rpc00033-LjA-2.2 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00033

Lotus japonicus LjA 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, 1 mg/L 2,4-D, 0.1 mg/L kinetin, 0.9% (w/v)
agar, pH 5.7 (medium no. 29) [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: “Lotus japonicus LjA cell line(rpc00033) 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
LjA cell line was established from a shoot apex of a seedling of Lotus japonicus (Regel)
K.Larsen accession Gifu B-129. The LjA cells are grown on a Murashige and Skoog(MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1
mg/L kinetin, and solidiﬁed with 0.9% (w/v) agar, pH 5.7. Our LjA cell culture has been
maintained in the dark at 27℃ and subcultured at 28-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·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/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) Kinetin (0.2 mg/mL)
Kinetin
0.2 mg/mL
Dissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled waterG) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
H) KOH (1 N)

2Glassware and equipment
A) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use
Preparation of MS medium (medium no. 29)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mixSucrose

1 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)
Kinetin (0.2 mg/mL)

1 mL
2.5 mL5 mL
0.5 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 40 mL of the medium into a 100-mL ﬂask containing 0.36 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 LjA 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 LjA callus culture stably, it is essential to observe the growth ofcells carefully. Because proliferation of LjA 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 pieces of LjA callus (about 5-mm indiameter) on 40 mL of MS medium in a 100-mL ﬂask, and culture them for 28 days.

3• 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. 29)
ChemicalConcentration (mg/L)

KNO3

1900

NH4 NO3

1650

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

Nicotinic acid

0.5

Pyridoxine·HCl

0.5

Thiamine·HCl

0.1

Glycine2

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

1.18

Kinetin

0.1

Agar90005