rpc00003: Vitis VR 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, 0.05 mg/L 2,4-D, 0.2 mg/L kinetin, 1.2% (w/v) agar, pH 6.1 (medium no. 3)
  • Culture conditions: 27°C, dark
  • Subculture: 56-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: “Vitis VR cell line (rpc00003) was provided by the RIKEN BRC which is participating in the National BioResource Project of the MEXT/AMED, Japan.”

Introduction

Grape VR cell lines was established from anthers of a Vitis interspecific hybrid cultivar Bailey Alicante A (Yamakawa et al. 1983a [1], b [2]). The VR callus cells accumulate higher amount of anthocyanins than VW callus cells (rpc00004). The VR cells are grown on a modified Linsmaier and Skoog (mLS) medium supplemented with 0.05 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/L kinetin and solidified with 1.2% (w/v) agar, pH 6.1. Our VR cell culture has been maintained in the dark at 27°C and subcultured at 56-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

  • 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)

  • Kinetin (0.2 mg/mL)

    Chemical Concentration (mg/mL)
    Kinetin 0.2

    Dissolve kinetin in small volume of KOH (1 N), and fill up with distilled water

  • Agar, powder

  • KOH (1 N)

Glassware and equipment

  • Erlenmeyer flask (300 mL), capped with two layers of aluminum foil
  • Forceps, sterilized before use

Preparation of mLS medium (medium no. 3)

  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)
    LS_VT_modified 2.5
    2,4-D (0.2 mg/mL) 0.25
    Kinetin (0.2 mg/mL) 1
  3. Adjust the pH of the solution to 6.1 with KOH (1 N), and fill up to 1 L with distilled water.

  4. Pour 100 mL of the medium into a 300-mL flask containing 1.2 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 56-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 VR 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 VR callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of VR 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 two to three pieces of VR callus (about 8-mm in diameter) on 100 mL of mLS medium in a 300-mL flask, and culture them for 56 days.
  • It is important to subculture good healthy cells. Yellow and pink VR cells are the most suitable, but red cells are too aged.

Maintenance history

References

[1]Yamakawa T, Ishida K, Kato S, kodama T, Minoda Y (1983a) Formation and identification of anthocyanins in cultured cells of Vitis sp. Agricultural and Biological Chemistry 47: 997–1001. DOI: 10.1080/00021369.1983.10865764
[2]Yamakawa T, Kato S, Ishida K, Kodama T, Minoda Y (1983b) Production of anthocyanins by Vitis cells in suspension culture. Agricultural and Biological Chemistry 47: 2185–2191. DOI: 10.1080/00021369.1983.10865 938