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Visualization of the 3D structure of the graft union of grapevine using X-ray tomography
Scientia Horticulturae 144 (2012) 130–140
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Visualization of the 3D structure of the graft union of grapevine using X-ray tomography Mayeul Milien a,1 , Anne-Sophie Renault-Spilmont a,∗,1 , Sarah Jane Cookson b , Amélie Sarrazin a , Jean-Luc Verdeil c a
Institut Franc¸ais de la Vigne et du Vin (IFV), Domaine de l’Espiguette, 30240 Le Grau du Roi, France INRA, ISVV, EGFV, UMR 1287, F-33140 Villenave d’Ornon, France c CIRAD-Bios, UMR AGAP, PHIV, TA A-108/02 Avenue Agropolis, 34 398 Montpellier Cedex 5, France b
a r t i c l e
i n f o
Article history: Received 2 December 2011 Received in revised form 18 June 2012 Accepted 29 June 2012 Keywords: X-ray tomography Graft interface Graft quality 3D imaging Vascular connections Grapevine (Vitis vinifera)
a b s t r a c t Successful grafting in plants requires the development of a functional vascular system between the scion and the rootstock. Understanding the spatial organization of the graft interface is important to the evaluation of new rootstock genotypes and to the development of new grafting technologies. Until now the graft interface has only been studied using 2D classical histology and low resolution 3D magnetic resonance imaging. Here we investigate the ability of X-ray tomography to examine the graft interface of Vitis vinifera in high resolution and in 3D. Data were collected using a Skyscan 1076, scanning parameters, such as, X-ray energy, filter selection, pixel size and rotation angles, were optimized to study the particularities of the graft interface. The X-ray tomography technique was then used to evaluate graft quality. Two young vines were compared; one graft was classified as of ‘good’ quality, whereas the other was classified as of ‘bad’ quality. We were able to distinguish the “omega cut”, the pith, the phloem and the xylem vessels in the images. The analysis shows several differences between the two vines. In the good graft, tissues appear well-connected in the wood and phloem, and had a regular structure; the wood appears homogenous with a lot of vessels that form a compact mass. By contrast, in the bad graft, the structures appear disorganized and not completely connected. Numerous new vessels, continuous between the scion and the rootstock, are visible in the “good graft” whereas only few ones are visible in the “bad one”. It is the first time, to our knowledge, that 3D imaging of the graft interface and the vascular connections across it have been reported, opening new avenues for graft quality assessment in woody plants. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Routine grafting of grapevine in the agricultural systems of Europe began at the end of the 19th century to combat the devastating yield losses caused by the introduction of phylloxera from the USA (Ravaz, 1930). This practice has now been adopted in approximately 80% of vineyards world-wide (Pouget, 1990). In contrast, vegetable grafting on a commercial scale is relatively recent, beginning in Asia about 30 years ago and beginning in Europe and the USA in the 1990s (Lee et al., 2010). Despite the generalization of grafting in viticulture and the increase in vegetable grafting worldwide,
Abbreviations: 2D, two dimensions; 3D, three dimensions; GG, “good graft”; BG, “bad graft”; PAS, NBB. ∗ Corresponding author. Tel.: +33 4 66 51 17 54; fax: +33 4 66 53 29 16. E-mail address: [email protected] (A.-S. Renault-Spilmont). 1 These authors have contributed equally to this study. 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.06.045
little is known about the early mechanisms involved in grafting and how the structure of the graft union develops during graft union formation in any plant species. Grafting knowledge and techniques are essentially based on practical experience rather than scientific study. The general cellular events which occur after the grafting are quite well known and common to woody and non-woody plants. Successful grafting is a complex biochemical and structural processes that begins with an initial wound response, followed by callus formation, creation of a continuous cambium and the establishment of a functional vascular system between the two grafting partners (reviewed by Pina and Errea, 2005; Martinez-Ballesta et al., 2010). Grafting success requires numerous developments at the graft interface such as cell recognition and communication, the initiation of cell cycle, cell proliferation, cell differentiation and plasmodesmata formation (Estrada-Luna et al., 2002; Ermel et al., 1997; Pina et al., 2009; Kollmann and Glockmann, 1991). The cellular events at the graft interface have been well
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characterized by histological studies in various woody plants, such as, Picea spp., apples and Prunus spp. (Weatherhead and Barnett, 1986; Soumelidou et al., 1994; Olmstead et al., 2006). These histological studies (using light, confocal and electron microscopy) give beautiful and detailed images of the graft interface, but only in one plane. Understanding spatial tissue organization between the two partners of a graft union is of paramount importance to management and selection of future rootstock genotypes as well as being of scientific interest. To date graft union morphology has been studied with magnetic resonance imaging (MRI) in pine trees (Leszczynski et al., 2000) and in grapevine (Bahar et al., 2010). Unfortunately, these MRI studies are of relatively low resolution and only present 2D virtual sections of the graft interface. Fluorescent dyes and 14 Csucrose markers have been employed to characterize the functional developments of the phloem at the graft interface in tomato but the morphological characterization was also destructive and only provided 2D information (Schöning and Kollmann, 1997). X-ray tomography is a minimally invasive structural imaging method that allows 3D reconstruction of scanned objects (as reviewed by Larabell and Nugent, 2010). It was first used as a medical diagnostic tool in 1970s. It has since been applied to the study of animals and minerals; the use of X-ray tomography began in plants in the late 1990s (e.g. Pierret et al., 1999). X-ray tomography has been applied to the study of the anatomy of stem wood samples of trees (Fromm et al., 2001; Steppe et al., 2004; Longuetaud et al., 2005) and recently to the study of vessel dimensions and intervessel connections in grapevine (Brodersen et al., 2011). Other higher resolution tomography technologies also exist; these technologies can visualize and 3D reconstruct to the single cell level, but only in small plant samples such as seeds (e.g. Cloetens et al., 2006; Smith et al., 2009). Although graft incompatibility is rare in grapevine, grafting success can be very variable (as reviewed by May, 1994). The objective of this study was to evaluate the potential of X-ray tomography to study the internal structures and the 3D organization of graft zone in grapevine. After different tests and optimization of the scanning parameters, the method was applied to young vines with differing degrees of grafting success in order to understand how grapevine tissues and structures organize in response to the grafting. In this manuscript we present the study of the 3D organization of the graft interface in grapevine, this method could open new avenues to assess graft quality in woody plants.
2. Materials and methods 2.1. Plant material and growing conditions 2.1.1. Grafting Vitis vinifera were grafted onto the rootstocks 110R (110 Richter) or SO4 (Selection Oppenheim 4). One-bud cuttings were made for scions and two node de-budded cuttings were made for rootstocks shortly before grafting. Mechanical omega grafting was performed on scion/rootstock pairs of approximately the same diameter. Grafts were briefly dipped into melted wax and settled for callusing for 21 days at 28 ◦ C, 95% humidity. After callusing and rooting, grafted plants were planted in sandy soil in a nursery (Domaine de l’Espiguette, Grau du Roi, France).
