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In vitro growth of seedlings

来源:本站原创  2003/11/2 3:13:00   访问量:2607
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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

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