Nov 15, 2019
Azolla filiculoides sporocarp cryo-preservation
- Paul Brouwer1,
- Henriette Schluepmann1
- 1Utrecht University
External link: https://doi.org/10.1111/nph.12708
Protocol Citation: Paul Brouwer, Henriette Schluepmann 2019. Azolla filiculoides sporocarp cryo-preservation. protocols.io https://dx.doi.org/10.17504/protocols.io.9bdh2i6
Manuscript citation:
Brouwer P, Bräutigam A, Külahoglu C, Tazelaar AO, Kurz S, Nierop KG, van der Werf A, Weber AP, Schluepmann H. A zolla domestication towards a biobased economy?. New Phytologist. 2014 May;202(3):1069-82. https://doi.org/10.1111/nph.12708
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 in our group and it is working.
Created: November 15, 2019
Last Modified: November 15, 2019
Protocol Integer ID: 29765
Keywords: Azolla filiculoides, Nostoc azollae, cryopreservation, megasporocarps, microsporocarps
Abstract
Method extracted from https://doi.org/10.1111/nph.12708
Brouwer P, Bräutigam A, Külahoglu C, Tazelaar AO, Kurz S, Nierop KG, van der Werf A, Weber AP, Schluepmann H. A zolla domestication towards a biobased economy?. New Phytologist. 2014 May;202(3):1069-82.
Attachments
nph.12708.pdf
1.7MB
Materials
Sporulating A. filiculoides Lam. was collected in mid-October 2012 from a ditch in Utrecht, the Netherlands (52°04′24″N; 5°08′53″E) and kept in demineralized water in a glasshouse at 5–15°C and 14 h days with a light intensity of at least 70 micromol/ s.m2 Photosynthetic Photon Flux Density (PPFD).
Mostly fertilized megasporocarps from sediment accumulating at the bottom of the containers in which sporulating cultures were kept was used for the cryo-preservation procedure. To purify these, the method according to Toia et al. (1987;https://doi.org/10.1111/j.1469-8137.1987.tb00142.x) was used except that the sorbitol: water step gradient was layered on top of the residue of the 200-microm mesh size sieve to obtain a clear layer of fertilized megasporocarps.
Unfertilized megasporocarps were also collected according to Toia et al. (1987;https://doi.org/10.1111/j.1469-8137.1987.tb00142.x) with modifications: sporulating plants were placed on top of a stack of sieves with 1000-, 500- and 200- lm mesh sizes and megasporocarps were detached from the stems using a strong water jet. Residue recovered on the 200- microm mesh size sieve was then layered on a 3 M sorbitol: water step gradient and centrifuged at 400 g for 10 min, resulting in a clear layer of pure megasporocarps. Megasporocarps were washed three times by centrifugation with 50 ml water before use for cross fertilization experiments.
For crosses, mature microsporocarps were plucked manually from the plants.
Preliminary knowledge: cryo-preservatives were not effective but drying was.
The cryopreservation protocols were explored on batches of 20-50 megasporocarps. Each condition was evaluated in duplicate.
For cryopreservation involving cryoprotectant pre-treatment, 1 ml of the cryoprotective solution was added to the megasporocarps immediately before snap-freezing in liquid nitrogen (LN). Batches were kept for 5 min in LN, then thawed on a heating plate set at 30°C for 1.5 min and washed thrice in 1 ml medium.
To test cryopreservation using a drying pre-treatment, megasporocarps were dried for 1, 4 and 8 d in the fume hood at RT and then snap frozen in LN without added fluid. Batches were kept for 5 min in LN then thawed for 1.5 min at 30°C before adding 1 ml medium.
For each cryopreservation protocol, a treatment control was included that was not frozen but still exposed to the cryopreservation pre-treatment; additionally two controls were included that received no treatment.
Table 1 Cryo-preservatives were not effective but drying was.
Drying of megasporocarps collected from sediments (with massulae attached) were tested using batches of 247-391megasporocarps.
Table 2 Constant temperature drying at 26 degrees for seven days yielded high viability after the freeze-thaw cycle.
Drying at constant temperature of 26 degrees Celsius
Use mature megasporocarps with sporocarps attached (we used what was found in the culture sediment), place these in an oven at 26 oC for 7 days, transfer to tubes fit for cryo-preservation.
Snap freeze, then transfer to -80 degree freezer for indefinite storage- we tried up until 4 years and this yielded the same viability.
Thaw the frozen megasporocarps with massulae attached by transfer into 400 micro liter of Azolla growth medium at pH 5.5 (Watanabe et al., 1992#) in a growth cabinet set at 25°C day: 15°C nights with 12 h light (40– 70 micromol/ s m2 PPFD). This can be up-scaled in small petri-dishes, but small volumes with small batches of megaspores allow for more controlled temperatures.
Spores may be scored for germination over a period of 6 wk. Morphological changes to the megasporocarps can be tracked under a binocular Leica Axioskop light microscope, using either x 10 or x 5 objectives.
We used a Leica SP2 confocal laser scanning fluorescence microscope, equipped with either x 16 or x 40 objectives and a helium–neon laser with excitation wavelength of 543 nm, to visualize N. azollae fluorescence in the range 630–670 nm. We visualized plant tissue fluorescence in the range 560–630 nm and/or 680–750 nm.
This is IRRI medium.AZOLLAIRRI.pdf
AZOLLAIRRI.pdf Good luck!
Please report tests of the method on other Azolla species here.
The aim of such endeavor should be to eventually generate a frozen biodiversity collection of Azolla ferns*.
* We have yet to verify whether the preservation method preserves other bacteria persistently associated with Azolla ferns (Dijkhuizen LW, Brouwer P, Bolhuis H, Reichart GJ, Koppers N, Huettel B, Bolger AM, Li FW, Cheng S, Liu X, Wong GK. Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify. New Phytologist. 2018 Jan;217(1):453-66).
Note: we found that megasporocarps and microsporocarps may also be stored wet- simply in water- in the 4oC fridge in our laboratory for up to 4 years.