rpc00046-GCR237-3.2 (2015.11.26; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00046

Solanum lycopersicum GCR237 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, 0.2 mg/L 2,4-D, 2 mg/L NAA, 0.2 mg/L
zeatin, pH 5.8 (medium no. 39) [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 scientiﬁcjournal, it should be cited in the following manner: “Solanum lycopersicum
GCR237 cell line (rpc00046) was provided by the RIKEN BRC through theNational BioResource Project of the MEXT, Japan.”Experimental Plant Division
RIKEN BioResource Research Center (BRC)
Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074
Japan
FAX: +81 29 836 9053
E-mail: plant.brc@riken.jp
http://epd.brc.riken.jp/en/

1

Introduction
Tomato GCR237 cell line was established from a hypocotyl of Solanum lycopersicum
L. cultivar Craigella accession GCR237 (Tm-1/Tm-1) (Ishibashi et al. 2007). The accession GCR237 is nearly isogenic to tomato accession GCR26, but the GCR237 has ahomozygous Tm-1 gene, which confers resistance to tomato mosaic virus infection (Motoyoshi and Oshima 1977). The GCR237 cells are grown in a Murashige and Skoog (MS)medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg/L 1naphthaleneacetic acid (NAA) and 0.2 mg/L zeatin, pH 5.8. Our GCR237 cell culture has
been maintained in 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 Chemical
Corporation (#392-00591)
B) SucroseC) MS_VT
Nicotinic acid
Pyridoxine·HClThiamine·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) NAA (1 mg/mL)
NAA·K
1.2 mg/mL
Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation(#161-04021)2G) Zeatin (1 mg/mL)trans-Zeatin
1 mg/mL
Dissolve trans-zeatin in small volume of KOH (1 N), and ﬁll up with distilledwaterH) 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 minPreparation of MS medium (medium no. 39)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mix
Sucrose1 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)
NAA (1 mg/mL)
Zeatin (1 mg/mL)

1 mL
2.5 mL
1 mL
2 mL
0.2 mL

3. Adjust the pH of the solution to 5.8 with KOH (1 N), and ﬁll up to 1 L with distilledwater.
4. Pour 75 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 1–4 mL of cell suspension to 75 mL offresh MS medium with a pipette.
2. Incubate cell cultures on a rotary shaker at 120 rpm under the dark condition at27℃.

3Notes• For domestic customers: We send GCR237 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 GCR237 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 GCR237 cell suspension cultures stably, it is essential to transfer
an adequate amount of cells to fresh MS medium in every subculture. The amountof cells may vary from one lab to another, because proliferation of GCR237 cells
is aﬀected by culture conditions, such as a room temperature, rotation speed of arotary 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; https://www.shinpoly.co.jp/en/product/product/medical/plugs.html).

References
Ishibashi K, Masuda K, Naito S, Meshi T, Ishikawa M (2007) An inhibitor of viral RNA
replication is encoded by a plant resistance gene. Proceedings of National Academyof Sciences of USA 104: 13833–13838. DOI: 10.1073/pnas.0703203104
Motoyoshi F, Oshima N (1977) Expression of genetically controlled resistance to tobacco
mosaic virus infection in isolated tomato leaf mesophyll protoplasts. Journal of General Virology 34: 499–506. DOI: 10.1099/0022-1317-34-3-499

4Appendix A: Formulation of culture medium
Table A.1. Murashige and Skoog medium(medium no. 39)
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

KI0.83Na2 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 acid0.5

Pyridoxine·HCl

0.5

Thiamine·HCl

0.1

Glycine2

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.236

NAA·K

2.4

trans-Zeatin

0.25