============================================================== rpc00100: *Athyrium yokoscense* AY-01 cell suspension culture ============================================================== :download:`Download a PDF version (143 KB) <../cell_lines/rpc00100_AY-01_1_3.pdf>` .. 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 (\ :doc:`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 .. csv-table:: :header: "Chemical", "Concentration (mg/mL)" :widths: auto "Nicotinic acid", "0.5" "Pyridoxine·HCl", "0.5" "Thiamine·HCl", "0.1" "Glycine", "2" * MS_inositol .. csv-table:: :header: "Chemical", "Concentration (mg/mL)" :widths: auto "*myo*-Inositol", "40" * Kinetin (0.2 mg/mL) .. csv-table:: :header: "Chemical", "Concentration (mg/mL)" :widths: auto "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) --------------------------------------------- :doc:`/media/medium_60` #. Dissolve the following chemicals in approximately 800 mL of distilled water. .. csv-table:: :header: "Chemical", "Amount" :widths: auto "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. .. csv-table:: :header: "Stock solution", "Volume (mL)" :widths: auto "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\ :sup:`−2` s\ :sup:`−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 =========== :doc:`/genotyping/g_rpc00100` Maintenance history ==================== :doc:`/maintenance/m_rpc00100` References =========== .. [1] 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/s00299-004-0877-9 `_ Protocols ========== * Genotyping of plant cell lines: :doc:`/protocols/genotyping/athyrium` External links =============== * `World Flora Online: Athyrium yokoscense (Franch. & Sav.) Christ `_ * `Wikipedia: Athyrium yokoscense `_ .. index:: single: Athyrium yokoscense single: AY-01