The overall goal of this procedure is to learn how to generate and manipulate parathesia development and ask APO discharge in culture. This is accomplished by first inducing and actively growing culture to initiate sexual development. The second step is to assay for active spore discharge, then recombinant parathesia are generated.
The final step is to evaluate the progeny for phenotypes that indicate whether or not recombination occurred. Ultimately, the results can be used to screen for mutants in sexual development or to generate multiple mutations within one strain. Visual demonstration of this method is critical as the steps are hard to describe, and although simple, they must be done precisely to obtain optimal spore discharge and consistent parathesia production.
Begin by center inoculating carrot auger in a six centimeter Petri dish with a fertile strain of f gramine. Arum incubate the inoculated dishes under bright standard commercial. Use fluorescent lighting at room temperature.
Allow fungi to grow for three to five days until mycelium has reached the outer edge of the dish. At the hood. Gently scrape the surface of the culture with the sterile toothpick to remove the aerial mycelia.
Then apply one milliliter of surfactant to the auger and rub into surface with the bent or bulbous end of a sterile glass rod. Without applying perfil. Place the plates back under the lights.
After 24 hours, the surface of the plates should have a shiny appearance. If mycelia reappear res scrape, surface, reapply tween 60 solution and bathe them in light for an additional 24 hours. Now observe the development of the parathesia for a week.
Young parathesia are visible as black grains on the auger surface. As compared to mature parathesia. There should be nearly no superficial mycelia.
By day seven mature spores accumulate on the plate lid, and by day nine, the spores are so dense that they're visible without magnification. To harvest the intermediate stage, parathesia gently scrape the surface of the auger with a scalpel and transfer the parathesia to a cryo tube. The tube can then be flash frozen for RNA extraction six days after applying the surfactant.
Cut a one centimeter diameter circle out of the auger with a wood, boer, or scalpel. Slice the circle in half and mount the halves on a glass microscope slide orient the pieces so the surface containing the fruiting bodies is perpendicular to the surface of the slide, and the spores are fired down the length of the slide. At this time, if an acro spore inhibitor is desired, apply it to the back of the auger block.
This technique can be used to screen chemicals for discharge inhibitors and to screen genetic mutants for loss of the ability to fire spores, we have successfully done both. Now, incubate the slide overnight in a simple illuminated room temperature humidified chamber. As the fruiting bodies erupt, spores accumulate on the slide and will be visible there in the morning.
Accumulated spores can now be washed off the slide collected in a clean dish and quantified. Begin by setting up a genetic cross between two strains of f gramine arum by inoculating them on opposite sides of a carrot auger petri dish. As previously described, grow the fungi until hyphie have filled the surface.
Scrape off the aerial portions and apply T between 60 solution. In this scenario, mark the plate where the hyphie meet. Return the cultures to the light and incubate them for about seven days when the parathesia have developed and Siri begin to form from the parathesia along the intersection between the cultures.
This technique can be used to combine multiple mutations into one strain. It's also worth noting that both knit plus and knit minus can be selected for making this a very useful selectable marker. Under the dissecting microscope, bring into view the parathesia along the intersection of the two cultures.
Then find the sear eye. Now dip a sterilized insect pin into sterile water and touch the pin to a serous formed at the border between the cultures. The SRIs should stick to the pin's moisture.
Then rinse the tip of the pin in 200 microliters of sterile water contained in a 1.5 milliliter micro centrifuge tube. Flame the pin, moisten it again, and pick another sru. Pick between three to five Siri and transfer them all to their own tube.
Now briefly vortex. Each tube containing suspended Siri for each tube. Spread 60 microliters of spore suspension on a nine millimeter Petri dish.
With MMTS medium, incubate the plates at room temperature with or without lights, and store the remaining spore suspension at four degrees Celsius for up to a week. After three to five days, very small colonies appear on the plate. One phenotype can be scored under a microscope.
Knit negative colonies will have sparser hyphy than knit positive colonies. Discard those plates with only one of the phenotypes. The Siri from recombinant parathesia have both colony phenotypes in approximately equal proportion.
Keep these plates. Sometimes other phenotypes result due to segregation of other factors such as colonies, which are intermediate between knit positive and knit negative for storage purposes, transfer the colonies from the recombinant plates to a V eight or carrot auger plate to continue the study by examining other phenotypes plate the colonies on Chap X dock's auger and on plates with PDA containing chlorate. Parathesia were grown into a black velvet like lawn without any mycelia or spores.
24 hours after the application of tween, the surface of the auger had a slight sheen. If Mycelia had reappeared, the plate may not have been scraped enough to induce parathesia development. Several blocks of auger with fruiting bodies from a wild type strain were set up to fire their spores on glass slides to observe.
Firing proper illumination was prepared. Parathesia that were too young or too old often did not fire. When too old, numerous hyphy appeared over the surface of the culture, giving it a whitish hue.
When too young, the fruiting bodies did not fire, and the experiment was repeated. 24 hours later when 10 Siri were collected from a cross, at least six were typically recombinant as scored by a mix of knit negative and knit positive phenotypes. Very occasionally a mutation prevented a strain from mating.
If one of the parents had an apparent growth phenotype, then more than two phenotypes were present among the colonies on MMTS. When attempting this procedure, it's important to remember to start with a culture that has been well-maintained and not sequentially transferred over time. Always start with a culture that's been grown from a stable freezer stock.
