Sep 24, 2019
Marchantia spores sterilisation
- 1University of Cambridge;
- 2Plant Sciences, University of Cambridge, OpenPlant
- OpenPlant Project
Protocol Citation: Linda Silvestri, Eftychis Frangedakis, Marius Rebmann, Susana Sauret-Gueto 2019. Marchantia spores sterilisation. protocols.io https://dx.doi.org/10.17504/protocols.io.4wbgxan
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: June 28, 2019
Last Modified: September 24, 2019
Protocol Integer ID: 25251
Keywords: marchantia spore sterilisation milton
Guidelines
Use 2-4 sporangia per transformation planned
Add one Milton tablet into 25 mL of sterile water to prepare the "sterilization solution".
Add the sporangia into a 1.5 mL centrifuge tube and add 0.5 mL of sterilization solution into the tube (B in Figure)
Use sterile metal tweezers to crush the sporangia (C in Figure).
Place a 40 μM cell strainer into a 50 mL Falcon tube and pour the 0.5 mL suspension of crushed spore heads onto the filter and then wash the strainer with another 1 mL of sterilization solution (D in Figure).
Once filtered, aliquot the spore solution in 1.5 mL Eppendorf tubes (1.5 mL per tube) and incubate at room temperature for 10 min (the exposure to the sterilization solution should not exceed 25 min) (E in Figure).
10m
Centrifuge for 2 min at 15800 xg at room temperature, discard the supernatant and re-suspend the spores in sterilization solution (150 μl per transformation planned) (F in Figure).
2m
Spread 150 μl of re-suspended spores on a plate containing growth media (G in Figure), seal the lid with micropore tape and place under continuous light at 21 °C 150 μmol/m2/s for 7 days (H in Figure). Incubate with the bottom side up, which will prevent rhizoids from growing into the agar, making the removal of sporelings from the plate easier by simply using a sterile scalpel.