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Cryopreservation of Marchantia polymorpha spermatozoa

Abstract

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.

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Acknowledgements

We thank Kiyoshi Naruse, Ai Akimoto-Kato and Kagayaki Kato (National Institute for Basic Biology, Japan), Yoshihiko Hosoi (Kindai University) for technical advice, and Yoriko Matsuda, Aino Komatsu (Kyoto University), Yoshihiro Takikawa (Kindai University) for technical assistance. This work was supported by the Interuniversity Bio-Backup Project (IBBP) (Collaborative Study Project).

Author information

Correspondence to Katsuyuki T. Yamato.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 6342 KB)

Motility of spermatozoa before cryogenic treatment (MP4 8981 KB)

Motility of spermatozoa before cryogenic treatment (MP4 8981 KB)

Motility of spermatozoa after cryogenic treatment with sucrose (MP4 4631 KB)

Motility of spermatozoa after cryogenic treatment with sucrose (MP4 4631 KB)

Motility of spermatozoa after cryogenic treatment with sucrose SGY (MP4 8926 KB)

Motility of spermatozoa after cryogenic treatment with sucrose SGY (MP4 8926 KB)

Spermatozoa immediately after being released into SGY. Each spermatozoon remains motionless in the cell wall from spermatid (MP4 4640 KB)

Spermatozoa immediately after being released into SGY. Each spermatozoon remains motionless in the cell wall from spermatid (MP4 4640 KB)

‘Hatched’ spermatozoa after replacing SGY with water (MP4 4659 KB)

‘Hatched’ spermatozoa after replacing SGY with water (MP4 4659 KB)

Spermatozoa cryopreserved at –80˚C for a week (MP4 4732 KB)

Spermatozoa cryopreserved at –80˚C for a week (MP4 4732 KB)

Motility of spermatozoa after cryogenic treatment with SSY (MP4 4766 KB)

Motility of spermatozoa after cryogenic treatment with SSY (MP4 4766 KB)

Motility of spermatozoa after cryogenic treatment with SAY (MP4 4608 KB)

Motility of spermatozoa after cryogenic treatment with SAY (MP4 4608 KB)

Motility of spermatozoa after cryogenic treatment with SMY (MP4 4658 KB)

Motility of spermatozoa after cryogenic treatment with SMY (MP4 4658 KB)

Extraction of spermatozoa trajectories from a recorded movie by TrackMate (MP4 1554 KB)

Extraction of spermatozoa trajectories from a recorded movie by TrackMate (MP4 1554 KB)

Supplementary material 12 (PDF 197 KB)

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Togawa, T., Adachi, T., Harada, D. et al. Cryopreservation of Marchantia polymorpha spermatozoa. J Plant Res 131, 1047–1054 (2018). https://doi.org/10.1007/s10265-018-1059-0

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Keywords

  • Antherozoid
  • Bryophyte
  • Liquid nitrogen
  • Spermatozoid
  • Ultra-low temperature