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rpc00044: *Nicotiana glauca* G89 callus culture
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  :download:`Download a PDF version (139 KB) <../cell_lines/rpc00044_G89_2_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: MS medium, 1 mg/L IAA, 0.9% (w/v) agar, pH 5.7 (\ :doc:`medium no. 35 </media/medium_35>`\ )

     * Culture conditions: 27°C, dark

     * Subculture: 28-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: “*Nicotiana glauca* G89 cell line (rpc00044) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”

Introduction
=============

  Tobacco G89 cell line was established from a seedling stem of *Nicotiana glauca* Graham (Syōno and Fujita 1994 [1]_). The G89 cells are grown on a Murashige and Skoog (MS) medium supplemented with 1 mg/L indole-3-acetic acid (IAA), and solidified with 0.9% (w/v) agar, pH 5.7. Our G89 cell culture has been maintained in the dark at 27°C and subcultured at 28-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"

* IAA (1 mg/mL)

    .. csv-table::
       :header: "Chemical", "Concentration (mg/mL)"
       :widths: auto

       "IAA·K", "1.217"

    Potassium 3-indoleacetate, FUJIFILM Wako Pure Chemical Corporation (#160-07531)

* Agar, powder

* KOH (1 N)

Glassware and equipment
------------------------

* Erlenmeyer flask (100 mL), capped with two layers of aluminum foil

* Forceps, sterilized before use

Preparation of MS medium (medium no. 35)
-----------------------------------------

  :doc:`/media/medium_35`

#. Dissolve the following chemicals in approximately 800 mL of distilled water.

    .. csv-table::
       :header: "Chemical", "Amount"
       :widths: auto

       "MS salt mix", "1 bag (1 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", "1"
       "MS_inositol", "2.5"
       "IAA (1 mg/mL)", "1"

#. Adjust the pH of the solution to 5.7 with KOH (1 N), and fill up to 1 L with distilled water.

#. Pour 40 mL of the medium into a 100-mL flask containing 0.36 g of agar.

#. Autoclave the flask at 121°C for 20 min.

Methods
========

#. Pick up an appropriate amount of callus cells from a 28-day-old culture with a forceps and place the cells onto fresh MS medium.

#. Incubate cell cultures under the dark condition at 27°C.

Notes
======

* We send G89 cells on semi-solid MS medium in a 9-cm disposable Petri dish. The cells should be subcultured to fresh MS medium immediately after arrival.

* In order to maintain G89 callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of G89 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 three pieces of G89 callus (about 5-mm in diameter) on 40 mL of MS medium in a 100-mL flask, and culture them for 28 days.

Genotyping
===========

  :doc:`/genotyping/g_rpc00044`

Maintenance history
====================

  :doc:`/maintenance/m_rpc00044`

References
===========

.. [1] Syōno K, Fujita T (1994) Habituation as a tumorous state that is interchangeable with a normal state in plant cells. International Review of Cytology 152: 265--299. DOI: `10.1016/S0074-7696(08)62559-0 <https://doi.org/10.1016/S0074-7696(08)62559-0>`_

Protocols
==========

* Genotyping of plant cell lines: :doc:`/protocols/genotyping/nicotiana`

External links
===============

* `World Flora Online: Nicotiana glauca Graham <http://www.worldfloraonline.org/taxon/wfo-0001023776>`_

* `Wikipedia: Nicotiana glauca <https://en.wikipedia.org/wiki/Nicotiana_glauca>`_

.. index::
   single: Nicotiana glauca
   single: Tobacco
   single: G89