===================================================== rpc00017: *Spinacia oleracea* Spi-I-1 callus culture ===================================================== :download:`Download a PDF version (140 KB) <../cell_lines/rpc00017_Spi-I-1_3_2.pdf>` .. note:: * Components * A 9-cm plastic Petri dish, 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: mMS medium, 0.5 mg/L NAA, 1 mg/L BAP, 1.2% (w/v) agar, pH 6.5 (\ :doc:`medium no. 68 `\ ) * Culture conditions: 27°C, dark * Subculture: 28--56-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: “*Spinacia oleracea* Spi-I-1 cell line (rpc00017) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.” Introduction ============= Spinach Spi-I-1 cell line was established from *Spinacia oleracea* L. (Nakagawa *et al*. 1985 [1]_). The Spi-I-1 cells are grown on a modified Murashige and Skoog (mMS) medium supplemented with 0.5 mg/L 1-naphthaleneacetic acid (NAA) and 1 mg/L 6-benzylaminopurine (BAP), and solidified with 1.2% (w/v) agar, pH 6.5. Our Spi-I-1 cell culture has been maintained in the dark at 27°C and subcultured at 28--56-day intervals. Materials ========== Chemicals and stock solutions ------------------------------ (All stock solutions are stored at 4°C) * KCl * L-Glutamine * NZ-Amine Type A * CaCl\ :sub:`2`\ ·2H\ :sub:`2`\ O * MgSO\ :sub:`4`\ ·7H\ :sub:`2`\ O * KH\ :sub:`2`\ PO\ :sub:`4`\ * Sucrose * MS_micro_1 .. csv-table:: :header: "Chemical", "Concentration (mg/mL)" :widths: auto "H\ :sub:`3`\ BO\ :sub:`3`", "0.62" "MnSO\ :sub:`4`\ ·5H\ :sub:`2`\ O", "2.41" "ZnSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "0.86" "KI", "0.083" "Na\ :sub:`2`\ MoO\ :sub:`4`\ ·2H\ :sub:`2`\ O", "0.025" "CuSO\ :sub:`4`\ ·5H\ :sub:`2`\ O", "0.0025" "CoCl\ :sub:`2`\ ·6H\ :sub:`2`\ O", "0.0025" * MS_micro_2 .. csv-table:: :header: "Chemical", "Concentration (mg/mL)" :widths: auto "FeSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "2.78" "Na\ :sub:`2`\ -EDTA", "3.73" Heat at 80°C for 3--4 hours for chelating Fe * 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" * NAA (1 mg/mL) .. csv-table:: :header: "Chemical", "Concentration (mg/mL)" :widths: auto "NAA·K", "1.2" Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation (#161-04021) * BAP (1 mg/mL) .. csv-table:: :header: "Chemical", "Concentration (mg/mL)" :widths: auto "6-Benzylaminopurine", "1" Dissolve 6-benzylaminopurine in small volume of KOH (1 N), and fill up with distilled water * Agar, powder * NaOH (1 N) Glassware and equipment ------------------------ * Erlenmeyer flask (100 mL), capped with two layers of aluminum foil * Forceps, sterilized before use Preparation of mMS medium (medium no. 68) ------------------------------------------ :doc:`/media/medium_68` #. Dissolve the following chemicals in approximately 800 mL of distilled water. .. csv-table:: :header: "Chemical", "Amount" :widths: auto "KCl", "1.4 g" "L-Glutamine", "1 g" "NZ-Amine Type A", "1 g" "CaCl\ :sub:`2`\ ·2H\ :sub:`2`\ O", "0.44 g" "MgSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "0.37 g" "KH\ :sub:`2`\ PO\ :sub:`4`", "0.17 g" "Sucrose", "20 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_micro_1", "10" "MS_micro_2", "10" "MS_VT", "1" "MS_inositol", "2.5" "NAA (1 mg/mL)", "0.5" "BAP (1 mg/mL)", "1" #. Adjust the pH of the solution to 6.5 with NaOH (1 N), and fill up to 1 L with distilled water. #. Pour 40 mL of the medium into a 100-mL flask containing 0.48 g of agar. #. Autoclave the flask at 121°C for 20 min. Methods ======== #. Pick up an appropriate amount of callus cells from a 28--56-day-old culture with a forceps and place the cells onto fresh mMS medium. #. Incubate cell cultures under the dark condition at 27°C. Notes ====== * We send Spi-I-1 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. * In order to maintain Spi-I-1 callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of Spi-I-1 cells is affected by culture conditions, such as a room temperature, aeration conditions of the culture and so on, an amount of cells transferred to fresh medium and the subculture intervals may vary from one lab to another. We usually inoculate four to seven pieces of Spi-I-1 callus (about 3--5-mm in diameter) on 40 mL of mMS medium in a 100-mL flask, and culture them for 28--56 days. Genotyping =========== :doc:`/genotyping/g_rpc00017` Maintenance history ==================== :doc:`/maintenance/m_rpc00017` References =========== .. [1] Nakagawa H, Tanaka H, Oba T, Ogura N, Iizuka M (1985) Callus formation from protoplasts of cultured *Spinacia oleracea* cells. Plant Cell Reports 4: 148--150. DOI: `10.1007/BF00571303 `_ Protocols ========== * Genotyping of plant cell lines: :doc:`/protocols/genotyping/spinacia` External links =============== * `World Flora Online: Spinacia oleracea L. `_ * `Wikipedia: Spinacia oleracea `_ .. index:: single: Spinacia oleracea single: Spinach single: Spi-I-1