Microseparation And In Vitro Culture Of Zygotic Embryos From Polyembryony Citrus

Polyembryony is a serious handicap in Citrus breeding. It results in the abortion of the zygotic embryo, due to the competition with the nucellar embryos, which are often more vigorous. Within this context, microseparation and in vitro culture of early zygotic embryos from polyembryony Citrus is very important because it makes possible the use of immature hybrid embryos and accelerates the sexual hybridization breeding in Citrus.The systems of microseparation and in vitro culture of zygotic embryos from polyembryony Citrus and identification of Citrus hybrids were preliminary established in this research. Some early zygotic embryos of Citrus were microseparated and regenerated into seedlings successfully by this way. The results are as follows:1,The best stage to separate zygotic embryos was 50-55 days after pollination. It can boost the efficiency of zygotic embryos separation to use steel needles with 50μm large diameter needlepoint under the micromanipulator.2,MS, White and MT basic medium are all suitable for the culture of zygotic embryos. The addition of 1mg/Lof IAA and 20mg/L of BAto the medium provides best conditions for the mature culture of zygotic embryos of Citrus. It is favourable for the development of early zygotic embryos to increase the osmotic pressures of media and supplement lactalbumin hydrolysate (LH). The suitable concentration of sucrose was 10% and LH was 400mg/L.3,Cultured in solid or liquid medium are all suitable for the development of early zygotic embryos of Citrus. But zygotic embryos cultured in solid medium can be more ease to be observed and controlled.4,It doesn't need to change the combination of IAA and BA when developed zygotic embryos change to differentiation culture. Seedlings can be regenerated from mature zygotic embryos when the concentration of sucrose decreased to 2% and cultured in illumination, and it can also accelerate the speed of regeneration with addition of 0．2mg/L of ZT in this way. The best stage to change to differentiation culture is the stage when cotyledons came out from the zygotic embryos cultured in solid medium or the cotyledons of the zygotic embryos cultured in liquid medium are 1-1．5 cm long.5,The method of isolation of genomic DNA from Citrus and the optimal amplification system and program for RAPD-PCR were established after iterative experiments. 61 zygotic seedlings were identificated from 73 regenerated seedlings by random amplified polymorphic DNA(RAPD) with 6 primers of 10 bases. The proportion of zygotic seedlings is 83．6%.All results above showed that it is a efficiency method to get rid of the inhibition from nucellar embryos and accelerate the sexual hybridization breeding in polyembryony Citrus to microseparate the zygotic embryos of Citrus by micromanipulator and culture them in vitro into regenerated seedlings.