rpc00035: Nicotiana tabacum Xan-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: B5 medium, 1 µM 2,4-D, 0.04 µM kinetin, 0.9% (w/v) agar, pH 5.7 (medium no. 30)
  • 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: “Nicotiana tabacum Xan-1 cell line (rpc00035) was provided by the RIKEN BRC which is participating in the National BioResource Project of the MEXT/AMED, Japan.”

Introduction

Tobacco Xan-1 cell line was established from a leaf of Nicotiana tabacum L. cultivar Xanthi NC (Taguchi et al. 2003 [1]). The Xan-1 cells are grown on a Gamborg’s B5 medium supplemented with 1 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.04 µM kinetin and solidified with 0.9% (w/v) agar, pH 5.7. Our Xan-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)
  • B5 salt mix

    Gamborg’s B5 Medium Salt Mixture, Wako Pure Chemical Industries (#399-00621)

  • Sucrose

  • B5_VT

    Chemical Concentration (mg/mL)
    Nicotinic acid 0.4
    Pyridoxine·HCl 0.4
    Thiamine·HCl 4
    myo-Inositol 40
  • 2,4-D (1 mM)

    Chemical Concentration (mg/mL)
    2,4-D sodium monohydrate 0.261

    (2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich (D6679)

  • 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

  • Agar, powder

  • KOH (1 N)

Glassware and equipment

  • Erlenmeyer flask (200 mL), capped with two layers of aluminum foil
  • Forceps, sterilized before use

Preparation of B5 medium (medium no. 30)

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

    Chemical Amount
    B5 salt mix 1 bag (1 L)
    Sucrose 20 g
  2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution Volume (mL)
    B5_VT 2.5
    2,4-D (1 mM) 1
    Kinetin (1 mM) 0.04
  3. Adjust the pH of the solution to 5.7 with KOH (1 N), and fill up to 1 L with distilled water.

  4. Pour 80 mL of the medium into a 200-mL flask containing 0.72 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 B5 medium.
  2. Incubate cell cultures under the dark condition at 27°C.

Notes

  • We send Xan-1 cells on semi-solid B5 medium in a 9-cm plastic Petri dish. The cells should be subcultured to fresh B5 medium immediately after arrival.
  • In order to maintain Xan-1 callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of Xan-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 three pieces of Xan-1 callus (about 3-mm in diameter) on 80 mL of B5 medium in a 200-mL flask, and culture them for 28 days.

Maintenance history

References

[1]Taguchi F, Shimizu R, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2003) Post-translational modification of flagellin determines the specificity of HR induction. Plant & Cell Physiology 44: 342–349. DOI: 10.1093/pcp/pcg042