medium no. 15: AA medium, 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.2 mg/L kinetin, 1.2% (w/v) agar, pH 5.8

Plant cell lines

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • 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

  • AA_1

    Chemical

    Concentration (mg/mL)

    KCl

    29.4

    CaCl2·2H2O

    4.4

    MgSO4·7H2O

    3.7

    KH2PO4

    1.7

  • AA_4

    Chemical

    Concentration (mg/mL)

    Nicotinic acid

    0.05

    Pyridoxine·HCl

    0.05

    Thiamine·HCl

    0.01

    myo-Inositol

    10

    Store at -20°C

  • AA_5

    Chemical

    Concentration (mg/mL)

    L-Glutamine

    8.77

    L-Aspartic acid

    2.66

    L-Arginine

    2.28

    Glycine

    0.75

    Store at -20°C

  • 2,4-D (0.2 mg/mL)

    Chemical

    Concentration (mg/mL)

    2,4-D sodium monohydrate

    0.236

    (2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich (D6679)

  • 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

  • Agar, powder

  • KOH (1 N)

Medium preparation

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

    Chemical

    Amount

    Sucrose

    30 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

    AA_1

    100

    AA_4

    10

    AA_5

    100

    2,4-D (0.2 mg/mL)

    5

    Kinetin (0.2 mg/mL)

    1

  3. Adjust the pH of the solution to 5.8 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)

L-Glutamine

877

L-Aspartic acid

266

L-Arginine

228

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

75

myo-Inositol

100

Sucrose

30000

2,4-D sodium monohydrate

1.18

Kinetin

0.2

Agar

12000