rpc00054: Coptis japonica Cj 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: LS medium, 100 µM NAA, 1 µM BAP, 1% (w/v) agar, pH 6.0 (medium no. 42)

    • Culture conditions: 27°C, dark

    • Subculture: 56-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: “Coptis japonica Cj cell line (rpc00054) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

Cj cell line was established from a root of Coptis japonica (Thunb.) Makino var. dissecta (Yatabe) Nakai ex Satake (Fukui et al. 1982 [1], Sato et al. 1993 [2]). The Cj callus cells exclusively accumulate berberine in the vacuoles. The Cj cells are grown on a Linsmaier and Skoog (LS) medium supplemented with 100 µM 1-naphthaleneacetic acid (NAA) and 1 µM 6-benzylaminopurine (BAP), and solidified with 1% (w/v) agar, pH 6.0. Our Cj cell culture has been maintained in the dark at 27°C and subcultured at 56-day intervals.

Materials

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • MS salt mix

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

  • Sucrose

  • LS_VT

    Chemical

    Concentration (mg/mL)

    Thiamine·HCl

    0.16

    myo-Inositol

    40

  • NAA (10 mM)

    Chemical

    Concentration (mg/mL)

    NAA·K

    2.243

    Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation (#161-04021)

  • BAP (1 mM)

    Chemical

    Concentration (mg/mL)

    6-Benzylaminopurine

    0.225

    Dissolve 6-benzylaminopurine in small volume of KOH (1 N), and fill up with distilled water

  • Agar, powder

    Agar, powder, Junsei Chemical (#24440-1201)

  • KOH (1 N)

Glassware and equipment

  • Erlenmeyer flask (100 mL), capped with two layers of aluminum foil

  • Forceps, sterilized before use

Preparation of LS medium (medium no. 42)

  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)

    LS_VT

    2.5

    NAA (10 mM)

    10

    BAP (1 mM)

    1

  3. Adjust the pH of the solution to 6.0 with KOH (1 N), and fill up to 1 L with distilled water.

  4. Pour 40 mL of the medium into a 100-mL flask containing 0.4 g of agar.

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

Methods

  1. Pick up an appropriate amount of callus cells from a 56-day-old culture with a forceps and place the cells onto fresh LS medium.

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

Notes

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

  • In order to maintain Cj callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of Cj 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 four pieces of Cj callus (about 7-mm in diameter) on 40 mL of LS medium in a 100-mL flask, and culture them for 56 days.

Genotyping

Maintenance history

References

Protocols