rpc00008: Arabidopsis thaliana T87 cell suspension culture

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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: JPL medium, 1 µM NAA %, pH %5.7 (medium no. 5)

  • Culture conditions: 22°C, continuous light, 120 rpm

  • Subculture: 14-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: “Arabidopsis thaliana T87 cell line (rpc00008) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction

Arabidopsis T87 cell line was established from a seedling of Arabidopsis thaliana (L.) Heynh. accession Columbia (Axelos et al. 1992 [1]). The T87 cell culture is green and composed of small near-uniform aggregates of cells (Figure 1). The T87 cells are grown in a Jouanneau and Péaud-Lenoël (JPL) medium supplemented with 1 µM 1-naphthaleneacetic acid (NAA). Our T87 cell culture has been maintained under the continuous light at 22°C with rotary shaking at 120 rpm and subcultured at 14-day intervals (Figure 2).

Materials

Chemicals and stock solutions

(All stock solutions are stored at 4°C)

• JPL_Aʼ

  Chemical	Concentration (mg/mL)
  KNO3	65.5
  CaCl2·2H2O	4.4
  MgSO4·7H2O	3.7
  KH2PO4	1.7

• JPL_B

  Chemical	Concentration (mg/mL)
  H3BO3	6.2
  MnSO4·5H2O	24.1
  ZnSO4·7H2O	10.6
  KI	0.83
  Na2MoO4·2H2O	0.25
  CuSO4·5H2O	0.025
  CoCl2·6H2O	0.025

• JPL_C

  Chemical	Concentration (mg/mL)
  FeSO4·7H2O	2.78
  Na2-EDTA	3.73

  Heat at 80°C for 3–4 hours for chelating Fe

• JPL_D

  Chemical	Concentration (mg/mL)
  Glycine	0.2
  myo-Inositol	10

• JPL_VT

  Chemical	Concentration (mg/mL)
  Nicotinic acid	0.5
  Pyridoxine·HCl	0.5
  Thiamine·HCl	0.4

• JPL_P

  Stock solution	Volume (mL)
  200 mM KH2PO4	19.5
  200 mM Na2HPO4	30.5
  H2O	50

• NAA (1 mM)

  Chemical	Concentration (mg/mL)
  NAA·K	0.224

  Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation (#161-04021)

• Sucrose

• Casein hydrolysate, vitamin free

  Casamino acids vitamin assay, Difco (#228820)

• KOH (1 N)

• HCl (1 N)

Glassware and equipment

(All are sterilized by autoclaving at 121°C for 20 min)

• Erlenmeyer flask (300 mL), capped with two layers of aluminum foil

• Pipette (10 mL; large tip opening) and a bulb

• Stainless sieve (diameter, 5 cm; pore size, 1 mm) set on a tall beaker (200 mL), capped with two layers of aluminum foil

Preparation of JPL medium (medium no. 5)

medium no. 5: Jouanneau and Péaud-Lenoël medium, 1 µM 1-naphthaleneacetic acid

1. Prepare three solutions as follows.

• JPL mineral solution (1 L)

  Stock solution	Volume (mL)
  JPL_Aʼ	37.5
  JPL_B	0.375
  JPL_C	2.5

  Adjust the pH to 5.7 with KOH (1 N)

• JPL organic solution (100 mL)

  Chemical/Stock solution	Amount
  JPL_D	10 mL
  JPL_VT	1 mL
  Casein hydrolysate	0.1 g

  Adjust the pH to 5.7 with HCl (1 N)

• JPL sucrose solution (100 mL)

  Chemical/Stock solution	Amount
  JPL_P	1 mL
  NAA (1 mM)	1 mL
  Sucrose	15 g

1. Autoclave the solutions at 121°C for 20 min.

2. Add 800 mL of JPL mineral solution, 100 mL of JPL organic solution, and 100 mL of JPL sucrose solution asceptically.

3. Pour 80 mL of the medium into a sterile 300-mL flask.

Methods

1. Filter a 14-day-old cell suspension through a stainless sieve (Figure 3).

2. Agitate the filtrate well and transfer 2–3 mL of cell suspension to 80 mL of fresh JPL medium with a pipette.

3. Incubate cell cultures on a rotary shaker at 120 rpm under the continuous light condition (photosynthetic photon flux density 40–42 µmol m^-2 s^-1) at 22°C.

Notes

• For domestic customers: We send T87 cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh JPL medium immediately after arrival.

• For overseas customers: We send T87 cells placed on semi-solid JPL medium in a 250-mL disposable Erlenmeyer flask. The cells should be transferred to fresh JPL 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 T87 cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh JPL medium in every subculture. The amount of cells may vary from one lab to another, because proliferation of T87 cells is affected by culture conditions, such as a room temperature, rotation speed of a rotary shaker, and aeration condition of the culture. T87-cell clumps occasionally develop into large aggregates, which causes a decrease in the amount of cells passed through a 1-mm sieve.

• 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).

• T87 cells may be maintained in Gamborgʼs B5 medium (Yamada et al. 2004 [3]) or Murashige and Skoog medium (Cunillera et al. 2000 [2]) other than JPL medium.

Genotyping

Genotyping: rpc00008

Maintenance history

Maintenance history: rpc00008

References

[1]

Axelos M, Curie C, Mazzolini L, Bardet C, Lescure B (1992) A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures. Plant Physiology and Biochemistry 30: 123–128.

[2]

Cunillera N, Arró M, Forés O, David Manzano, Ferrer A (2000) Characterization of dehydrodolichyl diphosphate synthase of Arabidopsis thaliana, a key enzyme in dolichol biosynthesis. FEBS Letters 477: 170–174. DOI: 10.1016/S0014-5793(00)01798-1

[3]

Yamada H, Koizumi N, Nakamichi N, Kiba T, Yamashino T, Mizuno T (2004) Rapid response of Arabidopsis T87 cultured cells to cytokinin through His-to-Asp phosphorelay signal transduction. Bioscience, Biotechnology, and Biochemistry 68: 1966–1976. DOI: 10.1271/bbb.68.1966

Protocols

• Cryopreservation of Arabidopsis T87 suspension cell cultures

• Genotyping of plant cell lines: Arabidopsis thaliana cell lines

External links

• World Flora Online: Arabidopsis thaliana (L.) Heynh.

• Wikipedia: Arabidopsis thaliana

Figures

Figure 1

Arabidopsis thaliana T87 cell suspension culture

1. Two-week-old cell suspension culture.

2. Cell clumps. The size of cell clumps are varied, but most of them are below 1 mm. Scale bar = 1 mm

3. Morphology of T87 cells. T87 cells were observed by using differential interference contrast microscopy. The cells contain many chloroplasts. Scale bar = 50 µm

Figure 2

Growth profile of T87 cells

Figure 3

Procedure for subculturing T87 cells

1. Two-week-old cell suspension culture.

2. Pass the culture through a stainless sieve (pore size, 1 mm).

3. Transfer 2 mL of the filtrate to 80 mL of fresh JPL medium.