===========================================================================================================
medium no. 15: AA medium, 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.2 mg/L kinetin, 1.2% (w/v) agar, pH 5.8
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Plant cell lines
=================

* :doc:`/cell_lines/rpc00020`

Chemicals and stock solutions
==============================

(All stock solutions are stored at 4°C)

* 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

* AA_1

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

       "KCl", "29.4"
       "CaCl\ :sub:`2`\ ·2H\ :sub:`2`\ O", "4.4"
       "MgSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "3.7"
       "KH\ :sub:`2`\ PO\ :sub:`4`", "1.7"

* AA_4

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

       "Nicotinic acid", "0.05"
       "Pyridoxine·HCl", "0.05"
       "Thiamine·HCl", "0.01"
       "*myo*-Inositol", "10"

    Store at −20°C

* AA_5

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

       "L-Glutamine", "8.77"
       "L-Aspartic acid", "2.66"
       "L-Arginine", "2.28"
       "Glycine", "0.75"

    Store at −20°C

* 2,4-D (0.2 mg/mL)

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

       "2,4-D sodium monohydrate", "0.236"

    (2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich (D6679)

* 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

* Agar, powder

* KOH (1 N)

Medium preparation
===================

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

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

       "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_micro_1", "10"
       "MS_micro_2", "10"
       "AA_1", "100"
       "AA_4", "10"
       "AA_5", "100"
       "2,4-D (0.2 mg/mL)", "5"
       "Kinetin (0.2 mg/mL)", "1"

#. Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.

#. Add 12 g of agar to the medium.

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

Formulation
============

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

       "KCl", "2940"
       "L-Glutamine", "877"
       "L-Aspartic acid", "266"
       "L-Arginine", "228"
       "CaCl\ :sub:`2`\ ·2H\ :sub:`2`\ O", "440"
       "MgSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "370"
       "KH\ :sub:`2`\ PO\ :sub:`4`", "170"
       "H\ :sub:`3`\ BO\ :sub:`3`", "6.2"
       "MnSO\ :sub:`4`\ ·5H\ :sub:`2`\ O", "24.1"
       "ZnSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "8.6"
       "KI", "0.83"
       "Na\ :sub:`2`\ MoO\ :sub:`4`\ ·2H\ :sub:`2`\ O", "0.25"
       "CuSO\ :sub:`4`\ ·5H\ :sub:`2`\ O", "0.025"
       "CoCl\ :sub:`2`\ ·6H\ :sub:`2`\ O", "0.025"
       "FeSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "27.8"
       "Na\ :sub:`2`\ -EDTA", "37.3"
       "Nicotinic acid", "0.5"
       "Pyridoxine·HCl", "0.5"
       "Thiamine·HCl", "0.1"
       "Glycine", "75"
       "*myo*-Inositol", "100"
       "Sucrose", "30000"
       "2,4-D sodium monohydrate", "1.18"
       "Kinetin", "0.2"
       "Agar", "12000"