rpc00036: Nicotiana tabacum BY-2H 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, 0.9% (w/v) agar, pH 5.8 (medium no. 31)

    • Culture conditions: 27°C, dark

    • Subculture: 28-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-2H cell line (rpc00036) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

Tobacco BY-2H cell line is a habituated BY-2 cell line that have capacity to proliferate without 2,4-dichlorophenoxyacetic acid (2,4-D) (Syōno and Fujita 1994 [2]). The parent cell line BY-2 (rpc00001) was established from a callus induced from a seedling of Nicotiana tabacum L. cultivar Bright Yellow 2 in the presence of 2,4-D (Nagata et al. 1992 [1]). The BY-2H cells are grown on a phytohormone-free modified Linsmaier and Skoog (mLS) medium solidified with 0.9% (w/v) agar, pH 5.8. Our BY-2H 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, 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

  • Agar, powder

    Agar, powder, Junsei Chemical (#24440-1201)

  • 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. 31)

  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

  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 0.36 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 mLS medium.

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

Notes

  • We send BY-2H 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 BY-2H callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of BY-2H 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 BY-2H callus (about 5–7-mm in diameter) on 40 mL of mLS medium in a 100-mL flask, and culture them for 28 days.

  • It is important to subculture good healthy cells. Brownish BY-2H cells near the upper part of a callus should not be used.

Genotyping

Maintenance history

References

Protocols