===========================================================================================
medium no. 2: modified Lin and Staba medium, 0.5 µM 2,4-dichlorophenoxyacetic acid, pH 5.7
===========================================================================================

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

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

(All stock solutions are stored at 4°C)

* KNO\ :sub:`3`\

* NH\ :sub:`4`\ Cl

* CaCl\ :sub:`2`\ ·2H\ :sub:`2`\ O

* MgSO\ :sub:`4`\ ·7H\ :sub:`2`\ O

* Sucrose

* Lin_and_Staba_1

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

       "H\ :sub:`3`\ BO\ :sub:`3`", "2.4"
       "MnSO\ :sub:`4`\ ·5H\ :sub:`2`\ O", "10.18"
       "ZnSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "4.05"

* Lin_and_Staba_2

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

       "FeSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "5.56"
       "Na\ :sub:`2`\ -EDTA", "7.46"

    Heat at 80°C for 3--4 hours for chelating Fe

* Lin_and_Staba_3

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

       "KI", "0.375"
       "Na\ :sub:`2`\ MoO\ :sub:`4`\ ·2H\ :sub:`2`\ O", "0.127"
       "CuSO\ :sub:`4`\ ·5H\ :sub:`2`\ O", "0.01"
       "CoCl\ :sub:`2`\ ·6H\ :sub:`2`\ O", "0.01"

* Lin_and_Staba_4

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

       "Nicotinic acid", "5"
       "Pyridoxine·HCl", "0.5"
       "Thiamine·HCl", "3"

* Lin_and_Staba_5

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

       "KH\ :sub:`2`\ PO\ :sub:`4`", "68"

* 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)

* KOH (1 N)

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

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

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

       "KNO\ :sub:`3`", "5.56 g"
       "NH\ :sub:`4`\ Cl", "0.268 g"
       "CaCl\ :sub:`2`\ ·2H\ :sub:`2`\ O", "0.22 g"
       "MgSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "0.185 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

       "Lin_and_Staba_1", "1"
       "Lin_and_Staba_2", "5"
       "Lin_and_Staba_3", "1"
       "Lin_and_Staba_4", "1"
       "Lin_and_Staba_5", "1"
       "2,4-D (1 mM)", "0.5"

#. Adjust the pH of the solution to 5.7 with KOH (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

       "KNO\ :sub:`3`", "5560"
       "NH\ :sub:`4`\ Cl", "268"
       "CaCl\ :sub:`2`\ ·2H\ :sub:`2`\ O", "220"
       "MgSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "185"
       "KH\ :sub:`2`\ PO\ :sub:`4`", "68"
       "H\ :sub:`3`\ BO\ :sub:`3`", "2.4"
       "MnSO\ :sub:`4`\ ·5H\ :sub:`2`\ O", "10.18"
       "ZnSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "4.05"
       "KI", "0.375"
       "Na\ :sub:`2`\ MoO\ :sub:`4`\ ·2H\ :sub:`2`\ O", "0.127"
       "CuSO\ :sub:`4`\ ·5H\ :sub:`2`\ O", "0.01"
       "CoCl\ :sub:`2`\ ·6H\ :sub:`2`\ O", "0.01"
       "FeSO\ :sub:`4`\ ·7H\ :sub:`2`\ O", "27.8"
       "Na\ :sub:`2`\ -EDTA", "37.3"
       "Nicotinic acid", "5"
       "Pyridoxine·HCl", "0.5"
       "Thiamine·HCl", "3"
       "Sucrose", "20000"
       "2,4-D sodium monohydrate", "0.131"