Cryopreservation of Marchantia polymorpha spermatozoa

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.