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 less than 1000 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 (this is day 1).

11. Transfer seeds into 50 ml Falcon tube with 10ml liquid medium at day 3.

12. Shake under lights. Seeds will germinate after a few days.

13. Change fresh liquid medium at day 13.

14. Add chitin at day 14.

* You can sterilize as many as 1000 seeds in one 1.5 ml Eppendorf tube. But after cold treatment, they have to be seperated into different falcon tubes (50 seeds/tube, 10ml liquid medium).

For me, I calculate the number of samples I will need for chitin treatment first. Say, if you want to do 5 treatments for the seeds from the same line, you will need (5+1)*50=300 seeds (See below for the reason I add 1). I count the seed number, it should be final number*120%, that is 360 seeds, because you will lose some seeds during sterilization. These seeds can be sterilized in a single 1.5 ml Eppendorf tube. After cold treatment, I transfer the seeds

into a 15ml falcon tube, add liquid medium to 6ml. Prepare 5 50ml falcon tubes, preadd 9ml liquid medium. Mix the seeds well by pippett the solution and transfer them to the 50ml falcon tube, 1ml/tube. It will be much easier to control the seed number you get by add 1 more sample and use 6ml instead of 5ml. (if you have 10 samples you will need to prepare seeds for 12 samples at the beginning)