Studies on Cryopreservation of Two Diospyros spp.Germplasm by Modified Droplet Vitrification

The factors were studied that effected the cryopreservation of Diospyros lotus L.and D.kaki Thunb.in vitro shoot-tips by modified droplet vitrification,such as cold hardening method,preculture time,PVS₂ treatment time etc.A suitable procedure was established:shoot-tips of in vitro axillaries were cultured on 1/2MS medium and followed by alternating temperatures(25±1)℃ in daytime/(4±1)℃ at night cold-hardening for 6 weeks,precultured with 0.5 mol/L sucrose for 5 days,which were loaded in loading solution for 20 min at 20℃,and incubated in PVS₂ for 1.5 h at 0℃ prior to direct plunge into liquid nitrogen.After rapid thawing at 40℃ warm water bath and rinsed five to six times,the shoot-tips were plated on the regeneration medium supplemented with 1.0 mg/L TDZ,0.6 g/L PVP,30 g/L sucrose and 7 g/L agar for one week in dark prior to exposure to the normal condition(The result is found in optimizing of regeneration system of Diospyros spp.by tissue culture).Using this procedure,the survival rate of ‘Eshi 1’,‘Xiangxi-tianshi’and D.lotus L.in vitro shoot-tips was 79.6%,67.4% and 60.9%,respectively.