This procedure begins with plating arabidopsis seeds on Ms.Agar on day 14. After the seeds germinate, the seedlings are collected and sliced. The plant material is then digested in an enzyme solution breaking down the cell wall.
The resulting mixture is sift through a cheesecloth to separate the protoplasts from other plant material. The collected protoplasts are further purified on a sucrose density gradient by layering them with W five solution. The protoplasts concentrate at the interface of the enzyme and W five solution.
They are collected and washed before experimenting. Hi, I'm Jang Ja from the laboratory of Lina Wauk in the Department of Crop and Soil Sciences at Cornell University. Today we'll show you a procedure for isolation of intact photographs from 14 days old TH of aro Ana.
We use this procedure in our laboratory to study the subcellular localization of the proteins of interest for isolation of intact organelles and for the functional analysis of gene using double stranded RNI interference. So let's get started. We start the procedure by preparing 0.5 times MS plates supplemented with 1%sucrose and 0.7%agar for one liter of medium.
Weigh out 2.15 grams of MS powder. Add into 800 milliliters of water in a 1.5 liter autoclavable bottle with a stir bar in it, and stir the medium on a stirring plate to dissolve the powder while stirring. Add drop by drop one normal potassium hydroxide to adjust the pH to 5.7.
Continue the stirring and add 10 grams of sucrose. Next, add seven grams of agar, which does not dissolve, but will solubilize later during autoclaving. And finally, adjust the volume to 1000 milliliters.
Sterilize the MS medium by autoclaving at 121 degrees Celsius for 20 minutes. Keeping the stir bar in the bottle after autoclaving. Stir the medium on a stirring plate to cool it down to approximately 60 degrees Celsius.
Stirring while chilling prevents the agar from precipitating at the bottom of the bottle. If one is able to keep one's hands on the bottle for 10 seconds, then the medium has cooled down sufficiently and the plates can be poured. Move to the hood in order to pour the medium into 150 by 15 millimeter Petri dishes.
90 milliliters per plate. Store the plates at four degrees Celsius. Now that the plates were growing, the plants are ready.
Let's prepare the Arabidopsis seeds. Start growing the plants by sterilizing the Arabidopsis seeds for plating on one Petri dish. Place seeds in an einor micro centrifuge tube in an amount equivalent to 50 microliters of volume.
First sterilize with ethanol by adding one milliliter of 70%ethanol and mixing. Well incubate with the ethanol for two minutes during which time the seeds will sediment. Take out as much of the ethanol as possible using a pipetter next sterilize with bleach by adding one milliliter of a bleach tween solution and mix for 10 minutes by vortexing every two to three minutes.
To facilitate removing the bleach solution, give the seeds a quick spin down and pipe it out as much of the solution as possible. The final sterilization step is done in a laminar flow hood. Add one milliliter of sterile water to the seeds and mix.Well.
Give the seeds a quick spin down and aspirate the water. Repeat this water wash four more times. Now that the seeds are sterilized, spread them on the MS plate.
After spreading the seeds, keep the seeded plates for 24 hours at four degrees Celsius in the dark for stratification. Following the 24 hours of incubation, transfer the plants to a growth and grow them for 14 days. At eight hours light and 16 hours dark, the 14 day old seedlings are then ready for harvesting.
Now that the seedlings grew for two weeks, it is time to prepare the protoplasts in the hood. Use a fresh razor blade to slice two grams of seedlings in 15 milliliters of filter sterilized TVL solution in a Petri dish. Next, transfer the chopped tissues into a 200 milliliter beaker wrapped in aluminum foil.
Add 20 milliliters of freshly prepared filter sterilized enzyme solution to break down the plant cell wall. Swirl the beaker to mix and cover with biofilm and aluminum foil to keep the tissue in the dark. Proceed to shake the plant tissues in the enzyme solution for 16 hours at room temperature.
At the end of the incubation with the enzyme solution, collect the released protoplasts into a 50 milliliter falcon tube by sing the mixture through eight layers of cheesecloth pre-wet in W five solution sive the remaining protoplasts by washing the cheesecloth slowly with 10 milliliters of W five solution. The protoplasts are now collected in the falcon tube. Carefully overlay the protoplasts with five milliliters of WW five solution.
Be careful not to disturb the sugar gradient formed by adding the W five solution. Next centrifuge the protoplasts for seven minutes at 100 Gs.The protoplasts collect at the interface of the enzyme solution and the W five solution. Collect 10 milliliters of liquid at the interface.
Transfer the collected protoplasts to a new 50 milliliter falcon tube and add 15 milliliters W five solution. Then centrifuge for five minutes at 60 gs. After the centrifugation, remove the supernatant and resuspend the protoplasts in 15 milliliters of W five solution.
Again, centrifuge the protoplasts for five minutes at 60 gs. After centrifugation, remove the snat and resus. Suspend the pelleted protoplasts in one to two milliliters of W five solution.
Finally, evaluate the protoplasts yield by cell counting with a hemo cytometer under the microscope. The culturing procedure described here yields healthy arabidopsis seedlings suitable for isolating protoplasts after digestion and separation on a density gradient. Harvesting one gram of fresh seedlings typically yields five to 10 times 10 to the sixth intact protoplasts.
We have show you how to isolate protoplasts from 14 days old ceiling of aosis. When doing this procedure is very important to remember, success depends on healthy plants. Therefore, it is important to maintain a steep to plate ratio and specifies in this protocol for net mills, ms.
Medium per plate. Finally, remember, the port plus are very fragile. Once the cell is removed, handle put bus carefully mixing by typing instead of vortexing and preparing up and down.
That's it. Thank you for watching. Good luck with your experiment.
