rpc00062: Nicotiana tabacum BY-TIPG transgenic cell suspension culture

Note

  1. Components
  • Domestic delivery: Two 50-mL tubes, containing 25 mL of cell suspension
  • Overseas delivery: Two 250-mL flasks, 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, pH 5.8 (medium no. 1)
  • Culture conditions: 27°C, dark, 130 rpm
  • Subculture: 7-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 BY-TIPG cell line (rpc00062) was provided by the RIKEN BRC which is participating in the National BioResource Project of the MEXT/AMED, Japan.”

Introduction

Tobacco BY-TIPG cell line is a transgenic BY-2 cell line expressing Green Fluorescent Protein (GFP) fused with a tobacco gamma tonoplast intrinsic protein, NtTIP1;1 (Okubo-Kurihara et al. 2009 [3]). GFP fluorescence is observed in vacuolar membrane 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 [2]). The BY-TIPG cells are grown in a modified Linsmaier and Skoog (mLS) medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), pH 5.8. Our BY-TIPG cell culture has been maintained in the dark at 27°C with rotary shaking at 130 rpm and subcultured at 7-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)

  • KOH (1 N)

Glassware

  • Erlenmeyer flask (300 mL), capped with two layers of aluminum foil
  • Pipette (10 mL; large tip opening) and a bulb, sterilized by autoclaving at 121°C for 20 min

Preparation of mLS medium (medium no. 1)

  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 95 mL of the medium into a 300-mL flask.

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

Methods

  1. Agitate a 7-day-old culture well and transfer 3 mL of cell suspension to 95 mL of fresh mLS medium with a pipette.
  2. Incubate cell cultures on a rotary shaker at 130 rpm under the dark condition at 27°C.

Notes

  • For domestic customers: We send BY-TIPG cell suspension in 50-mL disposable tubes. The cells should be transferred to fresh mLS medium immediately after arrival. Transfer settled cells to Erlenmeyer flasks containing fresh liquid medium with a pipette.
  • For overseas customers: We send BY-TIPG cells placed on semi-solid mLS medium in 250-mL disposable flasks. The cells should be transferred to fresh mLS medium immediately after arrival. Collect the cells from the semi-solid medium with a spatula and transfer them to Erlenmeyer flasks containing fresh liquid medium.
  • In order to maintain BY-TIPG cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh mLS medium in every subculture. The amount of cells may vary from one lab to another, because proliferation of BY-TIPG cells is affected by culture conditions, such as a room temperature, rotation speed of a rotary shaker, and aeration condition of the culture.
  • A low growth rate of the parent BY-2 cells is sometimes caused by poor aeration (Kumagai-Sano et al. 2007 [1]). In order to obtain good aeration of a suspension culture, a silicone cap may be used instead of the aluminum foil cap.

Maintenance history

References

[1]Kumagai-Sano F, Hayashi T, Sano T, Hasezawa S (2007) Cell cycle synchronization of tobacco BY-2 cells. Nature Protocols 1: 2621–2627. DOI: 10.1038/nprot.2006.381
[2]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
[3]Okubo-Kurihara E, Sano T, Higaki T, Kutsuna N, Hasezawa S (2009) Acceleration of vacuolar regeneration and cell growth by overexpression of an aquaporin NtTIP1;1 in tobacco BY-2 cells. Plant & Cell Physiology 50: 151–160. DOI: 10.1093/pcp/pcn181