medium no. 30: Gamborgʼs B5 medium, 1 µM 2,4-dichlorophenoxyacetic acid, 0.04 µM kinetin, 0.9% (w/v) agar, pH 5.7

Plant cell lines

• rpc00035: Nicotiana tabacum Xan-1 callus culture

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

• B5 salt mix

  Gamborgʼs B5 Medium Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#399-00621)

• Sucrose

• B5_VT

  Chemical	Concentration (mg/mL)
  Nicotinic acid	0.4
  Pyridoxine·HCl	0.4
  Thiamine·HCl	4
  myo-Inositol	40

• 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)

• 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

• KOH (1 N)

Medium preparation

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

   Chemical	Amount
   B5 salt mix	1 bag (1 L)
   Sucrose	20 g

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

   Stock solution	Volume (mL)
   B5_VT	2.5
   2,4-D (1 mM)	1
   Kinetin (1 mM)	0.04

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

4. Add 9 g of agar to the medium.

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

Formulation

Chemical	Concentration (mg/L)
KNO3	2500
(NH4)2SO4	134
CaCl2·2H2O	150
MgSO4·7H2O	250
NaH2PO4·H2O	150
H3BO3	3
MnSO4·H2O	10
ZnSO4·7H2O	2
KI	0.75
Na2MoO4·2H2O	0.25
CuSO4·5H2O	0.025
CoCl2·6H2O	0.025
FeSO4·7H2O	27.8
Na2-EDTA	37.3
Nicotinic acid	1
Pyridoxine·HCl	1
Thiamine·HCl	10
myo-Inositol	100
Sucrose	20000
2,4-D sodium monohydrate	0.261
Kinetin	0.0086
Agar	8000