rpc00091-TBY2-31/ST-1.2 (2022.9.12; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00091

Nicotiana tabacum TBY2-31/ST transgenic 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, 110 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 TBY231/ST cell line (rpc00091) 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
Japan
FAX: +81 29 836 9053
E-mail: plant.brc@riken.jp
http://epd.brc.riken.jp/en/

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Introduction
Tobacco TBY2-31/ST cell line is a transgenic BY-2 cell line expressing Green FluorescentProtein (GFP) fused with Arabidopsis SYP31 and monomeric Red Fluorescent Protein(mRFP) fused with rat sialyltransferase (Ito et al. 2012). GFP ﬂuorescence is observed incis-Golgi, and mRFP ﬂuorescence is in trans-Golgi, using a ﬂuorescence microscope. The
parent cell line BY-2 (rpc00001) was established from a callus induced from a seedlingof Nicotiana tabacum L. cultivar Bright Yellow 2 (Nagata et al. 1992). The TBY2-31/ST
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 TBY2-31/ST cell culture has
been maintained in the dark at 27℃ with rotary shaking at 110 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) BY2_P
KH2 PO480 mg/mLD) LS_VT_modiﬁed
Thiamine·HCl
myo-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 (100 mL), capped with two layers of aluminum foil2B) Pipette (2 mL; large tip opening) and a bulb, sterilized by autoclaving at 121℃ for
20 minPreparation 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 30 mL of the medium into a 100-mL ﬂask.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Agitate a 7-day-old culture well and transfer 0.4 mL of cell suspension to 30 mL offresh mLS medium with a pipette.
2. Incubate cell cultures on a rotary shaker at 110 rpm under the dark condition at27℃.

Notes• For domestic customers: We send TBY2-31/ST 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 TBY2-31/ST cells placed on semi-solid mLS
medium in a 250-mL disposable Erlenmeyer ﬂask. The cells should be transferred
to fresh mLS medium immediately after arrival. Collect the cells from the semisolid medium with a spatula and transfer them to Erlenmeyer ﬂasks containing freshliquid medium.
• In order to maintain TBY2-31/ST cell suspension cultures stably, it is essential totransfer an adequate amount of cells to fresh mLS medium in every subculture. The3amount of cells may vary from one lab to another, because proliferation of TBY231/ST cells is aﬀected by culture conditions, such as a room temperature, rotation
speed of a rotary shaker, and aeration condition of the culture.
• A low growth rate of the parent BY-2 cells is sometimes caused by poor aeration(Kumagai-Sano 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
Ito Y, Uemura T, Shoda K, Fujimoto M, Ueda T, Nakano A (2012) cis-Golgi proteins
accumulate near the ER exit sites and act as the scaﬀold for Golgi regeneration
after brefeldin A treatment in tobacco BY-2 cells. Molecular Biology of the Cell 23:
3203–3214. DOI: 10.1091/mbc.E12-01-0034
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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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

8.6KI0.83

Na2 MoO4 ·2H2 O

0.25CuSO4 ·5H2 O

0.025

CoCl2 ·6H2 O

0.025

FeSO4 ·7H2 O

27.8

Na2 -EDTA

37.3

Thiamine·HCl1

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.2365