Protonema induction, transient transformation, and protoplast regeneration in the peat moss Sphagnum papillosum

Sphagnum mosses are important carbon sequesters and emerging model organisms. However, induction and long-term cultivation of thalloid protonema in several species was not achievable so far. Here, we provide protocols for a set of new tools relevant for Sphagnum molecular biology: a new way for Sphagnum protoplast isolation and regeneration, and a first protocol for transient protoplast transformation. Together, these protocols will support the emerging Sphagnum research community in basic and applied science. Advancing molecular research in climate-critical plants.


Introduction
Peat mosses of the genus Sphagnum comprise most of the biomass in northern peatlands, storing over 500 Gt of carbon, while covering only 3% of the earth's land mass (Gorham 1991;Clymo et al. 1998;Yu et al. 2021). As climatecritical plants, Sphagnum mosses have gained interest in recent years (Beike et al. 2015;Weston et al. 2018). While there are a number of axenic Sphagnum laboratory strains (Heck et al. 2021a) and cultivation techniques available (Batra et al. 2003;Beike et al. 2015;Zhao et al. 2019;Heck et al. 2021b), the toolbox for working with Sphagnum mosses under axenic conditions is still small. While Sphagnum gametophores are easy to propagate (Beike et al. 2015;Heck et al. 2021a, b), juvenile protonema tissue, from which the gametophores arise, is naturally formed only after spore germination (Clymo and Hayward 1982;Beike et al. 2015). Upon wounding, gametophores will eventually form thalloid protonemata, a method described in Zhao et al. (2019), where the protonemata of S. squarrosum had to be sub-cultivated regularly to prevent gametophore formation. So far, protoplasts were isolated from gametophore apices of S. fallax (Batra et al. 2003). This is laborious and contrary to other mosses, where protoplasts are isolated from protonemata as an "easier to handle" material, as protonemata are grown in suspension culture in different volumes (Hohe and Reski 2002; 1 3 Schween et al. 2003;Hohe et al. 2004). So far, regeneration of Sphagnum protoplasts has been achieved only with a chlorophyll-free Solanum hybrid clone as feeder (Batra et al. 2003) which hampers subsequent analyses. Here, we report on an efficient method for protonema induction, which works in different Sphagnum species, as well as long-term cultivation, preventing differentiation of gametophores, and allowing growth of homogenous, thalloid protonemata in suspension. Moreover, we provide protocols for protoplast isolation from protonemata, the first protocol for transformation of Sphagnum cells and protoplast regeneration without feeder cells.

Media, moss lines and cultivation
-Axenic Sphagnum strains are available from the International Moss Stock Center (www. moss-stock-center. org) and published in Heck et al. (2021a). -All cultivation techniques used for Sphagnum gametophores are described in Beike et al. (2015) and Heck et al. (2021a). -Suspension culture for protonema induction and preculture prior to protoplast isolation were performed in 50 mL and 200 mL of media. -Media: (1) Knop ME pH 5.4: standard moss medium, described in Beike et al. (2015). Osmolarity Osmolarity should be checked using an osmometer with several measurements. For this, a freezing point depression osmometer was used. Adjust osmolarity via mannitol concentration ± 5 mOsm.

General advice
While growth conditions for Sphagnum gametophores have been established for many species, induction of protonemata can be difficult. We advise always using more than one sample when trying to induce protonemata or isolate protoplasts. Sphagnum protoplasts are very sensitive to mechanical stress; use wide-mouth pipettes, transfer them slowly and be sure to use the correct osmolarity in the desired media. Cultures should be checked for contaminations regularly as described in Heck et al. (2021a). All steps should be performed under axenic conditions with sterilized material, while working in a laminar flow hood.