2.1.2. Plant material Three types of plant material were used in this study. (1) Two plants of Syrah were grafted onto 110R in April 2009 and were then harvested for immediate analysis 18 months later
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in October 2010 (Supplementary Data 1A). They were used for X-ray tomography and classical histology (microtome) (2) The graft union of a 14-year-old grapevine plant (Syrah grafted onto the rootstock SO4) was collected from the vineyard (Domaine de l’Espiguette Grau du Roi, France) in October 2010 (Supplementary Data 1B) and immediately scanned. (3) Various self-hybrids of Syrah were grafted on the 110R rootstock in the beginning of May 2011 and planted in a nursery (Domaine de l’Espiguette, Grau du Roi, France); grafting success was evaluated in December 2011 (8 months after grafting). Two genotypes displaying differing degrees of grafting success were selected for analysis (Supplementary Data 1C and D). The first vine was selected from a vigorous genotype that showed normal development of the scion and roots: this plant is termed “good graft” or “GG”. The second vine was chosen from a genotype that had reduced scion development: this plant is termed “bad graft” or “BG”. After the plants were harvested, they were pruned (scion and roots) and the graft zones and scions were briefly dipped into wax. The plants were kept in a cold damp room in bags until being scanned. Before the scan, wax was removed from the graft zone, and rootstock was protected by parafilm. After scanning, these two vines were used for histological analysis. 2.2. Histological analysis 2.2.1. Sample preparation After being scanned, young wood samples (8 months and 18 months after grafting) were cut with a band saw in order to obtain serial sections of wood of approximate 5 mm thick. Samples were fixed in a solution of 1% acrolein (v/v) and 4% glutaraldehyde (v/v) in 0.2 M phosphate buffer (pH 7.2) for 72 h at room temperature (according to Buffard-Morel et al., 1992). Samples were then washed with ethanol 70%. 2.2.2. Sample treatment for classical histology The prepared wood samples were dehydrated in a graded ethanol series (70% ethanol for 30 min followed by 90% ethanol for 1 h) and then transferred to ethanol/butanol (50/50, v/v) for 3 days. The samples were then transferred to butanol (100%) for 15 days, before being gradually embedded in a glycomethacrylate resin (Technovit 7100, Kulzer); the samples were placed in butanol/resin (50/50, v/v) for 21 days and then transferred to 100% resin. Samples were conserved at 4 ◦ C, on a shaker for impregnation for a minimum of 3 months. Transverse sections were cut with a thickness of 6 m on a rotary microtome (Leica RM 2255). Sections were double-stained with Periodic Acid Schiff (PAS) and Naphtol Blue Black (NBB) (Fisher, 1968; Buffard-Morel et al., 1992). Materials were mounted using a microscope slide media (Isomont2000, LABOnord) and examined using a Leica DM 4500 B light microscope (Leica, Germany). Images were taken using a camera: Retiga 2000R (QIMAGING FAST 1494, Canada) and Volocity software (Improvision, UK). 2.2.3. Sample treatment for epi-fluorescence microscopy Transverse sections were obtained from prepared wood samples using a vibratome (Microm HM 650V). Then the samples were softened before being cut into successive slides of 30 m and mounted in VectaMount (Vector Laboratories, Inc.). Autofluorescence of these samples was observed using a Leica DM 6000 light microscope (Leica, Germany,) and the filter A (450–490 nm). Images
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were taken using a camera: Retiga 2000R (QIMAGING FAST 1494, Canada) and Volocity software (Improvision, UK). 2.3. High-resolution computed tomography 2.3.1. Acquisition Graft zones were scanned at the Montpellier RIO Imaging Center (France) using a micro-tomography SkyScan 1076 (SkyScan, Belgium), the configuration was adapted from a protocol used to analyze minerals. Electrons, emitted from the source (sealed Xray tube 20–100 kV, spot size < 5 m, 10 W) were accelerated (59 or 70 kV) and focused on an anode where they generate X-ray photons which can be filtered (Table 1). Then, the X-ray photons impacted the sample, which was positioned precisely within the X-ray beam. The X-ray shadow images were acquired by a sensitive X-ray camera (4000 × 2672 pixels, 12 bit, fiber-optically coupled to a scintillator). This produced a longitudinal X-ray photograph through the plant body by moving the X-ray source and the camera in opposite directions during the exposure. Rotation steps were chosen (Table 1) between 0.3◦ and 0.44◦ (over 210◦ ). Scans were made over 2 cm for the 18 and the 8 month after grafting samples and over 8 cm for the 14 years after grafting sample. A typical scan of 2 cm in axial length required 7–50 min and generated 0.5–13 GB of data (Table 1). 2.3.2. Image processing Raw 2D tomographic projection images were reconstructed using NRecon software (SkyScan, Belgium) with the Felkamp algorithm, to create cross-section image stacks (8-bit or 16-bit series, tagged image file). ImageJ software (http://rsbweb.nih.gov/ij/) was used to analyze images (2D, virtual slices) and to measure the development of new wood tissue produced after grafting on the 8 month after grafting samples. In the scion and the rootstock, every 900 m, a region of interest was drawn on virtual transverse section to measure newly formed wood. 3D analysis was done with the Volocity software (Improvision, UK). This software provides 3D surface rendering, and allows us to make virtual cuts (or crops) and to change the image brightness (by changing the thresholds). Pseudocolors can be attributed according to desired parameters, such as, density. The software also provides a specific interface to generate 3D movies (5 frames s−1 ). 3D rendering was also done on Imaris software (Bitplane, Switzerland). It was used to study vascular organization. To identify the vascular organization and the pith, segmentation had been done using threshold on gray level and particle size. Pseudocolors were attributed according structures (e.g. the vessels in green, and pith in red) and a virtual section was added. 3D movies were generated (5 frames s−1 ). Before this visualization, images were treated with ImageJ software: image resolution was reduced to 72 m to allow Imaris to work. 3. Results 3.1. Optimizing the parameters for X-ray tomography at the graft interface in grapevine The use of X-ray tomography to study the graft interface in grapevine began by testing 24 parameters. The most important of which are given in Table 1. Voltage was optimized in order to give the best possible contrast between the plant tissues, air and water; it was 59 kV for 18 months and 70 kV for 14 years after grafting samples. To improve the signal to noise ratio, low filters were chosen to stop X-rays with low energy. Filter selection was an important factor in the acquisition of well-contrasted images (result not
shown). Preliminary investigations of the graft interface of the 14-year-old plant showed that the older plant had greater variations in tissue density. For this reason a slightly thicker filter was required. In this work, three different resolutions were tested (high 9 m, medium 18 m and low 36 m) on the 18 months old plant: illustrations are given in Fig. 1B and C for the highest and lowest resolutions. All parameters were adjusted to produce a suitably contrasted image according to the sample specificity (diameter and size). On the young plant, with a full field of view at 9 m, resolution allowed the entire cross-sectional area of the graft zone to be scanned at a rate of 1 cm of stem every 24 min (at a 36 m resolution 1 cm was scanned every 3 min). The capacity of Skyscan 1076 allowed us to adapt this technology to study a large wood sample (8 cm long × 6.6 cm maximum diameter) using steps of 2 cm in length (in a sample with large differences in tissue density). 3.2. 3D imaging by X-ray tomography as an alternative to classical histology in young tissue Images obtained by X-ray tomography (Fig. 1B and C) can be compared to a classical histology specimen (Fig. 1A). In the classical histology specimen, periodic acid Schiff (PAS) was used to color polysaccharides in red (e.g. cellulosic compounds and starch) and Naphtol Blue Black (NBB) colored proteins in blue (e.g. the proteins of the nucleus). This double staining aided the visualization of the different structures of the wood; the xylem (vessels, rays and annual rings), phloem (vessels and rays), phellogene and phelloderme could be observed. Xylem ray appears purple due to the presence of starch granules. The pith is indicated by the light tones of these large empty cells and the characteristic forms of their cell walls. The phloem is strongly colored in pink/purple, due to the presence of many polysaccharides in cytoplasm and cell wall. The rays of the phloem are aligned with those of the xylem; due to the division of cambium cells (cambium is located between the xylem and phloem). On this section of classical histology, it is possible to observe up to three years of phloem development. In the X-ray tomography images the bright pixels represent the densest tissues that stop/slow down X-ray photons more than the surrounding tissue, therefore, it is the differential Xray attenuation that produces image contrast (Fig. 1B and C). The attenuation of the X-ray photons by a material is dependent on the energy of the X-ray radiation and the following parameters of the material studied: thickness, density and atomic number. For example, oxygen and carbon have slightly different X-ray attenuations; so as a consequence, water and cellulose have different X-ray attenuations that produce different pixel greyscale intensities. With a high resolution of 9 m, structures and tissues can be observed (Fig. 1B). Xylem, phloem, pith, annual rings, ray tissues and pericycle fibers can be identified. We are able to distinguish clearly the early and the late wood depending on the diameters of the vessels. Two growth rings can also be distinguished in the wood and the pith, which appears dark. In the X-ray tomography images, the presence of contaminating paraffin and grains of sand on the outside of the sample can be reduced with the beam hardening correction option. Fig. 1 clearly shows that if the X-ray tomography is done with high resolution (9 m and rotation step of 0.3◦ ), the tissue structure is as visible by tomography as by classical microscopy (low magnification). However, it is the limit of the resolution whereas more details can be observed by microscopy in increasing the magnification. By contrast, the reconstruction of the scans at 36 m
