Nov 28, 2019
Marchantia Cryopreservation of Gemmae
Forked from Marchantia cryopreservation of gemmae
- Marius Rebmann1,
- Marta Marta Tomaselli2,
- Linda Silvestri3,
- Susana Sauret-Gueto1,
- Michelle Lim4
- 1Plant Sciences, University of Cambridge, OpenPlant;
- 2University of Cambridge, Open Plant;
- 3University of Cambridge;
- 4Plant Sciences, University of Cambridge, OpenPlant,
- OpenPlant Project
Protocol Citation: Marius Rebmann, Marta Marta Tomaselli, Linda Silvestri, Susana Sauret-Gueto, Michelle Lim 2019. Marchantia Cryopreservation of Gemmae. protocols.io https://dx.doi.org/10.17504/protocols.io.9vxh67n
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 28, 2019
Last Modified: November 28, 2019
Protocol Integer ID: 30359
Keywords: Cryopreservation, Marchantia, Gemmae
Abstract
Simplified cryopreservation protocol for Marchantia polymorpha gemmae, based on (Tanaka et al. 2016, Plant and Cell Physiology). Enables long term storage of gemmae at -80°C.
Materials
MATERIALS
AgarMelfordCatalog #A20021
Abscisic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1049
Sodium AlginateDuchefa BiochemieCatalog #S1320
Calcium chlorideVWR International (Avantor)Catalog #22328.262
SucroseFisher ScientificCatalog #10634932
GlycerolMerck MilliporeSigma (Sigma-Aldrich)Catalog #G5516
Gamborg B5 Medium with VitaminsDuchefa BiochemieCatalog #G0210
- Petridish, 9cm/4.5cm or 6/12-well transparent multi-well plates
- Liquid nitrogen (N2)
Reagent setup:
Preculture plates
1/2 strength Gamborg B5, pH 5.8 + 1.2% agar + 0.3 M sucrose + 10µM ABA
Autoclave media without ABA, add ABA from a filter sterilised stock solution just before pouring plates. 9cm/4.5cm petridishes or 6/12-well transparent multi-well plates can be used for plates, depending on desired throughput.
Alginate Solution
1/2 strength Gamborg B5 + 3% Sodium alginate
CaCl2 Solution
1/2 strength Gamborg B5 + 0.1M CaCl2
Loading solution
1/2 strength Gamborg B5 + 2M glycerol, + 1M sucrose
Thawing solution
1/2 strength Gamborg B5 + 1M sucrose
Rinse solution
1/2 strength Gamborg B5 + 1% sucrose
Preculture
Preculture
Collect fresh gemmae and plate on preculture plates, incubate 1-3 days under normal growth conditions (e.g. 21°C constant light)
Encapsulation
Encapsulation
Place small drops (approx. 40μL) of alginate solution on an empty 4.5cm petridish/multi-well plate to form beads.
Use forceps to transfer gemmae from preculture plates, place 1-5 gemmae inside each bead.
Proceed quickly to step 4 to avoid gemmae sinking to the bottom or edge of the bead.
Add a drop of CaCl2 solution to each bead, make sure to not displace gemmae from the bead.
Allow beads to solidify for 10min.
Use a serological pipette to fill the plate with loading solution, submerging the beads. Saok beads for 30min.
Use a serological pipette to remove the loading solution without disrupting the beads.
Remove lid and air dry beads in flowhood for >2h.
Use forceps or scalpels to transfer beads into 1.5mL Eppendorf tubes (max. 10 beads/tube).
Flash freeze tubes in liquid nitrogen for >2 minutes.
Move tubes to -80°C for long term storage.
Thawing and Recovery
Thawing and Recovery
Remove tubes from freezer and immediately place in a 37ºC water bath or heat block for 2 minutes.
Add 1.5mL thawing solution to each tube, incubate at room temperature for 10 minutes.
Replace thawing solution with rinse solution. Incubate at room temperature for 10 minutes.
Remove rinse solution, transfer beads on 1/2 Gamborg plates, incubate under normal growth conditions (e.g. 21°C constant light) until thalli grow