rpc00020: Oryza sativa OS-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: AA medium, 1 mg/L 2,4-D, 0.2 mg/L kinetin, 1.2% (w/v) agar, pH 5.8 (medium no. 15)
- Culture conditions: 27°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: “Oryza sativa OS-1 cell line (rpc00020) was provided by the RIKEN BRC which is participating in the National BioResource Project of the MEXT/AMED, Japan.”
Rice OS-1 cell line was established from Oryza sativa L. (Nakasone et al. 1986 ). The OS-1 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°C and subcultured at 28-day intervals.
Chemicals and stock solutions¶
(All stock solutions are stored at 4°C)
Chemical Concentration (mg/mL) H3BO3 0.62 MnSO4·5H2O 2.41 ZnSO4·7H2O 0.86 KI 0.083 Na2MoO4·2H2O 0.025 CuSO4·5H2O 0.0025 CoCl2·6H2O 0.0025
Chemical Concentration (mg/mL) FeSO4·7H2O 2.78 Na2-EDTA 3.73
Heat at 80°C for 3–4 hours for chelating Fe
Chemical Concentration (mg/mL) KCl 29.4 CaCl2·2H2O 4.4 MgSO4·7H2O 3.7 KH2PO4 1.7
Chemical Concentration (mg/mL) Nicotinic acid 0.05 Pyridoxine·HCl 0.05 Thiamine·HCl 0.01 myo-Inositol 10
Store at -20°C
Chemical Concentration (mg/mL) L-Glutamine 8.77 L-Aspartic acid 2.66 L-Arginine 2.28 Glycine 0.75
Store at -20°C
2,4-D (0.2 mg/mL)
Chemical Concentration (mg/mL) 2,4-D sodium monohydrate 0.236
(2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich (D6679)
Kinetin (0.2 mg/mL)
Chemical Concentration (mg/mL) Kinetin 0.2
Dissolve kinetin in small volume of KOH (1 N), and fill up with distilled water
KOH (1 N)
Glassware and equipment¶
- Erlenmeyer flask (100 mL), capped with two layers of aluminum foil
- Forceps, sterilized before use
Preparation of AA medium (medium no. 15)¶
Dissolve the following chemicals in approximately 800 mL of distilled water.
Chemical Amount (g) Sucrose 30
Add following stock solutions, and fill up to approximately 950 mL with distilled water.
Stock solution Volume (mL) MS_micro_1 10 MS_micro_2 10 AA_1 100 AA_4 10 AA_5 100 2,4-D (0.2 mg/mL) 5 Kinetin (0.2 mg/mL) 1
Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.
Pour 40 mL of the medium into a 100-mL flask containing 0.48 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 AA medium.
- Incubate cell cultures under the dark condition at 27°C.
- We send OS-1 cells on semi-solid AA medium in a 9-cm plastic Petri dish. The cells 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 of cells 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 amount of 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 in diameter) on 40 mL of AA medium in a 100-mL flask, and culture them for 28 days.
|||Nakasone S, Minami E, Imai T, Akiyama F, Ohashi Y (1993) Synchronous cell division in rice 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|