PDF(Pubmed)
Abstract：
BACKGROUND QUERCUS: 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.
摘要：
背景：在常规条件下，难以干燥和冻结温度的种子不能储存在基因库中。然而,某些顽固种子物种的种质可以存储在液氮（-196°C）中。不幸的是,对于许多物种来说，其中几乎是整个钉螺属，一种有效的冷冻保存方法仍然未知。在这项研究中,我们提出了一种成功的冷冻存储协议（Matt。)利伯。使用在铝低温板上冷冻的羽毛（胚胎的茎尖分生组织）的种质。结果：从10个种源的橡子中分离出的羽状体预先储存在0.5M蔗糖溶液中（18h）。为了形成藻酸盐珠(每个珠一个小珠)，将管形管置于低温板的孔中，并包埋在海藻酸钙凝胶中。对于冷冻保护，在25°C下，将包封的管体浸入含有2.0M甘油和不同浓度的蔗糖（0.8-1.2M）的冷冻保护剂溶液中40分钟，并在层流柜下干燥1.0-4.0小时。将带有管体的冷冻板直接浸入液氮中，然后冷冻保存30分钟。为了复温，将具有管形的低温板浸入1.0M蔗糖溶液中，并在25°C下再水化15分钟。在1.0M蔗糖溶液中冷冻保护并干燥2h后，成活率从25.8到83.4不等。冷冻保存的烟羽的体外再生长率因种源而异，为26-77％。结论：本研究提出，第一次,一个成功的,可用于基因库的Q.petraea种质的冷冻保存简单有效的方案。该实验成功地重复了来自各种来源的种子，每个产生相似的，良好的结果。然而,种子质量和收获后的储存时间是冷冻保存后胚珠再生的重要因素。
