Cao Dai Minh, Bui Anh Lan, Bui Le Van, Quach Phuong Ngo Diem
Plant Biotechnology Laboratory, Department of Plant Biotechnology and Biotransformation, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Viet Nam; Vietnam National University Ho Chi Minh City, Viet Nam.
Cryobiology. 2025 Mar;118:105186. doi: 10.1016/j.cryobiol.2024.105186. Epub 2024 Dec 4.
The valuable anti-cancer and anti-inflammatory secondary metabolite, imperatorin, has been found in the hairy roots (HRs) of Urena lobata. However, an increasing number of problems related to cryo-injury and cryoprotectant toxicity could potentially reduce the quality of root clones, highlighting the need to develop a reliable technique for long-term preservation. Based on the impact of the successive steps of the initial droplet-vitrification procedure employed for cryopreservation of HRs using the histological evaluation of plasmolysis, various selected factors were independently investigated. The maximum plasmolysis was observed after the preculture with 0.5 M sucrose (46.59 %) and the dehydration treatment (48.32 %). In the improved cryopreservation procedure, when prolonged preculture for 72 h in liquid WPM with 0.3 M sucrose and dehydration in the appropriate vitrification solution, which included 30 % (w/v) glycerol and 50 % (w/v) sucrose, for 10 min at 0 °C, the plasmolysis in the two steps was significantly reduced in comparison with the untreated control. The cryopreserved HRs could increase their regeneration to 93.3 % and regenerated root length to 4.65 cm. Their growth and imperatorin production were almost the same as those of untreated controls after three to five subsequent subcultures. Our results have demonstrated that our new approach, which only focuses on the key factors that significantly increase the plasmolysis, modifies their level to fit with the HR cells of U. lobata. The early application of the plasmolysis evaluation method may immediately screen the impact of factors on individual root cells instead of spending time and cost evaluating the recovery of root tips at each step to develop an efficient cryopreservation procedure.
在梵天花的毛状根中发现了具有抗癌和抗炎作用的珍贵次生代谢产物欧前胡素。然而，与冷冻损伤和冷冻保护剂毒性相关的问题越来越多，这可能会降低根克隆的质量，凸显了开发可靠的长期保存技术的必要性。基于采用组织学质壁分离评估法对梵天花毛状根进行冷冻保存时初始玻璃化法连续步骤的影响，对各种选定因素进行了独立研究。在用0.5M蔗糖预培养（46.59%）和脱水处理（48.32%）后观察到最大质壁分离。在改进的冷冻保存程序中，当在含0.3M蔗糖的液体WPM中延长预培养72小时，并在0°C下于适当的玻璃化溶液（包括30%（w/v）甘油和50%（w/v）蔗糖）中脱水10分钟时，与未处理的对照相比，这两个步骤中的质壁分离明显减少。冷冻保存的毛状根再生率可提高到93.3%，再生根长度达到4.65厘米。在随后进行三到五次继代培养后，它们的生长和欧前胡素产量与未处理的对照几乎相同。我们的结果表明，我们的新方法仅关注显著增加质壁分离的关键因素，并调整其水平以适应梵天花的毛状根细胞。质壁分离评估方法的早期应用可以立即筛选各因素对单个根细胞的影响，而不必花费时间和成本在每个步骤评估根尖的恢复情况来开发高效的冷冻保存程序。
