In virto growth

Sterilisation of seeds:

1. rinse with 70% EtOH for 30 sec
2. put in 1% bleach (sodium hypochlorite, supplemented by a few drops of Tween-20) for 5 min
3. wash 2-4x with sterile H₂O for 1 min

 

Plating and growth

1. resuspend in sterile 0.1% agarose
2. plate 4 ml in petridish (0.8% agarose supplemented with 1/2 MS10) in sterile hood
3. let dry for 1-2 h in the hood
4. wrap with 3M micropore tape
5. keep at 4°C for 4 days
6. 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