medium no. 33: modified Linsmaier and Skoog medium, 1 µM indole-3-acetic acid, 10 µM kinetin, 1% (w/v) agar, pH 6.3

Plant cell lines

• rpc00038: Lithospermum erythrorhizon OM callus 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

• CuSO₄ (1.2 mM)

  Chemical	Concentration (mg/mL)
  CuSO4·5H2O	0.3

• LS_VT

  Chemical	Concentration (mg/mL)
  Thiamine·HCl	0.16
  myo-Inositol	40

• IAA (1 mM)

  Chemical	Concentration (mg/mL)
  IAA·K	0.213

  Potassium 3-indoleacetate, FUJIFILM Wako Pure Chemical Corporation (#160-07531)

• Kinetin (1 mM)

  Chemical	Concentration (mg/mL)
  Kinetin	0.215

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

• Agar, powder

  Agar, powder, Junsei Chemical (#24440-1201)

• 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)
   CuSO4 (1.2 mM)	1
   LS_VT	2.5
   IAA (1 mM)	1
   Kinetin (1 mM)	10

3. Adjust the pH of the solution to 6.3 with NaOH (1 N), and fill up to 1 L with distilled water.

4. Add 10 g of agar to the medium.

5. 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	0.325
CoCl2·6H2O	0.025
FeSO4·7H2O	27.8
Na2-EDTA	37.3
Thiamine·HCl	0.4
myo-Inositol	100
Sucrose	30000
IAA·K	0.213
Kinetin	2.15
Agar	10000