rpc00103-MM2d-1.2 (2023.3.15; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00103

Arabidopsis thaliana MM2d 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, 0.5 mg/L NAA, 0.05 mg/L kinetin, pH 5.8(medium no. 61) [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 MM2dcell line (rpc00103) 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
Arabidopsis MM2d cell line was established from a stem of Arabidopsis thaliana (L.)
Heynh. accession Landsberg erecta (Menges and Murray 2002). The MM2d cell line canbe used for cell cycle synchronization and genetic transformation (Menges and Murray
2002, Kono et al. 2006). The MM2d cells are grown in a Murashige and Skoog (MS)medium supplemented with 0.5 mg/L 1-naphthaleneacetic acid (NAA) and 0.05 mg/L
kinetin, pH 5.8. Our MM2d cell culture has been maintained in the dark at 27℃ withrotary 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) MS_VT
Nicotinic acid
Pyridoxine·HClThiamine·HClGlycine

0.5 mg/mL
0.5 mg/mL
0.1 mg/mL2 mg/mLD) MS_inositol
myo-Inositol

40 mg/mL

E) NAA (0.5 mg/mL)
NAA·K
0.6 mg/mL
Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation(#161-04021); Sterilize the solution by membrane ﬁltration, and store at −20℃
F) Kinetin (0.05 mg/mL)
Kinetin0.05 mg/mL
Dissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled water;
Sterilize the solution by membrane ﬁltration, and store at −20℃
G) KOH (1 N)

2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 minPreparation of MS medium (medium no. 61)
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

1 mL
2.5 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.
6. Add the following phytohormones to the medium just before subculturing.
NAA (0.5 mg/mL)
Kinetin (0.05 mg/mL)

50 µL
50 µL

Methods
1. Agitate a 7-day-old culture well and transfer 2 mL of cell suspension to 50 mL offresh MS 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 cell suspension in a 50-mL disposable
conical centrifuge tube. The cells should be transferred to fresh MS medium immediately after arrival.

3• For overseas customers: We send MM2d 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 MM2d cell suspension cultures stably, it is essential to transfer
an adequate amount of cells to fresh MS medium in every subculture. The amountof cells may vary from one lab to another, because proliferation of MM2d cells is
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
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
Kono A, Ohno R, Umeda-Hara C, Uchimiya H, Umeda M (2006) A distinct type of cyclin
D, CYCD4;2, involved in the activation of cell division in Arabidopsis. Plant CellReports 25: 540–545. PMID: 16408177. DOI: 10.1007/s00299-005-0075-4

4Appendix A: Formulation of culture medium
Table A.1. Murashige and Skoog medium(medium no. 61)
ChemicalConcentration (mg/L)

KNO31900

NH4 NO31650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO36.2

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

Sucrose

30000

NAA·K

0.6

Kinetin0.055