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.

Introduction

The liverwort Marchantia polymorpha L. belongs to the group of basal land plants and is an excellent reference organism to understand the evolution of land plants (Bowman et al. 2007). Many of the regulatory systems found in angiosperms, such as responses to plant hormones and light, are conserved in M. polymorpha (Tougane et al. 2010, Ishizaki et al. 2012, Komatsu et al. 2014, Kubota et al. 2014, Eklund et al. 2015, Kanazawa et al. 2016, Kato et al. 2015). However, ongoing genomic analysis strongly suggests that the redundancy of regulatory genes in M. polymorpha is remarkably low. Furthermore, M. polymorpha is haploid during most of its life cycle, which is a significant advantage regarding mutagenesis and subsequent genetic analyses (Ishizaki et al. 2013b, Ishizaki et al. 2016). Marchantia polymorpha propagates not only sexually but also asexually, which enables clonal maintenance and propagation of lines of interest.

Agrobacterium-mediated transformation of M. polymorpha is simple and efficient (Ishizaki et al. 2008, Kubota et al. 2013), and it is applicable for gene targeting using homologous recombination (Ishizaki et al. 2013a) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated) technology (Sugano et al. 2014). The biolistic transformation procedure is available not only for the nuclear genome (Takenaka et al. 2000, Chiyoda et al. 2008) but also for the plastid genome (Chiyoda et al. 2007). Consequently, the number of transformants and mutants generated is increasing, and maintaining and sharing such plant resources is of interest to current and prospective M. polymorpha users.

Seeds are one of the most successful developments occurring after the emergence of land plants. Because seeds are naturally designed for long-term survival, they are routinely used for the stock and distribution of angiosperm model plants. In contrast, bryophytes do not have such physiologically robust, multicellular tissues for reproduction, and instead produce single-celled spores that can survive for long periods of time under conditions unfavourable for growth. Properly desiccated spores of M. polymorpha can be stored for years in a refrigerator or freezer (Nakazato et al. 1999). However, the collection of spores requires crossing, which is a process which takes several months. In addition, crossing generates genetic variation in the F1 spores, which could be a problem for subsequent molecular genetic analyses (Ishizaki et al. 2016).

The most convenient and frequently used method for the maintenance of M. polymorpha plants is the subculture of gemmae, and >100 gemmae are formed in a mature cupule. Thalli are another good source for subculture, as apical meristems grow continuously with occasional bifurcation. Even without apical meristems, thalli can be regenerated and grown into intact plants under regular culture conditions (Nishihama et al. 2015). In most cases, thalli grown aseptically on agar plates can be conveniently and reliably preserved at 4°C for several months; however, the survival rate of thalli after extended storage (>1 year) is unpredictable. Furthermore, the time and labor required for managing subcultures increase as the number of plants increases, and frequent subculture may cause accumulation of spontaneous mutations, which can compromise experimental reproducibility. Therefore, reliable long-term preservation methods that allow little or no plant growth are desirable.

Pence (1998) described a method for the preservation of M. polymorpha thalli in liquid nitrogen. Thalli were first pre-cultured in medium containing 10 µM ABA for 1 week, cut into small pieces and encapsulated in alginate beads. The beads were dehydrated in Murashige and Skoog (MS) medium containing 0.75 M sucrose for 18 h, air-dried for 3–4 h and then placed in liquid nitrogen. The requirement for pre-culture in the presence of ABA is consistent with the observation that M. polymorpha accumulates late embryogenesis abundant proteins in response to the drought signal mediated by ABA and acquires desiccation tolerance (Hatanaka et al. 2014).

Marchantia polymorpha and many other bryophyte species can propagate asexually by gemmae, which are clones of the plant that produced them. Gemmae can be readily recovered from cupules on the surface of thalli, and are at the same developmental stage, since they are dormant and do not start growing in the absence of water. These features make gemmae suitable for a range of experiments and for preservation. Gemmae of M. polymorpha can survive several months of desiccation at room temperature (Takezawa 2011). When slowly desiccated in a Petri dish, gemmae on thalli survive, whereas thalli themselves do not, suggesting that desiccated gemmae are adequate for long-term preservation. However, desiccation is a long process that has not been critically evaluated.

Freezing is a common method to store biological materials. However, freezing generally causes lethal damage to cells because of intracellular ice formation. Therefore, vitrification, i.e. solidification by cooling without ice formation, is critical in order to cryopreserve living materials alive. For successful vitrification, the intracellular solute concentration must be elevated via dehydration and then cooled rapidly enough to prevent the formation of ice crystals (Engelmann 2000).

