rpc00037: Lithospermum erythrorhizon M18-1 callus culture¶
- Two 9-cm Petri dishes, containing cells placed on semi-solid medium
- Subculture the cells to fresh medium immediately after arrival.
- Do not store the cell culture in a refrigerator and a freezer.
- Maintain aseptic conditions of the cell culture, and work in a laminar flow cabinet.
- Culture medium: LS medium, 1 µM IAA, 10 µM kinetin, 1% (w/v) agar, pH 6.3 (medium no. 32)
- Culture conditions: 23°C, dark
- Subculture: 28–42-day intervals
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 which is participating in the National BioResource Project of the MEXT/AMED, Japan.”
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 a high capacity for producing shikonin under the induction conditions. The M18-1 cells are grown 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°C and subcultured at 28–42-day intervals.
Chemicals and stock solutions¶
(All stock solutions are stored at 4°C)
MS salt mix
Murashige and Skoog Plant Salt Mixture, Wako Pure Chemical Industries (#392-00591)
Chemical Concentration (mg/mL) Thiamine·HCl 0.16 myo-Inositol 40
IAA (1 mM)
Chemical Concentration (mg/mL) IAA·K 0.213
Potassium 3-indoleacetate, Wako Pure Chemical Industries (#160-07531)
Kinetin (1 mM)
Chemical Concentration (mg/mL) Kinetin 0.215
Dissolve kinetin in small volume of KOH (1 N), and fill up with distilled water
NaOH (1 N)
Glassware and equipment¶
- Erlenmeyer flask (100 mL), capped with two layers of aluminum foil
- Forceps, sterilized before use
Preparation of LS medium (medium no. 32)¶
Dissolve the following chemicals in approximately 800 mL of distilled water.
Chemical Amount MS salt mix 1 bag (1 L) Sucrose 30 g
Add following stock solutions, and fill up to approximately 950 mL with distilled water.
Stock solution Volume (mL) LS_VT 2.5 IAA (1 mM) 1 Kinetin (1 mM) 10
Adjust the pH of the solution to 6.3 with NaOH (1 N), and fill up to 1 L with distilled water.
Pour 40 mL of the medium into a 100-mL flask containing 0.4 g of agar.
Autoclave the flask at 121°C for 20 min.
- Pick up an appropriate amount of callus cells from a 28–42-day-old culture with a forceps and place the cells onto fresh LS medium.
- Incubate cell cultures under the dark condition at 23°C.
- We send M18-1 cells on semi-solid LS medium in a 9-cm plastic Petri dish. The cells 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 growth of 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, an amount of cells transferred to fresh medium and the subculture intervals may vary from one lab to another. We usually inoculate seven to nine pieces of M18-1 callus (about 3–5-mm in diameter) on 40 mL of LS medium in a 100-mL flask, and culture them for 28–42 days.
|||Mizukami H, Konoshima M, Tabata M (1978) Variation in pigment production in Lithospermum erythrorhizon callus cultures. Phytochemistry 17: 95–97. DOI: 10.1016/S0031-9422(00)89687-9|
|||Yazaki K, Kunihisa M, Fujisaki T, Sato F (2002) Geranyl diphosphate:4-hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon: Cloning and characterization of a key enzyme in shikonin biosynthesis. Journal of Biological Chemistry 277: 6240–6246. DOI: 10.1074/jbc.M106387200|