rpc00037-M18-1-2.3 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00037

Lithospermum erythrorhizon M18-1 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: LS medium, 1 µM IAA, 10 µM kinetin, 1% (w/v) agar, pH
6.3 (medium no. 32) [Materials III]
• Culture conditions: 23℃, dark [Methods II]• Subculture: 28–42-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
M18-1 cell line (rpc00037) was provided by the RIKEN BRC through theNational 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 M18-1 cell line was established from a seedling of Lithospermum erythrorhizon
Siebold & Zucc. (Mizukami et al. 1978, Yazaki et al. 2002). The M18-1 callus cells have ahigh capacity for producing shikonin under the induction conditions. The M18-1 cells aregrown on a Linsmaier and Skoog (LS) 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 M18-1 cell
culture has been maintained in the dark at 23℃ and subcultured at 28–42-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) LS_VTThiamine·HCl
myo-Inositol

0.16 mg/mL
40 mg/mLD) IAA (1 mM)
IAA·K
0.213 mg/mL
Potassium 3-indoleacetate, FUJIFILM Wako Pure Chemical Corporation(#160-07531)
E) Kinetin (1 mM)
Kinetin0.215 mg/mL
Dissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled waterF) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
G) NaOH (1 N)
Glassware and equipment
A) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use

2Preparation of LS medium (medium no. 32)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mix
Sucrose1 bag (1 L)
30 g

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

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 30 mL of the medium into a 100-mL ﬂask containing 0.3 g of agar.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Pick up an appropriate amount of callus cells from a 28–42-day-old culture with aforceps and place the cells onto fresh LS medium.
2. Incubate cell cultures under the dark condition at 23℃.

Notes
• We send M18-1 cells on semi-solid LS medium in a 9-cm disposable Petri dish. Thecells should be subcultured to fresh LS medium immediately after arrival.
• In order to maintain M18-1 callus culture stably, it is essential to observe the growthof cells carefully. Because proliferation of M18-1 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 seven to nine pieces of M18-1 callus(about 3–5-mm in diameter) on 30 mL of LS medium in a 100-mL ﬂask, and culture
them for 28–42 days.

References
Mizukami H, Konoshima M, Tabata M (1978) Variation in pigment production in Lithospermum erythrorhizon callus cultures. Phytochemistry 17: 95–97. DOI: 10.1016/S
0031-9422(00)89687-9

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Yazaki K, Kunihisa M, Fujisaki T, Sato F (2002) Geranyl diphosphate:4-hydroxybenzoate
geranyltransferase from Lithospermum erythrorhizon: Cloning and characterizationof a key enzyme in shikonin biosynthesis. Journal of Biological Chemistry 277:6240–6246. DOI: 10.1074/jbc.M106387200

4Appendix A: Formulation of culture medium
Table A.1. Linsmaier and Skoog medium(medium no. 32)
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

KI0.83Na2 MoO4 ·2H2 O

0.25

CuSO4 ·5H2 O

0.025

CoCl2 ·6H2 O

0.025

FeSO4 ·7H2 O

27.8

Na2 -EDTA

37.3Thiamine·HCl

0.4

myo-Inositol

100

Sucrose

30000

IAA·K

0.213

Kinetin

2.15

Agar

100005