medium no. 51: modified Linsmaier and Skoog medium, 0.2 mg/L 2,4-dichlorophenoxyacetic acid, 200 mg/L kanamycin, 500 mg/L carbenicillin, 0.4% (w/v) gellan gum, pH 5.8

Chemicals and stock solutions

(All stock solutions are stored at 4°C)
  • MS salt mix

    Murashige and Skoog Plant Salt Mixture, Wako Pure Chemical Industries (#392-00591)

  • Sucrose

  • BY2_P

    Chemical Concentration (mg/mL)
    KH2PO4 80
  • LS_VT_modified

    Chemical Concentration (mg/mL)
    Thiamine·HCl 0.4
    myo-Inositol 40
  • 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)

  • Kanamycin (200 mg/mL)

    Chemical Concentration (mg/mL)
    Kanamycin sulfate 200

    Sterilize the solution by membrane filtration

  • Carbenicillin (500 mg/mL)

    Chemical Concentration (mg/mL)
    Carbenicillin disodium salt 500

    Sterilize the solution by membrane filtration

  • Gellan gum

  • KOH (1 N)

Medium preparation

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

    Chemical Amount
    MS salt mix 1 bag (1 L)
    Sucrose 30 g
  2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution Volume (mL)
    BY2_P 2.5
    LS_VT_modified 2.5
    2,4-D (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 4 g of gellan gum to the medium.

  5. Autoclave the medium at 121°C for 20 min.

  6. Add the following antibiotics to the medium just before agar solidification and swirl the medium gently to mix.

    Stock solution Volume (mL)
    Kanamycin (200 mg/mL) 1
    Carbenicillin (500 mg/mL) 1

Formulation

Chemical Concentration (mg/L)
KNO3 1900
NH4NO3 1650
CaCl2·2H2O 440
MgSO4·7H2O 370
KH2PO4 370
H3BO3 6.2
MnSO4·4H2O 22.3
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
Thiamine·HCl 1
myo-Inositol 100
Sucrose 30000
2,4-D sodium monohydrate 0.236
Kanamycin sulfate 200
Carbenicillin disodium salt 500
Gellan gum 4000