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

Summary

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.

Abstract

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'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'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.

Introduction

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 'pyramidalis' 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 also^9,11, 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.

Protocol

1. Preparation of Plant Material

1. 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.

2. Binary Vector Creation

1. 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.
2. Prepare a binary vector containing either a gene of interest knockdown or overexpression cassette and a β-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.
3. Transform binary vector into a disarmed strain of A. tumefaciens using electroporation or heat shock¹⁴ and store appropriately until needed for experimentation.
4. 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.

3. Preparation of A. tumefaciens for Inoculation

1. At least a week prior to experimentation, take a small amount (approximately 1 µl) 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 °C in an incubator to form small colonies and then store at 4 °C until needed.
2. 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 °C) LB media¹⁴ containing appropriate antibiotics for bacterial selection and agitate at 200 rpm on a shaker incubator for approximately 48 hr at 28 °C until mixture is very cloudy.
3. 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 °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.
4. Place diluted LB/A. tumefaciens mixture back on the shaker incubator and shake under the same conditions (200 rpm, 28 °C) until OD₆₀₀ is between 0.4-0.6 and remove.
5. Centrifuge LB/A. tumefaciens mixture for 15 min at 1,000 x g and 4 °C.
6. 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.

4. Inoculation of Plant Stem with A. tumefaciens​

1. 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.
2. Find a clear straight section of stem near the base of the plant and remove any leaves and branches.
3. Using a sharp scalpel (preferably No. 11) or razor blade, create a 1 cm² 'cambial window' 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.
4. 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 µl). Immediately reinsert the phloem strip.
5. Bind the phloem strip to the stem tightly with Parafilm.
6. 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° offset.
7. 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.
8. 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.

5. Harvesting for GUS Histological Assay

1. Excise the 'cambial window' from the stem removing any tissue not part of new growth within the cambial window.
   1. For studies relating to mature xylem and phloem cell/tissue morphology, peel the phloem from the xylem.
   2. 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.
2. 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.
3. Add 5 ml of GUS reagent (0.5 mM X-gluc (5-bromo-4-chloro-3-indolyl-β-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.
4. Incubate tubes for 10 min at 55 °C in the dark.
5. Incubate tubes for a further 12-16 hr on a shaker incubator at 37 °C in the dark using gentle agitation (between 30 and 60 rpm) to allow for mixing.
6. 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.
   1. 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.
7. Decant GUS reagent and replace with sufficient 70% ethanol to cover tissue.
8. Store at 4 °C until needed.

6. Identification of Transgenic Tissue

1. 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.
2. 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.
   1. 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).
   2. 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).
      1. 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.
3. Place only the tissue containing sectors back into the tube containing 70% ethanol and store until needed.
4. Repeat step 6.1 to 6.3 for any additional cambial windows including the positive and/or negative control.
5. 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).
6. 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.

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.

1. 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.
   1. From this step onwards please ensure lab coat, eye protection and gloves are used whenever woody tissue is being handled and treated.
   2. 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).
   3. Trim off excess xylem tissue leaving approximately 1 mm of xylem tissue to the tangential side of the sector (Figure 2b).
   4. Carefully cut transversely through the center of the sector using a fresh double edge razor blade (Figure 2c).
   5. 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.
   6. 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.
   7. Repeat steps 7.1.2 to 7.1.6 for any additional sectors including those from the negative control.
   8. 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).
   9. 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.
   10. 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.
   11. Repeat step 7.1.10 for the negative control.
       1. In cases where the positive control shows a low p-value (α <0.05), calculate the difference between the transgenic and adjacent non-transgenic cells/tissue for a morphological trait. Use this 'difference value' in an unpaired t-test to compare the effect of the gene of interest with the negative control to determine a p-value.
2. 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.
   1. 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 °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.
   2. Repeat for any additional sectors including those from the negative control.
   3. Remove liquid and replace with 25% ethanol: 75% LR white mixture for 2 days maintaining conditions.
   4. Repeat step 7.2.3 with 50% ethanol: 50% LR white, 25% ethanol: 75% LR white and then finally twice with 100% LR white.
   5. 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.
   6. Cut 5 µm 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.
   7. Add mounting media, place a cover slip and allow to set overnight.
   8. View under a light microscope between 100X and 600X magnification and capture image.
   9. Capture morphological traits from images using image measuring software.
      1. For statistical analysis of quantitative morphological traits, follow on from step 7.1.10.
      2. 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.
3. Analysis of Microfibril Angle (MFA) in Macerated Fibers Using Light Microscopy.
   1. 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.
   2. Complete steps 7.3.3 to 7.3.5 in a fume hood.
   3. Add 250 µl of hydrogen peroxide and 250 µl of glacial acetic acid to each tube.
   4. Place tube in heat block at 90 °C for 2 hr in fume hood.
   5. Remove tissue from tubes and carefully rinse with distilled water at least twice.
   6. Using a water soluble mounting media, mount tissue on a glass slide and tease apart with sharp forceps prior to affixing a cover slip.
   7. View under a light microscope and capture images of individual fibers at magnification greater than 400X.
   8. 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.
   9. For statistical analysis, follow on from step 7.1.10.