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Table 1 X-ray tomography parameters tested on the graft union of grapevines 8 months, 18 months and 14 years after grafting. Grapevine sample
Sample size (cm)
Scan diameter (cm)
Resolution (m)
Filter
Voltage (kV)
Current (A)
Rotation step (◦ )
Depth (bit)
Duration
Number Of Files
Data
18 months 18 months 18 months 14 years 14 years 8 months “good graft” 8 months “bad graft”
2 2 2 2 8 2 2
3.3 3.3 3.3 6.6 6.6 3.3 3.3
9 18 36 9 36 9 9
Ti 0.025 mm Ti 0.025 mm Ti 0.025 mm Al 1.0 mm Al 1.0 mm Ti 0.025 mm Ti 0.025 mm
59 59 59 70 70 59 59
167 167 167 141 141 167 167
0.300 0.400 0.440 0.300 0.300 0.300 0.300
16 16 16 16 16 16 16
0:49:14 0:11:24 0:06:11 01:30:52 01:48:28 00:48:28 00:48:40
660 498 453 >1294 2859 659 659
13.1 GB 2.46 GB 574 Mo >28.3 GB 6.98 GB 13.1 GB 13.1 GB
resolution with a rotation step of 0.44◦ produced images of insufficient quality to clearly identify the different tissues (Fig. 1C). The month in which the vines were analyzed (October) does not allow us to visualize the cambium, which was inactive and so reduced in size. With the tools used here (scanner and computer), the resolution is lower in X-ray tomography than in classical histology as we are not able to observe the structure at the cellular level. 3.3. The investigation of 14-year-old graft union X-ray tomography on a 14-year-old plant required the reoptimization of the analysis parameters. Preliminary investigations of the graft interface of the 14-year-old plant showed that the older plant had greater variations in tissue density. For this reason a slightly thicker filter was required (Al 1.0 mm). 3D reconstruction of the external surface of the 14-year-old plant revealed numerous details at the grapevine grafting zone, such as, pruning wounds (Fig. 2A). Fig. 2B shows a virtual longitudinal section through the middle of the graft interface shown in Fig. 2A. In this view, in full density, we can observe large differences in tissue density; the densest tissues are colored in white. With this virtual sectioning we can see the young xylem of the rootstock (bottom of image), the scion (the top of the image) and the complex tissues of the graft interface (Fig. 2B). A significant tissue degradation (dark gray) penetrating deeply into the interface between the two partners is observed, producing a deep gap between rootstock and scion (Fig. 2B). Fig. 2C is a whole view of the graft zone in pseudocolor with a threshold application; the densest tissues appear yellow, whereas the less dense appear blue. With this view, the dense organized xylem (colored yellow/green) of the rootstock can be seen on
the bottom right corner of the image and we can see that the xylem vessels span the entire graft interface (Fig. 2C). We can observe the complexity of the new tissue formed during graft union development and especially the complex organization of the vessels established through the graft union between the two partners. Furthermore, details like an old bud on the rootstock can be observed (bottom right of the image). This view clearly shows the connections between the two partners made from the tissue formed after grafting. A movie (Supplementary Data 3) made from the data in Fig. 2 reveals more clearly the details of the graft union in grapevine and the power of X-ray tomography. On the virtual transversal section we can observe that the pith and the central xylem are partially degraded (Supplementary Data 2). Some of the annual rings can also be observed (red arrows). 3.4. Comparative anatomy of differences in graft quality The X-ray tomography method was used to study two 8-monthold grapevines differing in graft quality; this was done in order to examine how the structures at the graft interface organize in a successful and unsuccessful graft. The conditions of the scan were adapted as indicated in Table 1. The main tissues of the graft interface are evident in the virtual longitudinal sections of the GG and BG (Fig. 3). The pith (black), the xylem (gray) with some individual vessels and the phloem (light gray) are visible. In the graft union some vessels are perpendicular to the longitudinal axis (or appear disrupted in their orientation). In both the vines, the “omega cut” is visible. Nevertheless numerous differences can be noted. The diameter of the graft union is much larger in the GG than in the BG, both at the scion and rootstock levels (Fig. 3). In the GG, it is
Table 2 Quantitative evaluation of the wood production on “good graft” and “bad graft” 8 months after grafting, using ImageJ software. “Good graft” Relative positiona Scion
Rootstock
a b
“Bad graft” Stem area (except phloem) (mm2 )
Neo-xylem produced/Total surface (%)b
2100 2000 1900 1800 1700 1600
85.8 87.9 95.2 109.0 134.6 162.4
67% 69% 71% 75% 80% 84%
600 500 400 300 200 100
126.8 105.5 90.7 79.4 73.6 69.9
76% 71% 64% 55% 55% 48%
Row of the slice, from the base of the stack (no units). Surface of the xylem produced after grafting reported to the global surface (without the phloem).
Relative positiona
Stem area (except phloem) (mm2 )
Neo-xylem produced/Total surface (%)b
2100 2000 1900 1800 1700 1600
22.4 21.8 21.2 21.5 22.7 25.6
36% 35% 35% 36% 43% 52%
600 500 400 300 200 100
46.7 44.2 41.7 40.6 39.8 39.0
21% 20% 17% 17% 14% 14%
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difficult to identify precisely the boundary between the rootstock the scion; the phloem appears completely continuous. By contrast, in the BG, the scion is not centered and the piths are misaligned. The omega cut remains very visible as little tissue was produced at the graft union and the connectivity between the scion and rootstock appears incomplete. The graft union of the BG is of low density and is composed of necrotic tissue: this was confirmed by histological analysis (Supplementary Data 4). The same samples of GG and BG were then analyzed in virtual transverse sections at different levels (the scion, graft union and rootstock levels, Fig. 4). It is possible to distinguish the different tissue structures such as the pith, the wood and the phloem in these images. Some xylem vessels and rays are also identifiable and the omega cut can be localized. In the GG, the partners are well aligned as shown by the pith position. The tissues were regularly produced in the graft union inducing a regular structure more or less oval (Fig. 4B). In the contact area, it is difficult to identify the boundary between the rootstock and the scion. The density of wood is very homogeneous and the rays are visible because of the presence of many dense granules aligned inside (especially in the scion and in the graft union virtual transverse sections). These granules correspond to starch as determined by complementary observations in classical histology (Supplementary Data 5). By contrast, in the BG, the scion and the rootstock are clearly misaligned (Fig. 4E) and the numbers of contacts between them remain limited. The wood also appears very different; it is heterogeneous with different levels of gray both in graft union and in the rootstock. The dark gray wood is less dense and corresponds to a partially necrotic wood. This is confirmed by analysis of the same sample with classical histology. In this area, no auto-fluorescence is observed which indicates that composition of the tissue is altered (Supplementary Data 4). In the omega zone, some poorly organized tissues between the scion and the rootstock appear dark gray and are probably necrotic. The xylem vessels appear enlarged in the BG, compared to the GG, even in the rootstock part which is exactly the same genotype. The quantity of wood produced seems greater in the GG than in the BG plant. To be able to evaluate this precisely, measurements of the wood surface area were done on the images at different levels (Table 2). These measurements could not be done in the omega cut/graft union zone because the vessels are in various orientations; this prevents us from precisely localizing the wood produced after and before the grafting. In the two vines, the percentage of the wood produced after the grafting increases near the omega cut and is greater in the scion than in the rootstock. The quantity of wood produced is systematically higher in the GG than in the BG, at all three levels. The new xylem produced after the grafting represents 48–84% of the surface in the GG, whereas it represents only 14–52% in the BG, indicating a significant variation in the cambial activity in these two cases.
Fig. 1. The comparison of transverse sections of grapevine stem tissue made with (A) classical histology (stained with PAS–NBB) and X-ray tomography at (B) 9 m and (C) 36 m resolution. The plant material was harvested from a grafted grapevine 18 months after grafting. The position of the cambium, which is not visible at the time of sampling, is localised between xylem and phloem. Scale bar = 500 m. Xy, xylem; Ph, phloem; R, ray; cambium (yellow arrows); and annual rings (red arrows).
