rpc00002-kurodagosun-2.3 (2017.10.19; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00002

Daucus carota kurodagosun 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: modiﬁed Lin and Staba medium, 0.2 mg/L 2,4-D, pH 5.7(medium no. 67) [Materials III]
• Culture conditions: 27℃, dark, 130 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: “Daucus carota kurodagosun
cell line (rpc00002) was provided by the RIKEN BRC through the NationalBioResource 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
Carrot kurodagosun cell line is an embryogenic cell culture induced from a hypocotylof Daucus carota L. cultivar Kurodagosun according to the method described by Fujimura and Komamine (1979). The cell clusters proliferate as undiﬀerentiated cells in thepresence of 2,4-dichlorophenoxyacetic acid (2,4-D), but develop into somatic embryos bytransferring them to phytohormone-free medium. Because the embryogenic competence
decreases during repeated subculturing, we have been establishing the kurodagosun cellculture at about 6-month intervals. The kurodagosun cells are grown in a modiﬁed Lin andStaba medium supplemented with 0.2 mg/L 2,4-D, pH 5.7. Our kurodagosun cell culture
has been maintained in the dark at 27℃ with rotary shaking at 130 rpm and subcultured
at 7-day intervals.

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) KNO3B) NH4 ClC) CaCl2 ·2H2 OD) MgSO4 ·7H2 OE) SucroseF) Lin_and_Staba_1
H3 BO3MnSO4 ·5H2 O
ZnSO4 ·7H2 O

2.4 mg/mL
10.18 mg/mL
4.05 mg/mLG) JPL_C
FeSO4 ·7H2 O
2.78 mg/mL
Na2 -EDTA
3.73 mg/mL
Heat at 80℃ for 3–4 hours for chelating FeH) Lin_and_Staba_3KI
Na2 MoO4 ·2H2 O
CuSO4 ·5H2 O
CoCl2 ·6H2 O

0.375 mg/mL
0.127 mg/mL
0.01 mg/mL
0.01 mg/mL2I) Lin_and_Staba_4
Nicotinic acid
Pyridoxine·HClThiamine·HCl

5 mg/mL
0.5 mg/mL
3 mg/mLJ) Lin_and_Staba_5
KH2 PO468 mg/mLK) 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)L) 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 modiﬁed Lin and Staba medium (medium no. 67)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
KNO3NH4 Cl
CaCl2 ·2H2 O
MgSO4 ·7H2 OSucrose

5.56 g0.268 g0.22 g0.185 g20 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
Lin_and_Staba_1
JPL_C
Lin_and_Staba_3
Lin_and_Staba_4
Lin_and_Staba_5
2,4-D (0.2 mg/mL)

1 mL
1 mL
1 mL
1 mL
1 mL
1 mL

3. Adjust the pH of the solution to 5.7 with KOH (1 N), and ﬁll up to 1 L with distilledwater.

34. Pour 80 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 10 mL of cell suspension to 80 mL offresh modiﬁed Lin and Staba medium with a pipette.
2. Incubate cell cultures on a rotary shaker at 130 rpm under the dark condition at27℃.

Notes• For domestic customers: We send kurodagosun cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh modiﬁed Linand Staba medium immediately after arrival.
• For overseas customers: We send kurodagosun cells placed on semi-solid modiﬁed
Lin and Staba medium in a 250-mL disposable Erlenmeyer ﬂask. The cells shouldbe transferred to fresh modiﬁed Lin and Staba medium immediately after arrival.
Collect the cells from the semi-solid medium with a spatula and transfer them toErlenmeyer ﬂasks containing fresh liquid medium.
• In order to maintain kurodagosun cell suspension cultures stably, it is essential totransfer an adequate amount of cells to fresh modiﬁed Lin and Staba medium inevery subculture. The amount of cells may vary from one lab to another, because
proliferation of kurodagosun 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; https://www.shinpoly.co.jp/en/product/product/medical/plugs.html).

References
Fujimura T, Komamine A (1979) Synchronization of somatic embryogenesis in a carrot
cell suspension culture. Plant Physiology 64: 162–164. DOI: 10.1104/pp.64.1.162

4Appendix A: Formulation of culture medium
Table A.1. modiﬁed Lin and Staba medium(medium no. 67)
ChemicalConcentration (mg/L)

KNO35560

NH4 Cl268

CaCl2 ·2H2 O

220

MgSO4 ·7H2 O

185

KH2 PO468

H3 BO3

2.4

MnSO4 ·5H2 O

10.18

ZnSO4 ·7H2 O

4.05

KI0.375

Na2 MoO4 ·2H2 O

0.127

CuSO4 ·5H2 O

0.01

CoCl2 ·6H2 O

0.01

FeSO4 ·7H2 O

2.78

Na2 -EDTA

3.73

Nicotinic acid5

Pyridoxine·HCl

0.5

Thiamine·HCl

3Sucrose

20000

2,4-D sodium monohydrate

0.2365