Hybrid Embryos Rescue And Early Identification Of Four Hybrid Citrus Combinations

Citrus hybrid breeding is one of the main methods of citrus breeding. first-filial generation showed a lot of quantitative character separate, is better than other way of breeding. But a lot of citrus has nucellar embryo,leed to Hybrid embryos involution or abortive,it is difficult to have hybrid seeding, Limit the cultivars of multiple embryo breeding. A lot of cultivars breed by single or less embryo of female parent, avoid the problem of polyembryony, but haven’t solve it,and let genetic genome narrow, excellent character can’t be assembly. the technology of saving Hybrid embryos can improve hybid ratio. but many character can be recognized in flower season or fruiting season. In hybrid identification, the traditional method don’t suitable for early identification. DNA molecular markers can be used in the identification. 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.This experiment comprises four cross combinations, Late JinCheng♀×Tanaka♂,Late JinCheng♀Tiancao♂,Taiwan Ponkan♀×Tarocco Blood Orange♂, Taiwan Ponkan♀×Late JinCheng♂.The development of hybrid embryos is observed regularly by paraffin method 50 days after pollination,and bead embryo sac invasion of heart time is judged timely.. After bead the heart invasion the embryo sac young embryo is taken regularly for in vitro culture, and the embryo total DNA is extracted through the trace law, the hybrid embryos is taken for early identification by molecular markers, then hybrid embryos is inducted into the plants. The results of the experiment are as follows:1 Determine the optimal time of hybrid embryos rescueAfter cross-Pollination 50 d, every 3 days take a materials, anatomy seeds Late JinCheng and Taiwan Ponkan in a microscope and the development of embryo partial seeds. The results show that Late JinCheng as hybrid materials,cross-pollination 60d can be observed embryo in ovule, andTaiwan Ponkan for the female parent mandarin materials,70d can be observed embryo formation. Since the 5th day after observing embryo, seed production paraffin section is taken every 5 days, observes the nucellus embryos when to embryo sac. The results find that Late JinCheng and Taiwan Ponkan respectively in cross-pollination 70d and 85 d began to invade the embryo sac. At the same time since observed embryo appeard on every 5 days making paraffin. The results show that since the 75 d Late JinCheng embryo shows cotyledons, since the 95 d Taiwan Ponkan showes cotyledons. Therefore, the Late JinCheng and Taiwan Ponkan hybrid embryo the best rescuing period respectively are 75 d and 95 d2 hybrid embryos rescueInoculate seed into diffenrent MS culture, and select the best culture.by IAA 0.5mg/L, 1mg/L,6-BA0.25mg/L,0.5mg/L, 1mg/L install combination. The results show that the culture that join in IAA 1 mg/L,6-BA 0.5 mg/L MS medium was the best one. finally received Late JinCheng×Tanaka 564embryos, Late JinCheng×Tiancao 249 embryos, Taiwan Ponkan×Late JinCheng 297 hybrid embryos, Taiw an and partial×Tarocco Blood Orange 6 young embryos.3 hybrid embryos identifyExtraction of hybrid parent materials DNA, from 20 pairs of SSR primer screening polymorphism of primers. The results show that 3 primers(SS4, SS8, Cssr53) can be used in Late JinCheng×Tanaka hybrid embryos identify; 2 primers (Cssr51 and Cssr53) can be used in Late JinCheng×Tiancao hybrid embryos identify; 6 primers SS12, SS15, SS18, Cssr38, SS16, Cssr53) can be used in Taiwan Ponkan×Late JinCheng and Taiwan Ponkan×Tarocco Blood Orange hybrid embryos identify.Extraction young embryo DNA, using the above polymorphism primers for hybrid embryos early identification.The Results showed that the Late JinCheng×Tanaka by hybrid embryos rescue get the 564 embryos and select 43 hybrid embryos. From the late JinCheng×Tiancao combination get 249 hybrid embryos material and identified 13 hybrid embryos; Taiwan Ponkan X Late JinCheng and Late JinCheng♀Tarocco Blood Orange♂combinations saved 297and 6 hybrid embryos, but no identified hybrid embryos.