rpc00018-Spi-12F-2.2 (2017.10.19; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00018

Spinacia oleracea Spi-12F cell suspension cultureComponents• Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cellsuspension• 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 [Notes I, II].
• Do not store the cell culture in a refrigerator and a freezer.
• Maintain aseptic conditions of the cell culture, and work in a laminar ﬂowcabinet.
Method• Culture medium: mMS medium, 0.5 mg/L NAA, 1 mg/L BAP, pH 6.5(medium no. 13) [Materials III]
• Culture conditions: 27℃, dark, 140 rpm [Methods II]• Subculture: 7-day intervals [Methods I]
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 NationalBioResource Project of the MEXT, Japan.”Experimental Plant Division
RIKEN BioResource Research Center (BRC)
Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074
Japan
FAX: +81 29 836 9053
E-mail: plant.brc@riken.jp
http://epd.brc.riken.jp/en/

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Introduction
Spinach Spi-12F cell line was established from Spinacia oleracea L. (Nakagawa et al.
1985). 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 at27℃ with rotary shaking at 140 rpm and subcultured at 7-day intervals.

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) KClB) L-Glutamine
C) NZ-Amine Type AD) CaCl2 ·2H2 O
E) MgSO4 ·7H2 OF) KH2 PO4G) SucroseH) MS_micro_1
H3 BO3MnSO4 ·5H2 O
ZnSO4 ·7H2 O
KINa2 MoO4 ·2H2 O
CuSO4 ·5H2 O
CoCl2 ·6H2 O

0.62 mg/mL
2.41 mg/mL
0.86 mg/mL
0.083 mg/mL
0.025 mg/mL0.0025 mg/mL
0.0025 mg/mLI) MS_micro_2
FeSO4 ·7H2 O
2.78 mg/mL
Na2 -EDTA
3.73 mg/mL
Heat at 80℃ for 3–4 hours for chelating FeJ) MS_VT
Nicotinic acid

0.5 mg/mL

2Pyridoxine·HCl
Thiamine·HCl
Glycine

0.5 mg/mL
0.1 mg/mL
2 mg/mLK) MS_inositol
myo-Inositol

40 mg/mLL) NAA (1 mg/mL)
NAA·K
1.2 mg/mL
Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation(#161-04021)
M) BAP (1 mg/mL)
6-Benzylaminopurine
1 mg/mL
Dissolve 6-benzylaminopurine in small volume of KOH (1 N), and ﬁll up withdistilled waterN) NaOH (1 N)
Glassware and equipmentA) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foilB) Pipette (10 mL; large tip opening) and a bulb, sterilized by autoclaving at 121℃ for20 minC) Plastic conical centrifuge tube (50 mL), sterile
Preparation of mMS medium (medium no. 13)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
KClL-Glutamine
NZ-Amine Type A
CaCl2 ·2H2 O
MgSO4 ·7H2 OKH2 PO4Sucrose

1.4 g
1g
1g
0.44 g0.37 g0.17 g20 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
MS_micro_1
MS_micro_2

10 mL
10 mL

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MS_VT
MS_inositol
NAA (1 mg/mL)
BAP (1 mg/mL)

1 mL
2.5 mL
0.5 mL
1 mL

3. Adjust the pH of the solution to 6.5 with NaOH (1 N), and ﬁll up to 1 L with distilledwater.
4. Pour 25 mL of the medium into a 100-mL ﬂask.
5. Autoclave the ﬂask at 121℃ 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 at27℃.

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 freshmMS medium immediately after arrival. Collect the cells from the semi-solid
medium with a spatula and transfer them to Erlenmeyer ﬂasks containing fresh liquidmedium.
• 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 amountof 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

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Polymer, Tokyo, Japan; https://www.shinpoly.co.jp/en/product/product/medical/plugs.html).

References
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

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Appendix A: Formulation of culture medium
Table A.1. modiﬁed Murashige and Skoog medium(medium no. 13)
ChemicalConcentration (mg/L)

KCl

1400

L-Glutamine

1000

NZ-Amine Type A

1000

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO3

6.2

MnSO4 ·5H2 O

24.1

ZnSO4 ·7H2 O

8.6

KI0.83Na2 MoO4 ·2H2 O

0.25

CuSO4 ·5H2 O

0.025

CoCl2 ·6H2 O0.025

FeSO4 ·7H2 O

27.8

Na2 -EDTA

37.3

Nicotinic acid

0.5

Pyridoxine·HCl

0.5

Thiamine·HCl

0.1

Glycine2

myo-Inositol

100

Sucrose

20000

NAA·K

0.6

6-Benzylaminopurine

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