rpc00017: Spinacia oleracea Spi-I-1 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: mMS medium, 0.5 mg/L NAA, 1 mg/L BAP, 1.2% (w/v) agar, pH 6.5 (medium no. 68)

    • Culture conditions: 27°C, dark

    • Subculture: 28–56-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: “Spinacia oleracea Spi-I-1 cell line (rpc00017) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

Spinach Spi-I-1 cell line was established from Spinacia oleracea L. (Nakagawa et al. 1985 [1]). The Spi-I-1 cells are grown on a modified Murashige and Skoog (mMS) medium supplemented with 0.5 mg/L 1-naphthaleneacetic acid (NAA) and 1 mg/L 6-benzylaminopurine (BAP), and solidified with 1.2% (w/v) agar, pH 6.5. Our Spi-I-1 cell culture has been maintained in the dark at 27°C and subcultured at 28–56-day intervals.

Materials

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • KCl

  • L-Glutamine

  • NZ-Amine Type A

  • CaCl2·2H2O

  • MgSO4·7H2O

  • KH2PO4

  • Sucrose

  • 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

  • MS_micro_2

    Chemical

    Concentration (mg/mL)

    FeSO4·7H2O

    2.78

    Na2-EDTA

    3.73

    Heat at 80°C for 3–4 hours for chelating Fe

  • 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

  • Agar, powder

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

  • NaOH (1 N)

Glassware and equipment

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

  • Forceps, sterilized before use

Preparation of mMS medium (medium no. 68)

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

    Chemical

    Amount

    KCl

    1.4 g

    L-Glutamine

    1 g

    NZ-Amine Type A

    1 g

    CaCl2·2H2O

    0.44 g

    MgSO4·7H2O

    0.37 g

    KH2PO4

    0.17 g

    Sucrose

    20 g

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

    Stock solution

    Volume (mL)

    MS_micro_1

    10

    MS_micro_2

    10

    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 40 mL of the medium into a 100-mL flask containing 0.48 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 28–56-day-old culture with a forceps and place the cells onto fresh mMS medium.

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

Notes

  • We send Spi-I-1 cells on semi-solid mMS medium in a 9-cm disposable Petri dish. The cells should be subcultured to fresh mMS medium immediately after arrival.

  • In order to maintain Spi-I-1 callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of Spi-I-1 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 to seven pieces of Spi-I-1 callus (about 3–5-mm in diameter) on 40 mL of mMS medium in a 100-mL flask, and culture them for 28–56 days.

Genotyping

Maintenance history

References

Protocols