rpc00032-Lj-2.3 (2017.10.19; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00032

Lotus japonicus Lj 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: MS medium, 1 mg/L 2,4-D, pH 5.7 (medium no. 27)[Materials III]
• Culture conditions: 27℃, dark, 120 rpm [Methods II]• Subculture: 7-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: “Lotus japonicus Lj cell line
(rpc00032) 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/

1

Introduction
Lj cell line was established from a shoot apex of a seedling of Lotus japonicus (Regel)
K.Larsen accession Gifu B-129. The Lj cells are grown in a Murashige and Skoog (MS)medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), pH 5.7. Our
Lj cell culture has been maintained in the dark at 27℃ with rotary shaking at 120 rpm andsubcultured at 7-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) MS_VT
Nicotinic acid
Pyridoxine·HClThiamine·HClGlycine

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

40 mg/mLE) 2,4-D (0.2 mg/mL)
2,4-D sodium monohydrate
0.236 mg/mL(2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich(D6679)
F) KOH (1 N)
GlasswareA) Erlenmeyer ﬂask (300 mL), capped with two layers of aluminum foilB) Pipette (10 mL; large tip opening) and a bulb, sterilized by autoclaving at 121℃ for
20 min2Preparation of MS medium (medium no. 27)
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.
MS_VT
MS_inositol
2,4-D (0.2 mg/mL)

1 mL
2.5 mL5 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 300-mL ﬂask.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Leave to stand a ﬂask containing a 7-day-old culture until the cells settle to the bottom of the ﬂask.
2. Transfer 25 mL of the sedimented cells to 80 mL of fresh MS medium with a pipette.
3. Incubate cell cultures on a rotary shaker at 120 rpm under the dark condition at27℃.

Notes• For domestic customers: We send Lj cell suspension in a 50-mL disposable conicalcentrifuge tube. The cells should be transferred to fresh MS medium immediately
after arrival.
• For overseas customers: We send Lj cells placed on semi-solid MS medium in
a 250-mL disposable Erlenmeyer ﬂask. The cells should be transferred to fresh
MS 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 Lj cell suspension cultures stably, it is essential to transfer anadequate amount of cells to fresh MS medium in every subculture. The amount ofcells may vary from one lab to another, because proliferation of Lj cells is aﬀected3

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).
• The parent accession Gifu B-129 is available from National BioResource Project
Lotus/Glycine (https://www.legumebase.brc.miyazaki-u.ac.jp/).

4Appendix A: Formulation of culture medium
Table A.1. Murashige and Skoog medium(medium no. 27)
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 O0.025

CoCl2 ·6H2 O

0.025

FeSO4 ·7H2 O

27.8

Na2 -EDTA

37.3

Nicotinic acid

0.5

Pyridoxine·HCl0.5

Thiamine·HCl

0.1

Glycine2

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

1.185