Cryopreservation of Gemmae from the Liverwort Marchantia polymorpha L

The liverwort Marchantia polymorpha L. is one of the key model plants in evo-devo studies, and an increasing number of transgenic and mutant lines have been established. For reliable long-term preservation of M. polymorpha plants, spores have been used, but crossing is indispensable to obtain them. Gemmae, however, are vegetative clones and readily available in large numbers without crossing, thereby enabling the clonal preservation and rapid propagation of transgenic or mutant lines. Here, we report a simple cryopreservation protocol for in vitro grown M. polymorpha gemmae using aluminum cryoplates. Gemmae were pre-cultured on sucrose-containing medium, embedded in calcium alginate gel on the surface of a cryoplate, moderately dehydrated and stored in liquid nitrogen. After rapid thawing, the stored gemmae showed a 100% survival rate. Our protocol does not require plant growth regulators such as ABA, and takes only 1 h to complete except for 1 d of pre-culture. Furthermore, gemmae treated as described above but then air-dried for 2 h can be stored at -80°C for at least 1 year without a significant decrease in survival rate, which is convenient for most laboratories that have a -80°C freezer but not a liquid nitrogen container for long-term storage. These preservation techniques for M. polymorpha should increase their availability in the research community.