Name: the use of induced somatic sector analysis (issa) for studying genes and promoters involved in wood formation and secondary stem development
Uploaded: 2016-10-05T15:00:00
Description: Watch this Scientific Journal Video about The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development at JoVE.com

The authors would like to acknowledge funding support for aspect of the work through Linkage Grants LP0776563 (GB, AS) and LP0211919 (GB) and industry partners Sappi and Mondi as well as Australian Postgraduate Award (EM) from the Australian Research Council and the Young Innovators and Scientist Award through the Australian Department of Agriculture (LT). We also like to thank the Zander Myburg, Qing Wang, Colleen MacMillan and Simon Southerton for the many discussions and ideas they put forward during the development of this protocol and to Martin Ranik, Minique De Castro, Julio Najera, Valerie Frassiant, Angelique Manuel and Noemie Defaix for assistance in laboratory related work.

1.植物材料的制备<ol><li>之前的实验，提高从种子或切割的优选树种新的幼苗和生长树/秒，直到在用于实验的区域中的杆的直径是直径约1厘米。 注：需要可能会有所不同，由于植物的生长速度，因此三至九个月这一步之间允许的时间。 </li></ol> 2.二进制向量创建<ol><li>从此部分进行工作，以在实验室或温室，其中根癌农杆菌可以处理和规定为实验室适当的个人防护...

<ol>
	<li>Spokevicius, A. V., Tibbits, J. F. G., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+plant+and+plant+part+transgenic+approaches+in+the+study+of+wood+formation+-+benefits+and+limitations.">Whole plant and plant part transgenic approaches in the study of wood formation - benefits and limitations.</a> TPJ. 1 (1), 49-59 (2007).</li><li>Chaffey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wood+formation+in+forest+trees:+from+Arabidopsis+to+Zinnia.">Wood formation in forest trees: from Arabidopsis to Zinnia.</a> Trends Plant Sci. 4 (6), 203-204 (1999).</li><li>Moller, R., McDonald, A. G., Walter, C., Harris, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+differentiation,+secondary+cell-wall+formation+and+transformation+of+callus+tissue+of+Pinus+radiata+D.+Don.">Cell differentiation, secondary cell-wall formation and transformation of callus tissue of Pinus radiata D. Don.</a> Planta. 217 (5), 736-747 (2003).</li><li>Spokevicius, A. V., Van Beveren, K., Leitch, M. M., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+in+vitro+transformation+of+wood-producing+stem+segments+in+eucalypts.">Agrobacterium-mediated in vitro transformation of wood-producing stem segments in eucalypts.</a> Plant Cell Rep. 23 (9), 617-624 (2005).</li><li>Plasencia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eucalyptus+hairy+roots,+a+fast,+efficient+and+versatile+tool+to+explore+function+and+expression+of+genes+involved+in+wood+formation.">Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.</a> Plant Biotech J. , (2015).</li><li>Van Beveren, K. S., Spokevicius, A. V., Tibbits, J., Wang, Q., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+cambial+tissue+in+vivo+provides+efficient+means+for+Induced+Somatic+Sector+Analysis+(ISSA)+and+gene+testing+in+stems+of+woody+plants+species.">Transformation of cambial tissue in vivo provides efficient means for Induced Somatic Sector Analysis (ISSA) and gene testing in stems of woody plants species.</a> Funct Plant Biol. 33 (7), 629-638 (2006).</li><li>Spokevicius, A. V., Van Beveren, K., Bossinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+transformation+of+dormant+lateral+buds+in+poplar+trees+reveals+developmental+patterns+in+secondary+stem+tissues.">Agrobacterium-mediated transformation of dormant lateral buds in poplar trees reveals developmental patterns in secondary stem tissues.</a> Funct Plant Biol. 33 (2), 133-139 (2006).