rpc00035-Xan-1-2.3 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00035

Nicotiana tabacum Xan-1 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: B5 medium, 1 µM 2,4-D, 0.04 µM kinetin, 0.9% (w/v) agar,
pH 5.7 (medium no. 30) [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 Xan-1
cell line (rpc00035) 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 Xan-1 cell line was established from a leaf of Nicotiana tabacum L. cultivar
Xanthi NC (Taguchi et al. 2003). The Xan-1 cells are grown on a Gamborg’s B5 medium
supplemented with 1 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.04 µM kinetin,and solidiﬁed with 0.9% (w/v) agar, pH 5.7. Our Xan-1 cell culture has been maintainedin the dark at 27℃ and subcultured at 28-day intervals.

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) B5 salt mix
Gamborg’s B5 Medium Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#399-00621)
B) SucroseC) B5_VT
Nicotinic acid
Pyridoxine·HCl
Thiamine·HCl
myo-Inositol

0.4 mg/mL
0.4 mg/mL
4 mg/mL
40 mg/mLD) 2,4-D (1 mM)
2,4-D sodium monohydrate
0.261 mg/mL(2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich(D6679)
E) Kinetin (1 mM)
Kinetin
0.215 mg/mL
Dissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled waterF) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
G) KOH (1 N)

2Glassware and equipment
A) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use
Preparation of B5 medium (medium no. 30)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
B5 salt mix
Sucrose1 bag (1 L)
20 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
B5_VT
2,4-D (1 mM)
Kinetin (1 mM)

2.5 mL
1 mL
0.04 mL

3. Adjust the pH of the solution to 5.7 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 B5 medium.
2. Incubate cell cultures under the dark condition at 27℃.

Notes• We send Xan-1 cells on semi-solid B5 medium in a 9-cm disposable Petri dish. Thecells should be subcultured to fresh B5 medium immediately after arrival.
• In order to maintain Xan-1 callus culture stably, it is essential to observe the growthof cells carefully. Because proliferation of Xan-1 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 Xan-1 callus (about3-mm in diameter) on 40 mL of B5 medium in a 100-mL ﬂask, and culture them for
28 days.

3References
Taguchi F, Shimizu R, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2003) Posttranslational modiﬁcation of ﬂagellin determines the speciﬁcity of HR induction.
Plant & Cell Physiology 44: 342–349. DOI: 10.1093/pcp/pcg042

4Appendix A: Formulation of culture medium
Table A.1. Gamborg’s B5 medium(medium no. 30)
ChemicalConcentration (mg/L)

KNO32500(NH4 )2SO4134CaCl2 ·2H2 O

150

MgSO4 ·7H2 O

250

NaH2 PO4 ·H2 O

150

H3 BO33MnSO4 ·H2 O

10ZnSO4 ·7H2 O

2KI0.75

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

Nicotinic acid1

Pyridoxine·HCl

1Thiamine·HCl

10

myo-Inositol

100

Sucrose20000

2,4-D sodium monohydrate

0.261

Kinetin0.0086

Agar8000

5