rpc00042: Nicotiana tabacum TBY2-AtRER1B transgenic callus culture

Note

  • Components

    • A 9-cm plastic Petri dish, 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, 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: 28–42-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 TBY2-AtRER1B cell line (rpc00042) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, 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 28–42-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)

  • 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

    Gellan gum, FUJIFILM Wako Pure Chemical Corporation (#073-03071)

  • KOH (1 N)

Glassware and equipment

  • Petri dish (9 cm diameter, 2 cm height), sterile

  • Forceps, sterilized before use

  • Surgical tape

    3M™ Micropore™ Surgical Tape, 12.5 mm × 9.1 m, 3M Japan Limited (#1530-0)

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. Add 4 g of gellan gum to the medium.

  5. Autoclave the medium 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 (mL)

    Kanamycin (200 mg/mL)

    1

    Carbenicillin (500 mg/mL)

    1

  7. Pour 30 mL of the medium into a 9-cm Petri dish.

Methods

  1. Pick up an appropriate amount of callus cells from a 28–42-day-old culture with a forceps and place the cells onto fresh mLS medium.

  2. Seal the Petri dishes using two rounds of surgical tape.

  3. 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 disposable 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 nine pieces of TBY2-AtRER1B callus (about 3-mm in diameter) on 30 mL of mLS medium in a 9-cm Petri dish, and culture them for 28–42 days.

  • GFP fluorescence of TBY2-AtRER1B cells can be weakened during long-term maintenance.

Genotyping

Maintenance history

References

Protocols