Some Factors Affecting the Efficiency of Hybrid Embryo Rescue in the 'Shiranuhi' Mandarin

The 'Shiranuhi' mandarin has attractive characteristics such as a high soluble solid content, a lack of seeds, and a rind that is easy to peel. Thus, it has been utilized as a genetic resource in citrus breeding programs. However, it has a polyembryony trait that causes problems when producing sexual hybrids. Therefore, methods need to be established to distinguish between zygotic and nucellar plants from cross offspring. In this study, the 'Shiranuhi' mandarin and the 'Sanguinelli' blood orange were crossed to produce immature embryos that were used to detect zygotic embryos at 90, 105, 125, 145, and 180 days after pollination (DAP). For germination, embryos at different developmental stages were cultured on Murashige and Skoog (MS), Murashige and Tucker (MT), and Gamborg B5 media supplemented with 500 mg.L-1 malt extract, 25 mg.L-1 adenine sulfate (ADS), 1 mg.L-1 gibberellic acid (GA(3)), 3% sucrose (w/v), and 0.8% phyto agar (w/v). The germination rate of immature embryos ranged from 36.4% to 74.9% depending on DAP, while germination rate was not significantly different by medium type. The rooting rate was high during the late embryo developmental stages and when there were low sucrose concentrations and high GA(3) concentrations. The percentage of zygotic plants per seed was measured using simple sequence repeat (SSR) markers and averaged 12.1% for 90 DAP, 7.6% for 105 DAP, 6.8% for 125 DAP, 1.0% for 145 DAP, and 4.1% for 180 DAP. These results indicated that embryo developmental stage was the greatest factor in relation to inorganic salt, sucrose, and GA(3) concentration for increasing the selection efficiency of sexual hybrids in crosses using polyembryony maternal parents by embryo rescue and SSR marker analysis.