Martínez M T, Ballester A, Vieitez A M
Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Apartado 122, 15080, Santiago de Compostela, Spain.
Cryobiology. 2003 Apr;46(2):182-9. doi: 10.1016/s0011-2240(03)00024-5.
Oak embryogenic cultures are generally maintained by repetitive embryogenesis. To facilitate management of embryogenic lines and limit the risks of somaclonal variation and contamination a cryopreservation protocol should be developed. In this work we investigated the ability of several pre-treatments to enable 4-6mg clumps (1.0-1.5mm) of globular-heart stage somatic embryos of Quercus robur to withstand freezing in liquid nitrogen. In the best of the two embryogenic culture lines used, 56% of clumps resumed embryogenesis after cooling when they had been pre-treated by successive pre-culture on 0.3 and 0.7M sucrose supplemented media followed by desiccation in the air flow of a laminar flow cabinet to water contents of 24-34%. In both lines, embryogenesis resumption rates of about 70% were achieved by pre-culture on 0.3M sucrose medium followed by application of a vitrification solution (PVS2) for 60-90min prior to rapid plunging in liquid nitrogen.
栎树胚性培养物通常通过重复胚胎发生来维持。为便于胚性系的管理并限制体细胞克隆变异和污染的风险，应制定冷冻保存方案。在这项工作中，我们研究了几种预处理方法使4-6毫克团块（1.0-1.5毫米）的欧洲栓皮栎球形-心形期体细胞胚耐受液氮冷冻的能力。在所使用的两个胚性培养系中，当在补充有0.3和0.7M蔗糖的培养基上连续预培养，然后在层流罩的气流中干燥至含水量为24-34%后进行预处理时，56%的团块在冷却后恢复了胚胎发生。在两个培养系中，通过在0.3M蔗糖培养基上预培养，然后在快速投入液氮之前应用玻璃化溶液（PVS2）60-90分钟，胚胎发生恢复率达到了约70%。
