For our troubleshooting experiment we were assigned the task of attempting to germinate A. thaliana on MS agar medium. We researched several publications and came to on consensus as a group as to what concentration of MS to use and what percentage of agar to use as well. Here is the protocol we came up with:
Germination of Seeds on Agar (MS Media) Protocol:
0.5 MS Media with 0.1% Agar and 2% Sucrose
1. Added 2.16 g MS Salts to 0.9 L of distilled H2O and let dissolve.
2. Measure pH. The pH should be around 5.7. Adjust if necessary.
3. Diluted to final volume of 1L and added agar (10g/L0
5. Added sucrose after media cooled before solution was poured in petri dishes. (20g Sucrose)
6. Sucrose dissolve(swirled the solution) before pouring into the petri dishes.
7. Poured approximately 85 mL of the solution into each petri dish.
8. MS media solidified overnight (in the hood)
Sterilization of Seeds
-15mL of Bleach + 15 mL dH2O = 0.5Bleach
-Added 15 microliters of Tween 20 to Bleach (0.05% Tween 20)
1. Washed Wild Type seeds with 0.5 Bleach, and then it was poured off.
2. Washed 4x with 10 mL sterile H2O (inverted 10-20 times each time)
3. Poured off water after seeds settled to the bottom
-Used 1000L pipet to suck up remaining water
4. Added 2 mL sterile H20 each time the seeds were plated
-Plate 1 –> 400 microliters H2O + seed on plate + 1 mL H2O onto plate
-Plate 2 –> extracted 300 microliters, added 2 mL H2O to tube, then distributed 1.7 mL onto plate
-Plate 3 & 4 –> Resuspend seeds & extract 2mL of H2O + seeds & distribute on plate
5. Tilted plates slightly and removed excess H2O from plate sides using widetip L200
-add water to make 2mL H2O into tube
-remove 300microLiters of seed + water and distribute onto plate
-add water to make complete 2mL H2O level in tube
-remove and distribute remaining 1.7L H2O of water + seeds onto plate
We found that adding 2mL of H2O made for an easier distribution of seeds on the plate. We did have some trouble with light issues though and that may have had an adverse effect on plant growth. The lights weren’t on for 16 hours like they should of been. Pictures of the plate with seeds that were germinated on it can be seen in the class photos section. We also found it very easy to remove the plants intact from the agar (including roots) after they had germinated. Pictures of this can also be seen in the class photos section.