Plant cell Research Institute of BIO-FD&C Co. Ltd., Incheon, 21990, South Korea.
Korea Research Institute of Bioscience and Biotechnology, South Korea.
Plant Physiol Biochem. 2024 Sep;214:108866. doi: 10.1016/j.plaphy.2024.108866. Epub 2024 Jul 9.
Plant calli, a perpetually undifferentiated cell culture, have defects in maintaining their genetic fidelity during prolonged tissue culture. Cryopreservation using ice-binding proteins (IBP) is a potential solution. Despite a few studies on cryopreservation using IBPs in plant calli, detailed insights into the intracellular metabolism during freezing, thawing, and re-induction remain sparse. This study investigated and employed IBP from polar yeast Leucosporidium sp. (LeIBP) in the cryopreservation process across diverse taxa, including gymnosperms, monocots, dicots, and woody plants. Molecular-level analyses encompassing reactive oxygen species levels, mitochondrial function, and ATP and lipophilic compounds content were conducted. The results across nine plant species revealed the effects of LeIBP on callus competency post-thawing, along with enhanced survival rates, reactive oxygen species reduction, and restored metabolic activities to the level of those of fresh calli. Moreover, species-specific survival optimization with LeIBP treatments and morphological assessments revealed intriguing extracellular matrix structural changes post-cryopreservation, suggesting a morphological strategy for maintaining the original cellular states and paracrine signaling. This study pioneered the comprehensive application of LeIBP in plant callus cryopreservation, alleviating cellular stress and enhancing competence. Therefore, our findings provide new insights into the identification of optimal LeIBP concentrations, confirmation of genetic conformity post-thawing, and the intracellular metabolic mechanisms of cryopreservation advancements in plant research, thereby addressing the challenges associated with long-term preservation and reducing labor-intensive cultivation processes. This study urges a shift towards molecular-level assessments in cryopreservation protocols for plant calli, advocating a deeper understanding of callus re-induction mechanisms and genetic fidelity post-thawing.
植物愈伤组织是一种持续未分化的细胞培养物，在长时间的组织培养过程中会出现遗传保真度缺陷。使用冰结合蛋白（IBP）进行冷冻保存是一种潜在的解决方案。尽管已经有一些关于植物愈伤组织使用 IBP 进行冷冻保存的研究，但对于冷冻、解冻和再诱导过程中的细胞内代谢仍缺乏详细的了解。本研究调查并在包括裸子植物、单子叶植物、双子叶植物和木本植物在内的多个分类群中使用来自极地酵母 Leucosporidium sp. 的 IBP（LeIBP）进行了冷冻保存过程。进行了包括活性氧水平、线粒体功能以及 ATP 和脂溶性化合物含量的分子水平分析。对九个植物物种的研究结果表明，LeIBP 对解冻后愈伤组织的能力有影响，同时提高了存活率、降低了活性氧水平，并使代谢活性恢复到新鲜愈伤组织的水平。此外，LeIBP 处理和形态评估的物种特异性存活优化揭示了冷冻保存后细胞外基质结构的有趣变化，这表明了一种维持原始细胞状态和旁分泌信号的形态策略。本研究率先全面应用 LeIBP 于植物愈伤组织的冷冻保存，减轻细胞应激并提高了细胞活力。因此，我们的研究结果为确定最佳 LeIBP 浓度、解冻后遗传一致性的确认以及植物研究中冷冻保存进展的细胞内代谢机制提供了新的见解，从而解决了长期保存的挑战并减少了劳动密集型的培养过程。本研究呼吁在植物愈伤组织的冷冻保存方案中进行分子水平的评估，以深入了解愈伤组织的再诱导机制和解冻后的遗传保真度。
