苹果休眠茎尖的超低温保存研究

A Study on Cryopreservation of Dormant Apple Shoot Tips

苹果金冠品种的休眠茎尖经３种不同方法（玻璃化法、包埋－干燥法和两步降温法）超低温保存后，均获得成功，存活率达到了８３％。休眠茎尖由于经受了冬季的深冷驯化和具有较低的含水量，因而成为超低温保存的良好试材。经过玻璃化液（ＰＶＳ３：５０％甘油＋５０％蔗糖）预处理８０ｍｉｎ（２５℃条件下）后或经蔗糖溶液预培养并于无菌空气中干燥脱水至含水量３０％左右后，休眠茎尖可直接投入液氮进行超低温保存。保存后的休眠茎尖经离体培养后可直接再生植株。试验进一步验证了试材生理状态的选择是超低温保存成功的一个关键因素，这不仅可以简化超低温保存的程序，而且可以提高此方法的实用性，为早日建立植物种质的超低温保存库奠定了基础。

Shoot rips of apple (Malus domestica Borkh.cv.Golden Dellicious)dormant buds were successfully cryopreserved by using three different methods(vitrification,encapsulation dehydration and two step freezing),the regeneration rate arrived to 83% after cooling to liquid nitrogen.As the dormant shoot tips have been cold hardened in winter and have lower water content,it became the value materials of cryopreservation.After pretreated in vitrification solution(PVS3: 50%glyceros+50%sucrose)for 80 min at 25℃ or desiccated in air flow for 4 h(the water content is about 30%)by using encapsulation dehydration method,the shoot tips can be plunged into LN directly.The dormant shoot tips could regrow directly after cryopreservation.The experiments demonstrated that the selecting of material physical state is a very important factor during cryopreservation,which could not only simplify the cryopreservation protocols,but also improve the practical useness of cryopreservation,in order that to establish the cryopreservation genebank of plant germplasm earlier.