In vitro growth of seedlings

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

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.

Materials