Rice seed germination

All ddH₂O used for this process should be sterilized.

1. Homogenization

Dehusked the seeds. Select only seeds that are well-developed, fully-filled, clean, and transparent.
Dry-incubate the seeds in dark under 45°C for 24 hours.

2. Sterilization

Soak the seeds in 3% bleach solution ( add one drop of detergent ) and gently inverting / shaking for 15 minutes.
Wash at least 6 times with ddH₂O, until the water is clear.

3. Priming and imbibition

Remove the water thoroughly with pipette.
(optional) Seed priming. Soak the seeds in 3% KNO₃ solution under room temperature for 24 hours;
Soak the seeds in 1 ~ 2% Plant Preservative Mixture™ ( PPM™ ) solution for 16 ~ 24 hours.

Add 300 µL PPM into 30 mL sterilized ddH₂O.
The PPM™ stock solution ( 100% ) is stored at 4°C (a).

4. Sowing

Remove the PPM solution.
Sow the seeds on suitable medium.

Optional: Add 0.05 ~ 0.2% ( v / v ) PPM™ to the medium, if it is difficult to control contamination. The PPM™ can be autoclaved together with the media.

Half-MS medium preparation

Chemicals	Stock Concentration	Addition	Final concentration	Location
MS basal salt	—	8.66 g	2.165 g / L	Ⅱ
Sucrose	—	10 g	2.5 g / L	Ⅰ
MES hydrate	—	2 g	0.05% (w/v)	Ⅲ
PPM™ (optional)	100%	2 mL	0.05%	4°C ( a )
KOH ^※	5 M	~ 710 µL	—	Ⅶ
ddH₂O	—	~ 4 L	—	—
Total volume		4 L

(optional) The gelling agent: Gelzan (G1910) or Phytagel ( P8169 ) ⟶ 1.5 g / L ( 0.15% ). Located at Ⅰ or Ⅸ. Generally, we make 400 mL per bottle, so weigh 0.6 g / 400 mL for each bottle.

The final pH should be around 5.7 ~ 5.8. Autoclave under 121°C for 20 mins.

^※ KOH is used for pH adjustment.
To prepare 5 M KOH solution, dilute 330 g of 85% KOH in 1 L ddH₂O. Since KOH releases heat during dissolution, it is important to add the KOH granules into the water gradually and slowly.