rpc00108: Calypogeia azurea SUN1001P 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: MSK-1410 medium, 0.2% (w/v) gellan gum, pH 5.7 (medium no. 66)

    • Culture conditions: 25°C, continuous light

    • 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: “Calypogeia azurea SUN1001P cell line (rpc00108) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

Liverwort SUN1001P cell line was established from gametophytes of Calypogeia azurea Stotler”, “Crotz (Nakagawara et al. 1992 [1]). The SUN1001P cells produce azulene derivatives. The SUN1001P cells are grown on a phytohormone-free modified Murashige and Skoog (MSK-1410) medium solidified with 0.2% (w/v) gellan gum, pH 5.7. Our SUN1001P cell culture has been maintained under the continuous light at 25°C and subcultured at 28-day intervals.

Materials

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

  • Fumalic acid

  • MS salt mix

    Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#392-00591)

  • D(+)-Glucose

  • MSK_VT

    Chemical

    Concentration (mg/mL)

    Pyridoxine·HCl

    1

    Thiamine·HCl

    1

    myo-Inositol

    100

    L-Ascorbic acid

    2

    Calcium (+)-pantothenate

    1

    Choline chloride

    1

    Nicotinamide

    1

    Folic acid

    0.4

    Riboflavin

    0.2

    p-Aminobenzoic acid

    0.02

    Cyanocobalamin

    0.02

    (+)-Biotin

    0.02

    Store at −20°C

  • MSK_F

    Chemical

    Concentration (mg/mL)

    Citric acid monohydrate

    43.8

    DL-Malic acid

    40

    Sodium pyruvate

    20

    Store at −20°C

  • MSK_G

    Chemical

    Concentration (mg/mL)

    D(−)-Fructose

    2.5

    D(+)-Cellobiose

    2.5

    D(−)-Mannitol

    2.5

    D(+)-Mannose

    2.5

    α-L(+)-Rhamnose monohydrate

    2.77

    D(−)-Ribose

    2.5

    D(−)-Sorbitol

    2.5

    D(+)-Xylose

    2.5

    Store at −20°C

  • Gellan gum

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

  • NaOH (5 N)

Glassware and equipment

  • Plant culture tube (25 × 120 mm; 9820TST-F25-120, IWAKI Scitech, AGC Techno Glass Co., Ltd)

  • Polypropylene cap (9988CAP25, IWAKI Scitech, AGC Techno Glass Co., Ltd)

  • Forceps, sterilized before use

Preparation of MSK-1410 medium (medium no. 66)

  1. Dissolve the following chemicals in approximately 300 mL of distilled water with NaOH (5 N).

    Chemical

    Amount

    Fumalic acid

    2.04 g

    NaOH (5 N)

    6 mL

  2. Fill up to approximately 800 mL with distilled water, and dissolve the following chemicals.

    Chemical

    Amount

    MS salt mix

    1 bag (1 L)

    D(+)-Glucose

    40 g

  3. Add following stock solutions, and fill up to approximately 950 mL with distilled water.

    Stock solution

    Volume (mL)

    MSK_VT

    1

    MSK_F

    1

    MSK_G

    1

  4. Adjust the pH of the solution to 5.7 with NaOH (5 N), and fill up to 1 L with distilled water.

  5. Add 2 g of gellan gum to the medium.

  6. Heat the medium in a microwave to dissolve the gellan gum.

  7. Pour 20 mL of the medium into a plant culture tube.

  8. Put a polypropylene cap on the plant culture tube.

  9. Autoclave the plant culture tube 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 MSK-1410 medium.

  2. Incubate cell cultures under the continuous light condition (photosynthetic photon flux density 17 µmol m-2 s-1) at 25°C for 7 days.

  3. Incubate cell cultures under the continuous light condition (photosynthetic photon flux density 50 µmol m-2 s-1) at 25°C.

Notes

  • We send SUN1001P cells on semi-solid MSK-1410 medium in a 9-cm disposable Petri dish. The cells should be subcultured to fresh MSK-1410 medium immediately after arrival.

  • In order to maintain SUN1001P callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of SUN1001P 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 one pieces of SUN1001P callus (about 3-mm in diameter) on 20 mL of MSK-1410 medium in a plant culture tube, and culture them for 28 days.

References

Genotyping

/genotyping/g_rpc00108

Maintenance history

Protocols