Name: a simple method for isolation of soybean protoplasts and application to transient gene expression analyses
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Description: Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

Dette arbejde blev støttet af plante Genome Research Program fra National Science Foundation (NSF-PGRP-IOS-1339388).

1. vækst af planter
<ol><li>So 5-10 sojabønner frø (Williams 82) i en 13 cm potte i drivhus betingelser længe-dag (16 h lys på 1.500 µmol m-2 s-1) på 25 ° C på den brugerdefinerede jord blanding for sojabønner (1:1:1 ratio af sand, jord, perlite og torpedo).</li>
</ol>2. forberedelse af Plasmid DNA
<ol><li>Med en steril pipette tip eller tandstikker, vælge en enkelt koloni eller frosne glycerol bestand af E. coli transporterer plasmid som indeholder ge...

<ol>
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Gaz. 141 (2), 157-164 (1980).</li><li>L&#246;rz, H., Baker, B., Schell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+to+cereal+cells+mediated+by+protoplast+transformation.">Gene transfer to cereal cells mediated by protoplast transformation.</a> Mol. Gen. Genet. 199 (2), 178-182 (1985).</li><li>Hayashimoto, A., Li, Z., Murai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polyethylene+glycol-mediated+protoplast+transformation+system+for+production+of+fertile+transgenic+rice+plants.">A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants.</a> Plant Physiol. 93 (3), 857-863 (1990).</li><li>Koop, H. -. 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Denne protokol til isolering af sojabønner protoplasts og programmet til forbigående udtryk undersøgelser er blevet grundigt testet og virker meget godt i vores laboratorium. Procedurerne, der er enkel og let og kræver almindelige udstyr og minimale omkostninger. Vores protokol giver store mængder af ensartet, høj kvalitet protoplasts i forhold til tidligere rapporteret metoder34,35,36,37...

Protoplasts er planteceller, der har cellevægge fjernet. Som de fleste af funktioner og aktiviteter af planteceller påstår de, protoplasts er en god modelsystem til at observere og vurdere forskellige cellulære begivenheder, og er værdifulde værktøjer til at studere somatiske hybridisering1 og plante regenerering2. Protoplasts har udnyttet også bredt for anlægget transformation3,4,5, da cellevægge ville ellers blokere passage af DNA i cellen. Protoplasts besidder nogle af de fysiologiske reaktioner og cellulære processer af intakt planter, derfor tilbyder grundlæggende værdi i grundlæggende forskning til at studere subcellulært protein lokalisering6,7,8, protein-protein interaktioner9,10, og promotor aktivitet11,12,13 i levende celler.
Isolering af planten protoplasts blev først rapporteret i 196014 og protokoller for både isoleret og transformation af protoplasts er blevet udviklet og optimeret. En standard procedure protoplast isolation indebærer opskæring af blade og Enzymatisk nedbrydning af cellevægge, efterfulgt af adskillelse af frigivne protoplasts fra ikke-fordøjet væv debris. Transformation strategier omfatter elektroporation15,16, mikroinjektion17,18, og funderede polyethylenglycol (PEG)4,5,19 metoder. En bred vifte af arter, der er blevet rapporteret succes for protoplast isolation, herunder Citrus20, Brassica21, Solanaceae22 og andre prydplante familier23,24. Mens forskellige vævstyper bruges i forskellige arter, har et system af forbigående udtryk i Arabidopsis mesofyllet protoplast (TEAMP) isoleret fra bladene af model planten Arabidopsis thaliana været veletablerede25 og bredt vedtaget til forskellige applikationer.
Sojabønner (Glycine max (L.) Merr.) er en af de vigtigste protein og olie afgrøder26. I modsætning til Arabidopsis og ris, er opnåelse af transgene sojabønner planter kendt for at være temmelig vanskeligt og lav effektivitet. Agrobacterium tumefaciens-medieret infiltration har været populært anvendes for forbigående gen expression undersøgelser i den epidermale celler i tobak27 og stiklinger i Arabidopsis28,29, hvorimod Agrobacterium rhizogenes har været brugt til transformering af behårede rødder i sojabønner30. Virus-induceret gene silencing tilgange har været udnyttet til downregulation af target gener31,32 og forbigående udtryk33 på en systemisk måde. Protoplasts giver en værdifuld og alsidig alternativ til disse tilgange. Protoplasts kan opnås fra sojas overjordiske materialer og giver mulighed for hurtig og synkroniseret transgen udtryk. Siden den oprindelige vellykkede isolering af sojabønner protoplasts i 198334, har der dog været begrænset rapporter om anvendelsen af protoplasts i sojabønner35,36,37, 38, primært som følge af forholdsvis lave udbytter af sojabønner protoplasts.
Her, beskriver vi en enkel og effektiv protokol for dyrkning af sojabønner protoplasts og dens anvendelse for forbigående gen expression undersøgelser. Ved hjælp af unge unifoliate blade fra sojabønner kimplanter, har vi kunnet opnå store mængder af afgørende protoplasts inden for et par timer. Derudover har vi optimeret en PIND-calcium-medieret transformation metode, der er enkel og billig at levere DNA i soja protoplasts med høj effektivitet.

