rpc00085-ATR-r-1.2 (2022.3.16; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00085

Nicotiana tabacum ATR-r cell suspension cultureComponents• Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cellsuspension• Overseas delivery: A 250-mL plastic Erlenmeyer ﬂask, containing cells
placed on semi-solid medium
Notice• Subculture the cells to fresh medium immediately after arrival [Notes I, II].
• 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, 10 µM NAA, 1 µM kinetin, pH 5.7 (mediumno. 55) [Materials III]
• Culture conditions: 24℃, continuous light, 90 rpm [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 ATR-rcell line (rpc00085) was provided by the RIKEN BRC through the NationalBioResource 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/

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Introduction
Tobacco ATR-r cell line is a mutant cell line resistant to photosynthesis-inhibiting
herbicides 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine) and 3-(3,4dichlorophenyl)-1,1-dimethylurea (Sato et al. 1988, Sigematsu et al. 1989). The ATR-rcell line was selected from a photoautotrophic NI cell line (rpc00084) that was establishedfrom a pith of Nicotiana tabacum L. cultivar Samsun NN (Yamada and Sato 1978). The
ATR-r cells are grown in a modiﬁed Linsmaier and Skoog (mLS) medium supplemented
with 10 µM 1-naphthaleneacetic acid (NAA) and 1 µM kinetin, pH 5.7. Our ATR-r cell
culture has been maintained under the continuous light at 24℃ with rotary shaking at 90rpm 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 ChemicalCorporation (#392-00591)
B) SucroseC) LS_VTThiamine·HCl
myo-Inositol

0.16 mg/mL
40 mg/mLD) NAA (1 mM)
NAA·K
0.224 mg/mL
Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation(#161-04021)
E) Kinetin (1 mM)
Kinetin
0.215 mg/mL
Dissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled waterF) KOH (1 N)
Glassware and equipment
A) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foilB) Laboratory dispensing spoon (head size, 1.2 × 0.8 cm), sterilized before use

2Preparation of mLS medium (medium no. 55)
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.
LS_VT
NAA (1 mM)
Kinetin (1 mM)

5 mL
10 mL
1 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 25 mL of the medium into a 100-mL ﬂask.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Transfer two to four spoons of cells from a 21-day-old culture into 25 mL of freshmLS medium with a laboratory dispensing spoon.
2. Incubate cell cultures on a rotary shaker at 90 rpm under the continuous light condition (photosynthetic photon ﬂux density 50–60 µmol m−2 s−1 ) at 24℃.

Notes
• For domestic customers: We send ATR-r cell suspension in a 50-mL disposable
conical centrifuge tube. The cells should be transferred to fresh mLS medium immediately after arrival.
• For overseas customers: We send ATR-r cells placed on semi-solid mLS medium in
a 250-mL disposable Erlenmeyer ﬂask. The cells should be transferred to freshmLS medium immediately after arrival. Collect the cells from the semi-solid
medium with a spatula and transfer them to Erlenmeyer ﬂasks containing fresh liquidmedium.
• In order to maintain ATR-r cell suspension cultures stably, it is essential to transfer
an adequate amount of cells to fresh mLS medium in every subculture. The amountof cells may vary from one lab to another, because proliferation of ATR-r cells isaﬀected by culture conditions, such as a room temperature, rotation speed of a rotary
shaker, and aeration condition of the culture.

3• In order to obtain good aeration of a suspension culture, a silicone sponge plug
may be used instead of the aluminum foil cap (e.g., cap-type Silicosen; Shin-Etsu
Polymer, Tokyo, Japan; https://www.shinpoly.co.jp/en/product/product/medical/plugs.html).

References
Sato F, Shigematsu Y, Yamada Y (1988) Selection of an atrazine-resistant tobacco cell line
having a mutant psbA gene. Molecular and General Genetics 214: 358–360. DOI:
10.1007/BF00337736
Sigematsu Y, Sato F, Yamada Y (1989) The mechanism of herbicide resistance in tobacco
cells with a new mutation in the QB protein. Plant Physiology 89: 986–992. DOI:
10.1104/pp.89.3.986
Yamada Y, Sato F (1978) The photoautotrophic culture of chlorophyllous cells. Plant &Cell Physiology 19: 691–699. DOI: 10.1093/oxfordjournals.pcp.a075640

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Appendix A: Formulation of culture medium
Table A.1. modiﬁed Linsmaier and Skoog medium(medium no. 55)
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

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

0.8

myo-Inositol

200

Sucrose

30000

NAA·K

2.24

Kinetin

0.215

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