Growing Arabidopsis on nutrient-rich media
Diep R Ganguly
Abstract
Notes on growing Arabidopsis seedlings on nutrient-rich media in plates.
Steps
Prepare media
Select and prepare autoclaved media of choice (see recipes below). Make sure to pH before adding gelling agent.
| A | B |
|---|---|
| MS basal salts | 2.15 – 4.33 g/L |
| Sucrose | 10 - 30 g/L |
| Vitamins (1000X) (optional) | 1 mL/L |
| MES (optional) | 0.1% (w/v) |
| KOH | pH 5.8 |
| Agar/Phytoblend | 8 g/L |
MS Media (Murashige & Skoog, 1962)
| A | B |
|---|---|
| Gamborg’s B-5 basal medium | 3.21 g/L |
| Sucrose | 10 - 30 g/L |
| KOH | pH 5.8 |
| Agar/Phytoblend | 8 g/L |
Gamborg B-5 Media (Gamborg et al 1968)
| A | B |
|---|---|
| Kanamycin | 30 - 50 uM |
| Basta (Glufosinate Ammonium) | 20 - 50 uM |
Supplement for transgenic selection - add to cooled autoclaved media immediately prior to pouring plates.
Pour room temperature media into plates in a biosafety cabinet - keep everything sterile! Allow media to cool and solidify, then package and store at 4 °C.
Surface sterilize seed
Collect small aliquots of desired Arabidopsis seeds into clean microfuge tubes.
Add 500 uL of a solution made of 40% (v/v) household bleach, and 0.01% (v/v) Tween20 dissolved in distilled water and incubate for 5 minutes.
Remove supernatant, then rinse seeds with 700 uL of 70% ethanol incubating for 5-10 minutes. If you did not perform bleach sterilization step, repeat ethanol wash an additional 1-2 times.
Remove supernatant then rinse with distilled water at least 3 times.
After final rinse, plate seeds out on media plates in sterile environment (e.g. BioSafety cabinet). Seeds can be stored at 4 °C for up to 1 month (maximum).