</li><li>Spokevicius, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-tubulin+affects+cellulose+microfibril+orientation+in+plant+secondary+fiber+cell+walls.">beta-tubulin affects cellulose microfibril orientation in plant secondary fiber cell walls.</a> Plant J. 51 (4), 717-726 (2007).</li><li>MacMillan, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fasciclin-like+arabinogalactan+protein+family+of+Eucalyptus+grandis+contains+members+that+impact+wood+biology+and+biomechanics.">The fasciclin-like arabinogalactan protein family of Eucalyptus grandis contains members that impact wood biology and biomechanics.</a> New Phytol. 206 (4), 1314-1327 (2015).</li><li>Creux, N. M., Bossinger, G., Myburg, A. A., Spokevicius, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+somatic+sector+analysis+of+cellulose+synthase+(CesA)+promoter+regions+in+woody+stem+tissues.">Induced somatic sector analysis of cellulose synthase (CesA) promoter regions in woody stem tissues.</a> Planta. 237 (3), 799-812 (2013).</li><li>Hussey, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SND2,+a+NAC+transcription+factor+gene,+regulates+genes+involved+in+secondary+cell+wall+development+in+Arabidopsis+fibers+and+increases+fiber+cell+area+in+Eucalyptus.">SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibers and increases fiber cell area in Eucalyptus.</a> BMC Plant Biology. 11, (2011).</li><li>Melder, E., Bossinger, G., Spokevicius, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+ARBORKNOX1+delays+the+differentiation+of+induced+somatic+sector+analysis+(ISSA)+derived+xylem+fiber+cells+in+poplar+stems.">Overexpression of ARBORKNOX1 delays the differentiation of induced somatic sector analysis (ISSA) derived xylem fiber cells in poplar stems.</a> Tree Genet. Genomes. 11 (5), (2015).</li><li>Baldacci-Cresp, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PtaRHE1,+a+Populus+tremula+x+Populus+alba+RING-H2+protein+of+the+ATL+family,+has+a+regulatory+role+in+secondary+phloem+fiber+development.">PtaRHE1, a Populus tremula x Populus alba RING-H2 protein of the ATL family, has a regulatory role in secondary phloem fiber development.</a> Plant J. 82 (6), 978-990 (2015).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A laboratory manual. , (2001).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bio-assays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bio-assays with tobacco tissue cultures.</a> Physiol. Plant. 15 (3), 473-497 (1962).</li><li>Hawkins, S., Pilate, G., Duverger, E., Boudet, A., Grima-Pettenati, J., Chaffey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+GUS+histochemistry+to+visualise+lignification+gene+expression+in+situ+during+wood+formation.">The use of GUS histochemistry to visualise lignification gene expression in situ during wood formation.</a> Wood formation in trees: Cell and Molecular Biology Techniques. , 271-295 (2002).</li><li>Hodal, L., Bochardt, A., Nielsen, J. E., Mattsson, O., Okkels, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+expression+and+specific+elimination+of+endogenous+beta-glucuronidase+activity+in+transgenic+and+nontransgenic+plants.">Detection, expression and specific elimination of endogenous beta-glucuronidase activity in transgenic and nontransgenic plants.</a> Plant Sci. 87 (1), 115-122 (1992).</li><li>Hansch, R., Koprek, T., Mendel, R. R., Schulze, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+protocol+for+eliminating+endogenous+beta-glucuronidase+background+in+barley.">An improved protocol for eliminating endogenous beta-glucuronidase background in barley.</a> Plant Sci. 105 (1), 63-69 (1995).</li></ol>

