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 greenhouse where Agrobacterium tumefaciens can be handled and that appropriate personal protective equipment prescribed for the laboratory is used.</li>
	<li>Prepare a binary vector containing either a gene of interest knockdown or overexpression cassette and a &#946;-glucuronidase (GUS) reporter gene cassette or a promoter of interest fused to a GUS gene depending on the type of study. 
	Note: There are a number of binary vector backbones that can be sourced commercially or through research networks as well as numerous techniques available to insert genes and promoters of interest into binary vectors. In the context of this protocol is up to the experimenter to make the decision on how to create a binary vector.</li>
	<li>Transform binary vector into a disarmed strain of A. tumefaciens using electroporation or heat shock14 and store appropriately until needed for experimentation.</li>
	<li>Repeat step 2.2 for any positive and/or negative controls. 
	Note: Negative controls should include a binary vector containing no gene of interest for a gene of interest study and a promoterless GUS for a promoter of interest study. Positive control for a promoter of interest study should include a Cauliflower Mosaic Virus 35s promoter (CaMV35s) fused to a GUS reporter.</li>
</ol>
3. Preparation of A. tumefaciens for Inoculation
<ol>
	<li>At least a week prior to experimentation, take a small amount (approximately 1 &#181;l) of A. tumefaciens prepared during step 2.2 and 2.3 and spread thinly on separate LB medium with agar14 plates containing appropriate antibiotics for bacterial selection. Grow at 28 &#176;C in an incubator to form small colonies and then store at 4 &#176;C until needed.</li>
	<li>48 hours prior to experimentation, transfer one colony of A. tumefaciens from each plate into separate 50 ml screw top tubes containing 5 ml of pre-warmed (28 &#176;C) LB media14 containing appropriate antibiotics for bacterial selection and agitate at 200 rpm on a shaker incubator for approximately 48 hr at 28 &#176;C until mixture is very cloudy.</li>
	<li>Between 4-6 hr prior to inoculation of plant stems (section 4) add 1 ml of the LB/A. tumefaciens mixture into a fresh 50 ml screw top tube containing 19 ml (1:20 dilution) of fresh warm (28 &#176;C) LB media with appropriate antibiotics for bacterial selection. If optical density (OD) at 600 nm (OD600, as measured by spectroscopy) is above 0.1, dilute further with warm LB until this is achieved.</li>
	<li>Place diluted LB/A. tumefaciens mixture back on the shaker incubator and shake under the same conditions (200 rpm, 28 &#176;C) until OD600 is between 0.4-0.6 and remove.</li>
	<li>Centrifuge LB/A. tumefaciens mixture for 15 min at 1,000 x g and 4 &#176;C.</li>
	<li>Decant liquid and immediately resuspend A. tumefaciens pellet into 1 ml of cooled MS media15, transfer to 2 ml microtube and store on ice until required for inoculation (section 4). This solution is referred to as Inoculation Media.</li>
</ol>
4. Inoculation of Plant Stem with A. tumefaciens&#8203;
<ol>
	<li>Start experimentation during late spring or early summer using plants created in section 1 that have an active and fast growing cambium. A good diagnostic for an active and fast growing cambium is that the phloem can be easily peeled from the xylem.</li>
	<li>Find a clear straight section of stem near the base of the plant and remove any leaves and branches.</li>
	<li>Using a sharp scalpel (preferably No. 11) or razor blade, create a 1 cm2 &#39;cambial window&#39; in a clear section of stem by introducing two vertical parallel incision through the phloem of 20 mm in length and one 5 mm apart then a horizontal cut that connects the two vertical cuts at their basal end.</li>
	<li>Peel the phloem strip created by the incision upwards exposing the developing xylem tissue and add sufficient Inoculation Media from step 3.6 to wet the surface of the exposed developing xylem using a pipette (typically 5-10 &#181;l). Immediately reinsert the phloem strip.</li>
	<li>Bind the phloem strip to the stem tightly with Parafilm.</li>
	<li>Repeat steps 4.2 to 4.5 for any additional cambial windows for the gene(s) or promoter(s) of interest creating any new cambial windows least 1 cm above or below other cambial windows and at a 90&#176; offset.</li>
