rpc00086-NaCl-r-1.2 (2022.3.16; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00086

Nicotiana tabacum NaCl-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, 0.2 M NaCl, pH
5.7 (medium no. 56) [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 NaCl-rcell line (rpc00086) 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
Japan
FAX: +81 29 836 9053
E-mail: plant.brc@riken.jp
http://epd.brc.riken.jp/en/

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Introduction
Tobacco NaCl-r cell line is a NaCl-adapted NI cell line, which can grow photoautotrophically in a NaCl-containing medium (Murota et al. 1994). The parent photoautotrophic NI
cell line (rpc00084) was established from a pith of Nicotiana tabacum L. cultivar Samsun
NN (Yamada and Sato 1978). The NaCl-r cells are grown in a modiﬁed Linsmaier andSkoog (mLS) medium supplemented with 10 µM 1-naphthaleneacetic acid (NAA), 1 µM
kinetin and 0.2 M NaCl, pH 5.7. Our NaCl-r cell culture has been maintained under thecontinuous light at 24℃ with rotary shaking at 90 rpm 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) LS_VT
Thiamine·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) NaClG) KOH (1 N)
GlasswareA) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foilB) Pipette (10 mL; large tip opening) and a bulb, sterilized by autoclaving at 121℃ for20 min2Preparation of mLS medium (medium no. 56)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mix
SucroseNaCl

1 bag (1 L)
30 g
11.69 g2. 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. Agitate a 21-day-old culture well and transfer 5–10 mL of cell suspension to 25 mLof fresh mLS medium with a pipette.
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 NaCl-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 NaCl-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 NaCl-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 NaCl-r cells is3aﬀected by culture conditions, such as a room temperature, rotation speed of a rotary
shaker, and aeration condition of the culture.
• 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
Murota K, Ohshita Y, Watanabe A, Aso S, Sato F, Yamada Y (1994) Changes related to salt
tolerance in thylakoid membranes of photoautotrophically cultured green tobacco
cells. Plant & Cell Physiology 35: 107–113. DOI: 10.1093/oxfordjournals.pcp.a078560
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. 56)
ChemicalConcentration (mg/L)

KNO3

1900

NH4 NO3

1650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O370

KH2 PO4170

H3 BO36.2

MnSO4 ·4H2 O22.3

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

27.8

Na2 -EDTA

37.3

Thiamine·HCl

0.8

myo-Inositol

200

Sucrose

30000

NAA·K

2.24

Kinetin

0.215

NaCl11.695