Vitrification of tissues in liquid nitrogen is a suitable preservation method for certain plant species, as reported by Sakai et al. (1990). In this process, dehydration tolerance is induced using appropriate treatments, and tissues are then dehydrated using a vitrification agent and placed in liquid nitrogen (Sakai et al. 1990). Tanaka et al. (2011) demonstrated the advantage of the droplet method, in which apices of in vitro grown black chokeberry are attached to an aluminum plate (cryoplate) and rapidly cooled in liquid nitrogen. Later, Yamamoto et al. (2011) showed that the droplet method using cryoplates improved the survival of a variety of plant species. In the present study, we optimized the vitrification-based cryoplate (V-Cryoplate) method developed by Yamamoto et al. (2011) to enable simple, fast and reliable long-term storage of M. polymorpha gemmae.

Results

Optimization of factors affecting cryopreservation

The V-Cryoplate method (Yamamoto et al. 2011) requires three critical steps prior to immersion in liquid nitrogen. First, plant materials to be cryopreserved are pre-cultured to induce dehydration tolerance. Next, the pre-cultured materials are encapsulated in alginate beads on a cryoplate to enable slow dehydration in the following step. Finally, the plant materials are dehydrated to prevent ice formation. Because bead encapsulation on cryoplates is a well-established method, the other two steps were optimized in this study.

Marchantia polymorpha gemmae were pre-cultured in medium containing 0.3 M sucrose for 0, 1 or 3 d, gel-encapsulated on a cryoplate, dehydrated in loading solution (LS; 2 M glycerol, 1 M sucrose) for 30 min, and cooled rapidly in liquid nitrogen. After rapid warming to 23°C in 1 M sucrose solution, gemmae were grown and their survival rate was evaluated. In the absence of pre-culture, 66.1% of gemmae survived storage in liquid nitrogen, whereas all pre-cultured gemmae survived (Fig. 1), indicating that pre-culture with sucrose for at least 1 d is essential and sufficient to achieve a survival rate of 100%.

Fig. 1

Effect of length of pre-culture. Marchantia polymorpha gemmae were pre-cultured on medium containing 0.3 M sucrose for the indicated days, embedded in alginate beads on cryoplates, dehydrated with loading solution (LS) for 30 min and subjected to cryopreservation (−196°C, 5 min). To thaw the cryopreserved gemmae, the cryoplates were soaked in 1 M sucrose at 23°C. Error bars represent the SEs of triplicate measurements. **P < 0.01 compared with samples without pre-culture (‘0’) (paired t-test).

Cryopreserved gemmae appeared normal after warming (Fig. 2A) and showed visible growth at 2 d after restoration (Fig. 2B). The growth and development of gemmae with or without cryopreservation were indistinguishable (Fig. 2C, D). Gemmae maintained in alginate beads and those separated from the beads after cryopreservation showed no significant difference in growth, indicating that the removal of alginate beads after cryopreservation is not necessary. This method was fully applicable to other M. polymorpha accessions, such as Takaragaike-2 (Tak-2; female) and those provided by the University of Oxford Botanic Garden.

Fig. 2

Cryopreservation does not affect the growth and development of M. polymorpha gemmae. Gemmae were pre-cultured on medium containing 0.3 M sucrose overnight, embedded in alginate beads on cryoplates, dehydrated with LS for 30 min and subjected to cryopreservation at −196°C for 5 min. To thaw the cryopreserved gemmae, the cryoplates were soaked in 1 M sucrose at 23°C. (A) A cryopreserved gemma encapsulated in an alginate bead after warming. (B) A cryopreserved gemma grown for 2 d. (C and D) Plants grown out of cryopreserved and fresh gemmae, respectively, after 30 d of culture.

Cryopreservation at higher temperature

To promote dehydration, gemmae that had been partially dehydrated with LS for 30 min as described above were further subjected to slow desiccation under continuous airflow and maintained at −80°C for 2 weeks without immersion in liquid nitrogen. The extent of dehydration affected the survival rate of gemmae stored at −80°C (Fig. 3A). The survival rate of gemmae air-dried for 120–200 min was 100%, whereas prolonged air-drying had a negative effect on survival. To evaluate the survival rate after longer storage periods, gemmae air-dried for 120 min were stored at −80°C for 10 d to 1 year (Fig. 3B). All of the cryopreserved gemmae resumed normal growth (Supplementary Fig. S1). Desiccated gemmae prepared as described above can be transferred from a −80°C freezer to liquid nitrogen without affecting the survival rate.

Fig. 3

Effect of desiccation time during storage at −80°C. (A) Gemmae were pre-cultured overnight, embedded in alginate beads, dehydrated with LS for 30 min and air-dried for various lengths of time, and their survival rate after storage at −80°C for 2 weeks was examined. To thaw the cryopreserved gemmae, the cryoplates were soaked in 1 M sucrose at 23°C. (B) Survival rate of gemmae air-dried for 120 min after storage at −80°C for the indicated length of time. All experiments were performed in triplicate, and error bars indicate the SEs.

