rpc00038: Lithospermum erythrorhizon OM 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: mLS medium, 1 µM IAA, 10 µM kinetin, 1% (w/v) agar, pH 6.3 (medium no. 33)
- Culture conditions: 23°C, dark
- Subculture: 28-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 OM cell line (rpc00038) was provided by the RIKEN BRC which is participating in the National BioResource Project of the MEXT/AMED, Japan.”
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°C and subcultured at 28-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)
CuSO4 (1.2 mM)
Chemical Concentration (mg/mL) CuSO4·5H2O 0.3
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 mLS medium (medium no. 33)¶
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) CuSO4 (1.2 mM) 1 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 30 mL of the medium into a 100-mL flask containing 0.3 g of agar.
Autoclave the flask at 121°C for 20 min.
- 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.
- Incubate cell cultures under the dark condition at 23°C.
- We send OM cells on semi-solid mLS medium in a 9-cm plastic Petri dish. The cells 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 of cells 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 amount of cells transferred to fresh medium and the subculture intervals may vary from one lab to another. We usually inoculate three pieces of OM callus (about 5-mm in diameter) on 30 mL of mLS medium in a 100-mL flask, and culture them for 28 days.