rpc00055-gnom-2.4 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00055

Arabidopsis thaliana gnom 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: mMS medium, 1 mg/L 2,4-D, 0.8% (w/v) agar, pH 5.8(medium no. 43) [Materials III]
• Culture conditions: 23℃, 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: “Arabidopsis thaliana gnom
cell line (rpc00055) 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 gnom cell line was established from a root of Arabidopsis thaliana (L.)
Heynh. gnom mutant (Ueda et al. 2004). GNOM encodes a GDP/GTP exchange factor
for small G-proteins of the ADP ribosylation factor class (Geldner et al. 2003). The gnommutant plants show severe growth arrest, but the gnom cell culture proliferate normally likewild-type cells (Ueda et al. 2004). The gnom cells are grown on a modiﬁed Murashigeand Skoog (mMS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid
(2,4-D), and solidiﬁed with 0.8% (w/v) agar, pH 5.8. Our gnom cell culture has been
maintained in the dark at 23℃ 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) KH2 PO4 (100 mg/mL)
KH2 PO4

100 mg/mLD) B5_VT
Nicotinic acid
Pyridoxine·HClThiamine·HCl
myo-Inositol

0.4 mg/mL
0.4 mg/mL
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) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
G) KOH (1 N)

2Glassware and equipment
A) Petri dish (9 cm diameter, 2 cm height), sterileB) Forceps, sterilized before useC) Surgical tape
3M™ Micropore™ Surgical Tape, 12.5 mm × 9.1 m, 3M Japan Limited(#1530-0)
Preparation of mMS medium (medium no. 43)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mix
Sucrose1 bag (1 L)
20 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
KH2 PO4 (100 mg/mL)
B5_VT
2,4-D (0.2 mg/mL)

3.4 mL
5 mL
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. Add 8 g of agar 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 28-day-old culture with a forceps and place the cells onto fresh mMS medium.
2. Seal the Petri dishes using two rounds of surgical tape.
3. Incubate cell cultures under the dark condition at 23℃.

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

3• In order to maintain gnom callus culture stably, it is essential to observe the growth ofcells carefully. Because proliferation of gnom 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 one
lab to another. We usually inoculate seven pieces of gnom callus (about 5-mm indiameter) on 30 mL of mMS medium in a 9-cm Petri dish, and culture them for 28days.

References
Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, UedaT, Nakano A, Jürgens G (2003) The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112:
219–230. DOI: 10.1016/S0092-8674(03)00003-5
Ueda T, Uemura T, Sato MH, Nakano A (2004) Functional diﬀerentiation of endosomesin Arabidopsis cells. Plant Journal 40: 783–789. DOI: 10.1111/j.1365-313X.2004.
02249.x4

Appendix A: Formulation of culture medium
Table A.1. modiﬁed Murashige and Skoog medium(medium no. 43)
ChemicalConcentration (mg/L)

KNO31900

NH4 NO31650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4510

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 acid2Pyridoxine·HCl

2Thiamine·HCl

20

myo-Inositol

200

Sucrose20000

2,4-D sodium monohydrate

1.18Agar

8000

5