rpc00017-Spi-I-1-3.3 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00017

Spinacia oleracea Spi-I-1 callus cultureComponents• A 9-cm plastic Petri dish, containing cells placed on semi-solid medium
Notice• Subculture the cells to fresh medium immediately after arrival [Notes I].
• 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, 1.2% (w/v)
agar, pH 6.5 (medium no. 68) [Materials III]
• Culture conditions: 27℃, dark [Methods II]• Subculture: 28–56-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-I-1
cell line (rpc00017) was provided by the RIKEN BRC through the National
BioResource Project of the MEXT, Japan.”Experimental Plant Division
RIKEN BioResource Research Center (BRC)
Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074
JapanFAX: +81 29 836 9053
E-mail: plant.brc@riken.jp
http://epd.brc.riken.jp/en/

1

Introduction
Spinach Spi-I-1 cell line was established from Spinacia oleracea L. (Nakagawa et al.
1985). 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℃ and subcultured at 28–56-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
KI
Na2 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/mL
0.0025 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·HClThiamine·HClGlycine

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 with
distilled waterN) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
O) NaOH (1 N)
Glassware and equipment
A) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use
Preparation of mMS medium (medium no. 68)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
KClL-Glutamine
NZ-Amine Type A
CaCl2 ·2H2 O
MgSO4 ·7H2 O
KH2 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

10 mL

3

MS_micro_2
MS_VT
MS_inositol
NAA (1 mg/mL)
BAP (1 mg/mL)

10 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 40 mL of the medium into a 100-mL ﬂask containing 0.48 g of agar.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Pick up an appropriate amount of callus cells from a 28–56-day-old culture with aforceps and place the cells onto fresh mMS medium.
2. Incubate cell cultures under the dark condition at 27℃.

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 growthof 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, anamount of cells transferred to fresh medium and the subculture intervals may varyfrom 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, andculture them for 28–56 days.

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. 68)
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

KI

0.83

Na2 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

1Agar

12000

5