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Morel Strain Creation
Morel Spore prints on a metal surface
Morel Spore Printing
Morel Spore Printing
Once you have collected morels, and you IDed the species, you can gather its spores to try and create your own new strains to use!
Leave the morel(s) in a mostly covered location, and leave for 24 hours undisturbed on an easy-to-manipulate surface (i.e. tin foil, an empty petri dish). If mature, you should come back to spores. The spores should be cream colored as shown in the picture to the left.
In a biosafety cabinet, 1.5 µL tubes, a morel spore print in a petri dish, a P1000 pipette and corresponding tips, and autoclaved deionized water.
Single Spore Isolation tools and equiptment:
Single Spore Isolation tools and equiptment:
Sterilized water
Enclosed, ventilated sterile environment, like a biosafety cabinet
P1000 pipette and fitting non-filtered tips
1.5-2mL tubes
~4 Potato Dextrose Agar (PDA) or Malt Yeast Extract Agar (MEYA) petri plates (These are the recommended media types)
Sterilizable Cell Spreader
Compound Microscope
Stainless Steel Biological Spatula
70% ethanol
Paper towels
Step 1:
Spore Mixture Prep
Bring your spores, tubes, pipette, and sterile water into your enclosed, ventilated sterile environment. In each of the tubes, add 900µL of the sterile water. Label the Top of the tubes: 1, 2, 3, 4
Tube 1 will have the highest concentration of spores at the end, and Tube 4 will have the lowest.
Step 2:
Creation of Spore Mixture
Lay out the morel spore print, and take 100µL of sterile water and place it on a section of the spore print. Using the pipette tip, mix the sporesup into the liquid, and pipette up and down to throughoutly encorporate the spores. Pipette back up the 100µL of spore-water mixture and add to Tube 1.
Step 3:
Serial Dilutions of Spore Mixture
Vortex Tube 1 for a couple of seconds, or flip to mix a couple of times to homogenize the spores evenly.
From Tube 1, Pipette 100µL of the mixture into Tube 2. Vortex Tube 2 or flip to mix and pipette 100µL into Tube 3.
Vortex Tube 3 or flip to mix, and pipette 100µL of the mixture into Tube 4.
Step 4:
Plating Spore Prep
Take the spore print, sterile water out of the sterile environment and clean the space with the 70% ethanol. Leave the 4 tubes, and the pipette.
Into the environment, bring in your agar media plates of choice (PDA or MEYA recommended), as well as a sterilizable cell spreader. The amount of plates you want to use is up to you and how many isolates you wish to make.
Step 5:
Plating Spore Mixture
Typically to get singular spore isolates, you will typically use only tubes 2,3,4, with tubes 3 and 4 the most successful.
To each plate you will pipette 100µL of a spore mixture onto the media plate and spread using a sterile spreader. Mark on the petri dish lid what species, the tube # you spread, the date, your initials, and a sample ID.
Sample ID is important so you can keep track of meta-data about the sample. Some example of information that you may want to collect is where the morel came from, the species, date the morel was found, ect.
Step 6:
Find Isolated Spores
Once it has been a ~24-36 hours, you can place the plate under a compound microscope. Typically spores can be found in the x10 objective. Anything hire you risk cracking the plate, and scratching the lenses.
Morel spores are typically elliptical or yellow/brown in color, and approximately 17-25µm in diameter. Once you find them, especially ones on their own and possibly starting to form hyphae, circle with a permanent marker them for ease of finding them later.
This spore in the picture is ideal as there is no other spores around it!
Step 7:
Replating Single Spores
Once you have found isolated spores, and they may have had some time to germinate, its time to move to their own media plate.
Working in sterile conditions, You can use the circles you did on the plate to find your single isolates, or as shown in the image above, you can see the germinated spores with your eye by catching the sheen if lighting is correct.
Use a sterile spatula and cut as close to the spore as possible, and transfer to a new media plate.
Parafilm or plastic cling wrap the two halves of the petri dish together, and store in a dry room temperature location.
Step 8:
Give Spores Germination Time
Store in a dry and room temperature environment for ~24-36 hours. Morel spores typically germinate quickly, and thus anywhere over 36 hours will risk losing isolates, as they may find a mate.
Step 9:
Mycelial Growth and Watch for Contamination
Step 9:
Mycelial Growth and Watch for Contamination
Watching for the speed of mycelial growth after re-plating is going to be important. For this study, we chose isolates that produced low melanin, had fast growth, and also had lots of microsclerotia
Watch for any discoloration. Morel mycelium is white with a possible slight brown tinge when young, turning more brown as it matures. If you see any other coloration, like green, black, yellow, and pinks, you have contamination. Early detection will be crucial to being able to hopefully save from the contamination taking over.
Step 10:
Morel Mating Type Identification
Step 10:
Morel Mating Type Identification
Once the mycelium has been grown out, and has reached the edges of the plate without contamination, identification of mating types can be important. Refresh your memory on morel mating types here
Into PCR tubes with extraction solution, take a small amount of mycelium (typically the size of a grain of sea salt) and place it into the tube. Using mating type primers for the specific species or phylum you are working with, as described by Chai et al. 2022, you can determine mating type!
In the photo above, Majority of the tested isolates were of MAT1-1, with the isolates 4, 8, and 7 suspected to be mated.
If you have suspected mated isolates, you can try PCR again to confirm it was not contaminated! If it occurs again, you have a poly-spore-isolate!
Step 11: {WILL ADD PICTURE SOON}
Mate selected isolates
Step 11: {WILL ADD PICTURE SOON}
Mate selected isolates
Poly-spore-isolation (isolates that have been mated already) and cross-mating type.
Mated isolates will be required to establish a viable, cultivatable strain!
Single-spore-isolates that are best cross are the ones that produce low melanin, had fast growth, and also produce lots of microsclerotia.
Poly-spore-isolates (as determined from the previous step) do not need to be cross-mated!
Morel strain SARE118 from the 2022-2023 growing season
Sources for Morel Spawn
Sources for Morel Spawn
He, P., Yu, M., Cai, Y., Liu, W., Wang, W., Wang, S., & Li, J. (2019). Effect of aging on culture and cultivation of the culinary-medicinal mushrooms morchella importuna and M. sextelata (Ascomycetes). International Journal of Medicinal Mushrooms, 21(11), 1089–1098. https://doi.org/10.1615/intjmedmushrooms.2019032891
Sources for Exogenous Nutrient Packs
Sources for Exogenous Nutrient Packs
Tan, H., Kohler, A., Miao, R., Liu, T., Zhang, Q., Zhang, B., Jiang, L., Wang, Y., Xie, L., Tang, J., Li, X., Liu, L., Grigoriev, I. V., Daum, C., LaButti, K., Lipzen, A., Kuo, A., Morin, E., Drula, E., … Martin, F. M. (2019). Multi‐omic analyses of exogenous nutrient bag decomposition by the Black Morel Morchella Importuna reveal sustained carbon acquisition and transferring. Environmental Microbiology, 21(10), 3909–3926. https://doi.org/10.1111/1462-2920.14741
Liu, Q., Ma, H., Zhang, Y., & Dong, C. (2017). Artificial cultivation of true morels: Current State, issues and perspectives. Critical Reviews in Biotechnology, 38(2), 259–271. https://doi.org/10.1080/07388551.2017.1333082
Xu, Y., Tang, J., Wang, Y. et al. Large-scale commercial cultivation of morels: current state and perspectives. Appl Microbiol Biotechnol 106, 4401–4412 (2022). https://doi.org/10.1007/s00253-022-12012-y
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