The overall goal of this assay is to determine the Circadian period in Arabidopsis lines by measuring luminescence emitted from transfected protoplasts. This method can help answer key questions in the field of Circadian biology, such as which genes feed back into the timekeeping mechanism itself. The main advantage of this technique is that it allows rapid analysis of the Circadian period of any Arabidopsis mutant without the need to generate stable transgenic lines.

To begin this procedure, label a petri dish and add to it 10 milliliters of filtered sterilized enzyme solution. Fix four strips of autoclave tape with the sticky side up on a clean lab surface, and mark the size of the petri dish on the strips. Collect plans from the growth room.

Cut leaves of four week old plants and press the upper epidermis onto the autoclave tape, so the lower epidermal surface is facing up. Put strips of magic adhesive tape onto the lower epidermal surface. Using a 15 milliliter conical tube, gently press the strips onto the leaves, taking care not to crush the leaf tissue.

Carefully pull the tape off to strip the lower epidermis and expose the mesophyll cells. Next, cut the autoclave tape to fit the petri dish, and use tweezers to move the tape into the petri dish, and float it on the enzyme solution, with the leaves facing down. Rotate at 60 RPM on a platform shaker for 60 minutes, during which the protoplasts will be released into the solution.

After 60 minutes, use tweezers to remove and discard the strips of autoclave tape. Using a wide bore pipette tip, pipette the solution containing the protoplasts into a 15 milliliter conical tube. Centrifuge the protoplasts at 100 gee for three minutes at four degrees Celsius, and remove the supernatant carefully, as the pellet is very loose.

Add 10 milliliters of W5 solution to the protoplasts, and resuspend by gently swirling the tube. Remove an aliquot of protoplasts for counting. Rest the protoplasts on ice for 30 to 60 minutes.

While the protoplasts are on ice, assess the yield by counting protoplasts in a hemocytometer. Collect the protoplasts by centrifugation at 100 gee for three minutes at four degrees Celsius. Remove the supernatant with care, as the pellet is very loose.

Lastly, resuspend the protoplasts in MMg solution to a concentration of 500, 000 protoplasts per milliliter. Begin the procedure for protoplast transfection by adding ten microliters of reporter plasmid to a 15 milliliter conical tube, followed by 400 microliters of protoplasts. Add one volume of polyethylene glycol, or PEG solution, and mix by inverting the tube gently 12 times to transfect the protoplasts.

Incubate for eight to 15 minutes at room temperature. The protoplasts must be incubated long enough to ensure efficient transfection, but not too long as that will reduce their viability. Next, dilute the protoplast DNA PEG mixture with four volumes of W5 solution, and mix by inverting gently six times.

Collect the transfected protoplasts by centrifugation at 100 gee for two minutes at room temperature. Remove the supernatant with a pipette, again taking great care as the pellet is very loose. Resuspend the protoplasts in 1, 250 microliters of imaging solution.

The protoplasts should resuspend fairly easily, indicating that they are intact after transfection. Aliquot 200 microliters per well into six replicate wells of a 96-well plate, and fill any empty wells with W5 solution. Seal the plate with an adhesive clear lid suitable for luminescent imaging.

To image the protoplasts, transfer the 96-well plate to any luminescent imaging setup suitable for plant imaging. Change the adhesive lid on the plate 10 to 15 minutes after transfer to avoid condensation and pressure differences among the wells. Start imaging.

Read the plate every 45 minutes for five days at room temperature. Subsequently, analyze the luminescence results using any analysis software, for example, the Biological Rhythms Analysis Software System. Mesophyll protoplasts isolated by this protocol are shown in this image at a magnification of 200 times.

Proplasts are wild type arabidopsis and the cca1/lhy mutant were transfected and imaged in constant light on a plate reader over five days. As expected, the free-running period of clock controlled gene expression is shortened in this mutant. In contrast, the period of Circadian oscillations in protoplasts of the ztl mutant is lengthened.

In protoplasts over-expressing CCA1, there were no oscillations in reporter construct expression because over-expression of CCA1 leads to arrhythmia. This bar chart shows the Circadian period estimates based on luminescence traces in the wild-type and clock mutant protoplasts. These Circadian periods are consistent with published data generated from whole plants using time-consuming transgenic approaches.

While attempting this procedure, it's important to use healthy plants and handle the protoplasts with care as they are fragile. Once mastered, this technique can be done in three to four hours if it performed properly. After watching this video, you should have a good understanding of how to isolate, transfect, and image Arabidopsis protoplasts.

These techniques can now be applied to any mutant line, or even be modified to image protoplasts of other plant species.

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