Finally, we evaluated the individual effects of LS treatment and air-drying on the survival rate of gemmae at −80°C (Fig. 4). Freshly collected gemmae without any treatment did not survive storage at either −196 or −80°C (Fig. 4 ‘direct LN2’ and ‘direct −80°C’, respectively), indicating that appropriate treatments are required prior to cryopreservation. Eliminating either LS treatment or air-drying (Fig. 4 ‘without LS’ and ‘without air-drying’, respectively) drastically reduced the survival rate at −80°C, indicating that both steps are indispensable for successful cryopreservation. A quick and reliable technique for storage at higher temperatures, such as −80°C, should make M. polymorpha even more attractive for molecular genetic studies.

Fig. 4

The two dehydration steps are indispensable for cryopreservation at −80°C. Freshly collected gemmae were processed differently and their survival rates were evaluated. ‘Direct LN2’, stored in liquid nitrogen for 5 min without any treatment. ‘Direct −80°C’, stored at −80°C for 2 weeks without any treatment. ‘Without LS’, pre-cultured on medium containing 0.3 M sucrose overnight, embedded in alginate beads on cryoplates, dehydrated by air-drying for 2 h and stored at –80°C for 2 weeks. ‘Without air-drying’, pre-cultured on medium containing 0.3 M sucrose overnight, embedded in alginate beads on cryoplates, dehydrated with LS for 30 min and stored at –80°C for 2 weeks. ‘Optimal’, pre-cultured on medium containing 0.3 M sucrose overnight, embedded in alginate beads on cryoplates, dehydrated by air-drying for 2 h and then with LS for 30 min, and stored at –80°C for 2 weeks. In all experiments, gemmae were soaked in 1 M sucrose at 23°C to thaw. Error bars represent the SEs of triplicate measurements.

Discussion

The optimized cryopreservation protocols described in this report are summarized in Fig. 5. As mentioned above, gemmae of M. polymorpha that have been slowly desiccated in culture plates can be preserved long term, although the process requires several weeks. In contrast, the V-Cryoplate method using liquid nitrogen described here requires overnight pre-culture and <1 h for encapsulation and dehydration. The other method requires an additional 2 h for air-drying. Both methods should enable the evaluation and optimization of cryopreservation of transgenic or mutant plants that might have lost desiccation tolerance.

Fig. 5

Schematic representation of the vitrification-based cryoplate procedures for cryopreservation of M. polymorpha gemmae.

Gemmae can be preserved at 4°C for several months, similar to thalli, and at lower temperatures after desiccation (Supplementary Fig. S2). Water evaporates slowly from agar plates sealed with porous and breathable tape, which results in the desiccation of plants. Gemmae on plants desiccated in this manner can often be preserved for months at room temperature (Takezawa 2011) and even years at −20 or −80°C (Supplementary Fig. S2). However, several weeks are necessary to achieve sufficient desiccation, and the survival of desiccated gemmae is not guaranteed.

Dehydration of plant materials, which is a critical step for successful cryopreservation, is performed in a highly hypertonic solution, such as PVS2 [plant vitrification solution 2; MS medium containing 0.4 M sucrose, 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide (DMSO)] (Sakai et al. 1990), or under airflow (Uragami et al. 1993). Although M. polymorpha gemmae cannot survive in liquid nitrogen without proper pre-treatment, our preliminary experiments showed that their cryopreservation does not require extensive dehydration using PVS2 (Supplementary Fig. S3). This differs from the original V-Cryoplate method Yamamoto et al. (2011). Less extensive dehydration using LS (2 M glycerol, 1 M sucrose) was effective for cryopreservation even without pre-culture (Fig. 1, ‘0’), whereas gemmae treated with LS containing a lower concentration of sucrose (0.4 M) died after storage in liquid nitrogen.

Although storage in liquid nitrogen is highly reliable, the storage of live plant samples at −80°C in a laboratory ultra-low temperature freezer would be more convenient for most researchers. In the protocol described above, gemmae to be preserved were only partially dehydrated and rapidly cooled to −196°C in liquid nitrogen to avoid intracellular ice formation and ensure vitrification. Intracellular water remains vitrified in liquid nitrogen because its glass transition temperature (Tg) is −134°C (McMillan and Los 1965). In general, the Tg of a given aqueous solution increases in correlation with increased solute concentration. Therefore, extensive dehydration, i.e. air drying (Figs. 4, 5), to raise the Tg of intracellular aqueous components is essential prior to vitrification of tissues at higher temperatures, such as −80°C.

