湿加松胚性愈伤组织的程序降温技术研究

Cryopreservation of embryogenic callus of Pinus elliottii × Pinus caribaea using control freezing

对湿加松胚性愈伤组织进行了超低温冷冻保存的研究。结果表明：继代培养9～12d的湿加松胚性愈伤组织经0．5mol／L梨醇预培养4d，在0．6mol／L山梨醇＋10％DMSO的冷冻保护液下0℃预处理20min，然后以-1℃min的降温速率降至-40℃，停留10min后再以-5℃／min的降温速率降至-90℃，投放入液氮中保存；复苏时于37℃水浴2min，1mol／L山梨醇的液体培养基清洗3次，以滤纸作支持物转到固体继代培养基再培养。在此程序处理下1个月内可获得生长状态良好的再生愈伤组织。

Cryopreservation of callus of Pinus elliottii ~ Pinus caribaea were conducted in this research. The results showed that the best process of cryopreservation for the embryogenic callus of P. elliottii ~ P. caribaea was as follows ： the callus were sub-cultured for 9 - 12 days in callus proliferation medium firstly and were pre-cultured in 0.5 mol/L sorbol for4 days, and then pretreated 20 minutes in buffer of 0.6 mol/L sorbol ＋ 10% DMSO, cooled to -40 ℃ at rate of -1 ℃/min, maintained at -40 ℃ for 10 min, preserved in iquid nitrogen after cooled to -90 ℃ at rate of -5 ℃/min. The resuscitate callus were kept in 37 ℃ water bath, clean three times in 1 mol/L sorbol liquid medium, passage in solid medium support by filter paper. New embryogenie callus will be produced within one month.