medium no. 2: modified Lin and Staba medium, 0.5 µM 2,4-dichlorophenoxyacetic acid, pH 5.7

Plant cell lines

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • KNO3

  • NH4Cl

  • CaCl2·2H2O

  • MgSO4·7H2O

  • Sucrose

  • Lin_and_Staba_1

    Chemical

    Concentration (mg/mL)

    H3BO3

    2.4

    MnSO4·5H2O

    10.18

    ZnSO4·7H2O

    4.05

  • Lin_and_Staba_2

    Chemical

    Concentration (mg/mL)

    FeSO4·7H2O

    5.56

    Na2-EDTA

    7.46

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

  • Lin_and_Staba_3

    Chemical

    Concentration (mg/mL)

    KI

    0.375

    Na2MoO4·2H2O

    0.127

    CuSO4·5H2O

    0.01

    CoCl2·6H2O

    0.01

  • Lin_and_Staba_4

    Chemical

    Concentration (mg/mL)

    Nicotinic acid

    5

    Pyridoxine·HCl

    0.5

    Thiamine·HCl

    3

  • Lin_and_Staba_5

    Chemical

    Concentration (mg/mL)

    KH2PO4

    68

  • 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)

  • KOH (1 N)

Medium preparation

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

    Chemical

    Amount

    KNO3

    5.56 g

    NH4Cl

    0.268 g

    CaCl2·2H2O

    0.22 g

    MgSO4·7H2O

    0.185 g

    Sucrose

    20 g

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

    Stock solution

    Volume (mL)

    Lin_and_Staba_1

    1

    Lin_and_Staba_2

    5

    Lin_and_Staba_3

    1

    Lin_and_Staba_4

    1

    Lin_and_Staba_5

    1

    2,4-D (1 mM)

    0.5

  3. Adjust the pH of the solution to 5.7 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

5560

NH4Cl

268

CaCl2·2H2O

220

MgSO4·7H2O

185

KH2PO4

68

H3BO3

2.4

MnSO4·5H2O

10.18

ZnSO4·7H2O

4.05

KI

0.375

Na2MoO4·2H2O

0.127

CuSO4·5H2O

0.01

CoCl2·6H2O

0.01

FeSO4·7H2O

27.8

Na2-EDTA

37.3

Nicotinic acid

5

Pyridoxine·HCl

0.5

Thiamine·HCl

3

Sucrose

20000

2,4-D sodium monohydrate

0.131