Kansas State University

MEDIUM FOR EMBRYO RESCUE (FOR 1 L)

1. Put 500 mL of distilled water in a 1-L beaker.
2. Add the following: 
   • Inorganic salts
   • Muraschige–Skoog Organic medium – 34.6 g
   • NaH2PO4H2O (stock 17 g/L) – 10-mL stock
   • Organic constituents 
     • ucrose (6 % final concentration) – 30 g
     • i-inositol (100 mg/L stock is 0.5 g/50 mL) – 10 mL
     • Thiamine (0.4 g/l stock is 2 g/50 mL) – 10 mL
     • Amino acids L-tyrosine, L-argenine, glycine – 10 mL (10 mg/L stock is 50 mg each aa/50 mL H₂O)
     • Kinetin (for very small enbryos only) – 2 mg
3. Dilute to 800 mL.
4. Adjust pH to 5.7 with NaOH..
5. Bring volume to 1,000 mL.
6. Add 8 grams Bacto® agar.
7. Melt agar in microwave for 3 minutes or dissolve while stirring on a hotplate.
8. Pour ~20 mL into Kimax culture tubes (50 mL) and cap.
9. Sterilize by autoclaving for 15 minutes.
10. Slant in tubes or pour into petri dishes.