rpc00038-OM-2.3 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00038

Lithospermum erythrorhizon OM 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: mLS medium, 1 µM IAA, 10 µM kinetin, 1% (w/v) agar,
pH 6.3 (medium no. 33) [Materials III]
• Culture conditions: 23℃, 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: “Lithospermum erythrorhizon
OM cell line (rpc00038) 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/

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Introduction
Puccoon OM cell line was established from a seedling of Lithospermum erythrorhizon
Siebold & Zucc. (Yamamoto et al. 2000). The OM callus cells are not capable of producing
shikonin even under the induction conditions. The OM cells are grown on a modiﬁed
Linsmaier and Skoog (mLS) medium supplemented with 1 µM indole-3-acetic acid (IAA)
and 10 µM kinetin, and solidiﬁed with 1% (w/v) agar, pH 6.3. Our OM cell culture has
been maintained in the dark at 23℃ and subcultured at 28-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) CuSO4 (1.2 mM)
CuSO4 ·5H2 O

0.3 mg/mLD) LS_VTThiamine·HCl
myo-Inositol

0.16 mg/mL
40 mg/mL

E) IAA (1 mM)
IAA·K
0.213 mg/mL
Potassium 3-indoleacetate, FUJIFILM Wako Pure Chemical Corporation(#160-07531)
F) Kinetin (1 mM)
Kinetin
0.215 mg/mL
Dissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled waterG) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
H) NaOH (1 N)

2Glassware and equipment
A) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use
Preparation of mLS medium (medium no. 33)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mixSucrose

1 bag (1 L)
30 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
CuSO4 (1.2 mM)
LS_VT
IAA (1 mM)
Kinetin (1 mM)

1 mL
2.5 mL
1 mL
10 mL

3. Adjust the pH of the solution to 6.3 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 0.4 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 mLS medium.
2. Incubate cell cultures under the dark condition at 23℃.

Notes
• We send OM cells on semi-solid mLS medium in a 9-cm disposable Petri dish. Thecells should be subcultured to fresh mLS medium immediately after arrival.
• In order to maintain OM callus culture stably, it is essential to observe the growth ofcells carefully. Because proliferation of OM 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 fromone lab to another. We usually inoculate three pieces of OM callus (about 5-mm3in diameter) on 40 mL of mLS medium in a 100-mL ﬂask, and culture them for 28days.

References
Yamamoto H, Inoue K, Yazaki K (2000) Caﬀeic acid oligomers in Lithospermum erythrorhizon cell suspension cultures. Phytochemistry 53: 651–657. DOI: 10.1016/S
0031-9422(99)00623-8

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Appendix A: Formulation of culture medium
Table A.1. modiﬁed Linsmaier and Skoog medium(medium no. 33)
ChemicalConcentration (mg/L)

KNO31900

NH4 NO31650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO3

6.2

MnSO4 ·4H2 O

22.3

ZnSO4 ·7H2 O

8.6

KI

0.83

Na2 MoO4 ·2H2 O

0.25

CuSO4 ·5H2 O

0.325

CoCl2 ·6H2 O

0.025

FeSO4 ·7H2 O

27.8

Na2 -EDTA

37.3

Thiamine·HCl

0.4

myo-Inositol

100

Sucrose

30000

IAA·K

0.213

Kinetin

2.15

Agar

10000

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