Medium:

20 g            Dextrose

1 pkg         Murashige’s and Skoog’s plant salt mixture

1 l               ddH2O

adjust pH to 5.7 with 10 N KOH (~ 250 µl)

autoclave for 15 min. When finished, remove promptly from autoclave.

1. Turn on hood and UV for ~ 30 min before start.

2. Wipe bench with EtOH.

3. Transfer about 50 seeds to 1.5 ml Eppendorf tube.

4. Add 200 ul 75% EtOH.

5. Immediately add 600ul sterile water, pipette 600 ul liquid off.

6. Repeat 5 for 3-4 times.

7. Add 200 ul bleach, mix well, set for 5min.

8. Add 600 ul sterile water, pipette 600 ul liquid off.

9. Repeat 8 for 5-6 times.

10. Cold treat for 2 days.

11. Transfer seeds into 50 ml Falcon tube with 10ml liquid medium.

12. Shake under lights. Seeds will germinate after a few days.   Cultures grow up in two weeks.

13. Change to fresh liquid medium before chitin treatment.