	<li>Complete steps 4.2 to 4.5 for the positive and negative control vectors (step 2.4) ensuring that each vector is added to the same stem of each plant used in the experiment.</li>
	<li>Monitor stem diameter growth periodically and harvest for GUS assay (section 5) when radial growth of at least 5 mm has been observed in stems. 
	Note: The amount of radial growth needed is dependent on the amount of tissue required for downstream analysis.</li>
</ol>
5. Harvesting for GUS Histological Assay
<ol>
	<li>Excise the &#39;cambial window&#39; from the stem removing any tissue not part of new growth within the cambial window.
	<ol>
		<li>For studies relating to mature xylem and phloem cell/tissue morphology, peel the phloem from the xylem.</li>
		<li>For studies where the cambial zone is required to remain intact or promoter expression patterns are to be assessed, slice the cambial window transversely using a razor blade or other sharp blade into discs of between 0.5 and 1 mm in thickness.</li>
	</ol>
	</li>
	<li>Place processed cambial window tissues in 14 ml round bottom tubes and rinse twice in 0.1 M phosphate buffer at pH 714. Ensure that the tissue is completely submerged, remains in solution for at least five minutes and all excess solution is removed at the end of the second rinse.</li>
	<li>Add 5 ml of GUS reagent (0.5 mM X-gluc (5-bromo-4-chloro-3-indolyl-&#946;-D-glucuronic acid), 10 mM EDTA, 0.5% Triton X-100 v/v, 0.5 mM potassium ferricyanide(III), 0.05 mM potassium ferrocyanide(II), made up to final volume with 0.1 mM phosphate buffer pH 7; see Hawkins et al.16) to each tube. If tissue is not completely submerged, add additional GUS reagent.</li>
	<li>Incubate tubes for 10 min at 55 &#176;C in the dark.</li>
	<li>Incubate tubes for a further 12-16 hr on a shaker incubator at 37 &#176;C in the dark using gentle agitation (between 30 and 60 rpm) to allow for mixing.</li>
	<li>Upon removal from shaker incubator randomly check the pH of GUS reagent in a small subset of tubes using litmus paper with a pH range 0-7.
	<ol>
		<li>If any of the tubes have a pH below 6 then label them. Continue to check the remainder of the tubes to confirm pH and label if pH is 6 or below. 
		Note: Samples with a pH below 6 may not be suitable for analysis. These samples have to be excluded from further analysis.</li>
	</ol>
	</li>
	<li>Decant GUS reagent and replace with sufficient 70% ethanol to cover tissue.</li>
	<li>Store at 4 &#176;C until needed.</li>
</ol>
6. Identification of Transgenic Tissue
<ol>
	<li>Using forceps, take out all the cambial window tissue from a tube and place in small tray or petri dish to allow for microscopic visualization.</li>
	<li>Using a dissecting microscope between 1X and 4X magnification, identify and tally the number of cells or tissues that have a bright blue staining. Blue stained cells or tissue are referred to as a sector from this point onwards. Tally them in the following ways.
	<ol>
		<li>For samples where the phloem has been removed (step 5.1.1), count the number of sectors in cambial tissue found on the surface of the developing xylem (cambial sector, Figure 1a).</li>
		<li>For samples that have been cut into discs (step 5.1.2), count the number sectors found in the different cell or tissue types (Figure 1b, 1c, 1d). Sectors can be found in the following tissue types: the periderm (Periderm sector, Figure 1e), phloem (Phloem sector, Figure 1f), cambial tissue (Cambial sector, Figure 1g, 1h, 1I), wound parenchyma (Wound Parenchyma Sector, Figure 1j) and in tyloses (Tylose sector, Figure 1k).
		<ol>
			<li>During microscopic assessment ensure that both sides of a disc have been viewed and that sectors that occur across two discs are matched so that numbers are not overestimated.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Place only the tissue containing sectors back into the tube containing 70% ethanol and store until needed.</li>
	<li>Repeat step 6.1 to 6.3 for any additional cambial windows including the positive and/or negative control.</li>
	<li>Calculate the average transformation efficiency for each sector type for each gene or promoter of interest and the controls separately by dividing the total number of sectors by the total number of 1 cm2 cambial windows to derive an Average Number of Transformation Events per cm2 of Cambium Inoculated (ATScm-2).</li>