在ISSA协议是在几个月的基因和所涉及的木材感兴趣的启动子的分析在空间中树种创造转基因茎组织的相对简单和有效的方法和茎的形成。举手之劳，除了保持植物活，需要成长接种后其鲜明的对比，其中需要大量的培养，以维持组织或植物，其中木材生产可能需要长达几年开始或者一个真正的体外方法转基因茎组织专干不创建1。 ISSA也被证明是适用于广泛范围的树种，包括杨树...

树茎包括行星生物量的显著量是巨大的生物，文化和商业上的重要性。二级茎由许多其他生命形式提供资源和庇护所创造的栖息地。他们以他们居住，并充当生产木材，纸浆和造纸等木材和非木材产品的可再生资源的生态系统提供多项服务。二次干发展并且更具体木材形成通过调节特定细胞类型的发展复杂的分子体系的约束，其细胞壁的生化成分，以及他们如何被布置为形成组织和器官。解剖专干的开发和木材形成的分子基础是由许多因素，包括木材和干性内部和之间的茎，长代次，异交交配系统，高杂合度，高遗传负荷，季节性休眠，长的可变性混淆成熟特点建立期和成熟的树木纯粹的物理尺寸。其结果是，干的发展相对于植物发育的分子控制的其它方面的详细知识次要的认识，仍处于起步阶段。 许多体外技术已被用于研究和了解二次干发展，尤其是木材和二级细胞壁的形成。这些协议包括使用整个植物或植物部分的系统，其中任一转基因植物产生或特定辅助细胞或组织转化为木材和/或仲干发展1的具体方面的研究。转基因植物可以从各种各样的植物组织和细胞类型的回收后的遗传转化，但是，进展缓慢，特别是在分析木纤维性状由于长期再生时和干成熟时间（年的顺序），高的技术和劳动德曼DS，可以通过低的候选基因，以及在传播一些木本植物物种困难。类似的技术已在非木质模型系统被开发，如拟南芥，成功地克服了这些限制，但不是所有的二次干细胞类型中在这些本茎和相关的季节性或长寿性状不能在这些物种2进行研究。备选地，植物部分系统，例如Pinu酒店小号辐射愈伤组织培养物3减少相关的时限。然而这些方法被限制于单个细胞类型的研究，并如所指出用于体外实验遭受类似的限制。同样，涉及整个茎段根尖干文化4显示的承诺，但目前还没有适用于特定基因或发起人的研究。最近，涉及毛状根替代协议已被开发用于桉树并已成功施加5，但是，这种方法仍需要在体外培养，涉及二次根而非茎和迄今它仅限于单一树种。 诱导的体部门分析（ISSA），如这里描述的，被开发来克服的一些问题，提供用于基因和启动子的高通量功能筛选工具的培养基中，在木材形成和二次茎组织发育疑似角色。 ISSA是被开发，以减少所产生的转基因细胞和组织在一个完整的辅助阀杆同时克服劳动时间在体内转化和筛选系统，体外方法中使用的常规技术和吞吐量的限制。这里所描述的协议允许用于同时建立数百独立地转化组织扇区和小区的一个短的时间期间内的次级茎，在树种和感兴趣组织而不克相对较短的时间框架和低廉的劳动力成本中enetic和/或环境的变化。 ISSA 体内技术首先被用于辅助阀杆6和芽7组织以来，通过参与形成层分化和基因和/或促进研究二次茎组织了细化说明，并有包括： 微管蛋白 （TUB）8，fasciclin样阿拉伯半乳聚糖 （ FLA）9， 纤维素酶 （CESA）10， 次生壁相关NAC域 （SND 2）11，ARBORKNOX（ARK1）12和非常有趣的新基因 （RING）H2蛋白13。在次级进行了这些研究茎杨树和桉树植物和提供见解细胞形态，细胞壁化学和基因表达。 这里描述的协议的目的是汇集经验第二，通过知识的ISSA从一系列在过去十年中发表和未发表的研究开发和应用获得的。他们专注于专干组织6 在体内转化，集中力量对涉及新疆杨“锥状&#39;克隆， 蓝桉以及11个桉×赤桉克隆研究。本文通过从植物和细菌，茎组织，生长和组织的收获，转基因细胞和组织的鉴定，为收集和分析数据的表型评估和方法编制转型的种植协议进行研究。虽然技术已成功应用于测量细胞壁的单糖组成也9,11，由于篇幅所限，本文集中用于二次ST测量细胞和组织的形态和理解基因表达模式技术只有EMS。因此，如所概述的协议是适合于那些希望获得进一步深入了解的基因连接于次级的作用和/或表达的茎使用成本低，在技术上容易，中到高通量方法。

