rpc00100-AY-01-1.3 (2022.3.16; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00100

Athyrium yokoscense AY-01 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: 1/2mMS medium, 1 mg/L kinetin, pH 5.8 (medium no. 60)[Materials III]
• Culture conditions: 24℃, continuous light, 100 rpm [Methods II]• Subculture: 14-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: “Athyrium yokoscense AY-01
cell line (rpc00100) 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
AY-01 cell line was established from a leaf of a metal hyper-tolerant fern Athyrium
yokoscense (Franch. & Sav.) Christ (Yoshihara et al. 2005). The AY-01 cells are grownin a 1/2 modiﬁed Murashige and Skoog (1/2mMS) medium supplemented with 1 mg/L
kinetin, pH 5.8. Our AY-01 cell culture has been maintained under the continuous light at24℃ with rotary shaking at 100 rpm and subcultured at 14-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/mL
2 mg/mLD) MS_inositol
myo-Inositol

40 mg/mL

E) Kinetin (0.2 mg/mL)
Kinetin
0.2 mg/mL
Dissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled waterF) KOH (1 N)
Glassware and equipmentA) Erlenmeyer ﬂask (300 mL), capped with two layers of aluminum foilB) Forceps, sterilized before use

2Preparation of 1/2mMS medium (medium no. 60)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mix
Sucrose0.5 bag (equivalent with 0.5 L)
30 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
MS_VT
MS_inositol
Kinetin (0.2 mg/mL)

0.5 mL1.25 mL5 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 100 mL of the medium into a 300-mL ﬂask.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Transfer seven to nine cell clumps from 14-day-old culture to 100 mL of fresh
1/2mMS medium with a forceps.
2. Incubate cell cultures on a rotary shaker at 100 rpm under the continuous light condition (photosynthetic photon ﬂux density 60 µmol m−2 s−1 ) at 24℃.

Notes
• For domestic customers: We send AY-01 cell suspension in a 50-mL disposable
conical centrifuge tube. The cells should be transferred to fresh 1/2mMS medium
immediately after arrival.
• For overseas customers: We send AY-01 cells placed on semi-solid 1/2mMS
medium in a 250-mL disposable Erlenmeyer ﬂask. The cells should be transferred
to fresh 1/2mMS medium immediately after arrival. Transfer the cell clumps toErlenmeyer ﬂasks containing fresh liquid medium with a forceps.
• In order to maintain AY-01 cell suspension cultures stably, it is essential to transfer
an adequate amount of cells to fresh 1/2mMS medium in every subculture. The
amount of cells may vary from one lab to another, because proliferation of AY-01
cells is aﬀected by culture conditions, such as a room temperature, rotation speed ofa 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
Yoshihara T, Tsunokawa K, Miyano Y, Arashima Y, Hodoshima H, Shoji K, Shimada
H, Goto F (2005) Induction of callus from a metal hypertolerant fern, Athyrium
yokoscense, and evaluation of its cadmium tolerance and accumulation capacity.
Plant Cell Reports 23: 579–585. DOI: 10.1007/s00299-004-0877-94Appendix A: Formulation of culture medium
Table A.1. 1/2 modiﬁed Murashige and Skoog medium(medium no. 60)
ChemicalConcentration (mg/L)

KNO3950NH4 NO3825

CaCl2 ·2H2 O

220

MgSO4 ·7H2 O

185

KH2 PO485

H3 BO3

3.1

MnSO4 ·4H2 O

11.15

ZnSO4 ·7H2 O

4.3KI

0.415

Na2 MoO4 ·2H2 O

0.125

CuSO4 ·5H2 O

0.0125

CoCl2 ·6H2 O

0.0125

FeSO4 ·7H2 O

13.9

Na2 -EDTA

18.65

Nicotinic acid

0.25

Pyridoxine·HCl

0.25

Thiamine·HCl

0.05

Glycine1

myo-Inositol

50

Sucrose

30000

Kinetin15