Development of a Cabbage Protoplast System for Studying Hypoxia Tolerance in Brassica

We're studying how cabbages react to low oxygen at a molecular level. Since cabbages are hard to obtain, we're developing a system using isolated cabbage cells. By exposing these spare cells, also known as protoplasts, to low-oxygen conditions, we can investigate their stress response easily.

Working with protoplast that's suspended in solution to study low-oxygen responses is tricky. The big challenge is keeping enough oxygen for our control samples. We figured out a way to pump out the oxygen in the liquid.

We have validated the method through reporter assays of hypoxia markers. We checked out different methods for handling low oxygen and found that the oxygen absorber pack works best. It's easy to use and effective, making it great for studying how protoplasts behave in low-oxygen conditions.

Studying the molecular mechanism in leafy crops under hypoxia conditions has been challenging due to the limitation of available tools, but with recent advancement, we've made great progress by using a cabbage protoplast system with hypoxia treatment. This new method is helping us understand how low oxygen impacts these plants at a molecular level. In the future, we plan to combine the protoplast system with immunoprecipitation and reporter assays to illustrate the molecular regulatory pathway involved in cabbage during flooding stress.

Our goal is to identify key regulators of flooding tolerance, which we hope we will aid in breeding a new cabbage cultivar with improved flooding tolerance. To begin, fill the round square-shaped holes of a 48-well plug tray with commercial plant substrate and sow Fuyudori and 228 cabbage seeds approximately one centimeter deep into the growing medium. To prepare 12.5 milliliters of enzyme solution, preheat a solution containing 10 millimolar MES and 0.6 molar mannitol at 55 degrees Celsius.

Then add 1.5%cellulase R-10 and 0.75%macerozyme R-10. Stir the solution and continue warming at 55 degrees Celsius for 10 minutes. Allow the enzyme solution to cool down to room temperature.

Then add 10 millimolar calcium chloride and 0.1%bovine serum albumin. Using a 0.22-micrometer syringe filter, sterilize the enzyme solution into a nine-centimeter Petri dish. After two to three weeks of growth, collect cabbage seedlings at the second leaf stage for mesophyll protoplast isolation.

Collect the second newly expanded true leaves from five to eight cabbage seedlings. Using a sharp razor blade, slice the leaves into 0.5 to 1.0-millimeter strips. Immediately transfer the leaf strips into the freshly prepared enzyme solution.

Subject the cabbage leaf strips to vacuum infiltration in the dark for 30 minutes. After vacuum infiltration, keep the leaf strips immersed in the enzyme solution in the dark for four to 16 hours. The next day, dilute the protoplasts'containing solution with an equal volume of W5 solution to stop the enzymatic digestion.

To release the protoplasts'suspension, gently swirl the mixture on an orbital shaker. Then filter the cell suspension through a 70-micrometer cell strainer into a 50-milliliter conical tube. Centrifuge the protoplast solution at 150 G for two minutes at four degrees Celsius.

Add 10 milliliters of W5 solution along the wall of the tube at a flow rate of approximately one milliliter per second to wash the pelleted protoplasts. After the last centrifugation, resuspend the protoplasts in the W5 solution and place the tube on ice for 30 minutes. Then remove one milliliter of supernatant at a time until all the supernatant is removed.

Resuspend the protoplasts in an ice pre-chilled MMG solution. Measure the protoplast concentration using a hemocytometer. Adjust the final concentration to 4 times 10 to the power of 5 protoplasts per milliliter using MMG solution.

Following the isolation and overnight digestion, the yield of protoplasts was 1.04 times 10 to the power of 7 for Fuyudori and 4.00 times 10 to the power of 6 protoplasts per gram fresh weight for 228. Protoplast transfection efficiency was above 40%in both Fuyudori and 228 cultivars, as demonstrated by the GFP reporter gene. After isolating cabbage protoplast, mix 100 microliters of 4 times 10 to the power of 4 protoplasts with 10 microliters of plasmid and place the mixture on ice for 10 minutes.

Add an equal volume of freshly prepared PEG solution into the protoplast solution and mix gently. Incubate the protoplast mixture at room temperature in the dark for 10 minutes. Then add 440 microliters of W5 solution to terminate the reaction.

Centrifuge the transfected protoplasts at 150 G for two minutes at four degrees Celsius. Resuspend the pellet in 750 microliters of oxygen-enriched W5 solution. Transfer the resuspended protoplasts to a six-well tissue culture plate pre-coded with 1%bovine serum albumin.

For oxygen consuming bag-induced hypoxia, place a plate containing W5 protoplast solution in a 3.5-liter anaerobic jar. Then add two oxygen absorber packs in the jar to create a hypoxic environment. After treatment, centrifuge the protoplasts at 150 G for two minutes at four degrees Celsius.

Discard the supernatant and freeze the collected protoplasts in liquid nitrogen before proceeding to dual luciferase assay. The oxygen absorber pack induced a 7.0 fold increase in BoADH1 promoter activity compared to the control, demonstrating the highest hypoxic response. The BoSUS1L promoter activity increased more than 5.6 fold, with the oxygen absorber pack treatment showing the greatest induction among treatments.