Cryopreservation Of In Vitro Prunus Humilis Bunge Shoot-tips And Analysis Of Genetic Variations

The purpose of the study was to establish the system of cryopreservation of Prunus humilis Bunge and then detect the variation of genetics and epigenetics. Two different methods were used in this study. The better one was chosen to establish the system of cryopreservation of Prunus humilis Bunge. After that, we established the system of cryopreservation of single sibling Prunus humilis Bunge, and then used this system to detect the variation of genetics and epigenetics.The result is shown as follows:(1) The regeneration of shoot-tips of Prunus humilis Bunge using vitrification is better than using encapsulation-vitrification.(2) The procedure on cryopreservation of shoot-tips of Prunus humilis Bunge by vitrification: 2.0 - 3. 0 cm in length, were pre-cultured on the B5 medium supplemented with 0.5 mg/L BA and 2 M Glycerol for 3 days. Then about 1.5 - 2.0 mm in length shoot-tips which from pre-cultured shoot-tips were loaded in a solution containing 60 % PVS2 for 10 - 20 min at room temperature, and incubated in PVS2 for 60 min at 0℃prior to a direct plunge into liquid nitrogen (LN) . After rapidly thawing in a water bath at 40℃, the shoot tips were washed fourth with 1.2 mol·L-1 sucrose solution and transferred on the same B5 medium as the previous one for 3d in dark prior to exposure to the light . The survival of shoot-tips was up to 83.9 % after 6 weeks and they grew and differentiated normally.(3) Two sites of Prunus humilis Bunge genome had changed after cryopreservation. The rate of changed sites was about 6.2 % out all detected sites.(4) The methylation of Prunus humilis Bunge genome had changed after cryopreservation. The total number of changes in bands pattern after the first cryopreservation was the same as the second cryopreservation. One third changes of bands pattern appeared no difference after first cryopreservation but they changed after second cryopreservation. Another one third changed sites of methylation status seems to have cumulative effects, and about another one third changed sites of methylation status can pass down to offspring via asexual reproduction.