Togawa Taisuke, Adachi Tohru, Harada Daijiro, Mitani Tasuku, Tanaka Daisuke, Ishizaki Kimitsune, Kohchi Takayuki, Yamato Katsuyuki T
Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa, Wakayama, 649-6493, Japan.
Institute of Advanced Technology, Kindai University, Kainan, Wakayama, 642-0017, Japan.
J Plant Res. 2018 Nov;131(6):1047-1054. doi: 10.1007/s10265-018-1059-0. Epub 2018 Jul 31.
The liverwort Marchantia polymorpha has become one of the model organisms, since it has less genetic redundancy, sexual and asexual modes of reproduction and a range of genomic and molecular genetic resources. Cryopreservation of fertile spermatozoa eliminates time, space and labor for growing and maintaining male plants in reproductive phase, and also provides an optional way to backup lines. Here we report a protocol to cryopreserve spermatozoa of M. polymorpha in liquid nitrogen. A cryoprotective solution containing sucrose, glycerol and egg yolk and controlled cooling and warming processes led to successful recovery of motile M. polymorpha spermatozoa after the cryogenic process. The survival rate and average motility of spermatozoa after cryopreservation were maintained at 71 and 54% of those before cryopreservation, respectively. Cryopreserved spermatozoa were capable of fertilization to form normal spores. The technique presented here confers more versatility to experiments using M. polymorpha and could be applied to preservation of plant spermatozoa in general.
地钱多形苔已成为模式生物之一，因为它具有较少的基因冗余、有性和无性繁殖方式以及一系列基因组和分子遗传资源。可育精子的冷冻保存消除了在生殖阶段种植和维护雄性植株所需的时间、空间和劳动力，还提供了一种备份品系的可选方法。在此，我们报告一种在地钱多形苔中冷冻保存精子于液氮中的方案。一种含有蔗糖、甘油和蛋黄的冷冻保护溶液以及控制的冷却和升温过程，使得冷冻保存后的地钱多形苔活动精子成功复苏。冷冻保存后精子的存活率和平均活力分别维持在冷冻保存前的71%和54%。冷冻保存的精子能够受精形成正常的孢子。这里介绍的技术为使用地钱多形苔的实验赋予了更多的通用性，并且一般可应用于植物精子的保存。
