Tomato seed TMV treatment and sterilization
NOTE: If you need the seeds to be sterile (e.g. you are sowing them in agar media), you should be working inside a Laminar Flow Hood to keep a sterile environment. If you don't need sterile seed, you can use regular DI water for rinsing.
- Eppie tubes or hystology cartridges depending on seed size.
- Sterile beaker & magenta box(es), MSMO plates, or phytatrays if sowing seed in agar media.
- Forceps
- 50% HCl
- 10% TSP (trisodium phosphate). TSP is inexpensive to purchase at hardware stores (~$10 for 4.5 lb); beware of mislabeling as some products labeled TSP have no TSP at all. Savogran brand TSP is 80% TSP, 20% sodium carbonate.
- 70% EtOH
- 50% bleach
- Sterile water
- MSMO media, if sowing seed in media.
MSMO (Murashige and Skoog Basal Medium) media (1L)
- 4.4g MS
- [optional] sucrose to desired concentration
- 7g (0.7%) bactoagar, or different amount depending on desired concentration
- Adjust pH to 5.7 with KOH
- Autoclave 35min
- Cool down in 65ºC water bath before pouring onto petri dishes
Treatment against TMV
You don’t have to do this part in the laminar flow hood, bench top is fine. But HCl can emit strong fumes, so you can do step 1 in the fume hood.
- Soak seeds in 50% HCl for 10 min
- Rinse with sterile water (1x)
- Soak seeds in 10% TSP for 15 min
- Rinse with sterile water (3x if you are stopping at this point, 1x if you are moving on to seed sterilization step)
- If you are stopping at this point, dry seeds on bench top before storing
Seed sterilization
Work inside a Laminar Flow Hood to keep a sterile environment.
- Soak seeds in 70% EtOH for 10 min
- Rinse with sterile water (1x)
- Soak seeds in 50% bleach for 20 min
- Rinse with sterile water (3x)
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