The moss Physcomitrella patens is a widely used model plant, and its cryopreservation method was established more than a decade ago (Schulte and Reski 2004). Gametophores of P. patens are first cultured in the presence of 8.7% mannitol, 10 µM ABA and 100 mM proline for 1 week. The pre-cultured plants are then cut into small pieces and incubated in a cryoprotectant containing 20% DMSO and 25% glucose for 1–3 h prior to cooling. For cryopreservation, the plant materials are cooled from 20°C to −35°C at a rate of −1°C min–1, maintained at −35°C for 10 min and then placed in gaseous nitrogen at −152°C for long-term storage. The cryopreservation of M. polymorpha gemmae requires fewer reagents and steps because they are inherently more tolerant to stress (Takezawa 2011).

In the cryopreservation method developed by Pence (1998), thalli were first pre-cultured in the presence of 1.5% sucrose and 10 µM ABA for 1 week, cut into 2 mm2 pieces, encapsulated in alginate beads, air-dried for 3–4 h, placed in cryovials and then rapidly cooled in liquid nitrogen. Tissues preserved in cryovials were thawed at room temperature for 20 min. However, when this method was applied to the M. polymorpha strains used in this study, the survival rate was 25% at the highest (5 out of 20 plants). In our study, gemmae pre-cultured without ABA were fully viable after cryopreservation, probably because gemmae are inherently tolerant to cryopreservation. In general, cryopreservation becomes more challenging as the number of cell types and the size of the tissue to be preserved increase (Baust et al. 2009). Gemmae of M. polymorpha are smaller and their structure is less complex than that of thalli, allowing uniform vitrification and making gemmae suitable for cryopreservation. For mutants that are deficient in gemma production, the meristematic tissue at the tip of thallus can be similarly cryopreserved. The survival rate is nearly 100% at the highest, but the protocol must be further optimized for that particular tissue.

The preservation technique described in the present study is characterized by the use of aluminum cryoplates. Gemmae to be cryopreserved are embedded in alginate beads on cryoplates. This allows a larger number of gemmae to be handled with ease and reproducibility, and damage caused by the direct handling of beads is minimized. The cryoplates used in this study are not commercially available at present, and thus can be provided by the authors upon request, though the number available is limited. Alternatively, one can make one’s own aluminum cryoplates according to Yamamoto et al. (2011). Aluminum foil or thin metal plate can substitute for cryoplates, but foil is too flexible and it may require skill to form gel-beads appropriately on a plate without pits. The least recommended alternative is to handle gel beads without cryoplates. In this case, it is difficult to keep dehydration and temperature conditions uniform among beads, which may cause a compromised survival rate. Finally, we found that gemmae that were dehydrated by PVS2 showed a 100% survival rate even without the use of cryoplates.

The techniques described in this study should contribute not only to the preservation and maintenance of standard, mutant and transgenic lines of M. polymorpha, but also to the establishment of a stock center for M. polymorpha, similar to the International Moss Stock Center for P. patens, and to the preservation of endangered bryophyte species after appropriate optimization.

Materials and Methods

Plant materials and growth conditions

Gemmae from a male accession of M. polymorpha, Takaragaike-1 (Tak-1), were used (Ishizaki et al. 2008), unless otherwise specified. Plants were grown aseptically on half-strength MS or B5 solid medium in cycles of 12 h of white fluorescent light (30 µmol m−2 s−1) and 12 h of darkness at 23°C, and maintained by subculturing gemmae of approximately 0.3 mm in diameter.

Treatments prior to cryopreservation

Gemmae were pre-cultured on half-strength MS agar plates containing 0.3 M sucrose at 23°C. Gemmae were then transferred onto pits (1 mm diameter) of cryoplates (37 mm×7 mm) (Yamamoto et al. 2011), and embedded in alginate beads by applying a drop of 3% sodium alginate solution and then pouring on 0.1 M CaCl2 solution. Calcium alginate beads were solidified for 15 min (Tanaka et al. 2004). The gel-encapsulated gemmae on the cryoplates were soaked in loading solution (2 M glycerol, 1 M sucrose; LS) for 30 min for dehydration. For cryopreservation at −80°C, LS-treated gemmae were air-dried under laminar airflow in a clean hood.

Cryopreservation and restoration

The cryoplates with gel-encapsulated gemmae were immersed in liquid nitrogen (−196°C) for 5 min. Cryoplates to be stored at −80°C were contained in cryovials and then placed in a −80°C freezer for the indicated times. For thawing, the cryoplates were soaked in 1 M sucrose at 23°C to elevate the temperature quickly. Once thawed, gemmae were transferred onto fresh solid medium without removing the alginate gel and grown as described above. The survival rate of cryopreserved gemmae was evaluated at 1 month after thawing, and the number of plants that showed growth was counted. Each experiment used 12 plants and was repeated three times.

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by the Interuniversity Bio-Backup Project (IBBP) [Collaborative Research Project].