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
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			<td height="21" style="height:21px;width:188px;">Plants</td>
			<td style="width:95px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">Please consult local nursery suppliers for plants as needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">Agrobacterium strain</td>
			<td style="width:95px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">There are many possible avenues to obtain Agrobactrium strains. We suggest you follow up within your local research community as there may be restrictions in obtaining the bacteria in your country and region.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:188px;">Binary vector (gene and promoter)</td>
			<td style="width:95px;">NA</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">We have developed a range of vectors to suite the ISSA protocol using a the Gateway Recombinase system. This include overexpression, RNAi knockouts and promoter fusion vectors based on modified pCAMBIA vectors and happy to provide as needed. In addition, there are many vectors avialable to the research community.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">LB media</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">L3022</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:188px;">LB media with agar</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">L2897</td>
			<td style="width:751px;">A like product could be sourced from another company</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:188px;">Antibiotics</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">NA</td>
			<td style="width:751px;">The catalog number will be dependent on the antibiotic you require as a range of antibiotic are used for bacterial selection in binary vectors. This product could be sourced from a&#160; range of companies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">50 ml Screw top tubes</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">2 ml Microtube</td>
			<td style="width:95px;">Watson Bio Lab</td>
			<td style="width:119px;">132-620C</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">MS Media</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">M9274</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Scalpel blade no 11</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">S2771</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Parafilm &#34;M&#34;</td>
			<td style="width:95px;">Bemis</td>
			<td style="width:119px;">PM996</td>
			<td style="width:751px;">This is the best product to use to bind the cambial window post creation&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">14 ml round bottom tubes</td>
			<td style="width:95px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">150268</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">EDTA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Triton</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">X100</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">X-Gluc</td>
			<td style="width:95px;">X-GLUC direct</td>
			<td style="width:119px;"></td>
			<td style="width:751px;">You will need to go to the website to order - http://www.x-gluc.com/index.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Potassium Ferricyanide (III)</td>
			<td style="width:95px;">Sigma</td>
			<td align="right" style="width:119px;">244023</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Potassium Ferrocyanide (II)</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">P9387</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Litmus paper</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">WHA10360300</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Single edge razor blade</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">L055</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Double edge razor blade</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">L056</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">SEM Pin Stub</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">GTP16111</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Sample vial with screw cap</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">L6204</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Ethanol</td>
			<td style="width:95px;">sigma</td>
			<td style="width:119px;">E7023</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">LR white</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">C025</td>
			<td style="width:751px;">The same product could be sourced from another company</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Embedding Mould</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">RL090</td>
			<td>We recommend this variety, however there are plenty of options available</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:188px;">Water Soulable mounting media</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">IA019</td>
			<td>One example of a mounting media that could be used however other options do exist and could be explored.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Hydrogen peroxide</td>
			<td style="width:95px;">Sigma</td>
			<td align="right" style="width:119px;">216763</td>
			<td>A like product could be sourced from another company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Glacial acetic acid</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:119px;">A9967</td>
			<td>A like product could be sourced from another company</td>
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			<td height="21" style="height:21px;width:188px;">Safranin O</td>
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			<td>A like product could be sourced from another company</td>
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		<tr height="63">
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			<td style="width:95px;">FEI</td>
			<td style="width:119px;"></td>
			<td>This is the instrument used at part of this study but any other SEM that has a low vacuum mode could be utilised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Image J imaging software</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td>&#160;can be sourced from the following URL http://rsbweb.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

the use of induced somatic sector analysis (issa) for studying genes and promoters involved in wood formation and secondary stem development

在树上二级茎生长和相关的木材形成都来自生物和商业的角度显著。然而，相对知之甚少的支配其发展的分子控制。这部分是由于经常与二次生长过程的研究相关的物理，资源和时间的限制。许多体外技术已被用于涉及两个木质和非木质植物物种或植物部分或整个植物系统。然而，关于它们的适用性进行二次茎的生长过程的研究问题，某些物种和劳动强度的顽抗往往望而却步中高吞吐量的应用。此外，在寻找专干发展，木材形成，当被调查的具体特征可能只历经数年的增长成为衡量一棵树的生命周期的后期。在解决体内 p这些挑战替代rotocols被开发出来，命名为诱导体部门分析，涉及直接在工厂的专干转基因体组织部门的创建。该协议的目的是提供一种有效的，简单和相对快速的方法来创建用于基因和启动子的功能分析，可在一个范围内树种可以利用转基因植物次生组织中。这里提出的结果表明，转基因次级干扇区可以在所有活的组织和细胞类型中创建二次茎的各种树种的木材形态特征的以及在次要启动子表达模式茎可容易地评估促进中到高通量功能特性。

使用该协议的所有活二次干细胞和组织类型已被证明是容易A.农杆菌转化并已确定为根据最初转化的细胞类型行业类型和其后续发展的增长模式。行业类型包括周皮，韧皮部，形成层，伤口实质和纤基乙酸钠（ 图1b，1C，1D）和一致的位置将在本段的剩余部分描述可以发现。一个周皮扇区包括发现只在周皮和从不延伸到所述韧皮?...

Watch this Scientific Journal Video about The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development at JoVE.com

