玻璃化法超低温保存荔枝胚性愈伤组织及其植株再生

采用玻璃化法对荔枝胚性愈伤组织超低温保存及植株再生进行了初步探讨.将继代保存15 d的荔枝胚性愈伤组织转接到预培养基[MS+50 mL.L-1二甲基亚砜(DMSO)+1 mg.L-12,4-D+20 g.L-1蔗糖+6 g.L-1琼脂糖]中,在5℃低温下预培养.将预培养后的愈伤组织于常温下用体积分数为60%玻璃化溶液(2PVS2)装载20 min,再于0℃下用PVS2溶液平衡40 min;换新鲜的PVS2溶液,迅速投入液氮中保存.1周后取出,于40℃温水浴中化冻,弃PVS2,用1.2 mol.L-1蔗糖液+MS基本培养基的洗涤液洗涤3次,再接种到新鲜的培养基中恢复培养,恢复培养后的胚性愈伤组织能正常进行体细胞胚胎发生、成熟和植株再生.

Cryopreservation of embryogenic calli by vitrification and plant regeneration via somatic embryogenesis in litchi

The procedure for cryopreservation by vitrification and plant regeneration from embryogenic calli were preliminarily developed in litchi.After subculturing for 15 d,the calli were precultured on the MS medium supplemented with 50 mL·L~(-1) DMSO,1 mg·L~(-1)2,4-D,20 g·L~(-1) sucrose and 6 g·L~(-1) agar at 5 ℃,and then the excised calli were loaded with 60% PVS_2 for 20 min at room temperature,and next they were exposed to PVS_2 for 40 min;after changing the solution with fresh PVS_2,the calli were put into liquid nitrogen.One week later,the calli were thawed rapidly in a 40 ℃ water bath;then they were washed with the MS medium with 1.2 mol·L~(-1) sucrose for 3 times and were further transferred to the fresh medium(MS with 1 mg·L~(-1) 2,4-D,20 g·L~(-1)sucrose and 6 g·L~(-1) agar) for growth.Plantlets regenerated normally via somatic embryogenesis from these cryopreservated calli.