Optimization Of Cryopreservation Technology Of Embryogenic Callus Of Fraxinus Mandshurica

The direct somatic embryogenesis system of Fraxinus mandshurica has been established,but there are still problems of low somatic embryo quality,poor synchronization effect,and high incidence of malformed embryos,which are not suitable for large-scale industrial seedling application.Therefore,it still faces the severe challenge of improving in vitro regeneration system and realizing large-scale production of tissue culture seedlings of F.mandshurica.Indirect somatic embryogenesis is more suitable for large-scale industrialized seedling breeding applications.However,in the process of long-term subculture and proliferation of embryogenic callus,it is inevitable that the rate of cell variation will increase and embryogenicity will lose,so it is difficult to achieve the purpose of long-term stable preservation.In order to maintain the genetic stability and morphogenesis potential of germplasm materials to the greatest extent,it is necessary to develop a cryopreservation method suitable for fine genetic materials of F.mandshurica.In this study,the embryogenic callus of F mandshurica was used as the material to optimize the cryopreservation technology of the embryogenic callus by program cooling,explore the method of cryopreservation by vitrification,and analyze the cell survival rate and physiological and biochemical differences in the process of cryopreservation.The main results are as follows:(1)Embryogenic callus with vigorous growth and good condition were pre cultured in 0.4 mol·L-1 sorbitol solution for 20 hours at 25℃,then dehydrated in 7.5%Dimethyl sulfoxide solution at 0℃ for 90 min,frozen in a-80℃ refrigerator for 2 h,and then quickly put into liquid nitrogen for preservation.After being frozen for 24 hours,thawed in a 40℃ water bath for 2 min,and finally resumed culture on WPM basic medium supplemented with 0.1 mg·L-16-BA and 0.15 mg·L-1 2,4-D.After cryopreservation by the program cooling method,the survival rate of callus cells was 80.82%,and it could reach 1.93 g when the fresh weight was restored for 60 days,the regeneration rate of callus was 100%,the proliferation coefficient of callus was 2.79,the differentiation rate was 56.83%,and the seedling emergence rate reached 23.59%.(2)Embryogenic callus that subcultured for 7-10 days with vigorous growth and good condition were selected.After cryopreservation by vitrification,the best pre culture method was to pre culture on the 0.5 mol·L-1 sucrose medium for 3 days,load and culture in the WPM liquid medium supplemented with 2 mol·L-1 glycerol and 0.4 mol·L-1 sucrose for 60 minutes,then dehydrate in 2 mL of PVS2(30%glycerol+15%DMSO+15%ethylene glycol+0.4 mol·L-1 sucrose+WPM liquid medium).After freezing in liquid nitrogen,it was thawed in a 40℃ water bath for 2 min.The highest cell survival rate was 61.94%,and the fresh weight could reach 1.82 g after 60 days of recovery.The proliferation coefficient of callus was 2.69,the differentiation rate was 53.87%,and the seedling emergence rate reached 20.97%.(3)The soluble sugar content of the programmed cooling method was higher than that of vitrification method(103.84 mg·g-1FW and 89.28 mg·g-1FW,respectively)during the pre-cultivation and recovery culture.In the dehydration process,the contents of proline,soluble protein and starch of the programmed cooling method were higher than those of vitrification method.During the thawing process,the content of malondialdehyde under the cryopreservation treatment of vitrification is higher than that of the programmed cooling method(3.56μmol·g-1FW,3.03 μmol·g-1FW,respectively).Therefore,the cryopreservation of programmed cooling method is more suitable for embryogenic callus of F.mandshurica compared with the cryopreservation of vitrification method.