medium no. 25: Linsmaier and Skoog medium, 0.1 µM 2,4-dichlorophenoxyacetic acid, 0.2 mg/L 6-benzylaminopurine, 1.2% (w/v) agar, pH 5.6

Plant cell lines

• rpc00029: Luffa cylindrica LcyD7 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)

• D(+)-Glucose

• LS_VT

  Chemical	Concentration (mg/mL)
  Thiamine·HCl	0.16
  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)

• BAP (1 mg/mL)

  Chemical	Concentration (mg/mL)
  6-Benzylaminopurine	1

  Dissolve 6-benzylaminopurine in small volume of KOH (1 N), and fill up with distilled water

• Agar, powder

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

• KOH (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)
   D(+)-Glucose	30 g

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

   Stock solution	Volume (mL)
   LS_VT	2.5
   2,4-D (1 mM)	0.1
   BAP (1 mg/mL)	0.2

3. Adjust the pH of the solution to 5.6 with KOH (1 N), and fill up to 1 L with distilled water.

4. Add 12 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	0.025
CoCl2·6H2O	0.025
FeSO4·7H2O	27.8
Na2-EDTA	37.3
Thiamine·HCl	0.4
myo-Inositol	100
D(+)-Glucose	30000
2,4-D sodium monohydrate	0.0261
6-Benzylaminopurine	0.2
Agar	12000