rpc00016-Spi-WT-2.2 (2017.10.19; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00016

Spinacia oleracea Spi-WT 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. 12) [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-WT
cell line (rpc00016) 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/

1

Introduction
Spinach Spi-WT cell line was established from Spinacia oleracea L. (Nakagawa et al.
1985). 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℃ 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) MS salt mix
Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical
Corporation (#392-00591)
B) SucroseC) MS_VT
Nicotinic acid
Pyridoxine·HCl
Thiamine·HCl
Glycine

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

40 mg/mL

E) NAA (1 mg/mL)
NAA·K
1.2 mg/mL
Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation(#161-04021)
F) BAP (1 mg/mL)
6-Benzylaminopurine
1 mg/mL
Dissolve 6-benzylaminopurine in small volume of KOH (1 N), and ﬁll up withdistilled waterG) NaOH (1 N)

2Glassware and equipment
A) 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℃ for
20 minC) 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.
MS salt mix
Sucrose1 bag (1 L)
20 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
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 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 at27℃.

3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 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-WT 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-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; 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

4Appendix A: Formulation of culture medium
Table A.1. modiﬁed Murashige and Skoog medium(medium no. 12)
ChemicalConcentration (mg/L)

KNO31900

NH4 NO31650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO36.2

MnSO4 ·4H2 O

22.3

ZnSO4 ·7H2 O

8.6

KI0.83Na2 MoO4 ·2H2 O

0.25

CuSO4 ·5H2 O

0.025

CoCl2 ·6H2 O

0.025

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

15