TITLE: Azolla filiculoides sporocarp cryo-preservation
AUTHORS: Paul Brouwer, Henriette Schluepmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Method extracted from </span><a href="https://doi.org/10.1111/nph.12708" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1111/nph.12708</span></a></div><div class = "text-block">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.</div></div>

[STEPS]
?. 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.Drying of megasporocarps collected from sediments (with massulae attached) were tested using batches of 247-391megasporocarps.
?. Drying at constant temperature of 26 degrees CelsiusUse 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.
?. 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.