Journal of Plant Research

, Volume 131, Issue 6, pp 1047–1054 | Cite as

Cryopreservation of Marchantia polymorpha spermatozoa

  • Taisuke Togawa
  • Tohru Adachi
  • Daijiro Harada
  • Tasuku Mitani
  • Daisuke Tanaka
  • Kimitsune Ishizaki
  • Takayuki Kohchi
  • Katsuyuki T. YamatoEmail author
Technical Note


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.


Antherozoid Bryophyte Liquid nitrogen Spermatozoid Ultra-low temperature 



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).

Supplementary material

10265_2018_1059_MOESM1_ESM.pdf (6.2 mb)
Supplementary material 1 (PDF 6342 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 SGY (MP4 8926 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)

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 SAY (MP4 4608 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)

10265_2018_1059_MOESM12_ESM.pdf (197 kb)
Supplementary material 12 (PDF 197 KB)


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Copyright information

© The Botanical Society of Japan and Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Taisuke Togawa
    • 1
  • Tohru Adachi
    • 1
  • Daijiro Harada
    • 1
  • Tasuku Mitani
    • 1
    • 2
  • Daisuke Tanaka
    • 3
    • 6
  • Kimitsune Ishizaki
    • 4
  • Takayuki Kohchi
    • 5
  • Katsuyuki T. Yamato
    • 1
    Email author
  1. 1.Faculty of Biology-Oriented Science and TechnologyKindai UniversityKinokawaJapan
  2. 2.Institute of Advanced TechnologyKindai UniversityKainanJapan
  3. 3.IBBP CenterNational Institute for Basic BiologyOkazakiJapan
  4. 4.Graduate School of ScienceKobe UniversityKobeJapan
  5. 5.Graduate School of BiostudiesKyoto UniversityKyotoJapan
  6. 6.Genetic Resources CenterNational Institute of Agrobiological SciencesTsukubaJapan

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