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">MES</td>
			<td style="width:181px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">M8250-100G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase CELF</td>
			<td>Worthington Biological Corporation</td>
			<td>LS002611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pectolyase Y-23</td>
			<td>BioWorld</td>
			<td>9033-35-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CELLULASE &#34;ONOZUKA&#34; R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACEROZYME R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td>ICN Biomedicals&#160;</td>
			<td>152540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher&#160;</td>
			<td>C79-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma Aldrich&#160;</td>
			<td>S7653-1kg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher&#160;</td>
			<td>P217-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma Aldrich&#160;</td>
			<td>M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEG4000</td>
			<td>Fluka</td>
			<td>81240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nylon mesh</td>
			<td>carolina</td>
			<td>652222N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Tissue Culture Plates&#160;</td>
			<td>USA Scientific</td>
			<td>CC7682-7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:181px;">Fisher</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hemacytometer</td>
			<td>hausserscientific</td>
			<td>1483</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:181px;">Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">EZNA plasmid miniprep kit</td>
			<td style="width:181px;">Omega</td>
			<td>D6942-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">GeneJET Plasmid Miniprep Kit</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>K0502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5810</td>
			<td style="width:181px;">eppendorf</td>
			<td style="width:119px;">5811000827</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5424</td>
			<td style="width:181px;">eppendorf</td>
			<td>22620401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Jencons Powerpette Plus Pipet Controller</td>
			<td style="width:181px;">Jencons</td>
			<td>14526-202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:256px;">Zeiss 710 Confocal Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:256px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td>
			<td style="width:181px;">Ambion</td>
			<td style="width:119px;">AM12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">15 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1018-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">50 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1060-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Newborn Calf Serum</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:119px;">16010159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Soil</td>
			<td style="width:181px;">Ingram&#39;s Nursery</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">perlite</td>
			<td style="width:181px;">Vigoro</td>
			<td style="width:119px;">100521091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Torpedo Sand</td>
			<td style="width:181px;">JKS Ventures</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LB Broth, Lennox (Powder)</td>
			<td style="width:181px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

Sojabønner (Glycine max (L.) Merr.) er en vigtig afgrøde arter og er blevet en bælgplante model for studier af genetiske og biokemiske veje. Det er derfor vigtigt at etablere en effektiv forbigående gen expression system i sojabønner. Vi rapporterer her, en enkel protokol for forberedelse af sojabønner protoplasts og dens anvendelse for forbigående funktionelle analyser. Vi fandt, at unge unifoliate blade fra sojabønner frøplanter resulterede i store mængder af høj kvalitet protoplasts. Ved at optimere en PIND-calcium-medieret transformation metode, opnåede vi høj transformation effektivitet ved hjælp af sojabønner unifoliate protoplasts. Dette system giver en effektiv og alsidig model til undersøgelse af komplekse regulerende og signalering mekanismer i live sojabønner celler og kan hjælpe til bedre forstå forskellige cellulære, udviklingsmæssige og fysiologiske processer i bælgplanter.

Forskellige organer i 10 - dage gamle sojabønner blev testet for protoplast forberedelse (figur 1) og udbyttet blev observeret under mikroskop (figur 2). Cellevægge fra hypocotyl og epicotyl var næppe fordøjes, og nogle celler blev knyttet til hinanden (figur 2B, 2 C). I Kimblad (figur 2D) og root (figur 2A), blev cellev...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Vi udviklet en enkel og effektiv protokol for forberedelse af store mængder af sojabønner protoplasts at studere komplekse regulerende og signalering mekanismer i levende celler.

En simpel metode til dyrkning af sojabønner Protoplasts og ansøgning til forbigående gen Expression analyser

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

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JoVE Journal

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