3.4.1. 3D visualization and vascular connections The 3D analysis of plants differing in graft quality was done with the Imaris software, which produced 3D surface rendering and digital images). The virtual dissection of the samples was done to visualize specific tissues and to study the internal structure. Segmentation was performed to highlight the vascular system and the pith; pseudocolors were respectively attributed (Fig. 5). The comparison of surface rendering images shows that the graft union is bigger and more regular in the GG rather in the BG. The scion and the rootstock diameters are also larger in the GG (Fig. 5A and D). The vessel spatial arrangement across the graft union is shown (Fig. 5B and E, Supplemental Data 6 and 7); the omega cut remains visible in both GG and BG. In the GG, many vessels organized in a
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compact mass are visible (Fig. 5B), whereas in the BG there seems to be little connectivity between the rootstock and scion (Fig. 5E). Examples of some vascular connections between the scion and the rootstock were visible (Fig. 5C and F and Supplemental Data 6 and 7). Vessels that form a continuous connection between the two partners correspond to newly formed vessels as shown by their location on the virtual transverse sections (Fig. 5C and F). The resolution used for the image analysis is not sufficient to study vessels individually thus it is not possible to quantify these vascular connections. The low density tissues of the graft interface were also analyzed (Fig. 5C and F). In the GG, the pith is easily identifiable (Fig. 5C), whereas in the BG (Fig. 5F) it is not possible to separate, the pith and the necrotic tissues associated with the omega cut by greyscale level threshold. 4. Discussion 4.1. X-ray tomography as a method to study the 3D structure of the graft interface of grapevines: development on grafted vines of various ages High quality 3D images of the graft interface of grapevine can be produced using X-ray tomography especially for the vines studied 8 months after grafting. The parameter optimization used allowed us to visualize the internal tissue organization of the graft interface, the resolution was close to what classical histology can produce in terms of global tissue organization. Xylem, phloem, pith, ray and necrotic tissues, as well as the omega cut, could be identified in the different vines tested with this technique. We were able to distinguish the main tissues essential to describe a graft union. By contrast, the cambium was never visible. This result is not surprising as the cambium is thinner and very difficult to observe by classical histological methods (i.e. light microscopy) at the period of the year in which the graft interface samples were harvested (in October, the end of the grapevine growing season in Europe). It may also be noted that, at the resolution used here, the smallest vessels are not clearly visible with the X-ray tomography method used. It could be due to the resolution of the images taken or due to the limitations of computer power available. Depending on the size of the object analyzed and the resolution chosen, scans take between 7 and 50 min. As is frequently the case in imaging studies, a compromise must be found between the time necessary to acquire an image and its resolution. The resolution of the images of the graft interface 14 years after grafting had to be sacrificed because otherwise the number of data files produced would have been beyond the computing capacity available to the project. The large size of the graft interface of the grapevine 14 years after grafting required successive scans at 4 levels to capture the entire structure, with a 36 m resolution it took one and a half hours and produced 7 gigabytes of data. Concerning the second part of the study on the 8-month-old vines, a very good resolution was obtained for the scans (which took 48 min each). However, to analyze the tissue organization in 3D using the Imaris software the image resolution had to be reduced to 72 m. This resolution was insufficient to analyze vessels individually: they appear too close to each other. Thus, it was not possible to quantify vessel connectivity. It can be assumed Fig. 2. 3D Reconstruction of the graft union of an old grapevine plant (14 years after grafting) imaged using X-ray tomography at 36 m resolution (using Volocity software): (A) an external view of surface of the graft interface; (B) a full density virtual longitudinal section through the graft interface, the dense tissues are in white and the degraded tissues that has developed between the rootstock and scion are in dark grey; (C) a pseudocolor threshold density view of the graft interface, the
densest tissues are yellow and the least dense tissues are blue. Scale bare = 2.5 cm Gz, graft zone; Ro, rootstock; Sc, scion; Bu: Bud; pruning wounds (red stars), tissue degradation (red triangles) and huge xylem vessels (yellow arrows). Movie of the 3D vascular structure corresponding to varying angles of view can be seen in Supplementary Data 3 (Film 1).
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Fig. 3. Comparative visualization of a (A) “good graft” and a (B) “bad graft” 8 months after grafting: longitudinal sections of the graft union extracted from the 3D X-ray tomography reconstruction. Virtual longitudinal section obtained using ImageJ software. Scale bar = 1 mm. Gz, grafting zone; Ro, rootstock; Sc, scion; , omega; Xyv, xylem vessel; Ph, phloem; P, pith; Sa, sand.
that with the use of a more powerful computer, not requiring a reduction in image resolution, the individual vessels may be studied. 4.2. Application to compare anatomy of a good and a bad grafted union The X-ray tomography method developed here to visualize in 3D the graft interface was applied to study grafting success in young grapevines. Our aim was to understand how grapevine tissues and structures organize in response to the grafting. Two genotypes displaying differing degrees of grafting success were selected for analysis. The first grapevine plant, termed GG, showed normal development of the scion and roots, whereas the second one, termed BG, had reduced scion development. The GG is characterized by a regular structure of large diameter consisting of well connected tissues, both in the wood and the phloem. The quantity of wood produced after grafting is an important indicator of cambial activity. Many starch granules are visible in the xylem rays and numerous continuous vessels between the scion and the rootstock were identified. By contrast, in the BG, the structures appear not so wellorganized and the tissues are not completely joined together. The density of the wood is heterogeneous with necrotic areas of lower density and only few continuous vessels were revealed in the BG. The irregular vessel orientation observed in the BG has also reported in cherries and apples grafted onto dwarfing rootstocks (Soumelidou et al., 1994; Olmstead et al., 2006). The xylem contains fewer vessels in the some examples of dwarfing rootstocks, which could be due to developments at the graft interface (Soumelidou et al., 1994; Olmstead et al., 2006). The union between the vascular elements of scion and rootstock is thought to be the critical event in the formation of a successful graft. The vessel connections were observed in the both BG and GG. Nevertheless there were fewer in the BG, which is coherent with its reduced scion development. The criteria defined here for the bad graft cannot be applied to all the cases of graft failure or
incompatibility; grafting failure is complex and could result from numerous factors. It is the first time, to our knowledge, that the junctions between scion and rootstock have been revealed in 3D. 4.3. Advantages and drawbacks of X-ray tomography compared to classical histology 4.3.1. Sample preparation One practical advantage of X-ray tomography is that no chemical fixation or sample preparation is needed, thus avoiding the complex and time-consuming process of obtaining good histological slides of woody material. In addition, artifacts such as cracks and cell wall damage are often formed during sample preparation for classical histology, especially on woody samples. It can so be used on material difficult or too precious to section for histological examination such as fossil plants (Smith et al., 2009). Unlike classical histology, X-ray tomography is not destructive or invasive so it can be used to image live plants. This method may also be used to follow the kinetics of tissue development at the graft interface or to detect potential flaws in the graft interface in plants used for commercial or scientific purposes. Although X-ray tomography is non-destructive, the potential impact of the ionizing effects of the X-rays on the living tissue has to be considered. Dhondt et al. (2010) observed a growth inhibition of Arabidopsis seedlings that were scanned with X-rays on a daily basis. This technique can also be applied on multiple specimens in identical conditions (image acquisition and analysis), which can allow some degree of automation (Brodersen et al., 2011). 4.3.2. Resolution and image quality of X-ray tomography images In this study the resolution of the images obtained with classic histology techniques was superior to those obtained with X-ray tomography, although they were comparable on a global tissue structure level. It is due to the limit of the scan resolution and the computer capacity for images analysis. The resolution used for the image analysis was not sufficient to study vessels individually.
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Fig. 4. Comparative visualization of a “good graft” (A–C) and a “bad graft” (D–F) 8 months after grafting: transverse sections of the graft union extracted from the 3D X-ray tomography reconstruction. Virtual transverse sections obtained using ImageJ software; sections were taken from the scion (A and D), the graft zone (B and E), the rootstock (C and F). Scale bar = 1 mm. On the insert, each virtual transverse section can be localised inside the volume, visualization with Imaris software (see arrows). Scale bar = 4 mm. Gz, grafting zone; Ro, rootstock; Sc, scion; Xy, xylem; Ph, phloem; S, starch; P, pith.
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Fig. 5. Comparative visualization of a “good graft” (A–C) and a “bad graft” (D–F) 8 months after grafting: 3D vasculature obtained after vessel segmentation. (A and D) External surface views. For tissue segmentation, higher density structures were classified and coloured according to their threshold and their volume: vascular elements are in green (B and E) pith in red and some isolated vascular elements in blue (C and F) (visualization with Imaris software). Scale bar = 4 mm. Animated 3D images of the vascular structure corresponding to varying angles of view for the “good graft” and “bad graft” are in Supplementary Data 6 and 7 respectively (films 2 and 3).
