Wasileńczyk Urszula, Wawrzyniak Mikołaj Krzysztof, Martins João Paulo Rodrigues, Kosek Paulina, Chmielarz Paweł
Kostrzyca Forest Gene Bank, Miłków, Poland.
Institute of Dendrology, Polish Academy of Sciences, Kórnik, Poland.
Plant Methods. 2024 Apr 12;20(1):53. doi: 10.1186/s13007-024-01161-y.
seeds that are recalcitrant to desiccation and freezing temperatures cannot be stored in gene banks under conventional conditions. However, the germplasm of some recalcitrant seeded species can be stored in liquid nitrogen (-196 °C). Unfortunately, for many species, among them for almost the whole genus Quercus, an effective cryostorage method is still unknown. In this study, we propose a successful cryostorage protocol for Quercus petraea (Matt.) Liebl. germplasm using plumules (a shoot apical meristem of an embryo) frozen on aluminium cryo-plates. RESULTS: The plumules isolated from the acorns of ten provenances were prestored in 0.5 M sucrose solution (for 18 h). To form alginate beads (one plumule per bead), the plumules were placed in the wells of a cryo-plate and embedded in calcium alginate gel. For cryoprotection, the encapsulated plumules were immersed in cryoprotectant solution containing 2.0 M glycerol and different concentrations of sucrose (0.8-1.2 M) for 40 min at 25 °C and desiccated under a laminar flow cabinet for 1.0-4.0 h. Cryo-plates with plumules were directly immersed in liquid nitrogen and then cryostored for 30 min. For rewarming, cryo-plates with plumules were immersed in 1.0 M sucrose solution and rehydrated for 15 min at 25 °C. Survival rates varied from 25.8 to 83.4 were achieved after cryoprotection in 1.0 M sucrose solution and the drying of plumules for 2 h. The in vitro regrowth rate of cryopreserved plumules varied among provenances and was 26-77%. CONCLUSIONS: This study presents, for the first time, a successful, simple and effective protocol for the cryopreservation of Q. petraea germplasm that could be used in gene banks. The experiment was successfully repeated on seeds from various provenances, each yielding similar, good results. However, seed quality and storage time after harvesting are important factors in plumule regrowth after cryopreservation.
对脱水和冷冻温度敏感的种子不能在常规条件下保存在基因库中。然而，一些顽拗性种子物种的种质可以保存在液氮（-196°C）中。不幸的是，对于许多物种，其中几乎包括整个栎属，一种有效的冷冻保存方法仍然未知。在本研究中，我们提出了一种成功的欧洲栓皮栎（Quercus petraea (Matt.) Liebl.）种质冷冻保存方案，该方案使用在铝制冷冻板上冷冻的胚芽（胚的茎尖分生组织）。结果：从十个种源的橡子中分离出的胚芽预先保存在0.5M蔗糖溶液中（18小时）。为了形成藻酸盐珠（每个珠一个胚芽），将胚芽放置在冷冻板的孔中并包埋在海藻酸钙凝胶中。为了进行冷冻保护，将包封的胚芽在25°C下浸入含有2.0M甘油和不同浓度蔗糖（0.8 - 1.2M）的冷冻保护剂溶液中40分钟，并在层流柜中干燥1.0 - 4.0小时。带有胚芽的冷冻板直接浸入液氮中，然后冷冻保存30分钟。为了复温，将带有胚芽的冷冻板浸入1.0M蔗糖溶液中，并在25°C下复水15分钟。在1.0M蔗糖溶液中进行冷冻保护并将胚芽干燥2小时后，存活率在25.8%至83.4%之间。冷冻保存的胚芽的体外再生率因种源而异，为26% - 77%。结论：本研究首次提出了一种成功、简单且有效的欧洲栓皮栎种质冷冻保存方案，可用于基因库。该实验在来自不同种源的种子上成功重复，每个种源都产生了相似的良好结果。然而，种子质量和收获后的储存时间是冷冻保存后胚芽再生的重要因素。
