rpc00098-topless3-GFP-1.0 (2024.8.28)
RIKEN BRC plant cell line documentation

rpc00098

Nicotiana tabacum topless3-GFP transgenic callus culture

Components• 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, 0.5% (w/v) gellan gum, pH
5.8 (medium no. 59) [Materials III]
• Culture conditions: 27℃, dark [Methods II]• Subculture: 21-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 topless3GFP cell line (rpc00098) 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/

1Introduction
Tobacco topless3-GFP cell line is a transgenic BY-2 cell line expressing Green FluorescentProtein (GFP) fused with NtTPL3 (Nagashima et al. 2019). GFP ﬂuorescence is observedin nuclei by using a ﬂuorescence microscope. The parent cell line BY-2 (rpc00001) wasestablished from a callus induced from a seedling of Nicotiana tabacum L. cultivar BrightYellow 2 (Nagata et al. 1992). The topless3-GFP cells are grown on a modiﬁed Linsmaier
and Skoog (mLS) medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid
(2,4-D), and solidiﬁed with 0.5% (w/v) gellan gum, pH 5.8. Our topless3-GFP cell culture
has been maintained in the dark at 27℃ and subcultured at 21-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) Gellan gum
Gellan gum, FUJIFILM Wako Pure Chemical Corporation (#073-03071)
G) KOH (1 N)

2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. 59)
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 5 g of gellan gum to the medium.
5. Autoclave the medium at 121℃ for 20 min.
6. Pour 35 mL of the medium into a 9-cm Petri dish.

Methods
1. Pick up an appropriate amount of callus cells from a 21-day-old culture with a forceps and place the cells onto fresh mLS medium.
2. Incubate cell cultures under the dark condition at 27℃.
3. Seal the Petri dishes using two rounds of surgical tape.

Notes• We send topless3-GFP cells on semi-solid mLS medium in a 9-cm disposable Petridish. The cells should be subcultured to fresh mLS medium immediately after ar-3

rival.
• In order to maintain topless3-GFP callus culture stably, it is essential to observe thegrowth of cells carefully. Because proliferation of topless3-GFP cells is aﬀected byculture conditions, such as a room temperature, aeration conditions of the culture
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 sixteen pieces oftopless3-GFP callus (about 1–2-mm in diameter) on 35 mL of mLS medium in a9-cm Petri dish, and culture them for 21 days.

References
Nagashima A, Higaki T, Koeduka T, Ishigami K, Hosokawa S, Watanabe H, Matsui K,Hasezawa S, Touhara K (2019) Transcriptional regulators involved in responses tovolatile organic compounds in plants. Journal of Biological Chemistry 294: 2256–
2266. DOI: 10.1074/jbc.RA118.005843
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

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Appendix A: Formulation of culture medium
Table A.1. modiﬁed Linsmaier and Skoog medium(medium no. 59)
ChemicalConcentration (mg/L)

KNO31900

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

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·HCl1

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.236

Gellan gum

5000

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