rpc00036-BY-2H-3.4 (2014.10.3; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00036

Nicotiana tabacum BY-2H 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, 0.9% (w/v) agar, pH 5.8 (medium no. 31)[Materials III]
• Culture conditions: 27℃, dark [Methods II]• Subculture: 28-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-2H
cell line (rpc00036) 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 BY-2H cell line is a habituated BY-2 cell line that have capacity to proliferate
without 2,4-dichlorophenoxyacetic acid (2,4-D) (Syōno and Fujita 1994). The parent cellline BY-2 (rpc00001) was established from a callus induced from a seedling of Nicotiana
tabacum L. cultivar Bright Yellow 2 in the presence of 2,4-D (Nagata et al. 1992). The BY2H cells are grown on a phytohormone-free modiﬁed Linsmaier and Skoog (mLS) medium
solidiﬁed with 0.9% (w/v) agar, pH 5.8. Our BY-2H cell culture has been maintained in
the dark at 27℃ and subcultured at 28-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/mL

E) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
F) KOH (1 N)Glassware and equipmentA) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foilB) Forceps, sterilized before use
Preparation of mLS medium (medium no. 31)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.

2

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

2.5 mL
2.5 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 40 mL of the medium into a 100-mL ﬂask containing 0.36 g of agar.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Pick up an appropriate amount of callus cells from a 28-day-old culture with a forceps and place the cells onto fresh mLS medium.
2. Incubate cell cultures under the dark condition at 27℃.

Notes
• We send BY-2H cells on semi-solid mLS medium in a 9-cm disposable Petri dish.
The cells should be subcultured to fresh mLS medium immediately after arrival.
• In order to maintain BY-2H callus culture stably, it is essential to observe the growthof cells carefully. Because proliferation of BY-2H cells is aﬀected by culture conditions, such as a room temperature, aeration conditions of the culture and so on, anamount of cells transferred to fresh medium and the subculture intervals may varyfrom one lab to another. We usually inoculate three pieces of BY-2H callus (about5–7-mm in diameter) on 40 mL of mLS medium in a 100-mL ﬂask, and culture themfor 28 days.
• It is important to subculture good healthy cells. Brownish BY-2H cells near theupper part of a callus should not be used.

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

3

Syōno K, Fujita T (1994) Habituation as a tumorous state that is interchangeable with anormal state in plant cells. International Review of Cytology 152: 265–299. DOI: 1
0.1016/S0074-7696(08)62559-0

4

Appendix A: Formulation of culture medium
Table A.1. modiﬁed Linsmaier and Skoog medium(medium no. 31)
ChemicalConcentration (mg/L)

KNO3

1900

NH4 NO31650

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·HCl1

myo-Inositol

100

Sucrose

30000

Agar

90005