medium no. 2: modified Lin and Staba medium, 0.5 µM 2,4-dichlorophenoxyacetic acid, pH 5.7

Plant cell lines

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

• KNO₃

• NH₄Cl

• CaCl₂·2H₂O

• MgSO₄·7H₂O

• Sucrose

• Lin_and_Staba_1

  Chemical	Concentration (mg/mL)
  H3BO3	2.4
  MnSO4·5H2O	10.18
  ZnSO4·7H2O	4.05

• Lin_and_Staba_2

  Chemical	Concentration (mg/mL)
  FeSO4·7H2O	5.56
  Na2-EDTA	7.46

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

• Lin_and_Staba_3

  Chemical	Concentration (mg/mL)
  KI	0.375
  Na2MoO4·2H2O	0.127
  CuSO4·5H2O	0.01
  CoCl2·6H2O	0.01

• Lin_and_Staba_4

  Chemical	Concentration (mg/mL)
  Nicotinic acid	5
  Pyridoxine·HCl	0.5
  Thiamine·HCl	3

• Lin_and_Staba_5

  Chemical	Concentration (mg/mL)
  KH2PO4	68

• 2,4-D (1 mM)

  Chemical	Concentration (mg/mL)
  2,4-D sodium monohydrate	0.261

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

• KOH (1 N)

Medium preparation

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

   Chemical	Amount
   KNO3	5.56 g
   NH4Cl	0.268 g
   CaCl2·2H2O	0.22 g
   MgSO4·7H2O	0.185 g
   Sucrose	20 g

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

   Stock solution	Volume (mL)
   Lin_and_Staba_1	1
   Lin_and_Staba_2	5
   Lin_and_Staba_3	1
   Lin_and_Staba_4	1
   Lin_and_Staba_5	1
   2,4-D (1 mM)	0.5

3. Adjust the pH of the solution to 5.7 with KOH (1 N), and fill up to 1 L with distilled water.

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

Formulation

Chemical	Concentration (mg/L)
KNO3	5560
NH4Cl	268
CaCl2·2H2O	220
MgSO4·7H2O	185
KH2PO4	68
H3BO3	2.4
MnSO4·5H2O	10.18
ZnSO4·7H2O	4.05
KI	0.375
Na2MoO4·2H2O	0.127
CuSO4·5H2O	0.01
CoCl2·6H2O	0.01
FeSO4·7H2O	27.8
Na2-EDTA	37.3
Nicotinic acid	5
Pyridoxine·HCl	0.5
Thiamine·HCl	3
Sucrose	20000
2,4-D sodium monohydrate	0.131