Downey Cassandra D, Golenia Gregory, Boudko Ekaterina A, Jones Andrew Maxwell P
Canopy Growth Corporation, Smiths Falls, ON K7A 0A8, Canada.
Gosling Research Institute for Plant Preservation, Department of Plant Agriculture, University of Guelph, Guelph, ON N1G 2W1, Canada.
Plants (Basel). 2021 Aug 28;10(9):1794. doi: 10.3390/plants10091794.
Cannabis has developed into a multi-billion-dollar industry that relies on clonal propagation of elite genetics with desirable agronomic and chemical phenotypes. While the goal of clonal propagation is to produce genetically uniform plants, somatic mutations can accumulate during growth and compromise long-term genetic fidelity. Cryopreservation is a process in which tissues are stored at cryogenic temperatures, halting cell division and metabolic processes to facilitate high fidelity germplasm preservation. In this study, a series of experiments were conducted to optimize various stages of cryopreservation and develop a protocol for long-term germplasm storage of . The resulting protocol uses a standard vitrification procedure to cryopreserve nodal explants from in vitro shoots as follows: nodes were cultured for 17 h in a pre-culture solution (PCS), followed by a 20-min treatment in a loading solution (LS), and a 60 min incubation in plant vitrification solution 2 (PVS2). The nodes were then flash frozen in liquid nitrogen, re-warmed in an unloading solution at 40 °C, and cultured on basal MS culture medium in the dark for 5 days followed by transfer to standard culture conditions. This protocol was tested across 13 genotypes to assess the genotypic variability. The protocol was successful across all 13 genotypes, but significant variation was observed in tissue survival (43.3-80%) and regrowth of shoots (26.7-66.7%). Plants grown from cryopreserved samples were morphologically and chemically similar to control plants for most major traits, but some differences were observed in the minor cannabinoid and terpene profiles. While further improvements are likely possible, this study provides a functional cryopreservation system that works across multiple commercial genotypes for long-term germplasm preservation.
大麻已发展成为一个价值数十亿美元的产业，该产业依赖于具有理想农艺和化学表型的优良基因的克隆繁殖。虽然克隆繁殖的目标是生产基因一致的植物，但体细胞突变可能在生长过程中积累并损害长期的遗传保真度。冷冻保存是一种将组织储存在低温下的过程，可停止细胞分裂和代谢过程，以促进高保真种质保存。在本研究中，进行了一系列实验以优化冷冻保存的各个阶段，并制定了一种用于长期种质保存的方案。所得方案采用标准玻璃化程序冷冻保存离体芽的节段外植体，具体如下：节段在预培养溶液（PCS）中培养17小时，然后在装载溶液（LS）中处理20分钟，接着在植物玻璃化溶液2（PVS2）中孵育60分钟。然后将节段在液氮中快速冷冻，在40℃的卸载溶液中复温，并在黑暗中于基础MS培养基上培养5天，随后转移至标准培养条件下。该方案在13个基因型上进行了测试，以评估基因型变异性。该方案在所有13个基因型上均成功，但在组织存活率（43.3 - 80%）和芽的再生长（26.7 - 66.7%）方面观察到显著差异。从冷冻保存样本中生长的植物在大多数主要性状上在形态和化学上与对照植物相似，但在次要大麻素和萜类化合物谱方面观察到一些差异。虽然可能还有进一步改进的空间，但本研究提供了一种适用于多种商业基因型的功能性冷冻保存系统，用于长期种质保存。