Recent advances in X-ray tomography could overcome this limitation, for example Steppe et al. (2004) used high resolution X-ray tomography to study the anatomy of the wood of beech and oak trees, and Dhondt et al. (2010) studied Arabidopsis hypocotyls with a resolution of 0.85 m that allowed the visualization of individual cells. The use of the synchrotron radiation X-ray tomographic microscopy in modern and fossil plants provides high-quality anatomical detail, up to the cell resolution, comparable to traditional histology (Cloetens et al., 2006; Smith et al., 2009). In the X-ray tomography study performed here, we were able to identify the phloem, the xylem vessels and the pith, but the cambium and the parenchyma were not easily distinguishable. This observation was also made by Brodersen et al. (2011) on both hydrated and dehydrated grapevine stems. The cambium was
not visible here because of the period of sampling. The phloem appears somewhat brighter than the xylem, as these tissues are spatially separated it does not affect our interpretation of the images. Nevertheless, use of some contrasting agents could improve their identification. Dhondt et al. (2010) reported the use of a contrasting agent (such as iodine) to increase the contrast within the soft Arabidopsis hypocotyl tissues, however, as the samples must be fixed and dehydrated it limits the usefulness of the X-ray tomography technique. 4.4. Imaging the 3D structure The great advantages of X-ray tomography are (1) the ability to obtain digital sections in multiple planes, (2) the possibility of
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virtually dissecting a plant to visualize specific tissues and study the internal structure, and (3) the 3D visualization (which is very difficult to obtain by histological techniques). This technique appears much more easy and rapid than the techniques of classical histology. The 3D imaging after reconstruction has been done previously for different woody tissue types (Steppe et al., 2004; Brodersen et al., 2011). 3D data can also be acquired using MRI (magnetic resonance imaging). MRI has been used to study the graft interface in apples, grapevine and in pine trees, but the resolution was very low (Warmund et al., 1993; Leszczynski et al., 2000; Bahar et al., 2010). The small cells of cambium appear bright on a MRI image due to their high water density (due to the absence of air and the absence of a thick cell wall); therefore MRI gives a good indication of cambium development at the graft interface. In addition, MRI is well adapted to the study of sap flows in xylem and phloem vessels (as reviewed by Dhondt et al., 2010) and can be considered as complementary to X-ray tomography as it can provide information about sap flow around and across the graft interface. The development of multimodal imaging, combining X-ray tomography and MRI, would allow us to study not only the 3D structure of the graft interface but also the functioning of the vasculature by examining the saps flows. 5. Conclusion: future potential applications New scion varieties and new rootstock genotypes are currently being selected for and field tested (particularly in terms of their resistance to fungal diseases and challenging climates); however, it is also essential that all new genotypes can be grafted successfully. Understanding the spatial organization of the grafting events is of paramount interest. The potential of X-ray tomography to study the complexity of the graft interface of grapevine in 3D was evaluated. The main important tissues in a graft union (the pith, xylem vessels, phloem and necrotic tissues) and even some compounds such as starch were identified. The main interest of X-ray tomography method is associated with the image analysis; we present a non-destructive 3D visualization of the graft interface that could provide new insights into the spatial tissue organization of the graft interface in grapevine. It is the first time, to our knowledge, that 3D imaging of the graft interface and vascular connections has been reported. This method could open new avenues to study graft quality assessment in woody plants. As this technique is non-destructive and non invasive, it may be of great interest to image live plants and study the kinetics of tissue development on the same graft plant overtime. While the X-ray tomography will not replace systematically classical histology, it does offer an improved technique with the ability to combine 2D analysis in multiple planes and 3D reconstruction. Thus it appears as an ideal tool to understand morphological developments at the graft interface and it could explain grafting success or failure and/or incompatibility in grapevines, but also for many other woody plants. Acknowledgements This work and the position of MM were funded by the ‘Consortium Inter-Régional sur le Dépérissement de la Syrah’ which included the Région Languedoc-Roussillon, the Région Provence Alpes-Côte d’Azur, the Région Rhône-Alpes, FranceAgriMer, InterRhône and the Centre du Rosé. The authors wish to thank the Montpellier RIO Imaging Center and specifically Renaud Lebrun for his helpful advice on Skyscan 1076 use
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and Marc Lartaud for his help on the Volocity and Imaris software.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.scienta.2012.06.045.
References Bahar, E., Korkutal, I., Carbonneau, A., Akcay, G., 2010. Using magnetic resonance imaging technique (MRI) to investigate graft connection and its relation to reddening discoloration in grape leaves. J. Food Agric. Environ. 8, 293–297. Brodersen, C.R., Lee, E.F., Choat, B., Jansen, S., Phillips, R.J., Shackel, K.A., McElrone, A.J., Matthews, M.A., 2011. Automated analysis of three-dimensional xylem networks using high-resolution computed tomography. New Phytol. 191, 1168–1179. Buffard-Morel, J., Verdeil, J., Pannetier, C., 1992. Embryogenèses somatique du cocotier (Cocos nucifera L.) à partir de tissus foliaires: étude histologique. Can. J. Bot. 70, 735–741. Cloetens, P., Mache, R., Schlenker, M., Lerbs-Mache, S., 2006. Quantitative phase tomography of Arabidopsis seeds reveals intercellular void network. PNAS 103, 14626–14630. Dhondt, S., Vanhaeren, H., Van Loo, D., Cnudde, V., Inze, D., 2010. Plant structure visualization by high-resolution X-ray computed tomography. Trends Plant Sci. 15, 419–422. Ermel, F.F., Poessel, J.L., Faurobert, M., Catesson, A.M., 1997. Early scion/stock junction in compatible and incompatible pear/pear and pear/quince grafts: a histo-cytological study. Ann. Bot. 79, 505–515. Estrada-Luna, A.A., Lopez-Peralta, C., Cardenas-Soriano, E., 2002. In vitro micrografting and the histology of graft union formation of selected species of prickly pear cactus (Opuntia spp.). Sci. Hortic. 92, 317–327. Fisher, D., 1968. Protein staining of ribboned epon sections for light microscopy. Histochemie 16, 92–96. Fromm, J.H., Sautter, I., Matthies, D., Kremer, J., Schumacher, P., Ganter, C., 2001. Xylem water content and wood density in spruce and oak trees detected by high-resolution computed tomography. Plant Physiol. 127, 416–425. Kollmann, R., Glockmann, C., 1991. Studies on graft unions. 3. On the mechanism of secondary formation of plasmodesmata at the graft interface. Protoplasma 165, 71–85. Larabell, C.A., Nugent, K.A., 2010. Imaging cellular architecture with X-rays. Curr. Opin. Struct. Bio. 20, 623–631. Lee, J.M., Kubota, C., Tsao, S.J., Bie, Z., Echevarria, P.H., Morra, L., Oda, M., 2010. Current status of vegetable grafting: diffusion, grafting techniques, automation. Sci. Hortic. 127, 93–105. Leszczynski, R., Byczkowski, B., Jurga, S., Korszun, S., 2000. NMR microimaging studies of the union between stock and scion. Appl. Magn. Reson. 18, 147–153. Longuetaud, F., Saint-Andre, L., Leban, J.M., 2005. Automatic detection of annual growth units on Picea abies logs using optical and X-ray techniques. J. Nondestruct. Eval. 24, 29–43. Martinez-Ballesta, M.C., Alcaraz-Lopez, C., Muries, B., Mota-Cadenas, C., Carvajal, M., 2010. Physiological aspects of rootstock–scion interactions. Sci. Hortic. 127, 112–118. May, P., 1994. Using Grapevine Rootstocks – The Australian Perspective. Grape and Wine Research Corporation, Adelaide. Olmstead, M.A., Lang, N.S., Ewers, F.W., Owens, S.A., 2006. Xylem vessel anatomy of sweet cherries grafted onto dwarfing and non-dwarfing rootstocks. J. Am. Soc. Hortic. Sci. 131, 577–585. Pierret, A., Capowiez, Y., Moran, C.J., Kretzschmar, A., 1999. X-ray computed tomography to quantify tree rooting spatial distributions. Geoderma 90, 307–326. Pina, A., Errea, P., 2005. A review of new advances in mechanism of graft compatibility–incompatibility. Sci. Hortic. 106, 1–11. Pina, A., Errea, P., Schulz, A., Martens, H.E., 2009. Cell-to-cell transport through plasmodesmata in tree callus cultures. Tree Physiol. 29, 809–818. Pouget, R., 1990. Histoire de la lutte contre le phylloxera de la vigne en France: 1968–1895. In: INRA, co-publisher Office International de la Vigne et du Vin, Paris, France, p. 158. Ravaz, L., 1930. De l’influence spécifique réciproque du sujet et du greffon – effets de la greffe. Extrait des Annales de l’Ecole nationale d’Agriculture de Montpelllier 20, 1–15. Schöning, U., Kollmann, R., 1997. Phloem translocation in regenerating in vitro heterografts of different compatibility. J. Exp. Bot. 48, 289–295. Smith, S.Y., Collinson, M.E., Rudall, P.J., Simpson, D.A., Marone, F., Stampanoni, M., 2009. Virtual taphonomy using synchrotron tomographic microscopy reveals cryptic features and internal structure of modern and fossil plants. PNAS 106, 12013–12018.
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Soumelidou, K., Battey, N.H., John, P., Barnett, J.R., 1994. The anatomy of the developing bud union and its relationship to dwarfing in apple. Ann. Bot. 74, 605–611. Steppe, K., Cnudde, V., Girard, C., Lemeur, R., Cnudde, J.P., Jacobs, P., 2004. Use of X-ray computed microtomography for non-invasive determination of wood anatomical characteristics. J. Struct. Biol. 148, 11–21.
