Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortunately, cryopreservation protocols still need to be developed that are capable of producing a sizeable number of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatization and soil-supported growth. The thermal properties of water within the embryos were monitored using differential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a minimum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seedlings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and significantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut cultivars and is useful for the routine cryopreservation of coconut genetic resources.

译文

椰子的冷冻保存可用作支持建立生活收藏品的策略,这些收藏品的维护成本高昂,并且不断受到生物和非生物因素的威胁。不幸的是,仍然需要开发能够生产大量田间种植植物的冷冻保存方案。因此,我们报告了一种改进的冷冻保存方案的开发,该方案可用于各种椰子品种。通过应用四个优化步骤 (即 :( i) 快速脱水; (ii) 快速冷却; (iii) 体外快速升温和恢复,以及 (iv) 适应和土壤支持的生长,实现了合子胚的冷冻保存及其对土壤生长的恢复。使用差示扫描量热法 (DSC) 监测胚胎中水的热性能,以确保将可冻结的组分保持在最低水平。使用澳大利亚的马来亚黄矮星 (MYD) 品种评估了该方案的可行性,然后在印度尼西亚新鲜收获和研究的一系列品种上进行了测试。最有效的方案是基于8小时快速脱水步骤,然后是快速冷却步骤。当使用快速升温步骤和优化的体外培养步骤时,可获得最佳回收率。按照该方案,可以将冷冻保存的所有MYD胚胎的20% (在收获后12天冷冻保存时) 和40% (在收获时冷冻保存时) 返回到在土壤中生长的正常幼苗中。DSC显示,该方案诱导胚胎鲜重下降至19%,并显著减少了可产生冰晶的剩余水量 (0.1%)。在测试的20个品种中,发现有16个在低温保存后产生10% 至40% 个正常幼苗,而四个在0% 至10% 个正常幼苗之间产生。此新协议适用于广泛的椰子品种,对于椰子遗传资源的常规冷冻保存很有用。

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