rpc00014-CRA-3.3 (2014.7.15; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00014

Catharanthus roseus CRA cell suspension cultureComponents• Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cellsuspension• 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 [Notes I, II].
• Do not store the cell culture in a refrigerator and a freezer.
• Maintain aseptic conditions of the cell culture, and work in a laminar ﬂowcabinet.
Method• Culture medium: MS medium, 0.5 mg/L 2,4-D, pH 6.2 (medium no. 10)[Materials III]
• Culture conditions: 27℃, dark, 120 rpm [Methods II]• Subculture: 7-day intervals [Methods I]
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.”Experimental Plant Division
RIKEN BioResource Research Center (BRC)
Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074
JapanFAX: +81 29 836 9053
E-mail: plant.brc@riken.jp
http://epd.brc.riken.jp/en/

1

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 2,4dichlorophenoxyacetic acid (2,4-D), pH 6.2. Our CRA cell culture has been maintainedin the dark at 27℃ with rotary shaking at 120 rpm and subcultured at 7-day intervals.

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) MS salt mix
Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure ChemicalCorporation (#392-00591)
B) SucroseC) MS_VT
Nicotinic acid
Pyridoxine·HCl
Thiamine·HClGlycine

0.5 mg/mL
0.5 mg/mL
0.1 mg/mL
2 mg/mLD) MS_inositol
myo-Inositol

40 mg/mLE) 2,4-D (0.2 mg/mL)
2,4-D sodium monohydrate
0.236 mg/mL(2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich(D6679)
F) KOH (1 N)
GlasswareA) Erlenmeyer ﬂask (300 mL), capped with two layers of aluminum foilB) Pipette (10 mL; large tip opening) and a bulb, sterilized by autoclaving at 121℃ for
20 min2Preparation of MS medium (medium no. 10)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mixSucrose

1 bag (1 L)
30 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
MS_VT
MS_inositol
2,4-D (0.2 mg/mL)

1 mL
2.5 mL
2.5 mL3. Adjust the pH of the solution to 6.2 with KOH (1 N), and ﬁll up to 1 L with distilledwater.
4. Pour 95 mL of the medium into a 300-mL ﬂask.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Agitate a 7-day-old culture well and transfer 4–5 mL of cell suspension to 95 mL offresh MS medium with a pipette.
2. Incubate cell cultures on a rotary shaker at 120 rpm under the dark condition at27℃.

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 tofresh MS medium immediately after arrival. Collect the cells from the semi-solid
medium with a spatula and transfer them to Erlenmeyer ﬂasks containing fresh liquidmedium.
• In order to maintain CRA cell suspension cultures stably, it is essential to transfer anadequate amount of cells to fresh MS medium in every subculture. The amount ofcells may vary from one lab to another, because proliferation of CRA cells is aﬀectedby culture conditions, such as a room temperature, rotation speed of a rotary shaker,
and aeration condition of the culture.

3• 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; https://www.shinpoly.co.jp/en/product/product/medical/plugs.html).

4Appendix A: Formulation of culture medium
Table A.1. Murashige and Skoog medium(medium no. 10)
ChemicalConcentration (mg/L)

KNO31900

NH4 NO31650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO3

6.2

MnSO4 ·4H2 O

22.3

ZnSO4 ·7H2 O

8.6

KI

0.83

Na2 MoO4 ·2H2 O

0.25

CuSO4 ·5H2 O

0.025

CoCl2 ·6H2 O

0.025

FeSO4 ·7H2 O

27.8

Na2 -EDTA

37.3

Nicotinic acid

0.5

Pyridoxine·HCl

0.5

Thiamine·HCl

0.1

Glycine2myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.595