Archived Protocol

<!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 3.2//JA"> <HTML> <HEAD> <Title>In vitro growth of seedlings</Title> <META NAME="description" CONTENT="This is the Schneitz lab home page at the University of Zuerich. We are studying organogenesis in plants using the ovules of Arabidopsis as a model system. Teaching is done in the area of plant developmental biology."><META NAME="keywords" CONTENT="Arabidopsis thaliana, biology, botany, cell biology, development, genetics, molecular biology, ovule, organogenesis, plant biology, science"> <META NAME="institution" CONTENT="Institut für Pflanzenbiologie der Universität Zürich, Schweiz, Institute of Plant Biology - University of Zurich, Switzerland"> <META NAME="Author" CONTENT="Kay Schneitz"> <META NAME="Publisher" CONTENT="Kay Schneitz"> <META NAME="Publisher-Email" CONTENT="Kay.Schneitz@access.unizh.ch"> <META NAME="Content-Language" CONTENT="en-US"> <meta name="generator" content="Frontier 6.1.1 WinNT"> <style type="text/css">  </style> <script language="JavaScript">  </script> </HEAD> <body bgcolor="#F5F5F5" alink="#FF3300" vlink="#330099" link="#CC0000" text="#0"> <A NAME="top"></A> <img src=http://www.unizh.ch/botinst/Cyto_Website/schneitzLab/images/schneitzLabTitleBar.gif height=105 width=600 usemap="#schneitzLabTitleBarMap" border=0> <map name="schneitzLabTitleBarMap"> <area coords="483,86,594,97" href="http://www.zuerich-online.ch/" alt="Zürich Online" shape="rect"> <area coords="362,86,455,97" href="http://www.unizh.ch/hbi/" alt="Bibliothek Uni ZH" shape="rect"> <area coords="251,84,349,98" href="http://www.unizh.ch/VVZV1/mnf/ENTRY013.html" alt="Vorlesungen" shape="rect"> <area coords="141,85,233,98" href="http://www.unizh.ch/botinst/" alt="Institute of Plant Biology Home Page" shape="rect"> <area coords="0,85,120,98" href="http://www.unizh.ch/" alt="U of Zürich Home Page" shape="rect"> </map>   <a href=http://www.unizh.ch/botinst/Cyto_Website/schneitzLab/index.html>Schneitz Lab Home Page</a> / <a href=http://www.unizh.ch/botinst/Cyto_Website/schneitzLab/Methods/index.html>Methods</a> / <a href=http://www.unizh.ch/botinst/Cyto_Website/schneitzLab/Methods/PlantWork/index.html>Plant Work</a> / In vitro growth of seedlings <TABLE BORDER="0" CELLPADDING="0" CELLSPACING="0" WIDTH="510"> <TR><TD COLSPAN="2" HEIGHT="30"> </TD></TR> <TR><TD WIDTH="30"> </TD><TD WIDTH="480"><TABLE WIDTH="480" BORDER="0" CELLPADDING="0" CELLSPACING="0"> <TR> <TD COLSPAN="3">In vitro growth of seedlings</TD> </TR> <TR> <TD COLSPAN="3" HEIGHT="8" VALIGN="MIDDLE"><img src=http://www.unizh.ch/botinst/Cyto_Website/schneitzLab/images/redHr.gif height=1 width=480 border=0></TD> </TR> <TR ALIGN="LEFT"> <TD WIDTH="249">Reference:  Schneitz Lab</TD> <TD WIDTH="103">Last updated: 2/16/00</TD> <TD WIDTH="106" ALIGN="RIGHT">By: Kay Schneitz</TD> </TR> <TR> <TD HEIGHT="40" WIDTH="249"> </TD> <TD HEIGHT="40" WIDTH="103"> </TD> <TD HEIGHT="40" WIDTH="106"> </TD> </TR> </TABLE></TD></TR> <TR><TD WIDTH="30"> </TD><TD WIDTH="480">sterilisation of seeds: <ol> <li>rinse with 70% EtOH for 30 sec</li> <li>put in 1% bleach (sodium hypochlorite, supplemented by a few drops of Tween-20) for 5 min</li> <li>wash 2-4x with sterile H2O for 1 min</li> </ol> plating and growth <ol> <li>resuspend in sterile 0.1% agarose</li> <li>plate 4 ml in petridish (0.8% agarose supplemented with 1/2 MS10) in sterile hood</li> <li>let dry for 1-2 h in the hood</li> <li>wrap with 3M micropore tape</li> <li>keep at 4°C for 4 days</li> <li>place the dishes under light</li> </ol> <h3>Solutions</h3> <table width="230" cellspacing="0" cellpadding="0" border="0"> <tr align="right" bgcolor="FF9900"><td width="140">1/2 MS10</td> <td width="90"> </td> </tr> <tr align="right" bgcolor="FFFFFF"><td width="140"> </td> <td