Warmund, M., Barritt, B., Brown, J., Schaffer, L., Jeong, B., 1993. Detection of vascular discontinuity in bud unions of ‘Jonagold’ apple on mark rootstock with magnetic resonance imaging. J. Amer. Soc. Hort. Sci. 118 (1), 92–96. Weatherhead, I., Barnett, J.R., 1986. Development and structure of unusual xylem elements during graft union formation in Picea sitchensis L. Ann. Bot. 57, 593–598.
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Visualization of the 3D structure of the graft union of grapevine using X-ray tomography Mayeul Milien a,1 , Anne-Sophie Renault-Spilmont a,∗,1 , Sarah Jane Cookson b , Amélie Sarrazin a , Jean-Luc Verdeil c a
Institut Franc¸ais de la Vigne et du Vin (IFV), Domaine de l’Espiguette, 30240 Le Grau du Roi, France INRA, ISVV, EGFV, UMR 1287, F-33140 Villenave d’Ornon, France c CIRAD-Bios, UMR AGAP, PHIV, TA A-108/02 Avenue Agropolis, 34 398 Montpellier Cedex 5, France b
a r t i c l e
i n f o
Article history: Received 2 December 2011 Received in revised form 18 June 2012 Accepted 29 June 2012 Keywords: X-ray tomography Graft interface Graft quality 3D imaging Vascular connections Grapevine (Vitis vinifera)
a b s t r a c t Successful grafting in plants requires the development of a functional vascular system between the scion and the rootstock. Understanding the spatial organization of the graft interface is important to the evaluation of new rootstock genotypes and to the development of new grafting technologies. Until now the graft interface has only been studied using 2D classical histology and low resolution 3D magnetic resonance imaging. Here we investigate the ability of X-ray tomography to examine the graft interface of Vitis vinifera in high resolution and in 3D. Data were collected using a Skyscan 1076, scanning parameters, such as, X-ray energy, filter selection, pixel size and rotation angles, were optimized to study the particularities of the graft interface. The X-ray tomography technique was then used to evaluate graft quality. Two young vines were compared; one graft was classified as of ‘good’ quality, whereas the other was classified as of ‘bad’ quality. We were able to distinguish the “omega cut”, the pith, the phloem and the xylem vessels in the images. The analysis shows several differences between the two vines. In the good graft, tissues appear well-connected in the wood and phloem, and had a regular structure; the wood appears homogenous with a lot of vessels that form a compact mass. By contrast, in the bad graft, the structures appear disorganized and not completely connected. Numerous new vessels, continuous between the scion and the rootstock, are visible in the “good graft” whereas only few ones are visible in the “bad one”. It is the first time, to our knowledge, that 3D imaging of the graft interface and the vascular connections across it have been reported, opening new avenues for graft quality assessment in woody plants. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Routine grafting of grapevine in the agricultural systems of Europe began at the end of the 19th century to combat the devastating yield losses caused by the introduction of phylloxera from the USA (Ravaz, 1930). This practice has now been adopted in approximately 80% of vineyards world-wide (Pouget, 1990). In contrast, vegetable grafting on a commercial scale is relatively recent, beginning in Asia about 30 years ago and beginning in Europe and the USA in the 1990s (Lee et al., 2010). Despite the generalization of grafting in viticulture and the increase in vegetable grafting worldwide,
Abbreviations: 2D, two dimensions; 3D, three dimensions; GG, “good graft”; BG, “bad graft”; PAS, NBB. ∗ Corresponding author. Tel.: +33 4 66 51 17 54; fax: +33 4 66 53 29 16. E-mail address: [email protected] (A.-S. Renault-Spilmont). 1 These authors have contributed equally to this study. 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.06.045
little is known about the early mechanisms involved in grafting and how the structure of the graft union develops during graft union formation in any plant species. Grafting knowledge and techniques are essentially based on practical experience rather than scientific study. The general cellular events which occur after the grafting are quite well known and common to woody and non-woody plants. Successful grafting is a complex biochemical and structural processes that begins with an initial wound response, followed by callus formation, creation of a continuous cambium and the establishment of a functional vascular system between the two grafting partners (reviewed by Pina and Errea, 2005; Martinez-Ballesta et al., 2010). Grafting success requires numerous developments at the graft interface such as cell recognition and communication, the initiation of cell cycle, cell proliferation, cell differentiation and plasmodesmata formation (Estrada-Luna et al., 2002; Ermel et al., 1997; Pina et al., 2009; Kollmann and Glockmann, 1991). The cellular events at the graft interface have been well
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characterized by histological studies in various woody plants, such as, Picea spp., apples and Prunus spp. (Weatherhead and Barnett, 1986; Soumelidou et al., 1994; Olmstead et al., 2006). These histological studies (using light, confocal and electron microscopy) give beautiful and detailed images of the graft interface, but only in one plane. Understanding spatial tissue organization between the two partners of a graft union is of paramount importance to management and selection of future rootstock genotypes as well as being of scientific interest. To date graft union morphology has been studied with magnetic resonance imaging (MRI) in pine trees (Leszczynski et al., 2000) and in grapevine (Bahar et al., 2010). Unfortunately, these MRI studies are of relatively low resolution and only present 2D virtual sections of the graft interface. Fluorescent dyes and 14 Csucrose markers have been employed to characterize the functional developments of the phloem at the graft interface in tomato but the morphological characterization was also destructive and only provided 2D information (Schöning and Kollmann, 1997). X-ray tomography is a minimally invasive structural imaging method that allows 3D reconstruction of scanned objects (as reviewed by Larabell and Nugent, 2010). It was first used as a medical diagnostic tool in 1970s. It has since been applied to the study of animals and minerals; the use of X-ray tomography began in plants in the late 1990s (e.g. Pierret et al., 1999). X-ray tomography has been applied to the study of the anatomy of stem wood samples of trees (Fromm et al., 2001; Steppe et al., 2004; Longuetaud et al., 2005) and recently to the study of vessel dimensions and intervessel connections in grapevine (Brodersen et al., 2011). Other higher resolution tomography technologies also exist; these technologies can visualize and 3D reconstruct to the single cell level, but only in small plant samples such as seeds (e.g. Cloetens et al., 2006; Smith et al., 2009). Although graft incompatibility is rare in grapevine, grafting success can be very variable (as reviewed by May, 1994). The objective of this study was to evaluate the potential of X-ray tomography to study the internal structures and the 3D organization of graft zone in grapevine. After different tests and optimization of the scanning parameters, the method was applied to young vines with differing degrees of grafting success in order to understand how grapevine tissues and structures organize in response to the grafting. In this manuscript we present the study of the 3D organization of the graft interface in grapevine, this method could open new avenues to assess graft quality in woody plants.
2. Materials and methods 2.1. Plant material and growing conditions 2.1.1. Grafting Vitis vinifera were grafted onto the rootstocks 110R (110 Richter) or SO4 (Selection Oppenheim 4). One-bud cuttings were made for scions and two node de-budded cuttings were made for rootstocks shortly before grafting. Mechanical omega grafting was performed on scion/rootstock pairs of approximately the same diameter. Grafts were briefly dipped into melted wax and settled for callusing for 21 days at 28 ◦ C, 95% humidity. After callusing and rooting, grafted plants were planted in sandy soil in a nursery (Domaine de l’Espiguette, Grau du Roi, France).
