rpc00020-OS-1-3.2 (2017.5.1; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00020

Oryza sativa OS-1 callus culture

Components• 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: AA medium, 1 mg/L 2,4-D, 0.2 mg/L kinetin, 1.2% (w/v)
agar, pH 5.8 (medium no. 15) [Materials III]
• Culture conditions: 27℃, dark [Methods II]• Subculture: 28-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: “Oryza sativa OS-1 cell line(rpc00020) 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
Rice OS-1 cell line was established from Oryza sativa L. (Nakasone et al. 1993). The OS1 cells are grown on a AA medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic
acid (2,4-D) and 0.2 mg/L kinetin, and solidiﬁed with 1.2% (w/v) agar, pH 5.8. Our OS-1
cell culture has been maintained in the dark at 27℃ and subcultured at 28-day intervals.

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) SucroseB) 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/mL
0.0025 mg/mL
0.0025 mg/mLC) 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 FeD) AA_1
KClCaCl2 ·2H2 O
MgSO4 ·7H2 O
KH2 PO429.4 mg/mL
4.4 mg/mL
3.7 mg/mL
1.7 mg/mLE) AA_4
Nicotinic acid
Pyridoxine·HCl
Thiamine·HCl
myo-Inositol
Store at −20℃

0.05 mg/mL
0.05 mg/mL
0.01 mg/mL
10 mg/mLF) AA_5

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L-Glutamine
L-Aspartic acid
L-Arginine
GlycineStore at −20℃

8.77 mg/mL
2.66 mg/mL
2.28 mg/mL
0.75 mg/mLG) 2,4-D (0.2 mg/mL)
2,4-D sodium monohydrate
0.236 mg/mL(2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich(D6679)H) Kinetin (0.2 mg/mL)
Kinetin
0.2 mg/mL
Dissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled waterI) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
J) KOH (1 N)
Glassware and equipment
A) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use
Preparation of AA medium (medium no. 15)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
Sucrose30 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
MS_micro_1
MS_micro_2
AA_1
AA_4
AA_5
2,4-D (0.2 mg/mL)
Kinetin (0.2 mg/mL)

10 mL
10 mL
100 mL
10 mL
100 mL
5 mL
1 mL

3. Adjust the pH of the solution to 5.8 with KOH (1 N), and ﬁll up to 1 L with distilledwater.

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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-day-old culture with a forceps and place the cells onto fresh AA medium.
2. Incubate cell cultures under the dark condition at 27℃.

Notes
• We send OS-1 cells on semi-solid AA medium in a 9-cm disposable Petri dish. Thecells should be subcultured to fresh AA medium immediately after arrival.
• In order to maintain OS-1 callus culture stably, it is essential to observe the growth ofcells carefully. Because proliferation of OS-1 cells is aﬀected by culture conditions,such as a room temperature, aeration conditions of the culture and so on, an amountof cells transferred to fresh medium and the subculture intervals may vary from one
lab to another. We usually inoculate two pieces of OS-1 callus (about 10-mm indiameter) on 40 mL of AA medium in a 100-mL ﬂask, and culture them for 28 days.

References
Nakasone S, Minami E, Imai T, Akiyama F, Ohashi Y (1993) Synchronous cell division inrice suspension cultures and cell cycle speciﬁc expression of histone H3 and PCNA
genes. Bulletin of National Institute of Agrobiological Resources (Japan) 8: 1–10.
(in Japanese) http://agriknowledge.affrc.go.jp/RN/2010490447

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Appendix A: Formulation of culture medium
Table A.1. AA medium(medium no. 15)
ChemicalConcentration (mg/L)

KCl

2940

L-Glutamine

877

L-Aspartic acid266

L-Arginine

228

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO36.2

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

Glycine75

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

1.18

Kinetin

0.2

Agar

12000

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