rpc00020: Oryza sativa OS-1 callus culture
Note
Components
A 9-cm plastic Petri dish, containing cells placed on semi-solid medium
Notice
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.
Method
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 through the National BioResource Project of the MEXT, Japan.”
Introduction
Rice OS-1 cell line was established from Oryza sativa L. (Nakasone et al. 1993 [1]). 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.
Materials
Chemicals and stock solutions
(All stock solutions are stored at 4°C)
Sucrose
MS_micro_1
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
MS_micro_2
Chemical
Concentration (mg/mL)
FeSO4·7H2O
2.78
Na2-EDTA
3.73
Heat at 80°C for 3–4 hours for chelating Fe
AA_1
Chemical
Concentration (mg/mL)
KCl
29.4
CaCl2·2H2O
4.4
MgSO4·7H2O
3.7
KH2PO4
1.7
AA_4
Chemical
Concentration (mg/mL)
Nicotinic acid
0.05
Pyridoxine·HCl
0.05
Thiamine·HCl
0.01
myo-Inositol
10
Store at −20°C
AA_5
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
Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
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
Sucrose
30 g
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.
Methods
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.
Notes
We send OS-1 cells on semi-solid AA medium in a 9-cm disposable 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.
Genotyping
Maintenance history
References
Protocols
Genotyping of plant cell lines: Oryza sativa (rice) cell lines