Protocol for overseas customers ================================================================================ Re-establishment of suspension cultures of tobacco BY-2 and related cell lines ================================================================================ :download:`Download a PDF version (3.5 MB) ` .. note:: * Cell lines * :doc:`/cell_lines/rpc00001` * :doc:`/cell_lines/rpc00039` * :doc:`/cell_lines/rpc00040` * :doc:`/cell_lines/rpc00041` * :doc:`/cell_lines/rpc00062` * :doc:`/cell_lines/rpc00091` * :doc:`/cell_lines/rpc00093` * :doc:`/cell_lines/rpc00095` * Culture medium * :doc:`/media/medium_1` Methods ======= Regrowth of BY-2 cells ---------------------- #. Recieve an 250-mL Erlenmeyer flask containing BY-2 cells. .. subfigure:: A|B :layout-sm: A|B :width: 350px :gap: 5px .. image:: flask_transport.jpg :alt: .. image:: flask_transport_large.jpg :alt: #. Loosen the screw cap slightly to keep good aeration. .. caution:: * Avoid microbial contamination. #. Incubate the BY-2 cells in the dark at 27°C for 1–2 weeks. .. subfigure:: A :layout-sm: A :width: 350px .. image:: flask_regrowth.jpg :alt: After 8 days. The red arrowheads indicate the growing BY-2 cells. .. subfigure:: A :layout-sm: A :width: 350px .. image:: cell_edge.jpg :alt: BY-2 cells around the edge are alive. .. subfigure:: A :layout-sm: A :width: 350px .. image:: cell_center.jpg :alt: BY-2 cells around the center are dead. #. Use the growing BY-2 cells for the induction of suspension cell cultures and for the maintenance of stock agar cultures. Induction of suspension cell cultures ------------------------------------- #. Transfer the BY-2 cells into a 100-mL Erlenmeyer flask containing 20 mL of a liquid culture medium. .. caution:: * Use at least the amount of BY-2 cells shown below. .. subfigure:: A :layout-sm: A :width: 350px .. image:: cell_suspension_induction.jpg :alt: #. Incubate the BY-2 cells in the dark at 27°C with rotary shaking at 130 rpm for 5–10 days. .. caution:: * Avoid microbial contamination. * Cover the flask mouth loosely with an aluminum foil cap for good aeration. .. note:: * A silicone sponge plug can be used instead of an aluminum foil cap (e.g., `Cap-type Silicosen; Shin-Etsu Polymer, Tokyo, Japan `_). .. subfigure:: A :layout-sm: A :width: 350px .. image:: cell_suspension_regrowth.jpg :alt: After 7 days #. Allow the Erlenmeyer flask to stand for a few minutes just before the first subculture of suspension cell culture. #. Transfer 1–2 mL of the sedimented cells into 95 mL of a fresh culture medium in a 300-mL Erlenmeyer flask. .. note:: * See :ref:`Appendix A ` for the BY-2-related cell lines. .. subfigure:: A :layout-sm: A :width: 350px .. image:: subculture.jpg :alt: #. Culture the BY-2 cells by the usual method. .. subfigure:: A :layout-sm: A :width: 350px .. image:: sunculture_regrowth.jpg :alt: After 7 days #. After 3–4 subculturing, the BY-2 suspension cell cultures grow stably. Maintenance of stock agar cultures ---------------------------------- #. Prepare cell culture dishes containing 30 mL of a culture medium solidified with 0.8% (w/v) agar. .. note:: * BY-2 cells can also be cultured on the media solidified with gellan gum. #. Transfer small pieces (3–5 mm in diameter) of BY-2 cells onto agar culture medium. .. subfigure:: A :layout-sm: A :width: 350px .. image:: callus_induction.jpg :alt: #. Seal culture dishes by using surgical tape to keep good aeration. #. Incubate the BY-2 cells in the dark at 27°C for 2–4 weeks. .. subfigure:: A :layout-sm: A :width: 350px .. image:: callus_regrowth.jpg :alt: After 4 weeks #. Subculture the BY-2 cells on fresh agar medium at 2–4-week intervals. .. _overseas_appendix_a: Appendix A: The first subculture of suspension cell cultures of BY-2-related cell lines ======================================================================================= .. csv-table:: :header: "BRC No.", "Cell line", "Transfer volume (mL) `*`", "Culture medium (mL)", "Erlenmeyer flask (mL)" :widths: auto "rpc00001", "BY-2", "1–2", "95", "300" "rpc00039", "GV7", "1–3", "95", "300" "rpc00041", "GT16", "3–6", "95", "300" "rpc00062", "BY-TIPG", "1–3", "95", "300" "rpc00091", "TBY2-31/ST", "0.4–0.8", "30", "100" "rpc00093", "TBY2-41/ST", "0.4–0.8", "30", "100" "rpc00095", "TBY2-R31", "0.4–0.8", "30", "100" `*` Subculture sedimented cells to a fresh culture medium. .. caution:: * For the transgenic BY-2 cell lines, check the expression of fluorescent proteins in re-established suspension cells before use. Appendix B: Preparation of BY-2 cell cultures for transportation ================================================================ Preparation of a cell culture ----------------------------- #. An agar-solidified culture medium was prepared in a 250-mL disposable Erlenmeyer flask. .. note:: * mLS medium (:doc:`medium no. 1 `) solidified with 1.4% (w/v) agar, 80 mL #. BY-2 cell suspension culture was prepared on day 7 of the culture. #. BY-2 cells were spread on the agar culture medium in the 250-mL disposable Erlenmeyer flask. #. The screw cap was loosely closed to keep good aeration. #. The BY-2 cells were pre-cultured in the dark at 27°C for 6 days. #. The screw cap was tightly closed and sealed with thermoplastic sealing film. Transportation test ------------------- #. The BY-2 cells was stored in the dark at 27°C for 7 days (simulating transport). #. The BY-2 cells were tested for regrowth.