rpc00020: Oryza sativa OS-1 callus culture

Note

  1. Components
  • Two 9-cm Petri dishes, containing cells placed on semi-solid medium
  1. 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.
  1. Summary
  • 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
  1. 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.”

Introduction

Rice OS-1 cell line was established from Oryza sativa L. (Nakasone et al. 1986 [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

  • 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)

  1. Dissolve the following chemicals in approximately 800 mL of distilled water.

    Chemical Amount (g)
    Sucrose 30
  2. 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
  3. Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.

  4. Pour 40 mL of the medium into a 100-mL flask containing 0.48 g of agar.

  5. Autoclave the flask at 121°C for 20 min.

Methods

  1. 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.
  2. 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 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.

Maintenance history

References

[1]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