Creation and identification hybrid population from Zhique × Trifoliate orange based on embryo rescue technology

Abstract: 【Objective】The research and utilization of resistant rootstock resources of Citrus in China started relatively late, and the breeding rootstock is still relatively delayed in comparison with that in the developed countries. In order to obtain hybrid offspring population of Zhique ( Citrus wilsonii Tanaka) Raf.) and Trifoliate orange[Poncirus trifoliata (L.) Raf], we conducted this study. Trifoliate orange is used as a kind of citrus rootstock widely in China. It has good affinity and general resistance. But the major defect is the insufficiency of iron absorption. Zhique is a kind of local citrus rootstock resource reported in Chenggu county, Shanxi province recently. It has good ability in using iron. In order to fully combine the beneficial properties, we carried out hybridization combined with embryo rescue technique to obtain hybrid offspring.【Methods】The species Zhique was planted in Chenggu county, Shanxi province. In this experiment, the Zhique's plants, leaves, flowers, fruits and other materials used for morphological description were all collected from a tree which was estimated more than 40 years old. Zhique was used as female parent and Trifoliate orange was used as male parent. The pollen of Trifoliate orange was collected in HZAU Germplasm Resources Nursery during morning. After the collection, the pollen was dried in dryer with no light at 27-29 ℃. We stored the pollen in 1.5 mL centrifuge tubes at4 ℃ when they were fully dried. We used acetic acid magenta to test the activity before the pollination and bagged the flowers after pollination. The hybrid fruits were picked 80-85 days after the pollination.The fruits were stored in 4 ℃ refrigerator before the embryo rescue was done. The embryos were inoculated on the shooting medium (MT+KT 0.5 mg · L-1+BA 0.5 mg · L-1+NAA 0.5 mg · L-1) under illumination culture until the bud grew up. The shoots were moved on rooting medium (l/2 MT+IBA 0.1 mg · L-1+NAA 0.5 mg · L-1+Activated Carbon 0.5 mg · L-1) until the root grew up. After subculture the plants were transfered into water for 1 month adaptation. Finally we put the well adapted seedlings into small pots with nutritive soil, and moved the pots in greenhouse. There were 1 133 progeny seedlings survival in the end, some of them were identified with morphology of the male parent, but the rest had to be identified by molecular markers. 8 of the primer pairs of the 10 possible SSR primer pairs were able to be used for the identification of the true hybrid seedlings.【Results】61 hybrid offspring were obtained.Among them 41 of the hybrid plants were identified with the trifoliate morphologic characteristics. Another 20 hybrid plants were identified by SSR molecular marker primer pair CS014 R-CS014 F and CS065 R-CS065 F.he hybrid rate was 4.1%.【Conclusion】Hybrid offsprings derived from the cross between Zhique and Trifoliate orange were successfully identified by means of morphology and SSR marker selection from the seedlings obtained with embryo rescue which created the materials for the further study of citrus rootstock breeding.