rpc00016: Spinacia oleracea Spi-WT cell suspension culture

Note

  • Components

    • Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cell suspension

    • Overseas delivery: A 250-mL plastic Erlenmeyer flask, 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: mMS medium, 0.5 mg/L NAA, 1 mg/L BAP, pH 6.5 (medium no. 12)

    • Culture conditions: 27°C, dark, 140 rpm

    • Subculture: 7-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: “Spinacia oleracea Spi-WT cell line (rpc00016) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

Spinach Spi-WT cell line was established from Spinacia oleracea L. (Nakagawa et al. 1985 [1]). The Spi-WT cells are grown in a modified Murashige and Skoog (mMS) medium supplemented with 0.5 mg/L 1-naphthaleneacetic acid (NAA) and 1 mg/L 6-benzylaminopurine (BAP), pH 6.5. Our Spi-WT cell culture has been maintained in the dark at 27°C with rotary shaking at 140 rpm and subcultured at 7-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

  • MS_VT

    Chemical

    Concentration (mg/mL)

    Nicotinic acid

    0.5

    Pyridoxine·HCl

    0.5

    Thiamine·HCl

    0.1

    Glycine

    2

  • MS_inositol

    Chemical

    Concentration (mg/mL)

    myo-Inositol

    40

  • NAA (1 mg/mL)

    Chemical

    Concentration (mg/mL)

    NAA·K

    1.2

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

  • BAP (1 mg/mL)

    Chemical

    Concentration (mg/mL)

    6-Benzylaminopurine

    1

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

  • NaOH (1 N)

Glassware and equipment

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

  • Pipette (10 mL; large tip opening) and a bulb, sterilized by autoclaving at 121°C for 20 min

  • Plastic conical centrifuge tube (50 mL), sterile

Preparation of mMS medium (medium no. 12)

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

    Chemical

    Amount

    MS salt mix

    1 bag (1 L)

    Sucrose

    20 g

  2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution

    Volume (mL)

    MS_VT

    1

    MS_inositol

    2.5

    NAA (1 mg/mL)

    0.5

    BAP (1 mg/mL)

    1

  3. Adjust the pH of the solution to 6.5 with NaOH (1 N), and fill up to 1 L with distilled water.

  4. Pour 25 mL of the medium into a 100-mL flask.

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

Methods

  1. Centrifuge 7-day-old culture in a 50-mL conical centrifuge tube at 100 ×g for 5 min and discard the supernatant.

  2. Resuspend the pelleted cells in fresh mMS medium (total volume 30 mL).

  3. Transfer 15 mL of cell suspension to 25 mL of fresh mMS medium with a pipette.

  4. Incubate cell cultures on a rotary shaker at 140 rpm under the dark condition at 27°C.

Notes

  • For domestic customers: We send Spi-WT cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh mMS medium immediately after arrival.

  • For overseas customers: We send Spi-WT cells placed on semi-solid mMS medium in a 250-mL disposable Erlenmeyer flask. The cells should be transferred to fresh mMS medium immediately after arrival. Collect the cells from the semi-solid medium with a spatula and transfer them to Erlenmeyer flasks containing fresh liquid medium.

  • In order to maintain Spi-WT cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh mMS medium in every subculture. The amount of cells may vary from one lab to another, because proliferation of Spi-WT cells is affected by culture conditions, such as a room temperature, rotation speed of a rotary shaker, and aeration condition of the culture.

  • In order to obtain good aeration of a suspension culture, a silicone sponge plug may be used instead of the aluminum foil cap (e.g., cap-type Silicosen; Shin-Etsu Polymer, Tokyo, Japan).

Genotyping

Maintenance history

References

Protocols