cybrids have already displayed male sterility and seedlessness. By omics-based studies, the profiles of genes, proteins and microRNAs in the male sterile cybrid G1+HBP were uncovered, and the genes potentially responsible for the male sterility of G1+HBP are under further investigation. By SSR marker analysis, we also identified plenty of autotetraploids, haploids and dihaploids that were exploited spontaneously or regenerated from anther culture. These novel citrus germplasm resources hold great potential for seedless citrus breeding and rootstock improvement, as well as facilitating research on polyploid genomic variation and environmental adaptability.