medium no. 68: modified Murashige and Skoog medium, 0.5 mg/L 1-naphthaleneacetic acid, 1 mg/L 6-benzylaminopurine, 1.2% (w/v) agar, pH 6.5

Plant cell lines

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • KCl

  • L-Glutamine

  • NZ-Amine Type A

  • CaCl2·2H2O

  • MgSO4·7H2O

  • KH2PO4

  • Sucrose

  • MS_micro_1

    Chemical

    Concentration (mg/mL)

    H3BO3

    0.62

    MnSO4·5H2O

    2.41

    ZnSO4·7H2O

    0.86

    KI

    0.083

    Na2MoO4·2H2O

    0.025

    CuSO4·5H2O

    0.0025

    CoCl2·6H2O

    0.0025

  • MS_micro_2

    Chemical

    Concentration (mg/mL)

    FeSO4·7H2O

    2.78

    Na2-EDTA

    3.73

    Heat at 80°C for 3–4 hours for chelating Fe

  • 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

  • NAA (1 mg/mL)

    Chemical

    Concentration (mg/mL)

    NAA·K

    1.2

    Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation (#161-04021)

  • 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)

  • NaOH (1 N)

Medium preparation

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

    Chemical

    Amount

    KCl

    1.4 g

    L-Glutamine

    1 g

    NZ-Amine Type A

    1 g

    CaCl2·2H2O

    0.44 g

    MgSO4·7H2O

    0.37 g

    KH2PO4

    0.17 g

    Sucrose

    20 g

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

    Stock solution

    Volume (mL)

    MS_micro_1

    10

    MS_micro_2

    10

    MS_VT

    1

    MS_inositol

    2.5

    NAA (1 mg/mL)

    0.5

    BAP (1 mg/mL)

    1

  3. Adjust the pH of the solution to 6.5 with NaOH (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)

KCl

1400

L-Glutamine

1000

NZ-Amine Type A

1000

CaCl2·2H2O

440

MgSO4·7H2O

370

KH2PO4

170

H3BO3

6.2

MnSO4·5H2O

24.1

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

Nicotinic acid

0.5

Pyridoxine·HCl

0.5

Thiamine·HCl

0.1

Glycine

2

myo-Inositol

100

Sucrose

20000

NAA·K

0.6

6-Benzylaminopurine

1

Agar

12000