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红丽海棠茎尖超低温保存技术研究

彭颖 陈友锋 乔谦 刘燕

彭颖,陈友锋,乔谦,等. 红丽海棠茎尖超低温保存技术研究 [J]. 福建农业学报,2023,38(5):566−573 doi: 10.19303/j.issn.1008-0384.2023.05.008
引用本文: 彭颖,陈友锋,乔谦,等. 红丽海棠茎尖超低温保存技术研究 [J]. 福建农业学报,2023,38(5):566−573 doi: 10.19303/j.issn.1008-0384.2023.05.008
PENG Y, CHEN Y F, QIAO Q, et al. Cryopreservation of Malus Red Splendor Crabapple Shoots [J]. Fujian Journal of Agricultural Sciences,2023,38(5):566−573 doi: 10.19303/j.issn.1008-0384.2023.05.008
Citation: PENG Y, CHEN Y F, QIAO Q, et al. Cryopreservation of Malus Red Splendor Crabapple Shoots [J]. Fujian Journal of Agricultural Sciences,2023,38(5):566−573 doi: 10.19303/j.issn.1008-0384.2023.05.008

红丽海棠茎尖超低温保存技术研究

doi: 10.19303/j.issn.1008-0384.2023.05.008
基金项目: 雄安新区科技创新专项(2022XAGG0100);国家自然科学基金项目(31770741)
详细信息
    作者简介:

    彭颖(1998−),女,硕士,研究方向:植物种质资源保存(E-mail:pengying_hallo@163.com

    通讯作者:

    刘燕(1963−),女,博士,教授,研究方向:植物种质资源保存(E-mail:chblyan@163.com

  • 中图分类号: S 685.99

Cryopreservation of Malus Red Splendor Crabapple Shoots

  • 摘要:   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、400 μmol·L−1和0.04 μmol·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%。
  • 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1.5~2.5 mm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.  Survival rate of post-cryopreservation shoot tips

    A: Crabapple tissue culture; B: pre-culture; C: 1.5-2.5 mm cut of shoot tip; D: shoot tip segment in centrifugal tube; E: TTC stained red (left) and original (right) shoot tips; F: post-frozen shoot tips inoculated on recovery medium.

    图  2  预培养基蔗糖浓度对液氮冻存后茎尖存活率的影响

    不同小写字母表示不同差异显著(P<0.05),下同。

    Figure  2.  Effect of sucrose concentration in pretreatment medium on survival rate of post-cryopreservation shoot tips

    Different lowercase letters 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 pretreatment time on survival rate of post-cryopreservation shoot tips

    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 post-cryopreservation shoot tips

    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 post-cryopreservation shoot tips

    图  6  外源Eth对液氮冻存后茎尖存活率的影响

    Figure  6.  Effect of added Eth on survival rate of post-cryopreservation shoot tips

    图  7  外源CAT对液氮冻存后茎尖存活率的影响

    Figure  7.  Effect of added CAT on survival rate of post-cryopreservation shoot tips

    图  8  外源SNP对液氮冻存后茎尖存活率的影响

    Figure  8.  Effect of added SNP on survival rate of post-cryopreservation shoot tips

    图  9  外源GSH对液氮冻存后茎尖存活率的影响

    Figure  9.  Effect of added GSH on survival rate of post-cryopreservation shoot tips

    图  10  外源AsA对液氮冻存后茎尖存活率的影响

    Figure  10.  Effect of added AsA on survival rate of post-cryopreservation shoot tips

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出版历程
  • 收稿日期:  2022-12-20
  • 修回日期:  2023-03-22
  • 网络出版日期:  2023-05-24
  • 刊出日期:  2023-05-28

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