Study on cryopreservation technology for shoot tips of Malus ‘Red Splendor’
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摘要:
目的 为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，研究了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（CAT、AsA和GSH）和2种PCD抑制剂（Eth和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4～5mm带顶芽茎段，接种在含0.7mol.L-1蔗糖的预培养基后放入4 ℃冰箱冷锻炼2d；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20min，随后于0 ℃下PVS2溶液处理90min，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1min，再用Unloading溶液在室温下震荡洗涤2次，每次10min。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200U.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%。 Abstract:Objective In order to establish the cryopreservation technology system of Malus ‘Red Splendor’ sinensis and realize the preservation of its germplasm resources. Methods The effects of sucrose pretreatment concentration and time, Loading treatment time and PVS2 treatment time on the cryopreservation of Malus ‘Red Splendor’ shoot tips were investigated by adding three antioxidants (CAT, AsA and GSH) and two PCD inhibitors (Eth and SNP) to explore their effects on the post-freezing survival of shoot tips. Results (1) The procedure for the vitrification and preservation of Malus ‘Red Splendor’ shoot tips was as follows: firstly, Malus ‘Red Splendor’ histoponic seedlings were cut into 4-5 mm stem segments with terminal buds, inoculated in pre-culture medium containing 0.7 mol.L-1 sucrose and then placed in a 4 ℃ refrigerator for 2 d of cold exercise. Then they were cut into shoot tips of about 1.5-2 mm and put into Loading solution for 20 min at room temperature, then treated with PVS2 solution for 90 min at 0 ℃ and immediately freeze in liquid nitrogen; remove from the tank when required and quickly thawed in a water bath at 38°C for 1 min, they were washed twice with Unloading solution at room temperature by shaking for 10 min each time. By this method, the post-freezing survival rate of Malus ‘Red Splendor’ shoot tips was 66.58% and the recovery growth rate was 16.67%. (2) Different concentrations of exogenous additives had different effects on the post-freezing survival rate of Malus ‘Red Splendor’. Among them, the best added concentrations of CAT, AsA and GSH were 200 U.mL-1, 600 μmol.L-1 and 0.04 μmol.L-1, respectively, which increased the post-freezing survival rate by 20.28%, 6.75% and 27.61%, compared with the control, addition of Eth and SNP showed no significant positive effect. Conclusion Malus ‘Red Splendor’ shoot tips can be preserved at cryopreservation,exogenous addition of appropriate concentrations of GSH, CAT and AsA can increase the post-freezing rate of liquid nitrogen, with the most effective GSH increasing the recovery growth rate from 16.67% to 41.39%. -
图 1 茎尖玻璃化超低温保存及存活率检测
A.红丽海棠组培苗；B.预培养；C.切取1.5～2.5mm茎尖；D.茎尖在离心管中；E.经TTC染色变红（左）与未染色（右）的茎尖；F.冻后茎尖接种到恢复培养基中。
Figure 1. Shoot tip vitrification cryopreservation and viability testing
A. Tissue culture of Malus‘Red Splendor’; B. pre-culture; C. Cut the 1.5～2.5mm shoot tip; D. shoot tip in centrifugal tube;E. TTC stained red (left) and not red (right) shoot tips;F. Shoot tips were inoculated into recovery medium after freezing.
图 2 预培养基蔗糖浓度对液氮冻存后茎尖存活率的影响
不同小写字母（a，b，c，d）表示不同差异显著(P＜0.05)，下同。
Figure 2. Effect of sucrose concentration in premedium on survival rate of shoot tips after LN2 storage
Different lowercase letters (a, b, c, d) indicate different significant differences (P＜0.05), same as below.
图 3 预处理时间对液氮冻存后茎尖存活率的影响
Figure 3. Effect of pre-cultured time on survival rate of shoot tips after LN2 storage
图 4 Loading溶液处理时间对液氮冻存后茎尖存活率的影响
Figure 4. Effect of Loading solution treatment time on survival rate of shoot tips after LN2 storage
图 5 PVS2溶液处理时间对液氮冻存后茎尖存活率的影响
Figure 5. Effect of PVS2 treatment time on survival rate of shoot tips after LN2 storage
图 6 外源Eth对液氮冻存后茎尖存活率的影响
Figure 6. Effect of exogenous Eth on survival rate of shoot tips after LN2 storage
图 7 外源CAT对液氮冻存后茎尖存活率的影响
Figure 7. Effect of exogenous CAT on survival rate of shoot tips after LN2 storage
图 8 外源SNP对液氮冻存后茎尖存活率的影响
Figure 8. Effect of exogenous SNP on survival rate of shoot tips after LN2 storage
图 9 外源GSH对液氮冻存后茎尖存活率的影响
Figure 9. Effect of exogenous GSH on the survival rate of shoot tips after cryopreservation
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