rpc00111-YG1-c-1.1 (2024.3.13; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00111

Arabidopsis thaliana YG1-c 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: 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 YG1-ccell line (rpc00111) 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 YG1-c cell line was established by subculturing the YG1 suspension culture
on a semi-solid culture medium. The parent YG1 cell line (rpc00050) was derived from aseedling of Arabidopsis thaliana (L.) Heynh. accession Columbia (Nishikiori et al. 2011,
Yoshikawa et al. 2021). The YG1-c cells are grown on a modiﬁed Linsmaier and Skoog(mLS) medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), andsolidiﬁed with 0.4% (w/v) gellan gum, pH 5.8. Our YG1-c cell culture has been maintainedin 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 Chemical
Corporation (#392-00591)
B) SucroseC) BY2_P
KH2 PO480 mg/mLD) LS_VT_modiﬁed
Thiamine·HClmyo-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)
Glassware and equipment
A) Petri dish (9 cm diameter, 2 cm height), sterile2B) Forceps, sterilized before useC) Surgical tape
3M™ 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 YG1-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.
• In order to maintain YG1-c callus culture stably, it is essential to observe the growthof cells carefully. Because proliferation of YG1-c cells is aﬀected by culture conditions, such as a room temperature, aeration conditions of the culture and so on, an3amount of cells transferred to fresh medium and the subculture intervals may varyfrom one lab to another. We usually inoculate nine pieces of YG1-c callus (about3-mm in diameter) on 30 mL of mLS medium in a 9-cm Petri dish, and culture themfor 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
rpc00050 YG1.

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
Yoshikawa M, Han Y-W, Fujii H, Aizawa S, Nishino T, Ishikawa M (2021) Cooperative
recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA ampliﬁcation in Arabidopsis. Proceedings of National Academy of Sciences of USA 118:e2102885118. DOI: 10.1073/pnas.2102885118

4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·HCl

1

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.236Gellan gum

4000

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