# Rice seed germination
> All ddH2O used for this process should be sterilized.
## 1. Homogenization
1. Dehusked the seeds. Select only seeds that are well-developed, fully-filled, clean, and transparent.
2. Dry-incubate the seeds in dark under 45°C for 24 hours.
## 2. Sterilization
1. Soak the seeds in 3% bleach solution ( add one drop of detergent ) and gently inverting / shaking for 15 minutes.
2. Wash at least 6 times with ddH2O, until the water is clear.
## 3. Priming and imbibition
1. Remove the water thoroughly with pipette.
2. (optional) Seed priming. Soak the seeds in 3% KNO3 solution under room temperature for 24 hours;
3. 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
1. Remove the PPM solution.
2. 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.