Protonema induction and long-term cultivation
Sphagnum gametophores are disrupted into approx. 1 cm long pieces, using sterile forceps and cultivated for 6 weeks on solid Knop ME medium pH 5.4, flooded with PM, i.e. Knop ME pH 5.4 including 50 mM ammonium tartrate (10 mL per 9 cm petri dish). Apices and disrupted stems will form protonemata on wounded tissue ( Fig. 1A-D).
This thalloid tissue can be isolated using a stereoscope which was surface-sterilized while working in a laminar flow cabinet. Isolated protonema tissue can be maintained in suspension culture with protonema medium (PM).
Until growth of thalloid tissue is visible, the material should not be transferred to fresh media, to prevent protonema loss during the process.
Regular disruption using a dispersion tool (IKA T25 digital ULTRA-TURRAX©, IKA© Werke GmbH & Co. KG, Staufen, Germany; 1 min 15,000 rpm), at the start of inoculation with material from a plate once a month is sufficient. To obtain homogenous material, we recommend disruption and sub-cultivation of protonema material every 2-4 weeks (Fig. 1E, F). For rapid proliferation, weekly disruption and sub-cultivation is advised.
Long-term storage of protonemata in PM is possible (Fig. 1G, H), but culture densities should be kept below 1 g/L dry weight to prevent gametophore formation. Dry weight is estimated as described in Heck et al. (2021a), using 3 × 10 mL protonema suspension culture from a 200 mL culture (200-800 mg/L dry weight). It was possible to recover gametophores from over 1-year old protonema cultures without sub-cultivation after switching to Knop ME pH 5.4.
(2) Transfer protonemata to a petri dish (in our experience small plate diameters work better, e.g. 3-6 cm) and mix carefully with a 2% Driselase solution (5 mL for 6 cm petri dish), dissolved in PIM (see Hohe et al. 2004). Place in darkness on a tumble shaker at low speed for 18 h. All further steps follow the protocol described in Hohe et al. (2004) and have been adapted considering protoplast density and regeneration. (3) After 18 h, filtrate protoplasts by using a sieve with first 100 µm, then 50 µm pore size. Wash with PIM (without Driselase) (10 mL). (4) Sediment protoplasts by centrifugation (50 G max., slow brake/acceleration, 10 min) and wash by replacing supernatant with PIM (10 mL). (5) Repeat step 4 twice. (6) After the last washing step, concentrate all protoplasts in one centrifugation tube to assess protoplast density using a Fuchs-Rosenthal counting chamber. Expect low yields (5000-10,000/mL), with many dead cells being present. (7) Set protoplast density to 250,000/mL in TM for transformation. In our experience around 150,000 proto-plasts per 200 mL protonema culture with 100 mg/L dry weight can be expected.

Transformation
(1) 200 µL protoplast solution (50,000 protoplasts) per sample is used. (4) Mix gently by adding first 1 mL, then 2, 3, and 4 mL TM every 5 min. (5) Sediment cells by centrifugation (500 rpm, slow brake/ acceleration, 10 min) and discard the supernatant. (6) See protoplast regeneration step 1 for further cultivation. If you do not want to regenerate and only analyse transient transformation rates, you may use 2 mL of TM instead of RM. (7) Fluorescence of protoplasts can be checked 2 days after transformation using an appropriate microscope ( Fig. 2A-C).

Protoplast regeneration
(1) Add 2 mL of RM to protoplast pellets and transfer them to a 3 cm petri dish or 6-well plate, cultivating them in standard long-day conditions (16 h light/8 h dark) (Beike et al. 2015;Heck et al. 2021a).
(2) After 30 days, protoplasts will form an outgrowth from which further cell divisions start (Fig. 2D, E). (3) For the next 2 weeks, a quarter of the media should be replaced weekly with Knop ME. (4) 6 weeks after protoplast regeneration started (Fig. 2F), half of the supernatant media is discarded, while the remaining solution is plated onto solid Knop ME plates (volume should consider density of regenerating protoplasts and plate size).
(5) 2 weeks after plating, single protoplasts have regenerated into thalloid protonema structures, with filamentous protonemata growing downwards into the agar (Fig. 2G). (6) Around 3 months after protoplast isolation, a single gametophore has grown out of a single protoplast (Fig. 2H, I). In contrast to the low protoplast yield, regeneration of gametophores from protoplasts, once they started growing, works very well and a high percentage (~ 70%) of regenerants can be expected with the protocols presented here.

Transient transformation construct
We tested several promoters (Act5, CA200, 35S, AtUbq10) widely used in Physcomitrella research (Horstmann et al. 2004;Weise et al. 2006;Orellana-Escobedo et al. 2015;Peramuna et al. 2018) using citrine, GFP, YFP, and the luciferase reporter system. We did not detect any activity using different plasmid DNA concentrations and protoplast densities. The only promoter for which an activity in Sphagnum was detectable was the Arabidopsis-ubiquitin promoter AtUbq10 fused to YFP (Fig. 2B, C).

Problem Solution
Protoplasts do not form pellets after centrifugation Increase centrifugation time to 30 min (do not increase rpm). Make sure transformation tubes are devoid of any possible PEG residues, as this might lead to insufficient pellet formation of protoplasts Almost all/all protoplasts are dead Make sure to use wide-mouth pipettes, transfer slowly and make sure that your centrifugation steps are executed at slow acceleration and brake. Also check osmolarity and pH of your protoplast media Protoplasts form large aggregates during transformation/ regeneration Decrease density of protoplasts to about 50,000 per 2 mL regeneration medium or per transformation approach Differentiation of protonema to gametophores in suspension culture Decrease density of gametophores or increase concentration of ammonium tartrate in your suspension medium

Discussion
We present protocols which, for the first time, make "easy to take care of" cultivation of protonemata from different Sphagnum species possible. All techniques described so far for the cultivation of protonemata and protoplasts from Sphagnum plants showed inherent difficulties (Batra et al. 2003;Zhao et al. 2019). While protonema induction is still a difficult and low-yielding process, we improved the cultivation of protonemata, allowing efficient production of homogenous material for protoplast isolation (Hohe and Reski 2002) and long-term storage (Schulte and Reski 2004) in different species. We also provide the first protocol

Declarations
Conflict of interest All authors declare that they have no competing interests.
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