rpc00043-3n-3-2.2 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00043

Nicotiana tabacum 3n-3 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: MS medium, 0.9% (w/v) agar, pH 5.7 (medium no. 34)[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 3n-3cell line (rpc00043) 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 3n-3 cell line is a crown-gall line that was obtained by infecting mesophyll protoplasts of Nicotiana tabacum L. cultivar Petit Havana SR-1 with Agrobacterium tumefaciens
strain A208 (Nakagawa et al. 1987). The 3n-3 cells are grown on a phytohormone-free
Murashige and Skoog (MS) medium solidiﬁed with 0.9% (w/v) agar, pH 5.7. Our 3n-3cell culture has been maintained in the dark at 27℃ and subcultured at 28-day intervals.

MaterialsChemicals and stock solutions(All stock solutions are stored at 4℃)
A) MS salt mix
Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure ChemicalCorporation (#392-00591)
B) SucroseC) MS_VT
Nicotinic acid
Pyridoxine·HCl
Thiamine·HCl
Glycine

0.5 mg/mL
0.5 mg/mL
0.1 mg/mL
2 mg/mLD) MS_inositol
myo-Inositol

40 mg/mL

E) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
F) KOH (1 N)
Glassware and equipment
A) Erlenmeyer ﬂask (200 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use
Preparation of MS medium (medium no. 34)
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.
MS_VT
MS_inositol

1 mL
2.5 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 80 mL of the medium into a 200-mL ﬂask containing 0.72 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 MS medium.
2. Incubate cell cultures under the dark condition at 27℃.

Notes
• We send 3n-3 cells on semi-solid MS medium in a 9-cm disposable Petri dish. Thecells should be subcultured to fresh MS medium immediately after arrival.
• In order to maintain 3n-3 callus culture stably, it is essential to observe the growth ofcells carefully. Because proliferation of 3n-3 cells is aﬀected by culture conditions,such as a room temperature, aeration conditions of the culture and so on, an amountof cells transferred to fresh medium and the subculture intervals may vary from onelab to another. We usually inoculate three to ﬁve pieces of 3n-3 callus (about 10-mmin diameter) on 80 mL of MS medium in a 200-mL ﬂask, and culture them for 28days.

References
Nakagawa S, Tjokrokusumo DS, Sakurai A, Yamaguchi I, Takahashi N, Syōno K (1987)
Endogenous levels of gibberellins, IAA and cytokinins in tobacco crown gall tissuesof diﬀerent morphologies. Plant & Cell Physiology 28: 485–493. DOI: 10.1093/ox
fordjournals.pcp.a077319

3Appendix A: Formulation of culture medium
Table A.1. Murashige and Skoog medium(medium no. 34)
ChemicalConcentration (mg/L)

KNO31900

NH4 NO31650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO3

6.2

MnSO4 ·4H2 O

22.3

ZnSO4 ·7H2 O

8.6

KI0.83Na2 MoO4 ·2H2 O

0.25

CuSO4 ·5H2 O

0.025

CoCl2 ·6H2 O

0.025

FeSO4 ·7H2 O

27.8Na2 -EDTA

37.3

Nicotinic acid

0.5

Pyridoxine·HCl

0.5

Thiamine·HCl0.1

Glycine2

myo-Inositol

100

Sucrose

30000

Agar

90004