medium no. 57: Amino Acid medium, 0.02 µM 2,4-dichlorophenoxyacetic acid, 2 µM textit{N}-(2-chloro-4-pyridyl)-textit{N}$^prime$-phenylurea(4-CPPU), pH 6.2

Plant cell lines

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • Sucrose

  • CaCl2 (0.6 M)

    Chemical

    Concentration (mg/mL)

    CaCl2·2H2O

    88

  • AA_macro

    Chemical

    Concentration (mg/mL)

    KCl

    149

    MgSO4·7H2O

    37

    KH2PO4

    17

  • 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

  • Fe(III)-EDTA (20 mM)

    Chemical

    Concentration (mg/mL)

    Fe(III)-EDTA

    8.422

    Ethylenediamine-N,*N*,*N*′,*N*′-tetraacetic acid, iron (III), sodium salt, trihydrate, FUJIFILM Wako Pure Chemical Corporation (#343-01241)

  • 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

  • AA_VT

    Chemical

    Concentration (mg/mL)

    Nicotinic acid

    0.05

    Pyridoxine·HCl

    0.05

    Thiamine·HCl

    0.01

    Glycine

    0.16

    Store at -20°C

  • 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)

  • 4-CPPU (2 mM)

    Chemical

    Concentration (mg/mL)

    4-CPPU

    0.495

    N-(2-Chloro-4-pyridyl)-N′-phenylurea, Sigma-Aldrich (C2791); Dissolve 4-CPPU in ethanol and store at -20°C

  • NaOH (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)

    CaCl2 (0.6 M)

    5

    AA_macro

    10

    MS_micro_1

    10

    Fe(III)-EDTA (20 mM)

    5

    AA_5

    100

    AA_VT

    10

    2,4-D (1 mM)

    0.02

    4-CPPU (2 mM)

    1

  3. Adjust the pH of the solution to 6.2 with NaOH (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)

KCl

1490

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

Fe(III)-EDTA

42.11

Nicotinic acid

0.5

Pyridoxine·HCl

0.5

Thiamine·HCl

0.1

Glycine

76.6

Sucrose

30000

2,4-D sodium monohydrate

0.00522

4-CPPU

0.495