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

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

    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

    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