rpc00087-BsLs-1.2 (2022.3.16; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00087

Bruguiera sexangula BsLs 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: AA medium, 0.02 µM 2,4-D, 2 µM 4-CPPU, pH 6.2(medium no. 57) [Materials III]
• Culture conditions: 27℃, dark, 130 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: “Bruguiera sexangula BsLs
cell line (rpc00087) 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
Japan
FAX: +81 29 836 9053
E-mail: plant.brc@riken.jp
http://epd.brc.riken.jp/en/

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Introduction
BsLs cell line was established from a leaf of a mangrove species Bruguiera sexangula(Lour.) Poir. (Kura-Hotta et al. 2001). The BsLs suspension cells have high salt tolerance.
The BsLs cells are grown in a Amino Acid (AA) medium supplemented with 0.02 µM
2,4-dichlorophenoxyacetic acid (2,4-D) and 2 µM N-(2-chloro-4-pyridyl)-N ′ -phenylurea(4-CPPU), pH 6.2. Our BsLs cell culture has been maintained in the dark at 27℃ withrotary shaking at 130 rpm and subcultured at 21-day intervals.

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) SucroseB) CaCl2 (0.6 M)
CaCl2 ·2H2 O

88 mg/mLC) AA_macro
KCl
MgSO4 ·7H2 O
KH2 PO4149 mg/mL
37 mg/mL
17 mg/mLD) MS_micro_1
H3 BO3MnSO4 ·5H2 OZnSO4 ·7H2 OKINa2 MoO4 ·2H2 OCuSO4 ·5H2 O
CoCl2 ·6H2 O

0.62 mg/mL
2.41 mg/mL
0.86 mg/mL
0.083 mg/mL
0.025 mg/mL
0.0025 mg/mL
0.0025 mg/mLE) Fe(III)-EDTA (20 mM)
Fe(III)-EDTA
8.422 mg/mL′′
Ethylenediamine-N,N,N ,N -tetraacetic acid, iron (III), sodium salt, trihydrate,
FUJIFILM Wako Pure Chemical Corporation (#343-01241)
F) AA_5
L-Glutamine
L-Aspartic acid
L-Arginine

8.77 mg/mL
2.66 mg/mL
2.28 mg/mL

2GlycineStore at −20℃

0.75 mg/mLG) AA_VT
Nicotinic acid
Pyridoxine·HClThiamine·HClGlycine
Store at −20℃

0.05 mg/mL
0.05 mg/mL
0.01 mg/mL
0.16 mg/mLH) 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)
I) 4-CPPU (2 mM)
4-CPPU0.495 mg/mL
N-(2-Chloro-4-pyridyl)-N ′ -phenylurea, Sigma-Aldrich (C2791); Dissolve 4CPPU in ethanol and store at −20℃
J) NaOH (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℃ for
20 minPreparation of AA medium (medium no. 57)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
Sucrose30 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
CaCl2 (0.6 M)
AA_macroMS_micro_1
Fe(III)-EDTA (20 mM)
AA_5
AA_VT
2,4-D (1 mM)

5 mL
10 mL
10 mL
5 mL
100 mL10 mL
0.02 mL3

4-CPPU (2 mM)

1 mL

3. Adjust the pH of the solution to 6.2 with NaOH (1 N), and ﬁll up to 1 L with distilledwater.
4. Pour 20 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 2 mL of cell suspension to 20 mL offresh AA medium with a pipette.
2. Incubate cell cultures on a rotary shaker at 130 rpm under the dark condition at27℃.

Notes• For domestic customers: We send BsLs cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh AA medium immediately after arrival.
• For overseas customers: We send BsLs cells placed on semi-solid AA medium
in a 250-mL disposable Erlenmeyer ﬂask. The cells should be transferred tofresh AA 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 BsLs cell suspension cultures stably, it is essential to transfer anadequate amount of cells to fresh AA medium in every subculture. The amount ofcells may vary from one lab to another, because proliferation of BsLs cells is aﬀectedby 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
Kura-Hotta M, Mimura M, Tsujimura T, Nemoto-Washitani S, Mimura T (2001) High salt
treatment-induced Na+ extrusion and low salt treatment-induced Na+ accumulation4

in suspension-cultured cells of the mangrove plant, Bruguiera sexangula. Plant, Cell& Envrionment 24: 1105–1112. DOI: 10.1046/j.0016-8025.2001.00761.x

5Appendix A: Formulation of culture medium
Table A.1. Amino Acid medium(medium no. 57)
ChemicalConcentration (mg/L)

KCl

1490

L-Glutamine

877

L-Aspartic acid266

L-Arginine

228

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO36.2MnSO4 ·5H2 O

24.1

ZnSO4 ·7H2 O

8.6

KI

0.83

Na2 MoO4 ·2H2 O

0.25

CuSO4 ·5H2 O

0.025

CoCl2 ·6H2 O

0.025

Fe(III)-EDTA

42.11

Nicotinic acid

0.5

Pyridoxine·HCl

0.5

Thiamine·HCl

0.1

Glycine76.6

Sucrose30000

2,4-D sodium monohydrate

0.00522

4-CPPU

0.495

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