rpc00104-MM2d-LS-1.2 (2023.3.15; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00104

Arabidopsis thaliana MM2d-LS 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, 0.2 mg/L 2,4-D, pH 5.8 (medium no. 1)[Materials III]
• Culture conditions: 27℃, dark, 130 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: “Arabidopsis thaliana MM2dLS cell line (rpc00104) 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
Arabidopsis MM2d-LS cell line was established by subculturing the MM2d cells in adiﬀerent culture medium (Adachi et al. 2011). The parent MM2d cell line (rpc00103)
was established from a stem of Arabidopsis thaliana (L.) Heynh. accession Landsberg
erecta (Menges and Murray 2002). The MM2d-LS cell line can be used for cell cycle
synchronization and genetic transformation (Adachi et al. 2011). The MM2d-LS cells aregrown in a modiﬁed Linsmaier and Skoog (mLS) medium supplemented with 0.2 mg/L
2,4-dichlorophenoxyacetic acid (2,4-D), pH 5.8. Our MM2d-LS cell culture has been
maintained in the dark at 27℃ with rotary shaking at 130 rpm and subcultured 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) BY2_P
KH2 PO480 mg/mLD) LS_VT_modiﬁed
Thiamine·HCl
myo-Inositol

0.4 mg/mL
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℃ for20 min2Preparation of mLS medium (medium no. 1)
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.
BY2_P
LS_VT_modiﬁed
2,4-D (0.2 mg/mL)

2.5 mL
2.5 mL
1 mL

3. Adjust the pH of the solution to 5.8 with KOH (1 N), and ﬁll up to 1 L with distilledwater.
4. Pour 50 mL of the medium into a 300-mL ﬂask.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Agitate a 7-day-old culture well and transfer 1 mL of cell suspension to 50 mL offresh mLS 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 MM2d-LS 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 MM2d-LS cells placed on semi-solid mLS
medium in a 250-mL disposable Erlenmeyer ﬂask. The cells should be transferred
to fresh mLS medium immediately after arrival. Collect the cells from the semisolid medium with a spatula and transfer them to Erlenmeyer ﬂasks containing freshliquid medium.
• In order to maintain MM2d-LS cell suspension cultures stably, it is essential to transfer an adequate amount of cells to fresh mLS medium in every subculture. Theamount of cells may vary from one lab to another, because proliferation of MM2dLS cells is aﬀected by culture conditions, such as a room temperature, rotation speedof 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
Adachi S, Minamisawa K, Okushima Y, Inagaki S, Yoshiyama K, Kondou Y, Kaminuma
E, Kawashima M, Toyoda T, Matsui M, Kurihara D, Matsunaga S, Umeda M (2011)
Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis. Proceedings of National Academy of Sciences of USA 108: 10004–10009.
DOI: 10.1073/pnas.1103584108
Menges M, Murray JAH (2002) Synchronous Arabidopsis suspension cultures for analysisof cell-cycle gene activity. Plant Journal 30: 203–212. DOI: 10.1046/j.1365-313X.
2002.01274.x

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Appendix A: Formulation of culture medium
Table A.1. modiﬁed Linsmaier and Skoog medium(medium no. 1)
ChemicalConcentration (mg/L)

KNO3

1900

NH4 NO3

1650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O370

KH2 PO4370

H3 BO36.2

MnSO4 ·4H2 O22.3

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

1

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.2365