2.1.2. Plant material Three types of plant material were used in this study. (1) Two plants of Syrah were grafted onto 110R in April 2009 and were then harvested for immediate analysis 18 months later
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in October 2010 (Supplementary Data 1A). They were used for X-ray tomography and classical histology (microtome) (2) The graft union of a 14-year-old grapevine plant (Syrah grafted onto the rootstock SO4) was collected from the vineyard (Domaine de l’Espiguette Grau du Roi, France) in October 2010 (Supplementary Data 1B) and immediately scanned. (3) Various self-hybrids of Syrah were grafted on the 110R rootstock in the beginning of May 2011 and planted in a nursery (Domaine de l’Espiguette, Grau du Roi, France); grafting success was evaluated in December 2011 (8 months after grafting). Two genotypes displaying differing degrees of grafting success were selected for analysis (Supplementary Data 1C and D). The first vine was selected from a vigorous genotype that showed normal development of the scion and roots: this plant is termed “good graft” or “GG”. The second vine was chosen from a genotype that had reduced scion development: this plant is termed “bad graft” or “BG”. After the plants were harvested, they were pruned (scion and roots) and the graft zones and scions were briefly dipped into wax. The plants were kept in a cold damp room in bags until being scanned. Before the scan, wax was removed from the graft zone, and rootstock was protected by parafilm. After scanning, these two vines were used for histological analysis. 2.2. Histological analysis 2.2.1. Sample preparation After being scanned, young wood samples (8 months and 18 months after grafting) were cut with a band saw in order to obtain serial sections of wood of approximate 5 mm thick. Samples were fixed in a solution of 1% acrolein (v/v) and 4% glutaraldehyde (v/v) in 0.2 M phosphate buffer (pH 7.2) for 72 h at room temperature (according to Buffard-Morel et al., 1992). Samples were then washed with ethanol 70%. 2.2.2. Sample treatment for classical histology The prepared wood samples were dehydrated in a graded ethanol series (70% ethanol for 30 min followed by 90% ethanol for 1 h) and then transferred to ethanol/butanol (50/50, v/v) for 3 days. The samples were then transferred to butanol (100%) for 15 days, before being gradually embedded in a glycomethacrylate resin (Technovit 7100, Kulzer); the samples were placed in butanol/resin (50/50, v/v) for 21 days and then transferred to 100% resin. Samples were conserved at 4 ◦ C, on a shaker for impregnation for a minimum of 3 months. Transverse sections were cut with a thickness of 6 m on a rotary microtome (Leica RM 2255). Sections were double-stained with Periodic Acid Schiff (PAS) and Naphtol Blue Black (NBB) (Fisher, 1968; Buffard-Morel et al., 1992). Materials were mounted using a microscope slide media (Isomont2000, LABOnord) and examined using a Leica DM 4500 B light microscope (Leica, Germany). Images were taken using a camera: Retiga 2000R (QIMAGING FAST 1494, Canada) and Volocity software (Improvision, UK). 2.2.3. Sample treatment for epi-fluorescence microscopy Transverse sections were obtained from prepared wood samples using a vibratome (Microm HM 650V). Then the samples were softened before being cut into successive slides of 30 m and mounted in VectaMount (Vector Laboratories, Inc.). Autofluorescence of these samples was observed using a Leica DM 6000 light microscope (Leica, Germany,) and the filter A (450–490 nm). Images
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were taken using a camera: Retiga 2000R (QIMAGING FAST 1494, Canada) and Volocity software (Improvision, UK). 2.3. High-resolution computed tomography 2.3.1. Acquisition Graft zones were scanned at the Montpellier RIO Imaging Center (France) using a micro-tomography SkyScan 1076 (SkyScan, Belgium), the configuration was adapted from a protocol used to analyze minerals. Electrons, emitted from the source (sealed Xray tube 20–100 kV, spot size < 5 m, 10 W) were accelerated (59 or 70 kV) and focused on an anode where they generate X-ray photons which can be filtered (Table 1). Then, the X-ray photons impacted the sample, which was positioned precisely within the X-ray beam. The X-ray shadow images were acquired by a sensitive X-ray camera (4000 × 2672 pixels, 12 bit, fiber-optically coupled to a scintillator). This produced a longitudinal X-ray photograph through the plant body by moving the X-ray source and the camera in opposite directions during the exposure. Rotation steps were chosen (Table 1) between 0.3◦ and 0.44◦ (over 210◦ ). Scans were made over 2 cm for the 18 and the 8 month after grafting samples and over 8 cm for the 14 years after grafting sample. A typical scan of 2 cm in axial length required 7–50 min and generated 0.5–13 GB of data (Table 1). 2.3.2. Image processing Raw 2D tomographic projection images were reconstructed using NRecon software (SkyScan, Belgium) with the Felkamp algorithm, to create cross-section image stacks (8-bit or 16-bit series, tagged image file). ImageJ software (http://rsbweb.nih.gov/ij/) was used to analyze images (2D, virtual slices) and to measure the development of new wood tissue produced after grafting on the 8 month after grafting samples. In the scion and the rootstock, every 900 m, a region of interest was drawn on virtual transverse section to measure newly formed wood. 3D analysis was done with the Volocity software (Improvision, UK). This software provides 3D surface rendering, and allows us to make virtual cuts (or crops) and to change the image brightness (by changing the thresholds). Pseudocolors can be attributed according to desired parameters, such as, density. The software also provides a specific interface to generate 3D movies (5 frames s−1 ). 3D rendering was also done on Imaris software (Bitplane, Switzerland). It was used to study vascular organization. To identify the vascular organization and the pith, segmentation had been done using threshold on gray level and particle size. Pseudocolors were attributed according structures (e.g. the vessels in green, and pith in red) and a virtual section was added. 3D movies were generated (5 frames s−1 ). Before this visualization, images were treated with ImageJ software: image resolution was reduced to 72 m to allow Imaris to work. 3. Results 3.1. Optimizing the parameters for X-ray tomography at the graft interface in grapevine The use of X-ray tomography to study the graft interface in grapevine began by testing 24 parameters. The most important of which are given in Table 1. Voltage was optimized in order to give the best possible contrast between the plant tissues, air and water; it was 59 kV for 18 months and 70 kV for 14 years after grafting samples. To improve the signal to noise ratio, low filters were chosen to stop X-rays with low energy. Filter selection was an important factor in the acquisition of well-contrasted images (result not
shown). Preliminary investigations of the graft interface of the 14-year-old plant showed that the older plant had greater variations in tissue density. For this reason a slightly thicker filter was required. In this work, three different resolutions were tested (high 9 m, medium 18 m and low 36 m) on the 18 months old plant: illustrations are given in Fig. 1B and C for the highest and lowest resolutions. All parameters were adjusted to produce a suitably contrasted image according to the sample specificity (diameter and size). On the young plant, with a full field of view at 9 m, resolution allowed the entire cross-sectional area of the graft zone to be scanned at a rate of 1 cm of stem every 24 min (at a 36 m resolution 1 cm was scanned every 3 min). The capacity of Skyscan 1076 allowed us to adapt this technology to study a large wood sample (8 cm long × 6.6 cm maximum diameter) using steps of 2 cm in length (in a sample with large differences in tissue density). 3.2. 3D imaging by X-ray tomography as an alternative to classical histology in young tissue Images obtained by X-ray tomography (Fig. 1B and C) can be compared to a classical histology specimen (Fig. 1A). In the classical histology specimen, periodic acid Schiff (PAS) was used to color polysaccharides in red (e.g. cellulosic compounds and starch) and Naphtol Blue Black (NBB) colored proteins in blue (e.g. the proteins of the nucleus). This double staining aided the visualization of the different structures of the wood; the xylem (vessels, rays and annual rings), phloem (vessels and rays), phellogene and phelloderme could be observed. Xylem ray appears purple due to the presence of starch granules. The pith is indicated by the light tones of these large empty cells and the characteristic forms of their cell walls. The phloem is strongly colored in pink/purple, due to the presence of many polysaccharides in cytoplasm and cell wall. The rays of the phloem are aligned with those of the xylem; due to the division of cambium cells (cambium is located between the xylem and phloem). On this section of classical histology, it is possible to observe up to three years of phloem development. In the X-ray tomography images the bright pixels represent the densest tissues that stop/slow down X-ray photons more than the surrounding tissue, therefore, it is the differential Xray attenuation that produces image contrast (Fig. 1B and C). The attenuation of the X-ray photons by a material is dependent on the energy of the X-ray radiation and the following parameters of the material studied: thickness, density and atomic number. For example, oxygen and carbon have slightly different X-ray attenuations; so as a consequence, water and cellulose have different X-ray attenuations that produce different pixel greyscale intensities. With a high resolution of 9 m, structures and tissues can be observed (Fig. 1B). Xylem, phloem, pith, annual rings, ray tissues and pericycle fibers can be identified. We are able to distinguish clearly the early and the late wood depending on the diameters of the vessels. Two growth rings can also be distinguished in the wood and the pith, which appears dark. In the X-ray tomography images, the presence of contaminating paraffin and grains of sand on the outside of the sample can be reduced with the beam hardening correction option. Fig. 1 clearly shows that if the X-ray tomography is done with high resolution (9 m and rotation step of 0.3◦ ), the tissue structure is as visible by tomography as by classical microscopy (low magnification). However, it is the limit of the resolution whereas more details can be observed by microscopy in increasing the magnification. By contrast, the reconstruction of the scans at 36 m
