Cryopreservation Of Embryogenic Tissues And Genetic Stability Of Somatic Embryo In Quercus Variabilis

Quercus variabilis is a Quercus tree of Fagaceae, it is an important tree species for afforestation, Q. variabilis is propagated mainly by seeds. But, severe invasion by weevil, difficult to storage seeds over a long period of time, difficult to cutting, as well as offspring prone to genetic differentiation, have bad effects on the promotion of using the good genotypes. In recent years, the life activity of plant materials is minimized near to the zero in liquid nitrogen, So cryopreservation is the most safe and cost-effective method for long-term preservation of plant materials at an ultra-low temperature in liquid nitrogen. Vitrification is a main cryopreservation method because of its simple operating and simplified procedure. Effect of the low-temperature saving at5℃and vitrification and cryopreservation on embryogenic tissues in Q. variabilis, and different factors on cells viability were studied. And main steps of vitrification cryopreservation consisting of low-temperature preculture, frozen storage, thawing and recovery-medium were experimented by using the embryogenic tissues in Q. variabilis as the experimental materials. The method of the vitrification cryopreservation procedure of embryogenic tissues in Q. variabilis was established at a high cell survival rates. In addition, the survival cryopreserved materials were examined by molecular analysis, which confirmed their genetic stability. So vitrification cryopreservation was a practicable method for Q. variabilis germplasm storage. The main results were as follows:(1) The embryos in Q. variabilis were preserved for25、50、75、100days at5℃low-temperature. The survival rates and differentiation rates of the embryogenic tissues were tested after saving. The embryogenic tissues could develop normally after saving at5℃low-temperature, and5℃low-temperature preservation was a practicable method to store Q. variabilis germplasm.(2) In the vitrification cryopreservation, effect of vitrification and cryopreservation on embryogenic tissues in Q. variabilis and different factors on cells viability were studied. The result showed that the viability of cryopreserved materials was affected by the concentration of cryoprotectant and the treatment time. The cryoprotectant treatment time and the melting methods were very important to the cryopreserved materials survival. In addition, the cell survival rate did not change when tissues stored in liquid nitrogen during some days. The result of the vitrification cryopreservation procedure was as follows:The materials were percultured for3days at5℃low-temperature on the MS medium that contained6%DMSO after subculturing for10days, used60%PVS2to treat20min and100%PVS2to treat30min at0℃, then, put into liquid nitrogen(LN) rapidly after changing the solution with fresh PVS2, cultured on the MS medium after melting at40℃water bath, and we found that if the embryos in Q. variabilis after cryopreservation had been in darkness, the rate of recovery growth became faster and the survival rate after cryopreservation was also higher.(3) The embryos in Q. variabilis cryopreserved by vitrification could develop normally and RAPD analysis also showed no difference at DNA level. All the results suggested genetic stability of the survival tissues on molecular level after cryopreservation, and the vitrification cryopreservation was a practicable method to store Q. variabilis germplasm.