rpc00088: Rhizophora L46 callus culture

Note

  • Components

    • A 9-cm plastic Petri dish, containing cells placed on semi-solid medium

  • Notice

    • Subculture the cells to fresh medium immediately after arrival.

    • Do not store the cell culture in a refrigerator and a freezer.

    • Maintain aseptic conditions of the cell culture, and work in a laminar flow cabinet.

  • Method

    • Culture medium: AA medium, 0.2 µM 2,4-D, 0.2% (w/v) gellan gum, pH 6.2 (medium no. 58)

    • Culture conditions: 27°C, dark

    • Subculture: 63-day intervals

  • Citation of cell line

    When results obtained by using this cell line are published in a scientific journal, it should be cited in the following manner: “Rhizophora L46 cell line (rpc00088) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

L46 cell line was established from a leaf of a mangrove species Rhizophora sp. according to the method described by Mimura et al. 1997 [1]. The L46 callus cells have high salt tolerance. The L46 cells are grown on a Amino Acid (AA) medium supplemented with 0.2 µM 2,4-dichlorophenoxyacetic acid (2,4-D), and solidified with 0.2% (w/v) gellan gum, pH 6.2. Our L46 cell culture has been maintained in the dark at 27°C and subcultured at 63-day intervals.

Materials

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)

  • Gellan gum

    Gellan gum, FUJIFILM Wako Pure Chemical Corporation (#073-03071)

  • NaOH (1 N)

Glassware and equipment

  • Petri dish (9 cm diameter, 2 cm height), sterile

  • Forceps, sterilized before use

  • Scalpel, sterilized before use

  • Thermoplastic sealing film

Preparation of AA medium (medium no. 58)

  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)

    0.2

  3. Adjust the pH of the solution to 6.2 with NaOH (1 N), and fill up to 1 L with distilled water.

  4. Add 2 g of gellan gum to the medium.

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

  6. Pour 35 mL of the medium into a 9-cm Petri dish.

Methods

  1. Cut a 63-day-old callus into an appropriate size with a scalpel and a forceps.

  2. Place the callus pieces onto fresh AA medium.

  3. Seal the Petri dishes using one round of thermoplastic sealing film.

  4. Incubate cell cultures under the dark condition at 27°C.

Notes

  • We send L46 cells on semi-solid AA medium in a 9-cm disposable Petri dish. The cells should be subcultured to fresh AA medium immediately after arrival.

  • In order to maintain L46 callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of L46 cells is affected by culture conditions, such as a room temperature, aeration conditions of the culture and so on, an amount of cells transferred to fresh medium and the subculture intervals may vary from one lab to another. We usually inoculate six pieces of L46 callus (about 10-mm in diameter) on 35 mL of AA medium in a 9-cm Petri dish, and culture them for 63 days.

References

Genotyping

/genotyping/g_rpc00088

Maintenance history

Protocols