8. Analysis of Promoter Expression Patterns

1. Analysis of Promoter Expression Patterns in Secondary Stem Tissues.
   1. 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.
   2. 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.
      1. 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.
      2. 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).
2. Analysis of Promoter Expression Patterns During Cambial Derivative Development and Differentiation.
   1. 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).
   2. 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.
      1. See steps 8.1.2.1 and 8.1.2.2 for additional considerations.

Results

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 ...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plants	NA	NA	Please consult local nursery suppliers for plants as needed
Agrobacterium strain	NA	NA	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.
Binary vector (gene and promoter)	NA	NA	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.
LB media	Sigma	L3022	The same product could be sourced from another company
LB media with agar	Sigma	L2897	A like product could be sourced from another company
Antibiotics	Sigma	NA	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  range of companies
50 ml Screw top tubes	Fisher Scientific	14-432-22	The same product could be sourced from another company
2 ml Microtube	Watson Bio Lab	132-620C	The same product could be sourced from another company
MS Media	Sigma	M9274	The same product could be sourced from another company
Scalpel blade no. 11	Sigma	S2771	The same product could be sourced from another company
Parafilm "M"	Bemis	PM996	This is the best product to use to bind the cambial window post creation
14 ml round bottom tubes	Thermo Scientific	150268	The same product could be sourced from another company
EDTA	Sigma	E6758	The same product could be sourced from another company
Triton	Sigma	X100	The same product could be sourced from another company
X-Gluc	X-GLUC direct		You will need to go to the website to order - http://www.x-gluc.com/index.html
Potassium Ferricyanide(III)	Sigma	244023	The same product could be sourced from another company
Potassium Ferrocyanide(II)	Sigma	P9387	The same product could be sourced from another company
Litmus paper	Sigma	WHA10360300	The same product could be sourced from another company
Single edge razor blade	ProSciTech	L055	The same product could be sourced from another company
Double edge razor blade	ProSciTech	L056	The same product could be sourced from another company
SEM Pin Stub	ProSciTech	GTP16111	The same product could be sourced from another company
Sample vial with screw cap	ProSciTech	L6204	The same product could be sourced from another company
Ethanol	sigma	E7023	The same product could be sourced from another company
LR white	ProSciTech	C025	The same product could be sourced from another company
Embedding Mould	ProSciTech	RL090	We recommend this variety, however there are plenty of options available
Water Soulable mounting media	ProSciTech	IA019	One example of a mounting media that could be used however other options do exist and could be explored.
Hydrogen peroxide	Sigma	216763	A like product could be sourced from another company
Glacial acetic acid	Sigma	A9967	A like product could be sourced from another company
Safranin O	ProSciTech	C138	A like product could be sourced from another company
Quanta Environmental Scanning Electron Microscope	FEI		This is the instrument used at part of this study but any other SEM that has a low vacuum mode could be utilised
ImageJ imaging software			can be sourced from the following URL http://rsbweb.nih.gov/ij/