width="90"> </td> </tr> <tr align="right" bgcolor="FFCC66"><td width="140">MS Macro I</td> <td width="90">50.0 ml</td> </tr> <tr align="right" bgcolor="FFFFFF"><td width="140">MS Macro II</td> <td width="90">50.0 ml</td> </tr> <tr align="right" bgcolor="FFCC66"><td width="140">Myo</td> <td width="90">10.0 ml</td> </tr> <tr align="right" bgcolor="FFFFFF"><td width="140">FeNaEDTA</td> <td width="90">10.0 ml</td> </tr> <tr align="right" bgcolor="FFCC66"><td width="140">MS Vitamines</td> <td width="90">1.0 ml</td> </tr> <tr align="right" bgcolor="FFFFFF"><td width="140">MS Micronutrients</td> <td width="90">0.5 ml</td> </tr> <tr align="right" bgcolor="FFCC66"><td width="140">Sucrose</td> <td width="90">10.0 g </td> </tr> <tr align="right" bgcolor="FFFFFF"><td width="140">H2O</td> <td width="90">x.x ml</td> </tr> <tr align="right" bgcolor="FFCC66"><td width="140">Total</td> <td width="90">1000.0 ml</td> </tr> </table> adjust pH to 5.7 with 1 M KOH, alternatively MES (0.5g/l) can be used to ensure more stable pH during plant growth. <h3>Remarks</h3> It is essential that the medium is autoclaved in either a small autoclave or pressure cooker because prolonged exposure to heat will cause complex formation between sucrose and phytoagar which becomes visible as a yellow coloring of the medium. This medium will be softer after hardening than correctly autoclaved medium. The MS medium protocol is taken from Murashige and Skoog (1962) Physiologia Plantarum 15: 473-497. The recipe can be found <a href=http://www.unizh.ch/botinst/Cyto_Website/schneitzLab/Materials/MSmedium.html>here</a>. <h3>Materials</h3> <table width="480" cellspacing="0" cellpadding="0" border="0"> <tr align="center" bgcolor="CECFCE"><td width="202">Reagent/Tool</td> <td width="156">Supplier</td> <td width="114">Cat.-#</td> </tr> <tr align="center" bgcolor="FFFFFF"><td width="202"> </td> <td width="156"> </td> <td width="114"> </td> </tr> <tr align="center" bgcolor="FFFF9C"><td width="202">Agarose</td> <td width="156"><A HREF=http://www.quantum-appligene.com/>Quantum-Appligene</A></td> <td width="114">130022</td> </tr> <tr align="center" bgcolor="FFFFFF"><td width="202">MS media</td> <td width="156"><A HREF=http://www.phytotechlab.com/default.htm>Phytotechnology Laboratories</A></td> <td width="114"> </td> </tr> <tr align="center" bgcolor="FFFF9C"><td width="202">Petridish 145/20 mm</td> <td width="156"><A HREF=http://www.greiner-lab.com/>Greiner Labortechnik</A></td> <td width="114">639 102</td> </tr> <tr align="center" bgcolor="FFFFFF"><td width="202">Sodium hypochlorite 13%</td> <td width="156"><A HREF=http://www.sigma-aldrich.com>Fluka</A></td> <td width="114">71696</td> </tr> <tr align="center" bgcolor="FFFF9C"><td width="202">Tween-20</td> <td width="156"><A HREF=http://www.sigma-aldrich.com>Fluka</A></td> <td width="114">93773</td> </tr> <tr align="center" bgcolor="FFFFFF"><td width="202">3M Micropore tape</td> <td width="156"><A HREF=http://www.galenica.ch/>Galenica</A></td> <td width="114"> </td> </tr> </table> </TD></TR> </TABLE> <TABLE BORDER="0" CELLPADDING="0" CELLSPACING="0" WIDTH="510"> <TR><TD COLSPAN="2" HEIGHT="30"> </TD></TR> <TR><TD WIDTH="30"> </TD><TD WIDTH="480" VALIGN="BOTTOM" ALIGN="CENTER"><A HREF=http://www.unizh.ch/botinst/Cyto_Website/schneitzLab/Methods/PlantWork/invitroGrowthSeedlings.html#top>Top</A></TD></TR> <TR><TD COLSPAN="2" HEIGHT="20"> </TD></TR> </TABLE> <TABLE BORDER="0" CELLPADDING="0" CELLSPACING="0" WIDTH="600"><TR VALIGN=BOTTOM><TD COLSPAN="2"><HR size=1 NOSHADE></TD></TR> <TR><TD WIDTH="5"> </TD> <TD ALIGN=LEFT VALIGN=TOP WIDTH="595"> © Copyright 1997-2000, Kay Schneitz. Last updated 2/16/00; 3:08:58 PM. Send comments to the <a href=mailto:Kay.Schneitz@access.unizh.ch>webmeister</a>.</TD> </TR></TABLE> </body> </html>