================================================================================================================================
medium no. 57: Amino Acid medium, 0.02 µM 2,4-dichlorophenoxyacetic acid, 2 µM *N*-(2-chloro-4-pyridyl)-*N*′-phenylurea, pH 6.2
================================================================================================================================

Plant cell lines
=================

* :doc:`/cell_lines/rpc00087`

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

(All stock solutions are stored at 4°C)

* Sucrose

* CaCl\ :sub:`2` (0.6 M)

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

       "CaCl\ :sub:`2`\ ·2H\ :sub:`2`\ O", "88"

* AA_macro

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

       "KCl", "149"
       "MgSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "37"
       "KH\ :sub:`2`\ PO\ :sub:`4`", "17"

* 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"

* Fe(III)-EDTA (20 mM)

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

       "Fe(III)-EDTA", "8.422"

    Ethylenediamine-*N*,\ *N*,\ *N*′,\ *N*′-tetraacetic acid, iron (III), sodium salt, trihydrate, FUJIFILM Wako Pure Chemical Corporation (#343-01241)

* 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

* AA_VT

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

       "Nicotinic acid", "0.05"
       "Pyridoxine·HCl", "0.05"
       "Thiamine·HCl", "0.01"
       "Glycine", "0.16"

    Store at −20°C

* 2,4-D (1 mM)

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

       "2,4-D sodium monohydrate", "0.261"

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

* 4-CPPU (2 mM)

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

       "4-CPPU", "0.495"

    *N*-(2-Chloro-4-pyridyl)-*N*′-phenylurea, Sigma-Aldrich (C2791); Dissolve 4-CPPU in ethanol and store at −20°C

* NaOH (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

       "CaCl\ :sub:`2` (0.6 M)", "5"
       "AA_macro", "10"
       "MS_micro_1", "10"
       "Fe(III)-EDTA (20 mM)", "5"
       "AA_5", "100"
       "AA_VT", "10"
       "2,4-D (1 mM)", "0.02"
       "4-CPPU (2 mM)", "1"

#. Adjust the pH of the solution to 6.2 with NaOH (1 N), and fill up to 1 L with distilled water.

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

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

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

       "KCl", "1490"
       "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"
       "Fe(III)-EDTA", "42.11"
       "Nicotinic acid", "0.5"
       "Pyridoxine·HCl", "0.5"
       "Thiamine·HCl", "0.1"
       "Glycine", "76.6"
       "Sucrose", "30000"
       "2,4-D sodium monohydrate", "0.00522"
       "4-CPPU", "0.495"