medium no. 65: modified Murashige and Skoog medium, 4.5 µM 2,4-dichlorophenoxyacetic acid, pH 6.2

Plant cell lines

• rpc00107: Catharanthus roseus CrB cell suspension culture

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

• MS salt mix

  Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#392-00591)

• Sucrose

• AA_VT

  Chemical	Concentration (mg/mL)
  Nicotinic acid	0.05
  Pyridoxine·HCl	0.05
  Thiamine·HCl	0.01
  Glycine	0.16

  Store at −20°C

• 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)

• NaOH (1 N)

Medium preparation

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

   Chemical	Amount
   MS salt mix	1 bag (1 L)
   Sucrose	30 g

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

   Stock solution	Volume (mL)
   AA_VT	10
   2,4-D (1 mM)	4.5

3. Adjust the pH of the solution to 6.2 with NaOH (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	1900
NH4NO3	1650
CaCl2·2H2O	440
MgSO4·7H2O	370
KH2PO4	170
H3BO3	6.2
MnSO4·4H2O	22.3
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	1.6
Sucrose	30000
2,4-D sodium monohydrate	1.18