rpc00042-TBY2-AtRER1B-3.4 (2016.8.9; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00042

Nicotiana tabacum TBY2-AtRER1B transgenic callus cultureComponents• A 9-cm plastic Petri dish, containing cells placed on semi-solid medium
Notice• Subculture the cells to fresh medium immediately after arrival [Notes I].
• 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, 200 mg/L kanamycin,
500 mg/L carbenicillin, 0.4% (w/v) gellan gum, pH 5.8(medium no. 51) [Materials III]
• Culture conditions: 27℃, dark [Methods II]• Subculture: 28–42-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 TBY2AtRER1B cell line (rpc00042) 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/

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Introduction
Tobacco TBY2-AtRER1B cell line is a transgenic BY-2 cell line expressing Green Fluorescent Protein (GFP) fused with AtRer1B (Takeuchi et al. 2000, 2002). GFP ﬂuorescenceis observed in the Golgi apparatus by using a ﬂuorescence microscope. The parent cellline BY-2 (rpc00001) was established from a callus induced from a seedling of Nicotiana
tabacum L. cultivar Bright Yellow 2 (Nagata et al. 1992). The TBY2-AtRER1B cells aregrown on a modiﬁed Linsmaier and Skoog (mLS) medium supplemented with 0.2 mg/L
2,4-dichlorophenoxyacetic acid (2,4-D), 200 mg/L kanamycin and 500 mg/L carbenicillin,and solidiﬁed with 0.4% (w/v) gellan gum, pH 5.8. Our TBY2-AtRER1B cell culture has
been maintained in the dark at 27℃ and subcultured at 28–42-day intervals.

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) MS salt mixMurashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure ChemicalCorporation (#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) Kanamycin (200 mg/mL)
Kanamycin sulfate
200 mg/mL
Sterilize the solution by membrane ﬁltrationG) Carbenicillin (500 mg/mL)
Carbenicillin disodium salt500 mg/mL

2Sterilize the solution by membrane ﬁltration
H) Gellan gum
Gellan gum, FUJIFILM Wako Pure Chemical Corporation (#073-03071)
I) KOH (1 N)
Glassware and equipment
A) Petri dish (9 cm diameter, 2 cm height), sterileB) Forceps, sterilized before useC) Surgical tape
3M™ Micropore™ Surgical Tape, 12.5 mm × 9.1 m, 3M Japan Limited(#1530-0)
Preparation of mLS medium (medium no. 51)
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. Add 4 g of gellan gum to the medium.
5. Autoclave the medium at 121℃ for 20 min.
6. Add the following antibiotics to the medium just before agar solidiﬁcation and swirl
the medium gently to mix.
Kanamycin (200 mg/mL)
Carbenicillin (500 mg/mL)

1 mL
1 mL

7. Pour 30 mL of the medium into a 9-cm Petri dish.

3Methods
1. Pick up an appropriate amount of callus cells from a 28–42-day-old culture with aforceps and place the cells onto fresh mLS medium.
2. Seal the Petri dishes using two rounds of surgical tape.
3. Incubate cell cultures under the dark condition at 27℃.

Notes
• We send TBY2-AtRER1B cells on semi-solid mLS medium in a 9-cm disposable
Petri dish. The cells should be subcultured to fresh mLS medium immediately afterarrival.
• In order to maintain TBY2-AtRER1B callus culture stably, it is essential to observe
the growth of cells carefully. Because proliferation of TBY2-AtRER1B cells is affected by culture conditions, such as a room temperature, aeration conditions of theculture and so on, an amount of cells transferred to fresh medium and the subculture intervals may vary from one lab to another. We usually inoculate nine piecesof TBY2-AtRER1B callus (about 3-mm in diameter) on 30 mL of mLS medium ina 9-cm Petri dish, and culture them for 28–42 days.
• GFP ﬂuorescence of TBY2-AtRER1B cells can be weakened during long-term
maintenance.

References
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: 1
0.1016/S0074-7696(08)62452-3
Takeuchi M, Ueda T, Sato K, Abe H, Nagata T, Nakano A (2000) A dominant negative
mutant of sar1 GTPase inhibits protein transport from the endoplasmic reticulum
to the Golgi apparatus in tobacco and Arabidopsis cultured cells. Plant Journal 23:
517–525. DOI: 10.1046/j.1365-313x.2000.00823.x
Takeuchi M, Ueda T, Yahara N, Nakano A (2002) Arf1 GTPase plays roles in the protein
traﬃc between the endoplasmic reticulum and the Golgi apparatus in tobacco andArabidopsis cultured cells. Plant Journal 31: 499–515. DOI: 10.1046/j.1365-313X.2002.01372.x

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Appendix A: Formulation of culture medium
Table A.1. modiﬁed Linsmaier and Skoog medium(medium no. 51)
ChemicalConcentration (mg/L)

KNO3

1900

NH4 NO3

1650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4370

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

Thiamine·HCl

1

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.236

Kanamycin sulfate

200

Carbenicillin disodium salt

500

Gellan gum

4000

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