| 张茜.中华猕猴桃‘红阳’茎尖小滴玻璃化法超低温保存技术优化[J].中国南方果树,2026,55(2): |
| 中华猕猴桃‘红阳’茎尖小滴玻璃化法超低温保存技术优化 |
| Cryopreservation of shoot tip of Actinidia Chinesis L. ‘Hongyang’by droplet vitrification |
| 投稿时间：2025-05-15 修订日期：2025-11-27 |
| DOI：10.13938/j.issn.1007-1431.20250226 |
| 中文关键词: 猕猴桃 茎尖 小滴玻璃化法 超低温保存 |
| 英文关键词:Actinidia Chinesis shoot tip droplet-vitrification cryopreservation |
| 基金项目:四川省科技厅基本科研业务费项目（2021JDKY0023）；四川省“十四五”农作物及畜禽育种攻关项目（2021YFYZ0023-05）；四川省科技计划（2024ZHYS0012） |
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| 中文摘要: |
| 【目的】建立中华猕猴桃小滴玻璃化法茎尖超低温保存技术体系，为猕猴桃种质资源长期保存提供新途径。【方法】基于小滴玻璃化超低温保存法，以红肉中华猕猴桃‘红阳’为材料，通过单因子和双因子试验研究预处理培养基、预处理时间和脱水冷冻时间对猕猴桃茎尖超低温保存效果的影响，并通过染色体倍性检测保存后再生苗的遗传稳定性。【结果】优化后的中华猕猴桃茎尖小滴玻璃化法超低温保存技术流程：切取‘红阳’猕猴桃组培苗的顶芽茎尖（1.5-2 mm）；预培养基（MS+0.5 mol/L蔗糖）中振荡（100 rpm/min）预培养液中1 d；在加载液（MS+0.4 mol/L蔗糖+2 mol/L甘油）中加载20 min（0 ℃冰上）；在PVS2玻璃化液（MS+15% 乙二醇+15% DMSO+0.4 mol/L蔗糖）中脱水处理40 min（0 ℃冰上）；将脱水处理后的茎尖置于铝箔条上的PVS2小液滴中，快速浸入液氮中保持至少1 min，再转入液氮储存罐中进行保存；卸载液（MS+1.2 mol/L蔗糖）中解冻处理20 min（35 ℃水浴）；茎尖转入再生培养基（MS+1.0 mg/L ZT+0.1 mg/L NAA+30 g/L蔗糖+7.5 g/L琼脂）中暗培养；1周后转入新鲜培养基中，在正常光照下恢复培养。茎尖经该方法液氮冻存一个月后的成活率和再生率分别为55%和50%。倍性检测结果表明再生植株无染色体倍性变异，遗传稳定。【结论】本试验建立的中华猕猴桃茎尖小滴玻璃化法超低温保存技术简便高效，为中华猕猴桃种质资源冷冻保存库的建立提供技术依据。 |
| 英文摘要: |
| Abstract 【Objective】Establish a technical system for cryopreservation of shoot tips by the small drop vitrification method of Actinidia Chinesis, providing a new approach for the long-term preservation of kiwifruit germplasm resources.【Method】Based on the drop vitrification cryopreservation method, using the red-fleshed Actinidia Chinesis 'Hongyang' as the material, the effects of pretreatment medium, pretreatment time and dehydration freezing time on the cryopreservation effect of kiwifruit shoot tips were studied through single-factor and two-factor experiments, and the genetic stability of the regenerated seedlings after cryopreservation was detected by chromosomal ploidy.【Result】The optimized cryopreservation technical process of the shoot tip of Actinidia Chinesis, by drop vitrification: Cut the shoot tip of the terminal bud of the tissue culture seedlings of 'Hongyang' kiwifruit (1.5-2 mm); Shake in the pre-culture medium (MS+0.5 mol/L sucrose) at 100 rpm/min for 1 day in the pre-culture medium; Load in the loading solution (MS+0.4 mol/L sucrose +2 mol/L glycerol) for 20 minutes (on ice at 0 ℃); Dehydration treatment was carried out in PVS2 vitrification solution (MS+15% ethylene glycol +15% DMSO +0.4 mol/L sucrose) for 40 minutes (on ice at 0 ℃); The dehydrated shoot tips were placed in the small PVS2 droplets on the aluminum foil strips, quickly immersed in liquid nitrogen for at least 1 minute, and then transferred to the liquid nitrogen storage tank for preservation. Thawing treatment in the unloading solution (MS+1.2 mol/L sucrose) for 20 minutes (in a 35 ° C water bath); The shoot tips were transferred to the regenerative medium (MS+1.0 mg/L ZT+0.1 mg/L NAA+30 g/L sucrose +7.5 g/L AGAR) for dark culture; One week later, it was transferred to fresh culture medium and resumed culture under normal light. The survival rate and regeneration rate of the shoot tips after being frozen in liquid nitrogen by this method for one month were 55% and 50% respectively. The ploidy test results indicated that the regenerated plants had no chromosomal ploidy variation and were genetically stable.【Conclusion】The cryopreservation technology of Actinidia Chinesis shoot tip small drop vitrification established in this experiment is simple and efficient, providing a technical basis for the establishment of a cryopreservation bank for Actinidia Chinesis germplasm resources. |
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