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Table 4 Quantities of stock solutions and reagents required for the preparation of working buffers for wheat and barley mesophyll leaf protoplast isolation and transfection

From: A cell death assay in barley and wheat protoplasts for identification and validation of matching pathogen AVR effector and plant NLR immune receptors

Reagents/stock solutions Protoplast isolation buffera Wash buffera Transfection buffer 1a Transfection buffer 2a Regeneration buffera
0.8 M Mannitol 7.5 ml   12.5 ml 1.25 ml 11.25 ml
PEG 4000     2 g  
0.1 M MES pH 5.7 1 ml 1 ml 1 ml   600 µl
2 M KCl 100 µl 125 μl    150 µl
5 M NaCl   1.54 ml    
1 M MgCl2    375 µl   
Cellulase R10 150 mg     
Macerozyme R10 50 mg     
Heat to 55 °C for 10 min, cool to room temperature
1 M CaCl2 100 µl 6.25 ml   500 µl  
BSA 10 mg     
ddH2O Up to 10 ml Up to 50 ml Up to 25 ml Up to 5 ml Up to 15 ml
  Filter sterilize Filter sterilize    Filter sterilize
  1. aVolumes suitable for 12 independent transfections