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. Preparation of Plant Material
<ol>
	<li>Prior to experimentation, raise new seedlings of the preferred tree species from seed or cutting and grow tree/s until the diameter of the stem in the area intended for experimentation is approximately 1 cm in diameter. 
	Note: The time needed may vary due to plant growth rates therefore allow between three to nine months for this step.</li>
</ol>
2. Binary Vector Creation
<ol>
	<li>Conduct work from this section to section 7 in a laboratory or g...

<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>

The ISSA protocol is a relatively simple and efficient method for the creation of transgenic stem tissues in tree species in the space of a few months for analysis of genes and promoters of interest involved in wood and stem formation. Little effort, beyond keeping plants alive, is required to grow transgenic stem tissue following inoculation which stands in contrast to in vitro methods where extensive culturing is required to maintain tissue or plants, where wood production can take up to years to commence or w...

Tree stems comprise a significant amount of the planets biomass and are of immense biological, cultural and commercial importance. Secondary stems create habitat by providing resources and shelter for many other life forms. They deliver many other services to the ecosystems they inhabit and act as a renewable resource for the production of timber, pulp and paper and other wood and non-wood products. Secondary stem development and more specifically wood formation is governed by complex molecular system that regulate the development of specific cell types, the biochemical composition of their cell walls and how they are arranged to form tissues and organs. Dissecting the molecular basis of secondary stem development and wood formation is confounded by many factors including the variability of wood and stem properties within and between stems, long generation times, out-crossing mating systems, high heterozygosity, high genetic load, seasonal dormancy, long mature trait establishment periods and the sheer physical size of mature trees. As a result, understanding of secondary stem development relative to the detailed knowledge of most other aspects of the molecular control of plant development, is still in its infancy.
A number of in vitro techniques have been used to study and understand secondary stem development, in particular wood and secondary cell wall formation. These protocols involve the use of whole plant or plant part systems, where either transgenic plants are created or specific secondary cells or tissue are transformed for the study of specific aspects of wood and/or secondary stem development1. Transgenic plants can be recovered post genetic transformation from a wide variety of plant tissues and cell types, however, progress is slow, particularly when analyzing wood fiber traits due to long regeneration and stem maturation times (in the order of years), high technical and labor demands, low throughput of candidate genes as well as difficulties in propagating some woody plant species. Similar techniques have been developed in non-woody model system such as Arabidopsis that successfully overcome some of these limitations, but not all secondary stem cell types a present in these stems and traits related to seasonality or longevity cannot be studied in such species2. Alternatively, plant part systems, such as Pinus radiata callus cultures3 reduce the associated timeframes. These methods however are restricted to the study of an individual cell type and suffer similar constraints as noted for in vitro experiments. Similarly, apical stem cultures4 involving whole stem explants have shown promise but as yet have not been applied for the study of specific genes or promoters of interest. More recently, an alternative protocol involving hairy root cultures has been developed for eucalypts and has been successfully applied5, however, this method still requires in vitro cultivation, involves secondary roots rather than stems and to date it is limited to a single tree species.
Induced somatic sector analysis (ISSA), as described here, was developed to overcome some of these problems providing a medium to high throughput functional screening tool for genes and promoters with suspected roles in wood formation and secondary stem tissue development. ISSA is an in vivo transformation and screening system which was developed to reduce the time taken to produce transgenic cells and tissue in an intact secondary stem while overcoming labor, technical and throughput limitations of routinely used in vitro methods. The protocols described here allow for the simultaneous creation of hundreds of independently transformed tissue sectors and cells in secondary stems within a short period of time, in the tree species and tissue of interest without genetic and/or environmental variation within relatively short time frames and low labor cost. ISSA in vivo techniques were first described for secondary stem6 and bud7 tissues and have since been refined in secondary stem tissue through studies of genes and/or promoters involved in cambial differentiation and include: tubulin (TUB)8, fasciclin-like arabinogalactan (FLA)9, cellulose synthase (CESA)10, secondary cell wall-associated nac domain (SND2)11, ARBORKNOX (ARK1)12 and really interesting new gene (RING) H2 protein13. These studies were conducted in secondary stems of poplar and eucalypt plants and provided insights into cell morphology, cell wall chemistry and gene expression.
The protocols described here are intended to bring together the experience and knowledge gained through the development and application of ISSA from a range of published and unpublished studies over the last decade. They focus on the in vivo transformation of secondary stem tissues6 and concentrate on studies involving Populus alba &#39;pyramidalis&#39; clone, Eucalyptus globulus as well as 11 Eucalyptus globulus x camaldulensis clones. This document takes researchers through the protocol from the cultivation of plants and bacteria, transformation of stem tissues, growth and harvest of tissue, identification of transgenic cells and tissues, preparation for phenotypic assessments and methods for the collection and analysis of data. While techniques have successfully been applied to measure cell wall monosaccharide composition also9,11, due to space limitations, this document concentrates on techniques used for measuring cell and tissue morphology and understanding gene expression patterns in secondary stems only. Accordingly, the protocol as outlined is suited to those looking to gain further insights into the role and/or expression of genes linked to secondary stems using a low cost, technically easy, and medium to high throughput method.

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
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		<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>
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			<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>
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		<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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			<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>
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			<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:188px;">Safranin O</td>
			<td style="width:95px;">ProSciTech</td>
			<td style="width:119px;">C138</td>
			<td>A like product could be sourced from another company</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:188px;">Quanta Environmental Scanning Electron Microscope</td>
			<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>
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the use of induced somatic sector analysis (issa) for studying genes and promoters involved in wood formation and secondary stem development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

Using this protocol all live secondary stem cell and tissue types have been shown to be susceptible to A. tumefaciens transformations and have been defined into sector types based on the cell type initially transformed and it subsequent developmental growth pattern. Sector types include periderm, phloem, cambial, wound parenchyma and tylose (Figure 1b, 1c, 1d) and can be found in consistent locations describe in the remainder of ...

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.

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

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

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