rpc00018: Spinacia oleracea Spi-12F cell suspension culture

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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. 13)

  • 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-12F cell line (rpc00018) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

Spinach Spi-12F cell line was established from Spinacia oleracea L. (Nakagawa et al. 1985 [1]). The Spi-12F 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-12F 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)

• 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

• 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. 13)

medium no. 13: modified Murashige and Skoog medium, 0.5 mg/L 1-naphthaleneacetic acid, 1 mg/L 6-benzylaminopurine, pH 6.5

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 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 12 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-12F 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-12F 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-12F 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-12F 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

Genotyping: rpc00018

Maintenance history

Maintenance history: rpc00018

References

[1]

Nakagawa H, Tanaka H, Oba T, Ogura N, Iizuka M (1985) Callus formation from protoplasts of cultured Spinacia oleracea cells. Plant Cell Reports 4: 148–150. DOI: 10.1007/BF00571303

Protocols

• Genotyping of plant cell lines: Spinacia oleracea (spinach) cell lines

External links

• World Flora Online: Spinacia oleracea L.

• Wikipedia: Spinacia oleracea