CRUNC: a cryopreservation method for unencapsulated gemmae of Marchantia polymorpha

Plant materials and culture condition

Thalli of a WT strain (female BC3-38 strain) and four transgenic lines (TG#060-1, TG#066-5, TG#164-3, and TG#253-6) of Marchantia polymorpha were asexually cultured on half-strength B5 (1/2 B5) agar medium under 75 µmol photons m⁻² s⁻¹ continuous white light (FL40SW; NEC Corporation, Tokyo, Japan) in a culture room at 22 °C. The white light intensity was measured using an LI-250A light meter (LI-COR Biosciences, Lincoln, NE, USA). These four transgenic lines were previously reported (Table 1) (Kimura & Kodama, 2016; Sakata et al., 2019; Fujii et al., 2020). The transgenic lines were maintained at the G1 generation in the culture room, and G2 gemmae were used in the following experiments.

Sterilization of silica-gel beads

Silica-gel beads (6 mesh up, No. 19000535; Hayashi Pure Chemical Ind., Ltd., Osaka, Japan) in a glass Petri dish (MINIP-3; Sansyo Co., Ltd., Tokyo, Japan) were sterilized by dry heat in an oven (WFO-400; EYELA, Tokyo, Japan) at 180 °C for 1 h, and then placed in a laminar flow hood (VCB-1300; Oriental Giken Inc., Tokyo, Japan) until use.

Calculation of recovery rate and dehydration rate

The recovery rate (%R) was calculated by dividing the number of gemmae recovered (NR) by the number of all gemmae tested (NT): %R = (NR/NT) × 100. The dehydration rate (%D) was obtained by dividing the weight of gemmae after dehydration (WA) by the weight of gemmae before dehydration (WB): %D = (1-(WA/WB)) × 100. For quantitative analysis, average and standard deviation were calculated from three experiments (see raw data in Supplemental File 1).

Basic CRUNC procedure

All equipment, products, and reagents used in this study are listed in Tables 2 and 3. To cryopreserve gemmae by the CRUNC method, we collected the gemmae from two gemma cups (approximately 100–300 gemmae) using straight tweezers under a laminar flow hood (Fig. 1A). The gemmae were put into a 1.5-mL tube under the hood, and the weight of the gemmae was measured with an electric balance. To dry the gemmae, a bead of silica gel was added to the tube using straight tweezers under the hood (Fig. 1B), and the capped tube was incubated on the benchtop at 22 °C for 1 h. To evaluate the degree of drying, the silica-gel bead was removed as shown in Video 1 and the weight of the gemmae in the tube was measured again.

After the gemmae were subjected to the drying process, the tube containing the gemmae without the silica-gel bead was directly transferred into the freezer at −80 °C for cryopreservation. To recover the cryopreserved gemmae, the gemmae were removed from the tube using straight tweezers under the hood (Fig. 1C), thawed with 200 µL sterile ultra-pure water, spread with curved tweezers on 1/2 B5 agar medium (Fig. 1D; Video 2), and then cultured in the culture room with continuous white light (75 µmol photons m⁻² s⁻¹) at 22 °C. Growth of the recovered gemmae was comparable to that of untreated gemmae (Fig. 1E).

The CRUNC method can be used to store unencapsulated gemmae for a long time. For example, when gemmae from the same gemma cup were divided into two groups and cryopreserved at −80 °C for 1 month and 5 months, the recovery rate (abbreviated %R) was unchanged between the two storage times (Fig. 1F).

A higher dehydration rate gives a higher efficiency of recovery

To determine the appropriate degree of drying of the gemmae for maximum recovery and survival, we compared the recovery rates of the gemmae at various levels of dehydration (abbreviated as %D). After incubation with the silica gel for 0, 20, 40, and 60 min, average dehydration rates were 0, 39.2, 54.8, and 62.0%D, respectively (Fig. 2A). After −80 °C cryopreservation of the dehydrated gemmae for 1 day, the average recovery rates were 0, 10.1, 90.4, and 91.1%R, respectively, indicating that a higher dehydration rate confers a higher recovery rate (Fig. 2A). When the gemmae were incubated with the silica gel for 5 h, both dehydration and recovery rates remained high (Fig. 2B). A scatter plot of the average recovery rates (30 experiments) shows that 50–80%D was required to give recovery rates above 80%R (Fig. 2C).