	<li>Proceed to step 7.2 and 8 for analysis of promoter expression patterns and step 7.1, 7.2 or 7.3 for techniques to assess cell and tissue morphology in cambial tissue.</li>
</ol>
7. Analysis of Cell and Tissue Morphology in Cambial Tissue
Note: Below are a selection of techniques that have been used successfully for analysis of ISSA derived samples.
<ol>
	<li>Analysis of xylem cell area, lumen area, cell wall thickness and cell wall area using Scanning Electron Microscopy (SEM). 
	Note: This protocol requires minimal sample preparation and allows for relatively high throughput of sector analysis relating to the morphological traits outlined.
	<ol>
		<li>From this step onwards please ensure lab coat, eye protection and gloves are used whenever woody tissue is being handled and treated.</li>
		<li>Identify a cambial sector for analysis and make an incision approximately 0.5 mm to either side of it using a single edge razor blade to create a disc (Figure 2a).</li>
		<li>Trim off excess xylem tissue leaving approximately 1 mm of xylem tissue to the tangential side of the sector (Figure 2b).</li>
		<li>Carefully cut transversely through the center of the sector using a fresh double edge razor blade (Figure 2c).</li>
		<li>Make two additional shallow radial incisions on the transverse plane either side of sector to demarcate the transgenic tissue (Figure 2d). As GUS staining will not penetrate deep into the tissue follow along the radial files of cells to either side of stained tissue found at the cambial surface.</li>
		<li>Attach prepared cambial sector face up to stage of an SEM pin stub mount using SEM conductive tape (Figure 2e) and keep in desiccator until needed for SEM visualization.</li>
		<li>Repeat steps 7.1.2 to 7.1.6 for any additional sectors including those from the negative control.</li>
		<li>Using a SEM in a low vacuum mode (energy 5 kV, spot 3.0 nm), visualize and take photos of cells/tissue within the sector as well as the cells/tissue directly adjacent the sector (Figure 2f). The amount of magnification is dependent on the features of interest. For xylem fibers, 2,000X is suitable (Figure 2g).</li>
		<li>Once a cambial sector has been visualized and photographs taken at an appropriate magnification, measure xylem cell/tissue morphological traits of interest using image measuring software.</li>
		<li>For statistical analysis, undertake multiple pairwise analyses using paired t-tests to compare the difference between morphological measured in the transgenic sector and the adjacent non-transgenic control cells/tissue (measured within 0.5 mm from sector edge) for each sector to determine a p-value.</li>
		<li>Repeat step 7.1.10 for the negative control.
		<ol>
			<li>In cases where the positive control shows a low p-value (&#945; &#60;0.05), calculate the difference between the transgenic and adjacent non-transgenic cells/tissue for a morphological trait. Use this &#39;difference value&#39; in an unpaired t-test to compare the effect of the gene of interest with the negative control to determine a p-value.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Histochemical analysis of cambial cell/tissue morphology or promoter expression patterns in stem cells/tissues using light microscopy. 
	Note: While more time consuming, this method allows for more analysis options including aspects of cambial dynamics, e.g., cambial width as well as promoter expression patterns. In addition, if unable to access a SEM, this protocol can be used as an alternative for step 7.1.
	<ol>
		<li>Excise sector of interest using razor blade and some surrounding tissue as small blocks (no greater than 1 mm3) and place directly in 1-3 ml of 100% ethanol in a sample vial with screw cap for at least 2 days at 4 &#176;C on a shaker. Prepare the small block in a way that will allow for the surface of interest to be accessed by a microtome.</li>
		<li>Repeat for any additional sectors including those from the negative control.</li>
		<li>Remove liquid and replace with 25% ethanol: 75% LR white mixture for 2 days maintaining conditions.</li>
		<li>Repeat step 7.2.3 with 50% ethanol: 50% LR white, 25% ethanol: 75% LR white and then finally twice with 100% LR white.</li>
