Induction of doubled haploids in melon using a distant cross between melon and Cucumis africanus

Abstract:【Objective】China is the secondary origin of melon with abundant germplasm. Oriental melon (Cucumis melo ssp. agrestis) is favored by growers and consumers because of its special fruit flavor, wide applicability and higher yield. It is particularly vital to create various excellent oriental melon varieties both for the growers and consumers. However, traditional breeding methods are time and labor consuming, and have gradually failed to meet the needs of modern production of melon. Breeding efficiency can be greatly improved by using haploid to produce pure breeding line. In melon crops, haploids are obtained by means of radiation pollination induction techniques, in vitro unpollinated ovary/ ovule culture, and in vitro anther culture. Among those radiation pollination induction technique is widely adopted in breeding practice, although it is stringent in experiment condition and relatively low in haploid embryo (seedling) production ability. Subsequently, haploids are subjected to chromosome doubling by anti-mitotic agent such as colchicine to obtain doubled haploids (DH), which are usually accompanied by low efficiency. In present study, we used the pollen grains of wild species (C. africanus) to pollinate the oriental melon cultivar Chaotianxiaomaisu to induce regenerated DH melon directly with excellent traits. The characteristics of this DH were also systematically described herein.【Methods】The oriental melon Chaotianxiaomaisu was selected as the female parent and crossed with the wild species C. africanus. 25 mg ·L-1 forchlorfenuron solution was sprayed at the base of the ovary immediately after pollination, and 10 mg · L- 1 thidiazuron (TDZ) solution to the ovary 3-4 h after pollination. The fruits were harvested 4 weeks after pollination, and the relatively plump seeds were then plated in MS solid medium for embryo rescue. The ploidy of the regenerated seedling was determined using cell flow cytometry. And the 3 polymorphic SSR markers (DE1259, CMBR021 and CMBR026) and the botanical characters (stem length, stem width, leaf length, leaf width, male flower length, cotyledon length, cotyledon width) were comprehensively evaluated to identify its DH feature. Some fruit traits of DHs were also compared with those of the control.【Results】A total of 179 seeds were obtained from the cross between melon and C. africanus, and were immediately inoculated to MS medium for embryo culture in this study. After 15 days, the green seeds were picked and continued to inoculate on the solid MS medium. A single seedling was obtained, and the yield rate of regenerated embryos was 0.56%. The regenerated plant was detected to be diploid by cell flow cytometry. Gene loci, which were revealed to be polymorphic between parents by 3 pairs of SSR markers, were checked to be homozygous in the DH progenies. Subsequently, the 7 botanical characters, such as stem length, stem width, leaf length, leaf width, male flower length, cotyledon length, and cotyledon width were detected to be various insignificant among the 17 DH individuals. The data of the 7 botanical characters were found to be as normal distribution as well. Some important physiological indicators, including the soluble solids, ascorbic acid, and soluble sugar of the DH1 and the control were compared in detail, among them the soluble solid of the DH1 was higher than that of the control. It means that some quantitative genes related to the sugar content become homozygous rapidly during the DH formation. And the seed setting- and germinationrate of the DH were as normal as those of the control, which will bring great convenience both to the theoretical study and breeding practice of the doubled haploid. In addition, the appearance of the fruit (volume, pulp and peel color) changed greatly (from green to white) compared with that of the control, which will provide more selection opportunity in breeding practice. In a word, the DH technique of distant hybridization combined with embryo rescue used herein has many comparison advantages over radiation pollination induction technique, such as avoiding the process of haploid doubling, and escaping the adverse effects of anti-mitotic agents (i.e deformity or mixploidy after doubling, low doubling efficiency, and colchicine toxic effects on plants).【Conclusion】The excellent oriental melon DH was directly obtained by distant hybridization combined with embryo culture. And the fruits of the DH were sweeter than those of its original parents, and it could be preserved by seed propagation directly.