Team:UEA-JIC Norwich/pegtransformation

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1. Add 9ml of 8% mannitol to a petri dish.

2. Using a spatula, put 7 day old moss from 2-3 PPNH4 plates in the petri dish containing the mannitol.

3. Add 3ml of 2% driselase to the petri dish.

4. Incubate the petri dish at room temperature with gentle shaking for 1 hour.

5. Filter the protoplast suspension through a 100µm mesh.

6. Spin the filtered suspension at 250g for 5 minutes.Remove the supernatant. <p style="color:#000000">7. Resuspend the protoplasts very gently with 500µl of 8%mannitol.

8. Add 9.5ml 8% mannitol in the culture tube. Make sure the protoplasts are fully suspended.

9. Repeat the filtration and re-suspension steps (6,7,8) two more times.

10. Take 10µl of the protoplast solution and count the protoplasts using a haemocytometer.

11. Multiply the number of protoplasts in as 16 square area by 10,000 to obtain the amount of protoplastspts ml.

12. Spin the protoplast solution at 250g for 5 minutes. Remove the supernatant. <p style="color:#000000">13. Re-suspend protoplasts in MMg solution at the concentration 1.6 million protoplasts/ml.

14. Incubate the protoplast suspension at room temperature for 20 minutes.

15. Add 600µl of protoplast suspension into a culture tube containing 60µg DNA. Swirl the tube gently.

16. Add 700µl of PEG/Ca solution into the protoplast/DNA mixture. Swirl the tube gently until all the mixture homogeneous.

17. Incubate the mixture at room temperature for 30 minutes.

18. During this waiting period cover PRM-B plates with 80mm cellophane discs. Allow the cellophane to hydrate on the plate surface for at least 5 minutes.

19. With a spatula remove any air bubbles trapped between the cellophane and the plate.

20. Dilute the mixture with 3ml of W5 solution.

21. Spin the mixture at 250g for 5 minutes. Remove the supernatant.

22. Re-suspend protoplasts in melted 2ml of PRM-T. Plate 1ml of re-suspended protoplasts per PRM-B plate covered by cellophane. Wrap the plates with micropore tape and keep in a 25°C growth chamber.

Growth chamber is set to 16hrs light 8hrs dark cycle.

Move the cellophane onto a fresh selection plate 4 days after transformation.

Liu, Y., Vidali, L. 2011. Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens. J. Vis. Exp. (50), e2560, DOI: 10.3791/2560.