		<li>Place small block in Embedding Mold with the surface of interest aligned to short end and carefully cover with fresh LR white and polymerize as per manufacturer instructions.</li>
		<li>Cut 5 &#181;m sections from the surface of interest on a rotary microtome and mount on a glass slide. Use Safranin O (0.01%) staining to visualize cell wall and/or other features.</li>
		<li>Add mounting media, place a cover slip and allow to set overnight.</li>
		<li>View under a light microscope between 100X and 600X magnification and capture image.</li>
		<li>Capture morphological traits from images using image measuring software.
		<ol>
			<li>For statistical analysis of quantitative morphological traits, follow on from step 7.1.10.</li>
			<li>For qualitative analysis of morphological traits, compare and describe patterns observed for the gene or promoter of interest and the negative and/or positive control.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Analysis of Microfibril Angle (MFA) in Macerated Fibers Using Light Microscopy.
	<ol>
		<li>Excise transgenic xylem tissue directly from a sector as well as from non-transgenic tissue adjacent to it (within 0.5 mm, Figure 2h) and place in separate 1.5 ml tubes. Repeat for any additional sectors including those from the negative control.</li>
		<li>Complete steps 7.3.3 to 7.3.5 in a fume hood.</li>
		<li>Add 250 &#181;l of hydrogen peroxide and 250 &#181;l of glacial acetic acid to each tube.</li>
		<li>Place tube in heat block at 90 &#176;C for 2 hr in fume hood.</li>
		<li>Remove tissue from tubes and carefully rinse with distilled water at least twice.</li>
		<li>Using a water soluble mounting media, mount tissue on a glass slide and tease apart with sharp forceps prior to affixing a cover slip.</li>
		<li>View under a light microscope and capture images of individual fibers at magnification greater than 400X.</li>
		<li>To determine microfibril angle of fibers, measure the angle between the pit apertures and/or cell wall striations and the long axis of the fiber (Figure 2i) using image measuring software.</li>
		<li>For statistical analysis, follow on from step 7.1.10.</li>
	</ol>
	</li>
</ol>
8. Analysis of Promoter Expression Patterns
<ol>
	<li>Analysis of Promoter Expression Patterns in Secondary Stem Tissues.
	<ol>
		<li>Tally the frequency of each sector type for the promoter of interest as well as the positive and negative controls as noted in step 6.2.2.</li>
		<li>Compare the frequency of the different sector types for the promoter of interest with the positive control using Chi-square tests to establish p-values. Further analysis of cell/tissue specificity can be undertaken using step 7.2 as required.
		<ol>
			<li>If more than one promoter of interest is being investigated then repeat this analysis making comparison between all promoters of interest and the positive control.</li>
			<li>If sectors or blue staining are observed in the negative control then add this to the analysis or abandon depending on extent or if endogenous staining is suspected (see Discussion).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Analysis of Promoter Expression Patterns During Cambial Derivative Development and Differentiation.
	<ol>
		<li>Using a dissecting microscope, visualize all the cambial sectors (Figure 1g, 1h, 1i) identified in step 6.2 and determine the presence/absence of blue staining in three tissues types derived from the cambium; the phloem (P), developing xylem (X1) or developed xylem tissue (X2) (see Figure 3a).</li>
		<li>As per step 8.1.2, compare the frequency of presence/absence of GUS staining in the P, X1 and X2 regions of promoter of interest with the positive control using Chi-square tests to establish p-values. Further analysis of cell/tissue specificity can be undertaken using step 7.2 as required.
		<ol>
