红丽海棠茎尖超低温保存技术研究

Cryopreservation of Malus Red Splendor Crabapple Shoots

【目的】建立红丽海棠超低温保存技术体系,实现其种质资源保存。【方法】以红丽海棠茎尖为试材,研究蔗糖预处理浓度和时间、装载处理时间、PVS2处理时间对其超低温保存后的存活率的影响,并在此基础上,在超低温保存程序中的不同环节添加3种抗氧化剂[过氧化氢酶(Catalase,CAT)、抗坏血酸(Ascorbic acid,AsA)和谷胱甘肽(Glutathione,GSH)]和2种细胞程序性死亡(PCD)抑制剂[(乙烯利Ethephon,Eth)和NO供体(Sodium Nitroprusside,SNP)],探讨其对茎尖冻后存活率的影响。【结果】(1)红丽海棠茎尖的玻璃化保存程序为:首先将红丽组培苗切为4~5 mm带顶芽茎段,接种在含0.7 mol·L^(-1)蔗糖的预培养基后放入4℃冰箱冷锻炼2 d;再将其切成约1.5~2.0 mm的茎尖,置于Loading溶液中,在室温下处理20 min,随后于0℃下PVS2溶液处理90 min,立即投入液氮冻存;需用时将其从液氮罐取出后,快速放入38℃水浴中化冻1 min,再用Unloading溶液在室温下震荡洗涤2次,每次10 min。采用此方法红丽海棠茎尖液氮冻后存活率为66.58%,恢复生长率为16.67%。(2)不同浓度的外源添加剂对红丽海棠冻后存活率影响不同。其中,CAT、AsA和GSH最佳添加浓度分别为200 U·ml^(-1)、400μmol·L^(-1)和0.04μmol·L^(-1),较对照冻后存活率分别提高了20.28%、6.75%和27.61%,添加Eth和SNP没有显示明显的正向作用。【结论】红丽海棠茎尖可以实现超低温保存,外源添加适宜浓度GSH、CAT、AsA可以提高液氮冻存后率,效果最好的GSH可将恢复生长率从16.67%提高到41.39%。

【Objective】Cryopreservation technology was applied for the conservation of the ornamental Malus Red Splendor crabapple germplasm.【Methods】Sucrose concentration and time for pretreatment as well as conditions for the subsequent loading,PVS2,and unloading treatments on the shoot tips of Malus spp.were studied.Under the selected preservation conditions,additional improvement on the post-freezing survival of the shoots by application of antioxidants(i.e.,CAT,AsA,and GSH)and PCD inhibitors(i.e.,Eth and SNP)was explored.【Results】(1)The procedures employed for vitrification to preserve the shoots included cutting the histoponic seedlings into 4-5 mm segments containing terminal buds,inoculating the cut stems segments in a pre-culture medium containing 0.7 mol·L^(-1) sucrose at 4℃for 2 d,cutting the tips into approximately 1.5-2 mm in length and placing in a loading solution for 20 m at room temperature,treating with PVS2 solution for 90 m at 0℃followed by immediate submerging in liquid nitrogen,removing the cryogenically frozen tips to rapidly thaw them in a water bath at 38℃for 1 m,and rinsing twice with an unloading solution at room temperature with constant shaking for 10 m each time.The post-cryopreservation survival rate of the shoot tips was 66.58%,and 16.67%of them recovered to grow as normal.(2)The best improvement by the addition of various agents was found to be CAT at 200U·mL^(-1),AsA at 600μmol·L^(-1),and GSH at 0.04μmol·L^(-1),which increased the post-freezing survival rate by 20.28%,6.75%,and 27.61%,respectively,over control.On the other hand,the addition of Eth or SNP posted no significant effect on the tip survival.【Conclusion】The survival rate of the post-cryopreservation Malus Red Splendor shoot tips could be further improved by the addition of GSH,CAT or AsA in appropriate concentration.GSH could most significantly raise the rate of viable plants after the cryopreservation from 16.67%to 41.39%.