In a previous study that explored cryopreservation of unencapsulated gemmae, only dehydration time, but not dehydration rate, was evaluated (Wu et al., 2015). When Wu et al. (2015) dehydrated gemmae for 0, 3, 5, and 7 h, the recovery rates after cryopreservation for 1 day at −196 °C were 0, 68.4, 60.1, and 29.2%R, respectively. In their method, dehydration for 7 h significantly decreased the recovery rate (i.e., 29.2%R). In addition, a combination of dehydration for 5 h with cryopreservation for 2.5 months (75 days) also gave low recovery rates (18.9%R). In the CRUNC method, we focused not only on the dehydration time but also on the dehydration rate, and found that a higher dehydration rate gives a higher recovery rate (Figs. 2A–2C). When we tested dehydration of the gemmae for 24 h in the CRUNC method, the recovery rate was not decreased compared with shorter dehydration times (Fig. S1). Although the dehydration rates of the gemmae in the previous study are unknown (Wu et al., 2015), the recovery rate of the gemmae in the CRUNC method was higher than that of the previously described method. By contrast, a method for cryopreservation of encapsulated gemmae stably achieved 100% (Tanaka et al., 2016), while the CRUNC method achieved 100%R in only some tests (see raw data in Supplemental File 1). Therefore, the CRUNC method requires further improvement.

Gemmae from younger and older gemma cups are not suitable for the CRUNC method

Thalli of M. polymorpha grow radially with repeated dichotomous branching at the apex of each thallus, and a single gemma cup is generated on each branch of the thallus (Shimamura, 2016). Therefore, there are different aged gemma cups on multiple thalli of an individual M. polymorpha plant. To determine which gemma cups are most appropriate for the CRUNC method, we put a single gemma on agar medium, observed the growth and generation of gemma cups for 2 months, and then collected gemmae from the different aged gemma cups at the same time (Fig. 3A). The recovery rates of gemmae from younger (under 8 days after generation) and older (over 35 days after generation) gemma cups varied (Fig. 3B). The recovery rate was highest with gemmae from gemma cups at 10–33 days after generation (Fig. 3B). Therefore, gemmae taken from gemma cups at 10–33 days after generation should be used for the CRUNC method.

Dehydrated, unencapsulated gemmae can be cryopreserved in liquid nitrogen and frozen at −80 °C and −20 °C

Previous studies employed liquid nitrogen at −196 °C and a deep freezer at −80 °C to cryopreserve dehydrated unencapsulated and encapsulated gemmae (Wu et al., 2015; Tanaka et al., 2016). For the CRUNC method, we compared liquid nitrogen and freezer storage (deep freezer at −80 °C as well as a non-frost-free freezer at −20 °C). After dehydration of gemmae with a silica-gel bead, the unencapsulated gemmae were directly transferred to the three storage conditions and incubated for 1 day. There was no significant difference among the recovery rates in the three cryopreservation conditions (Fig. 4). These results show that dehydrated gemmae can be cryopreserved in liquid nitrogen (−196 °C), a deep freezer (−80 °C), or a freezer (−20 °C).

Gemmae cryopreserved by the CRUNC method can tolerate a freeze-thaw-freeze treatment

Freeze-thaw cycles can occur unexpectedly during a power outage. To determine the effect of thawing and refreezing of cryopreserved gemmae, we conducted a freeze-thaw-freeze treatment. After cryopreservation of the gemmae, the tube was thawed in an incubator at 28 °C for 1, 2, 3, and 4 h, followed by re-freezing at −80 °C. The recovery rate was unchanged by the thawing and refreezing treatment (Fig. 5), indicating that gemmae cryopreserved by the CRUNC method can survive a short-term thaw and refreezing. Note that this experiment mimics a power outage in non-frost-free freezers; because frost-free freezers undergo frequent freeze-thaw cycles, we recommend the use of non-frost-free freezers for the CRUNC method.

The recovery rate depends on the transgenic line

In the above experiments, WT gemmae of M. polymorpha (the BC3-38 strain) were used to develop the CRUNC method. Because the purpose of the present study is to improve the cryopreservation of genetically modified lines, we tested the CRUNC method on gemmae from four different transgenic lines (TG#060-1, TG#066-5, TG#164-3, and TG#253-6) that we reported previously (Table 1). We conducted six replicate experiments for each line and found that compared to the BC3-38 strain, the recovery rate of the transgenic gemmae varied, and was generally lower than that of the WT (Fig. 6). The varied recovery rate of transgenic gemmae suggested that the function(s) of the transgene(s) and/or the integration position of transfer-DNA in the genome might affect the fitness of the individual lines and thus their recovery rate. Therefore, for effective long-term storage of transgenic lines, a pre-check of the recovery rate is recommended.

Although the molecular mechanisms underlying the successful recovery of dehydrated and unencapsulated gemmae after cryopreservation remain to be determined, M. polymorpha does have natural dehydration and freezing resistance to survive dry and cold conditions during winter. Therefore, combining Agrobacterium-mediated genetic transformation with the CRUNC method might allow us to screen for genes involved in freezing resistance and recovery. These genes could be used to improve the survival of gemmae after dehydration and cryopreservation.