			<li>See steps 8.1.2.1 and 8.1.2.2 for additional considerations.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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><tbody><tr><td>Plants</td><td>NA</td><td>NA</td><td>Please consult local nursery suppliers for plants as needed</td></tr><tr><td>Agrobacterium strain</td><td>NA</td><td>NA</td><td>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><td>Binary vector (gene and promoter)</td><td>NA</td><td>NA</td><td>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><td>LB media</td><td>Sigma</td><td>L3022</td><td>The same product could be sourced from another company</td></tr><tr><td>LB media with agar</td><td>Sigma</td><td>L2897</td><td>A like product could be sourced from another company</td></tr><tr><td>Antibiotics</td><td>Sigma</td><td>NA</td><td>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&amp;nbsp; range of companies</td></tr><tr><td>50 ml Screw top tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td>The same product could be sourced from another company</td></tr><tr><td>2 ml Microtube</td><td>Watson Bio Lab</td><td>132-620C</td><td>The same product could be sourced from another company</td></tr><tr><td>MS Media</td><td>Sigma</td><td>M9274</td><td>The same product could be sourced from another company</td></tr><tr><td>Scalpel blade no. 11</td><td>Sigma</td><td>S2771</td><td>The same product could be sourced from another company</td></tr><tr><td>Parafilm &quot;M&quot;</td><td>Bemis</td><td>PM996</td><td>This is the best product to use to bind the cambial window post creation&amp;nbsp;</td></tr><tr><td>14 ml round bottom tubes</td><td>Thermo Scientific</td><td>150268</td><td>The same product could be sourced from another company</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td>The same product could be sourced from another company</td></tr><tr><td>Triton</td><td>Sigma</td><td>X100</td><td>The same product could be sourced from another company</td></tr><tr><td>X-Gluc</td><td>X-GLUC direct</td><td></td><td>You will need to go to the website to order - http://www.x-gluc.com/index.html</td></tr><tr><td>Potassium Ferricyanide(III)</td><td>Sigma</td><td>244023</td><td>The same product could be sourced from another company</td></tr><tr><td>Potassium Ferrocyanide(II)</td><td>Sigma</td><td>P9387</td><td>The same product could be sourced from another company</td></tr><tr><td>Litmus paper</td><td>Sigma</td><td>WHA10360300</td><td>The same product could be sourced from another company</td></tr><tr><td>Single edge razor blade</td><td>ProSciTech</td><td>L055</td><td>The same product could be sourced from another company</td></tr><tr><td>Double edge razor blade</td><td>ProSciTech</td><td>L056</td><td>The same product could be sourced from another company</td></tr><tr><td>SEM Pin Stub</td><td>ProSciTech</td><td>GTP16111</td><td>The same product could be sourced from another company</td></tr><tr><td>Sample vial with screw cap</td><td>ProSciTech</td><td>L6204</td><td>The same product could be sourced from another company</td></tr><tr><td>Ethanol</td><td>sigma</td><td>E7023</td><td>The same product could be sourced from another company</td></tr><tr><td>LR white</td><td>ProSciTech</td><td>C025</td><td>The same product could be sourced from another company</td></tr><tr><td>Embedding Mould</td><td>ProSciTech</td><td>RL090</td><td>We recommend this variety, however there are plenty of options available</td></tr><tr><td>Water Soulable mounting media</td><td>ProSciTech</td><td>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><td>Hydrogen peroxide</td><td>Sigma</td><td>216763</td><td>A like product could be sourced from another company</td></tr><tr><td>Glacial acetic acid</td><td>Sigma</td><td>A9967</td><td>A like product could be sourced from another company</td></tr><tr><td>Safranin O</td><td>ProSciTech</td><td>C138</td><td>A like product could be sourced from another company</td></tr><tr><td>Quanta Environmental Scanning Electron Microscope</td><td>FEI</td><td></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><td>ImageJ imaging software</td><td></td><td></td><td>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

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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Research

JoVE Journal

Genetics

8.7K Views.  The University of Melbourne. 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.

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

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

Environment

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

Biology

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Developmental Biology

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

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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