rpc00062: Nicotiana tabacum BY-TIPG transgenic cell suspension culture

Note

  • Components

    • Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cell suspension

    • Overseas delivery: A 250-mL plastic Erlenmeyer flask, 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: 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

  • 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 through the National BioResource Project of the MEXT, 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, FUJIFILM Wako Pure Chemical Corporation (#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 a 50-mL disposable conical centrifuge tube. 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 a 250-mL disposable Erlenmeyer flask. 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 sponge plug may be used instead of the aluminum foil cap (e.g., cap-type Silicosen; Shin-Etsu Polymer, Tokyo, Japan).

Maintenance history

References