rpc00112-At tom-c-1.1 (2024.3.13; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00112

Arabidopsis thaliana At tom-c transgenic callus cultureComponents• 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 ﬂow
cabinet.
Method• Culture medium: mLS medium, 0.2 mg/L 2,4-D, 0.4% (w/v) gellan gum, pH
5.8 (medium no. 71) [Materials III]
• Culture conditions: 27℃, dark [Methods II]• Subculture: 14–21-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: “Arabidopsis thaliana At tomc cell line (rpc00112) 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/

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Introduction
Arabidopsis At tom-c cell line was established by subculturing the At tom suspension culture on a semi-solid culture medium. The parent At tom cell line of Arabidopsis thaliana(L.) Heynh. accession Columbia a triple mutant that has tom1-2 (ethyl methanesulfonatemutagenesis), tom3-1 (ethyl methanesulfonate mutagenesis), and thh1-1 (T-DNA insertion) mutations (Nishikiori et al. 2011). The At tom protoplasts show complete inhibitionof tobamovirus multiplication. The At tom-c cells are grown on a modiﬁed Linsmaierand Skoog (mLS) medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid
(2,4-D), and solidiﬁed with 0.4% (w/v) gellan gum, pH 5.8. Our At tom-c cell culture has
been maintained in the dark at 27℃ and subcultured at 14–21-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 ChemicalCorporation (#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) Gellan gum
Gellan gum, FUJIFILM Wako Pure Chemical Corporation (#073-03071)
G) KOH (1 N)

2Glassware and equipment
A) Petri dish (9 cm diameter, 2 cm height), sterileB) Forceps, sterilized before useC) Surgical tape3M™ Micropore™ Surgical Tape, 12.5 mm × 9.1 m, 3M Japan Limited(#1530-0)
Preparation of mLS medium (medium no. 71)
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. Add 4 g of gellan gum to the medium.
5. Autoclave the medium at 121℃ for 20 min.
6. Pour 30 mL of the medium into a 9-cm Petri dish.

Methods
1. Pick up an appropriate amount of callus cells from a 14–21-day-old culture with aforceps and place the cells onto fresh mLS medium.
2. Seal the Petri dishes using two rounds of surgical tape.
3. Incubate cell cultures under the dark condition at 27℃.

Notes
• We send At tom-c cells on semi-solid mLS medium in a 9-cm disposable Petri dish.
The cells should be subcultured to fresh mLS medium immediately after arrival.

3• In order to maintain At tom-c callus culture stably, it is essential to observe thegrowth of cells carefully. Because proliferation of At tom-c cells is aﬀected by culture conditions, such as a room temperature, aeration conditions of the culture andso on, an amount of cells transferred to fresh medium and the subculture intervals
may vary from one lab to another. We usually inoculate seven pieces of At tom-ccallus (about 5-mm in diameter) on 30 mL of mLS medium in a 9-cm Petri dish,and culture them for 14–21 days.
• Suspension cultures can be derived from YU-1-c callus cultures by subculturing
them into a liquid culture medium. The suspension culture method is the same as
rpc00056 At tom.

References
Nishikiori M, Mori M, Dohi K, Okamura H, Katoh E, Naito S, Meshi T, Ishikawa M (2011)
A host small GTP-binding protein ARL8 plays crucial roles in tobamovirus RNA
replication. PLoS Pathogen 7: e1002409. DOI: 10.1371/journal.ppat.1002409

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Appendix A: Formulation of culture medium
Table A.1. modiﬁed Linsmaier and Skoog medium(medium no. 71)
ChemicalConcentration (mg/L)

KNO31900

NH4 NO31650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4370

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

Thiamine·HCl1

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.236

Gellan gum

40005