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

诱导体细胞行业分析（ISSA）的为研究木材形成和二次开发茎相关基因和推动者使用

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

The overall goal of Induced Somatic Sector Analysis is to provide a medium to high throughput technique for the functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. This method can help answer key questions around where and when danger expressed as well as their role in biologically important processes such as wood formation and secondary stem development. The main advantage of this technique is that it is efficient, easy and relatively a fast procedure that is applicable to a wide range of tree species.To begin, prepare a clean, 50 milliliter screw cap tube with 19 milliliters of fresh, 28 degree Celsius LB medium with the appropriate antibiotics for bacterial selection. Add one milliliter of A.tumefaciens culture and check the optical density, or OD, at 600 nanometres. If the OD is above 0.1, dilute further with warm LB.Place the A.tumefaciens culture into a shaking incubator until the OD 600 is between 0.4 and 0.6.Once this is achieved, centrifuge the cells for 15 minutes at four degrees Celsius and 1, 000 x g. Decant the liquid from the tube and immediately re-suspend the cell pellet with one milliliter of cooled MS medium. Transfer the suspension to a clean, two milliliter microtube and store on ice until required.Begin the experiment during the spring or early summer and select plants with an active and fast growing cambium. This is confirmed if the phloem can be easily peeled from the xylem. Identify a straight section of stem near the base of the plant and remove any leaves or branches.Using a sharp scalpel, cut a one square centimeter cambial window by creating two parallel, 20 millimeter incisions through the phloem at five millimeters apart and then a single, horizontal cut that bisects the two vertical cuts at the basal end. Heal the phloem strip upwards exposing the developing xylem tissue. And then, using a pipette, add sufficient inoculation medium to fully wet the surface of the exposed xylem.Immediately, reinsert the phloem strip into the stem and bind it into place tightly with parafilm. Repeat these inoculation steps for all genes or promoters of interest creating new cambial windows at least one centimeter above or below existing windows and at a 90 degree offset. Additionally, inoculate windows with the positive and negative control vectors on the same stem or stems, as the experimental inoculates.Monitor stem growth periodically and once radial growth of at least five millimeters has been observed, harvest the tissue for a beta-glucuronidase, or GUS assay. To harvest the test material, first excise the cambial window from the stem removing any tissue that is not part of new growth. For studies relating to mature xylem and phloem cell or tissue morphology, peel the phloem from the xylem.Alternatively, for investigations requiring that the cambial zone remain intact, or promoter expression patterns to be assessed, slice the cambial window transversely using a razor blade to create discs of between 0.5 and one millimeter thickness. Place the harvested cambial window tissues into individual, 14 milliliter round bottom tubes and rinse twice in 0.1 molar phosphate buffer at pH 7 for five minutes each time ensuring that the tissue is completely submerged. At the end of the second rinse, take care to remove all excess solution.Next, add five milliliters of GUS reagent to each tube. If this does not completely cover the tissue sample, add additional reagent. Incubate the tubes for ten minutes at 55 degrees Celsius in the dark.Following this, incubate the tubes for a further 12 to 16 hours on a shaker at 37 degrees Celsius in the dark using gentle agitation. Upon removal from the incubator, take a random subset of the tubes and check the pH of the GUS reagent using litmus paper with pH range of zero to seven. If any of them have a pH lower than six, label them and check the remainder of the tubes.Label and exclude any tubes with a pH lower than six. Decant the GUS reagent from the tubes and replace it with sufficient 70 percent ethanol to cover the tissue. Store the samples at four degrees Celsius until needed.Using a clean pair of forceps, remove all the cambial window tissue from a sample tube and place it onto a small tray or petri dish for microscopic visualization. Place the sample under the lens of a dissecting microscope between 1X and 4X magnification and identify and tally the number of cells or tissues displaying bright blue staining. In examples where the phloem was removed, count the number of sectors in cambial tissue found on the surface of the developing xylem.For samples cut into discs, count the number of sectors found in the different cell or tissue types. Ensure that both sides of the disc are viewed and that sectors that occur across two discs are matched to ensure that numbers are not overestimated. Place only the tissue containing sectors back into the tube containing 70 percent ethanol and store until needed.Repeat the analysis for any additional cambial windows harvested including the positive and negative controls. To aid in the accurate identification in the classification of sectors, we suggest you become familiar with secondary stem anatomy and development including stem responses to wounding. This figure shows several successful transformations seen here as regions of blue staining.An example phloem peel window displays multiple transformed sectors and discs harvested from two tree species show sectors of transformation and subsequent radial growth. Transformation in a range of tissue types can also be observed. Here stem sectors can be seen in the periderm, phloem, wound parenchyma, and several other tissue regions of white poplar.This image shows the results of gene promoter expression in cambial derivatives. In this example, we see the proportion of staining in each tissue type in a trial involving a range of CesA promoters from eucalyptus grandis transformed into stems of eucalypt hybrids. Expression in the phloem, developing and developed xylems is represented as a percentage.Average number of transformation events per centimeter of cambium inoculated can also be calculated for each promoter or gene of interest. This table shows the data for typical sectors identified in eucalypt and poplar subjects transformed with a positive control or promoter of interest analyzed using the disc harvesting method. Once mastered, this technique can provide a fast, efficient and widely applicable method for dissecting the complex nature of cambial differentiation with formation in secondary stem development.After watching this video, you should have a good understanding of how to prepare plants and bacteria, how to transform, grow and harvest stems as well as how to identify and classify transgenic tissue for phenotypic assessment.

Research

JoVE Journal

Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

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