medium no. 15: AA medium, 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.2 mg/L kinetin, 1.2% (w/v) agar, pH 5.8

Plant cell lines

• rpc00020: Oryza sativa OS-1 callus culture

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

• Sucrose

• MS_micro_1

  Chemical	Concentration (mg/mL)
  H3BO3	0.62
  MnSO4·5H2O	2.41
  ZnSO4·7H2O	0.86
  KI	0.083
  Na2MoO4·2H2O	0.025
  CuSO4·5H2O	0.0025
  CoCl2·6H2O	0.0025

• MS_micro_2

  Chemical	Concentration (mg/mL)
  FeSO4·7H2O	2.78
  Na2-EDTA	3.73

  Heat at 80°C for 3–4 hours for chelating Fe

• AA_1

  Chemical	Concentration (mg/mL)
  KCl	29.4
  CaCl2·2H2O	4.4
  MgSO4·7H2O	3.7
  KH2PO4	1.7

• AA_4

  Chemical	Concentration (mg/mL)
  Nicotinic acid	0.05
  Pyridoxine·HCl	0.05
  Thiamine·HCl	0.01
  myo-Inositol	10

  Store at −20°C

• AA_5

  Chemical	Concentration (mg/mL)
  L-Glutamine	8.77
  L-Aspartic acid	2.66
  L-Arginine	2.28
  Glycine	0.75

  Store at −20°C

• 2,4-D (0.2 mg/mL)

  Chemical	Concentration (mg/mL)
  2,4-D sodium monohydrate	0.236

  (2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich (D6679)

• Kinetin (0.2 mg/mL)

  Chemical	Concentration (mg/mL)
  Kinetin	0.2

  Dissolve kinetin in small volume of KOH (1 N), and fill up with distilled water

• Agar, powder

• KOH (1 N)

Medium preparation

1. Dissolve the following chemicals in approximately 800 mL of distilled water.

   Chemical	Amount
   Sucrose	30 g

2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

   Stock solution	Volume (mL)
   MS_micro_1	10
   MS_micro_2	10
   AA_1	100
   AA_4	10
   AA_5	100
   2,4-D (0.2 mg/mL)	5
   Kinetin (0.2 mg/mL)	1

3. Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.

4. Add 12 g of agar to the medium.

5. Autoclave the medium at 121°C for 20 min.

Formulation

Chemical	Concentration (mg/L)
L-Glutamine	877
L-Aspartic acid	266
L-Arginine	228
CaCl2·2H2O	440
MgSO4·7H2O	370
KH2PO4	170
H3BO3	6.2
MnSO4·5H2O	24.1
ZnSO4·7H2O	8.6
KI	0.83
Na2MoO4·2H2O	0.25
CuSO4·5H2O	0.025
CoCl2·6H2O	0.025
FeSO4·7H2O	27.8
Na2-EDTA	37.3
Nicotinic acid	0.5
Pyridoxine·HCl	0.5
Thiamine·HCl	0.1
Glycine	75
myo-Inositol	100
Sucrose	30000
2,4-D sodium monohydrate	1.18
Kinetin	0.2
Agar	12000