rpc00042: Nicotiana tabacum TBY2-AtRER1B transgenic 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: mLS medium, 0.2 mg/L 2,4-D, 200 mg/L kanamycin, 500 mg/L carbenicillin, 0.4% (w/v) gellan gum, pH 5.8 (medium no. 51)
  • 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 TBY2-AtRER1B cell line (rpc00042) was provided by the RIKEN BRC which is participating in the National BioResource Project of the MEXT/AMED, Japan.”

Introduction

Tobacco TBY2-AtRER1B cell line is a transgenic BY-2 cell line expressing Green Fluorescent Protein (GFP) fused with AtRer1B (Takeuchi et al. 2000 [2], 2002 [3]). GFP fluorescence is observed in the Golgi apparatus by using a fluorescence microscope. The parent cell line BY-2 (rpc00001) was established from a callus induced from a seedling of Nicotiana tabacum L. cultivar Bright Yellow 2 (Nagata et al. 1992 [1]). The TBY2-AtRER1B cells are grown on a modified Linsmaier and Skoog (mLS) medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 200 mg/L kanamycin and 500 mg/L carbenicillin and solidified with 0.4% (w/v) gellan gum, pH 5.8. Our TBY2-AtRER1B 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)
  • MS salt mix

    Murashige and Skoog Plant Salt Mixture, Wako Pure Chemical Industries (#392-00591)

  • Sucrose

  • BY2_P

    Chemical Concentration (mg/mL)
    KH2PO4 80
  • LS_VT_modified

    Chemical Concentration (mg/mL)
    Thiamine·HCl 0.4
    myo-Inositol 40
  • 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)

  • Kanamycin (200 mg/mL)

    Chemical Concentration (mg/mL)
    Kanamycin sulfate 200

    Sterilize the solution by membrane filtration

  • Carbenicillin (500 mg/mL)

    Chemical Concentration (mg/mL)
    Carbenicillin disodium salt 500

    Sterilize the solution by membrane filtration

  • Gellan gum

  • KOH (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. 51)

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

    Chemical Amount
    MS salt mix 1 bag (1 L)
    Sucrose 30 g
  2. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution Volume (mL)
    BY2_P 2.5
    LS_VT_modified 2.5
    2,4-D (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 0.16 g of gellan gum.

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

  6. Add the following antibiotics to the medium just before agar solidification and swirl the medium gently to mix.

    Stock solution Volume (µL)
    Kanamycin (200 mg/mL) 40
    Carbenicillin (500 mg/mL) 40

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 mLS medium.
  2. Incubate cell cultures under the dark condition at 27°C.

Notes

  • We send TBY2-AtRER1B 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 TBY2-AtRER1B callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of TBY2-AtRER1B 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 TBY2-AtRER1B callus (about 5-mm in diameter) on 40 mL of mLS medium in a 100-mL flask, and culture them for 28 days.
  • Very occasionally, GFP fluorescence of TBY2-AtRER1B cells can be weakened during long-term maintenance.

Maintenance history

References

[1]Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in the cell biology of higher plants. International Review of Cytology 132: 1–30. DOI: 10.1016/S0074-7696(08)62452-3
[2]Takeuchi M, Ueda T, Sato K, Abe H, Nagata T, Nakano A (2000) A dominant negative mutant of sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells. Plant Journal 23: 517–525. DOI: 10.1046/j.1365-313x.2000.00823.x
[3]Takeuchi M, Ueda T, Yahara N, Nakano A (2002) Arf1 GTPase plays roles in the protein traffic between the endoplasmic reticulum and the Golgi apparatus in tobacco and Arabidopsis cultured cells. Plant Journal 31: 499–515. DOI: 10.1046/j.1365-313X.2002.01372.x