The overall goal of this procedure is to transform SCO Metre Protoplasts. This is accomplished by first digesting the plants with Drizly. Next, the protoplasts are washed with mannitol to remove the drizly and are then incubated with DNA and peg.
Finally, the transformed protoplasts are spread onto plates and incubated. Ultimately, results can be obtained. That show transformed protoplasts expressing fluorescently labeled proteins through fluorescence microscopy Demonstrating the procedure will be Y lu, a postdoc from my laboratory.
The main advantage of this technique is that it is effective and consistently generates protoplasts that express the protein of interest To isolate moss Protoplasts begin by adding nine milliliters of 8%mannitol into a Petri dish using a spatula Place five to seven day old moss from two to three PP NH four plates into the mannitol. Please see the accompanying written protocol for this and additional reagent recipes. Next, add three milliliters of 2%drizly and incubate at room temperature with gentle shaking for one hour.
Filter the mixture through 100 micron mesh to obtain a green homogeneous suspension. Then spin the filtrate at 250 G for five minutes. Discard the supernatant very gently.
Resuspend the protoplasts in 500 microliters of 8%mannitol. Then add an additional 9.5 milliliters of 8%mannitol. Make sure that the protoplasts are fully suspended.
Perform the wash steps two more times by spinning the solution again at 250 G for five minutes. Discarding the supernatant and resus suspending the protoplasts. First in 500 microliters.
Then 9.5 milliliters of 8%mannitol using a hemo cytometer. Count the number of protoplasts in 10 microliters of the suspension to transform DNA into the isolated protoplasts. First, spin the protoplasts mixture at 250 G for five minutes.
Remove the supernatant resuspend the protoplasts at 1.6 million per milliliter in MMG solution. Incubate the suspension at room temperature for 20 minutes. Next, add 600 microliters of the Protoplasts solution into a culture tube containing 60 micrograms of DNA.Shake.
Gently add 700 microliters of PEG calcium solution and shake. Well incubate the sample at room temperature for 30 minutes during this waiting period. Cover PRMB plates with cellophane.
Then dilute the suspension with three milliliters of W five solution spin at 250 G for five minutes. To remove the PEG resuspend, the protoplasts in two milliliters of PRMT plate one milliliter per PRMB plate covered with cellophane. Keep the plates at 25 degrees Celsius in a growth chamber.
Finally, four days after transformation, move the cellophane onto fresh selection plates. This figure shows an example of 18 day old plants regenerated from protoplasts, transformed with 15 micrograms of super coiled plasma, DNA, carrying a hygromycin resistance cassette. Here, 30 micrograms of the same super coiled plasmid, DNA, were transformed into protoplasts.
60 micrograms of plaid DNA were transformed in this sample. And finally, these plants were regenerated from protoplasts that were transformed with 120 micrograms of the same super coiled plasma DNA carrying the hygromycin resistance cassette. The transformation efficiency is not affected by DNA concentrations of up to 60 micrograms.
However, higher concentrations of DNA are detrimental to this procedure. The data indicate that this method delivers consistent results over a wide range of DNA amounts Once semester. This technique can be done in three hours if it is performed properly.
Following this procedure, additional methods like transient RNAi and gene knockouts can be performed in order to answer additional questions about gene function.
