rpc00001-BY-2-3.2 (2014.2.20; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00001

Nicotiana tabacum BY-2 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: mLS medium, 0.2 mg/L 2,4-D, pH 5.8 (medium no. 1)[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: “Nicotiana tabacum BY-2
cell line (rpc00001) 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/

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Introduction
Tobacco BY-2 cell line is a fast-growing and highly-homogeneous cell line, and used for abroad range of plant studies around the world (Nagata et al. 2006). A suspension culture
was established from a callus induced from a seedling of Nicotiana tabacum L. cultivar
Bright Yellow 2 (Nagata et al. 1992). The BY-2 cells are grown in a modiﬁed Linsmaier
and Skoog (mLS) medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid
(2,4-D), pH 5.8. Our BY-2 cell culture has been maintained in the dark at 27℃ with rotary
shaking at 130 rpm and subcultured at 7-day intervals (Figure 1, 2).

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) BY2_P
KH2 PO480 mg/mLD) LS_VT_modiﬁedThiamine·HClmyo-Inositol

0.4 mg/mL
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 mLS medium (medium no. 1)
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.
BY2_P
LS_VT_modiﬁed
2,4-D (0.2 mg/mL)

2.5 mL
2.5 mL
1 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 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 1–1.2 mL of cell suspension to 95 mLof fresh mLS 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 BY-2 cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh mLS medium immediately after arrival.
• For overseas customers: We send BY-2 cells placed on semi-solid mLS medium ina 250-mL disposable Erlenmeyer ﬂask. The cells should be transferred to freshmLS 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 BY-2 cell suspension cultures stably, it is essential to transfer anadequate amount of cells to fresh mLS medium in every subculture. The amount ofcells may vary from one lab to another, because proliferation of BY-2 cells is aﬀectedby culture conditions, such as a room temperature, rotation speed of a rotary shaker,
and aeration condition of the culture.

3• A low growth rate of BY-2 cells is sometimes caused by poor aeration (KumagaiSano et al. 2007). 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
Kumagai-Sano F, Hayashi T, Sano T, Hasezawa S (2007) Cell cycle synchronization oftobacco BY-2 cells. Nature Protocols 1: 2621–2627. DOI: 10.1038/nprot.2006.381
Nagata T, Matsuoka K, Inze D (eds) (2006) Biotechnology in Agriculture and Forestry
58, Tobacco BY-2 cells: From cellular dynamics to omics. Springer-Verlag BerlinHeidelberg. DOI: 10.1007/3-540-32674-x
Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the “HeLa” cell in thecell biology of higher plants. International Review of Cytology 132: 1–30. DOI: 10.1016/S0074-7696(08)62452-3

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Figure 1. Nicotiana tabacum BY-2 cell suspension culture
A: Seven-day-old cell suspension culture.
B: Morphology of BY-2 cells after 3 day of subculture.
BY-2 cells were observed by using a diﬀerential interference contrast microscope.
Scale bar = 100 µm5Figure 2. Growth proﬁle of BY-2 cells

6Appendix A: Formulation of culture medium
Table A.1. modiﬁed Linsmaier and Skoog medium(medium no. 1)
ChemicalConcentration (mg/L)

KNO3

1900

NH4 NO31650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O370

KH2 PO4370

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 O0.025

FeSO4 ·7H2 O

27.8

Na2 -EDTA

37.3

Thiamine·HCl

1

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.2367