| In vitro growth of seedlings | ||
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| Reference: Schneitz Lab | Last updated: 2/16/00 | By: Kay Schneitz |
sterilisation of seeds:
- rinse with 70% EtOH for 30 sec
- put in 1% bleach (sodium hypochlorite, supplemented by a few drops of Tween-20) for 5 min
- wash 2-4x with sterile H2O for 1 min
plating and growth
- resuspend in sterile 0.1% agarose
- plate 4 ml in petridish (0.8% agarose supplemented with 1/2 MS10) in sterile hood
- let dry for 1-2 h in the hood
- wrap with 3M micropore tape
- keep at 4°C for 4 days
- place the dishes under light
Solutions
| 1/2 MS10 | |
| MS Macro I | 50.0 ml |
| MS Macro II | 50.0 ml |
| Myo | 10.0 ml |
| FeNaEDTA | 10.0 ml |
| MS Vitamines | 1.0 ml |
| MS Micronutrients | 0.5 ml |
| Sucrose | 10.0 g |
| H2O | x.x ml |
| Total | 1000.0 ml |
adjust pH to 5.7 with 1 M KOH, alternatively MES (0.5g/l) can be used to ensure more stable pH during plant growth.
Remarks
It is essential that the medium is autoclaved in either a small autoclave or pressure cooker because prolonged exposure to heat will cause complex formation between sucrose and phytoagar which becomes visible as a yellow coloring of the medium. This medium will be softer after hardening than correctly autoclaved medium.
The MS medium protocol is taken from Murashige and Skoog (1962) Physiologia Plantarum 15: 473-497. The recipe can be found here.
Materials
| Reagent/Tool | Supplier | Cat.-# |
| Agarose | Quantum-Appligene | 130022 |
| MS media | Phytotechnology Laboratories | |
| Petridish 145/20 mm | Greiner Labortechnik | 639 102 |
| Sodium hypochlorite 13% | Fluka | 71696 |
| Tween-20 | Fluka | 93773 |
| 3M Micropore tape | Galenica |