Name: isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana
Uploaded: 2009-08-17T00:00:00
Description: Watch this Scientific Journal Video about Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana at JoVE.com

这项工作是由美国康奈尔大学农业试验站（CUAES）NYC - 125433哈奇格兰特和康奈尔大学的支持，启动拨款，批到OKV

第1部分。制备固体培养基上生长的植物。 我们通常集中Murasige和Skoog（MS）培养基上的文化植物辅以1％蔗糖和0.7％的琼脂。它包含了所有必要的成分，保持健康的植物。 准备介质pH值5.7的MS，1L <ol><li>在1.5 - 2.0L高压灭菌瓶，加入到800毫升的水的MS粉2.15克。 </li><li>放置一个搅拌棒装入瓶中，并解散MS粉搅拌，搅拌板介质。 </li><li>边搅拌，加，...

<ol>
	<li>Cocking, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+the+Isolation+of+Plant+Protoplasts+and+Vacuoles.">A Method for the Isolation of Plant Protoplasts and Vacuoles.</a> Nature. 187, 962-963 (1960).</li><li>Chen, S., Halkier, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+glucosinolate+uptake+by+leaf+protoplasts+of+Brassica+napus.">Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.</a> J Biol Chem. 275, 22955-22960 (2000).</li><li>Robert, S., Zouhar, J., Carter, C., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+vacuoles+from+Arabidopsis+rosette+leaf-derived+protoplasts.">Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts.</a> Nat. Protocols. 2, 259-262 (2007).</li><li>Yoo, S. D., Cho, Y. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2, 1565-1572 (2007).</li><li>Zhai, Z., Sooksa-nguan, T., Vatamaniuk, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+RNAi+as+a+reverse+genetic+approach+for+gene+functional+analysis+in+protoplasts.">Establishing RNAi as a reverse genetic approach for gene functional analysis in protoplasts.</a> Plant Phys. 149, 642-652 (2009).</li></ol>

为了确保完整的原生质体的产量高，它是非常重要的启动与健康的植物。使用过滤消毒隔离原生质体解决方案。记住，原生质体是脆弱的。因此，当你正在处理的原生质体时，不要混用，吸管或涡他们大力，因为这将制动他们。相反，混合，通过缓慢旋转或胶带的离心管中的原生质体。所描述的过程产量完整的原生质体是可行的至少96小时。因此，原生质体，可用于研究各种细胞过程，如蛋白质?...

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<td>Murashige and Skoog Basal Salt Mixture (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5524</td>
<td>&nbsp;</td>
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<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1296</td>
<td>&nbsp;</td>
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<td>Petri Dishes (100x15mm)</td>
<td>&nbsp;</td>
<td>Kreckeler Scientific</td>
<td>82-4001</td>
<td>&nbsp;</td>
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<td>Cellulase (Onozuka R&#x2010;10)</td>
<td>&nbsp;</td>
<td>RPI Corp</td>
<td>C32200</td>
<td>&nbsp;</td>
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<td>Macerozyme (R10)</td>
<td>&nbsp;</td>
<td>RPI Corp</td>
<td>M22010</td>
<td>&nbsp;</td>
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<td>Cheesecloth wipes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>06-665-29</td>
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<td>Lab Rotator</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1167152Q</td>
<td>&nbsp;</td>
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<td>Allegra X&#x2010;15R Centrifuge</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>392932</td>
<td>&nbsp;</td>
<tr>
<td>Axioskop2 Plus Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
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isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana

原生质体是植物细胞有细胞壁酶去除的。超过40年前首次报道从不同植物组织的原生质体的分离<sup&gt; 1</sup&gt;，并一直适应研究各种细胞过程，如蛋白质的亚细胞定位，完整的细胞器的隔离和有针对性的双链RNA干扰（RNAi）基因失活，<sup&gt; 2-5</sup&gt;。原生质体分离协议大多采用成熟的拟南芥叶组织（例如35天的老厂）<sup&gt; 2-4</sup&gt;。我们采用14日龄的拟南芥幼苗修改现有的协议。在此过程中，一克14日龄的幼苗产生5 10<sup&gt; 6</sup&gt; -10<sup&gt; 7</sup&gt;原生质体至少96小时保持不变。从幼苗原生质体的产量是准备成熟的拟南芥叶相媲美，但原生质体的分离，而不是35-36天，在15天完成。这使得减少的时间和需要隔离时，使用成熟的植物原生质体的生长室空间，并加快下游研究，需要完整的原生质体。

Watch this Scientific Journal Video about Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana at JoVE.com

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

这个视频展示了从14日龄拟南芥幼苗组织中分离完整的原生质体的过程。此过程加快，原生质体保持不变的至少96小时，是从，而不是一月龄成熟的植物幼苗孤立的，需要完整的原生质体的检测。

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Research

JoVE Journal

Biology

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

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科研

生物学

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In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

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