rpc00100: Athyrium yokoscense AY-01 cell suspension culture
Note
Components
Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cell suspension
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.
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: 1/2mMS medium, 1 mg/L kinetin, pH 5.8 (medium no. 60)
Culture conditions: 24°C, continuous light, 100 rpm
Subculture: 14-day intervals
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.”
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 [1]). The AY-01 cells are grown in 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 at 24°C with rotary shaking at 100 rpm and subcultured at 14-day intervals.
Materials
Chemicals and stock solutions
(All stock solutions are stored at 4°C)
MS salt mix
Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#392-00591)
Sucrose
MS_VT
Chemical
Concentration (mg/mL)
Nicotinic acid
0.5
Pyridoxine·HCl
0.5
Thiamine·HCl
0.1
Glycine
2
MS_inositol
Chemical
Concentration (mg/mL)
myo-Inositol
40
Kinetin (0.2 mg/mL)
Chemical
Concentration (mg/mL)
Kinetin
0.2
Dissolve kinetin in small volume of KOH (1 N), and fill up with distilled water
KOH (1 N)
Glassware and equipment
Erlenmeyer flask (300 mL), capped with two layers of aluminum foil
Forceps, sterilized before use
Preparation of 1/2mMS medium (medium no. 60)
Dissolve the following chemicals in approximately 800 mL of distilled water.
Chemical
Amount
MS salt mix
0.5 bag (equivalent with 0.5 L)
Sucrose
30 g
Add following stock solutions, and fill up to approximately 950 mL with distilled water.
Stock solution
Volume (mL)
MS_VT
0.5
MS_inositol
1.25
Kinetin (0.2 mg/mL)
5
Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.
Pour 100 mL of the medium into a 300-mL flask.
Autoclave the flask at 121°C for 20 min.
Methods
Transfer seven to nine cell clumps from 14-day-old culture to 100 mL of fresh 1/2mMS medium with a forceps.
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°C.
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 to Erlenmeyer 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 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).
Genotyping
Maintenance history
References
Protocols
Genotyping of plant cell lines: Athyrium yokoscense cell lines