Early Rescue And Identification Of Citrus Hybrid Embryo In Vitro Via SSR Technology

The crossbreeding is still a main method used to cultivate fine citrus fruits variety, and plays the influential role in the citrus fruits breeding. However, nucellarembryony in most citrus varieties, which causes the hybrid degeneration and abortion, has prevented the development of hybrid breeding in many polyembryony varieties. Embryo rescue techniques hold great promise for obtaining zygote plants from inherently weak embryos, haploid plants as well as shortening the breeding cycle, and have been applied in citrus breeding. On the other hand, Molecular markers, including RAPD, AFLP, and SSR, which show the advantages of high accuracy and reliability in the identification of hybrid progeny, have been widely used in evaluation of citrus hybrid plants obtained via embryo rescue, but hardly in identification of hybrid embryo in the period of embryo culture. So it was necessary to improve a high efficient hybrid embryo rescue and identify method for solving the difficulties brought about by the nucellar embryo disturbance in a certain extent.In this study, using Jincheng orange×Red tangerine and Red tangerine×Jincheng orange as the research object, an efficient hybrid embryo rescue technology based on SSR analysis was developed via investigating the optimal time of embryo rescue, minimizing the sample volume of embryo material for extraction DNA, screening polymorphism primers and so on. The main results are as follows:1. Determine the optimal time of embryo rescueAfter cross-pollination 55 days, the growth of cultured embryo was observed under the microscope every 5 days, and then the optimal period of zygote embryo in vitro was identified. The study indicated that for Jincheng orange×Red tangerine and Red tangerine×Jincheng orange hybrid combinations, the optimal time of carrying on hybrid embryo rescue is after pollination 85 days and 90 days, respectively. 2. Minimizing the sample volume of citrus embryo material for extraction DNAUsing CTAB method, the extracted DNAs from different amounts of embryonic material (20 mg,10 mg,5 mg) were performed to PCR analysis. The result showed that only using 5 mg embryo material can extract enough genomic DNA for SSR analysis of early hybrid embryos.3. Screening of polymorphism primersTo select the polymorphism primers for early identification of hybrid embryo, the 20 pairs of SSR primers were evaluated using the DNA of the Jincheng orange and Red tangerine as the template, respectively. The result indicated that the SS4, Cssr036 and Cssr051 primers are suitable for the identification of hybrid embryo of Jincheng orange x Red tangerine, while the SS16, Cssr037 and Cssr051 primers are suitable for Red tangerine x Jincheng orange.4. Identification of Hybrid embryosDNAs of cultured embryo were extracted from the minimal sample volume (5mg) of citrus embryo material after about 45 d culture, and SSR-PCR analysis were performed to screening hybrid embryo in the early period of cultured embryo with the SSR primers selected. Our results showed that 9 and 7 hybrid embryos were identified from polyembryony of forty-six Jincheng orange×Red tangerine and from polyembryony of thirty-two Red tangerine x Jincheng orange, respectively.In summary, these results above demonstrated that using the improved embryo rescue technology in this paper, the identification of hybrid embryo could be accomplished in the early period of cultured embryo. And the technology will have great potential in the cross breeding of citrus.