medium no. 60: 1/2 modified Murashige and Skoog medium, 1 mg/L kinetin, pH 5.8

Plant cell lines

• rpc00100: Athyrium yokoscense AY-01 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

• MS_VT

  Chemical	Concentration (mg/mL)
  Nicotinic acid	0.5
  Pyridoxine·HCl	0.5
  Thiamine·HCl	0.1
  Glycine	2

• MS_inositol

  Chemical	Concentration (mg/mL)
  myo-Inositol	40

• 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

• KOH (1 N)

Medium preparation

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

   Chemical	Amount
   MS salt mix	0.5 bag (equivalent with 0.5 L)
   Sucrose	30 g

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

   Stock solution	Volume (mL)
   MS_VT	0.5
   MS_inositol	1.25
   Kinetin (0.2 mg/mL)	5

3. Adjust the pH of the solution to 5.8 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	950
NH4NO3	825
CaCl2·2H2O	220
MgSO4·7H2O	185
KH2PO4	85
H3BO3	3.1
MnSO4·4H2O	11.15
ZnSO4·7H2O	4.3
KI	0.415
Na2MoO4·2H2O	0.125
CuSO4·5H2O	0.0125
CoCl2·6H2O	0.0125
FeSO4·7H2O	13.9
Na2-EDTA	18.65
Nicotinic acid	0.25
Pyridoxine·HCl	0.25
Thiamine·HCl	0.05
Glycine	1
myo-Inositol	50
Sucrose	30000
Kinetin	1