Chlamydomonas is a single celled green alga that divides asexually under ideal growth conditions. When conditions deteriorate, the organism enters a sexual reproduction cycle. Gametogenesis can be induced by transferring strains from TAP auger to nitrogen deficient, and 10 auger cells are resuspended and agitated under strong light to generate modal MT plus and MT minus gat, which are then combined in a single flask for mating samples are spotted on A TAP auger plate and light, then wrapped in foil for zigas four maturation.
After five to seven days, vegetative cells are scraped off, exposing the zygotes, which are stuck tightly to the auger. These are transferred onto TAP DCO plates for germination. Under low light tetrads are then separated and grown under appropriate conditions.
Until visible clones appear, these can be picked for subsequent analysis. Hi, I am David Stern, president of Boy Thompson Institute for Plant Research. Today we're gonna show you procedures for mating and tetra dissection for the model green alga clam MOUs REINE hearty eye.
Many laboratories use these techniques to create vegetative diploids for analysis of dominance or following tread dissection to ascertain nuclear versus organelle inheritance. To test alm, to analyze epistasis, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organelle genotypes with particular nuclear genotypes.
I'd like to introduce you to my postdoctoral associate s Jan, who will take you through the details of the procedures. So let's get started. To start the experiment, transfer the MT plus and MT minus strains onto tris acetate phosphate or TAP plates Grow the cells in a one centimeter wide concentrated strip.
An intermediate transfer may be necessary for older stalks. Once a thick layer of growing cells appears, transfer the cells as a thick slab to N 10 plates. These plates are nitrogen deficient.
To induce gametogenesis incubate the cells for three days. After three days, check that the cells still appear dark green, discard any contaminated strains. Since they will not produce viable zygotes, the remaining strains are now ready to undergo gametogenesis.
Now that gametogenesis has been induced, start the mating by preparing two 50 milliliter flasks. Each containing 2.5 milliliters of sterile distilled water. Using a wire loop, transfer each strain to a separate flask and resand in the water.
Make sure there are no clumps. Gauge the cell concentration by eye. Add water to achieve equal cell concentrations.
Next, agitate the cells for two hours under strong light so that modal gametes will form. Check the motility under a light microscope. Remember that even poorly modal strains will mate.
In another 50 milliliter flask. Combine equal volumes of empty plus and empty minus strains to allow mating. Leave the gats under strong light, but do not agitate immediately check the mating under the light microscope.
Look for rapidly moving gamut pairs. Joined at the flagella, being careful not to agitate the mixture and disrupt mating pairs. Take a 300 microliter sample every hour for the next four hours and spot on a TAP auger plate.
Leave the unused mating mixture in the flask until the next day. Let the TAP auger plates dry in a sterile hood with the lids Aja until no surface liquid remains. Then leave the plates in the light for 18 hours.
Wrap the plates in foil since CIGA spore maturation occurs only in the dark and store the plates for one week. Finally agitate the flask containing the remaining mating mixture. If the medium appears clear, then zygotes remained attached to the surface of the flask, which indicates efficient mating.
If the agitation dislodges, the zygotes mating was inefficient or it failed during the following week, the zago spores will form on the plates and the tetras will be ready for dissection. After five days, the mated zygotes will have formed zago spores. So tetra dissection can begin using a dull scalpel.
Scrape off the vegetative cells, exposing the zygotes which stick tightly to the auger. Look under the microscope to distinguish zygotes from vegetative cells. Zygotes are larger, usually yellow, and have a black outline.
Next, prepare glass needles for manipulating the cells. Pull three millimeter glass rods an alcohol flame to generate a long thin thread. Break off the long thread.
Briefly heat the taper dent of the glass thread to bend it into a hook. Now use the glass needle to gather the zygotes into a small pile on the TAP auger plate. Form a square of auger containing the zygotes and excise it using a scalpel prior to transferring the zygotes.
For dissecting the tetrads, mark the bottom of a 1.5%TAP auger plate. Draw a horizontal line 1.5 centimeters from the top of the plate. Additionally, draw a grid with approximately 1.5 centimeters horizontal and 0.5 centimeters vertical spacing.
Then place the auger block face down on the top horizontal line drawn on the 1.5%TAP auger plate. Slide the auger block along the top horizontal line to distribute the zygotes. Finally, move individual zygotes to the intersections of the top horizontal lines with each vertical line ending with about 20 to 30 zygotes per plate.
Immediately after distributing the zygotes vert the plates for 30 seconds over a glass dish containing a thin layer of chloroform. Make sure the distance between the plate and the chloroform is five centimeters. And do not use too much chloroform.
This treatment will kill any vegetative cells that were transferred accidentally. Place the plates under low light for 16 hours to allow germination to occur. The next step will be tetrad dissection.
Now that the cells have finished incubating, look for germinated zygotes. These are swollen cells that have sometimes burst with daughter cells inside of them. If germination has occurred but the zago spore has not ruptured, gently touch it with a glass needle to break the wall.
Now begin separating the tetrads. Press the auger next to the daughter cells with the glass needle. The daughter cells are captured in the released liquid.
Drag the daughter cells in the puddle behind the needle to the grid points below the zygote. Now that the tetra is separated, look for a leftover shell remaining. After removing the daughter cells to make sure that a zygote was dissected and not a vegetative cell that divided twice, grow the dissected progeny until visible colonies can be picked for subsequent analysis.
Monitor the plates daily for contamination. When the protocol is performed correctly, the four dissected progeny from each zygote grow into visible clones. Two of the progeny are empty plus and two are empty, minus all of the progeny inherit the chloroplast, DNA of the MT plus parent.
We've just shown you standard methods for gametogenesis mating, zy germination, and tread dissection for clam dms. When using these techniques, it's important to make sure that the cells are healthy and without any infection. It is well known that laboratory adapted strains, which have not been crossed for long periods, may not mate well and water quality may also affect am mutagenesis.
By paying attention to these details and other technical details mentioned above, you should have much success with your climbing demon genetics. So that's it. Thanks for watching and good luck with your experiments.
