rpc00014: Catharanthus roseus CRA cell suspension culture
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: MS medium, 0.5 mg/L 2,4-D, pH 6.2 (medium no. 10)
Culture conditions: 27°C, dark, 120 rpm
Subculture: 7-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: “Catharanthus roseus CRA cell line (rpc00014) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”
Introduction
Periwinkle CRA cell line was established from Catharanthus roseus (L.) G.Don. The CRA cells are grown in a Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), pH 6.2. Our CRA cell culture has been maintained in the dark at 27°C with rotary shaking at 120 rpm and subcultured at 7-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
MS_VT
Chemical
Concentration (mg/mL)
Nicotinic acid
0.5
Pyridoxine·HCl
0.5
Thiamine·HCl
0.1
Glycine
2
MS_inositol
Chemical
Concentration (mg/mL)
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)
KOH (1 N)
Glassware
Erlenmeyer flask (300 mL), capped with two layers of aluminum foil
Pipette (10 mL; large tip opening) and a bulb, sterilized by autoclaving at 121°C for 20 min
Preparation of MS medium (medium no. 10)
Dissolve the following chemicals in approximately 800 mL of distilled water.
Chemical
Amount
MS salt mix
1 bag (1 L)
Sucrose
30 g
Add following stock solutions, and fill up to approximately 950 mL with distilled water.
Stock solution
Volume (mL)
MS_VT
1
MS_inositol
2.5
2,4-D (0.2 mg/mL)
2.5
Adjust the pH of the solution to 6.2 with KOH (1 N), and fill up to 1 L with distilled water.
Pour 95 mL of the medium into a 300-mL flask.
Autoclave the flask at 121°C for 20 min.
Methods
Agitate a 7-day-old culture well and transfer 4–5 mL of cell suspension to 95 mL of fresh MS medium with a pipette.
Incubate cell cultures on a rotary shaker at 120 rpm under the dark condition at 27°C.
Notes
For domestic customers: We send CRA cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh MS medium immediately after arrival.
For overseas customers: We send CRA cells placed on semi-solid MS medium in a 250-mL disposable Erlenmeyer flask. The cells should be transferred to fresh MS 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 CRA cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh MS medium in every subculture. The amount of cells may vary from one lab to another, because proliferation of CRA cells is affected by culture conditions, such as a room temperature, rotation speed of a rotary shaker, and aeration condition of the culture.
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).
Genotyping
Maintenance history
Protocols
Genotyping of plant cell lines: Catharanthus roseus (periwinkle) cell lines