Varis Saila, Ahola Susanna, Jaakola Laura, Aronen Tuija
Natural Resources Institute Finland (Luke), Green Technology, Finlandiantie 18, FI-58450 Punkaharju, Finland.
Climate Laboratory Holt, Department of Arctic and Marine Biology, UiT The Arctic University of Norway, Tromsø, Norway; NIBIO, Norwegian Institute of Bioeconomy Research, Ås, Norway.
Cryobiology. 2017 Jun;76:8-17. doi: 10.1016/j.cryobiol.2017.05.004. Epub 2017 May 10.
Somatic embryogenesis (SE) is considered as the most-effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). For mass propagation, a cryopreservation method able to handle large numbers of embryogenic tissues (ETs) reliably and at low costs is needed. The aim of the present study was to compare pretreatments, cryoprotectants and slow-cooling devices for cryopreservation of Norway spruce ETs, with 12 variations of methods and a total of 136 spruce genotypes. Secondly, possible applications for cold storage of mature somatic embryos were studied with the aim of developing a flexible time window for embling production. At best, 100% of the embryogenic lines were recovered following cryopreservation, but the results varied among the sets of lines. Also physiological condition of the tissues, pre-treatment and cryoprotectant applied, as well as the slow-cooling device used were found to affect the recovery. The best option for cryopreservation of Norway spruce is to select fresh growth from young ETs as samples, pretreat them on semi-solid medium with increasing sucrose concentration (0.1 M for 24 h; 0.2 M for another 24 h), apply a mixture of polyethylene glycol 6000, glucose, and dimethylsulfoxide, 10% w/v each, as cryoprotectant and use a programmable freezer with a slow cooling rate (0.17 °C/min). On average, 87% of the genotypes can be recovered, without any effect on their genetic fidelity, as shown by microsatellite markers and embryo production capacity. Mature somatic embryos of Norway spruce can also be safely cold-stored at +4 °C, without adverse effects on their germination ability.
体细胞胚胎发生（SE）被认为是挪威云杉（Picea abies L. Karst）营养繁殖最有效的方法。对于大规模繁殖，需要一种能够可靠且低成本处理大量胚性组织（ETs）的冷冻保存方法。本研究的目的是比较挪威云杉胚性组织冷冻保存的预处理、冷冻保护剂和慢速冷却装置，采用12种方法变体，共涉及136个云杉基因型。其次，研究了成熟体细胞胚胎冷藏的可能应用，目的是为苗木生产开发一个灵活的时间窗口。冷冻保存后，最多有100%的胚性系得以恢复，但不同组系的结果有所不同。研究还发现，组织的生理状态、所采用的预处理和冷冻保护剂以及使用的慢速冷却装置都会影响恢复率。挪威云杉冷冻保存的最佳选择是从幼胚性组织中选取新鲜生长部分作为样本，在半固体培养基上用逐渐增加的蔗糖浓度（0.1M处理24小时；0.2M再处理24小时）进行预处理，使用聚乙二醇6000、葡萄糖和二甲基亚砜各10%（w/v）的混合物作为冷冻保护剂，并使用具有慢速冷却速率（0.17℃/分钟）的程序降温冷冻仪。平均而言，87%的基因型能够恢复，微卫星标记和胚胎生产能力表明，这对它们的遗传保真度没有任何影响。挪威云杉的成熟体细胞胚胎也可以在4℃安全冷藏，对其萌发能力没有不利影响。