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Table 1 X-ray tomography parameters tested on the graft union of grapevines 8 months, 18 months and 14 years after grafting. Grapevine sample
Sample size (cm)
Scan diameter (cm)
Resolution (m)
Filter
Voltage (kV)
Current (A)
Rotation step (◦ )
Depth (bit)
Duration
Number Of Files
Data
18 months 18 months 18 months 14 years 14 years 8 months “good graft” 8 months “bad graft”
2 2 2 2 8 2 2
3.3 3.3 3.3 6.6 6.6 3.3 3.3
9 18 36 9 36 9 9
Ti 0.025 mm Ti 0.025 mm Ti 0.025 mm Al 1.0 mm Al 1.0 mm Ti 0.025 mm Ti 0.025 mm
59 59 59 70 70 59 59
167 167 167 141 141 167 167
0.300 0.400 0.440 0.300 0.300 0.300 0.300
16 16 16 16 16 16 16
0:49:14 0:11:24 0:06:11 01:30:52 01:48:28 00:48:28 00:48:40
660 498 453 >1294 2859 659 659
13.1 GB 2.46 GB 574 Mo >28.3 GB 6.98 GB 13.1 GB 13.1 GB
resolution with a rotation step of 0.44◦ produced images of insufficient quality to clearly identify the different tissues (Fig. 1C). The month in which the vines were analyzed (October) does not allow us to visualize the cambium, which was inactive and so reduced in size. With the tools used here (scanner and computer), the resolution is lower in X-ray tomography than in classical histology as we are not able to observe the structure at the cellular level. 3.3. The investigation of 14-year-old graft union X-ray tomography on a 14-year-old plant required the reoptimization of the analysis parameters. Preliminary investigations of the graft interface of the 14-year-old plant showed that the older plant had greater variations in tissue density. For this reason a slightly thicker filter was required (Al 1.0 mm). 3D reconstruction of the external surface of the 14-year-old plant revealed numerous details at the grapevine grafting zone, such as, pruning wounds (Fig. 2A). Fig. 2B shows a virtual longitudinal section through the middle of the graft interface shown in Fig. 2A. In this view, in full density, we can observe large differences in tissue density; the densest tissues are colored in white. With this virtual sectioning we can see the young xylem of the rootstock (bottom of image), the scion (the top of the image) and the complex tissues of the graft interface (Fig. 2B). A significant tissue degradation (dark gray) penetrating deeply into the interface between the two partners is observed, producing a deep gap between rootstock and scion (Fig. 2B). Fig. 2C is a whole view of the graft zone in pseudocolor with a threshold application; the densest tissues appear yellow, whereas the less dense appear blue. With this view, the dense organized xylem (colored yellow/green) of the rootstock can be seen on
the bottom right corner of the image and we can see that the xylem vessels span the entire graft interface (Fig. 2C). We can observe the complexity of the new tissue formed during graft union development and especially the complex organization of the vessels established through the graft union between the two partners. Furthermore, details like an old bud on the rootstock can be observed (bottom right of the image). This view clearly shows the connections between the two partners made from the tissue formed after grafting. A movie (Supplementary Data 3) made from the data in Fig. 2 reveals more clearly the details of the graft union in grapevine and the power of X-ray tomography. On the virtual transversal section we can observe that the pith and the central xylem are partially degraded (Supplementary Data 2). Some of the annual rings can also be observed (red arrows). 3.4. Comparative anatomy of differences in graft quality The X-ray tomography method was used to study two 8-monthold grapevines differing in graft quality; this was done in order to examine how the structures at the graft interface organize in a successful and unsuccessful graft. The conditions of the scan were adapted as indicated in Table 1. The main tissues of the graft interface are evident in the virtual longitudinal sections of the GG and BG (Fig. 3). The pith (black), the xylem (gray) with some individual vessels and the phloem (light gray) are visible. In the graft union some vessels are perpendicular to the longitudinal axis (or appear disrupted in their orientation). In both the vines, the “omega cut” is visible. Nevertheless numerous differences can be noted. The diameter of the graft union is much larger in the GG than in the BG, both at the scion and rootstock levels (Fig. 3). In the GG, it is
Table 2 Quantitative evaluation of the wood production on “good graft” and “bad graft” 8 months after grafting, using ImageJ software. “Good graft” Relative positiona Scion
Rootstock
a b
“Bad graft” Stem area (except phloem) (mm2 )
Neo-xylem produced/Total surface (%)b
2100 2000 1900 1800 1700 1600
85.8 87.9 95.2 109.0 134.6 162.4
67% 69% 71% 75% 80% 84%
600 500 400 300 200 100
126.8 105.5 90.7 79.4 73.6 69.9
76% 71% 64% 55% 55% 48%
Row of the slice, from the base of the stack (no units). Surface of the xylem produced after grafting reported to the global surface (without the phloem).
Relative positiona
Stem area (except phloem) (mm2 )
Neo-xylem produced/Total surface (%)b
2100 2000 1900 1800 1700 1600
22.4 21.8 21.2 21.5 22.7 25.6
36% 35% 35% 36% 43% 52%
600 500 400 300 200 100
46.7 44.2 41.7 40.6 39.8 39.0
21% 20% 17% 17% 14% 14%
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difficult to identify precisely the boundary between the rootstock the scion; the phloem appears completely continuous. By contrast, in the BG, the scion is not centered and the piths are misaligned. The omega cut remains very visible as little tissue was produced at the graft union and the connectivity between the scion and rootstock appears incomplete. The graft union of the BG is of low density and is composed of necrotic tissue: this was confirmed by histological analysis (Supplementary Data 4). The same samples of GG and BG were then analyzed in virtual transverse sections at different levels (the scion, graft union and rootstock levels, Fig. 4). It is possible to distinguish the different tissue structures such as the pith, the wood and the phloem in these images. Some xylem vessels and rays are also identifiable and the omega cut can be localized. In the GG, the partners are well aligned as shown by the pith position. The tissues were regularly produced in the graft union inducing a regular structure more or less oval (Fig. 4B). In the contact area, it is difficult to identify the boundary between the rootstock and the scion. The density of wood is very homogeneous and the rays are visible because of the presence of many dense granules aligned inside (especially in the scion and in the graft union virtual transverse sections). These granules correspond to starch as determined by complementary observations in classical histology (Supplementary Data 5). By contrast, in the BG, the scion and the rootstock are clearly misaligned (Fig. 4E) and the numbers of contacts between them remain limited. The wood also appears very different; it is heterogeneous with different levels of gray both in graft union and in the rootstock. The dark gray wood is less dense and corresponds to a partially necrotic wood. This is confirmed by analysis of the same sample with classical histology. In this area, no auto-fluorescence is observed which indicates that composition of the tissue is altered (Supplementary Data 4). In the omega zone, some poorly organized tissues between the scion and the rootstock appear dark gray and are probably necrotic. The xylem vessels appear enlarged in the BG, compared to the GG, even in the rootstock part which is exactly the same genotype. The quantity of wood produced seems greater in the GG than in the BG plant. To be able to evaluate this precisely, measurements of the wood surface area were done on the images at different levels (Table 2). These measurements could not be done in the omega cut/graft union zone because the vessels are in various orientations; this prevents us from precisely localizing the wood produced after and before the grafting. In the two vines, the percentage of the wood produced after the grafting increases near the omega cut and is greater in the scion than in the rootstock. The quantity of wood produced is systematically higher in the GG than in the BG, at all three levels. The new xylem produced after the grafting represents 48–84% of the surface in the GG, whereas it represents only 14–52% in the BG, indicating a significant variation in the cambial activity in these two cases.
Fig. 1. The comparison of transverse sections of grapevine stem tissue made with (A) classical histology (stained with PAS–NBB) and X-ray tomography at (B) 9 m and (C) 36 m resolution. The plant material was harvested from a grafted grapevine 18 months after grafting. The position of the cambium, which is not visible at the time of sampling, is localised between xylem and phloem. Scale bar = 500 m. Xy, xylem; Ph, phloem; R, ray; cambium (yellow arrows); and annual rings (red arrows).
3.4.1. 3D visualization and vascular connections The 3D analysis of plants differing in graft quality was done with the Imaris software, which produced 3D surface rendering and digital images). The virtual dissection of the samples was done to visualize specific tissues and to study the internal structure. Segmentation was performed to highlight the vascular system and the pith; pseudocolors were respectively attributed (Fig. 5). The comparison of surface rendering images shows that the graft union is bigger and more regular in the GG rather in the BG. The scion and the rootstock diameters are also larger in the GG (Fig. 5A and D). The vessel spatial arrangement across the graft union is shown (Fig. 5B and E, Supplemental Data 6 and 7); the omega cut remains visible in both GG and BG. In the GG, many vessels organized in a
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compact mass are visible (Fig. 5B), whereas in the BG there seems to be little connectivity between the rootstock and scion (Fig. 5E). Examples of some vascular connections between the scion and the rootstock were visible (Fig. 5C and F and Supplemental Data 6 and 7). Vessels that form a continuous connection between the two partners correspond to newly formed vessels as shown by their location on the virtual transverse sections (Fig. 5C and F). The resolution used for the image analysis is not sufficient to study vessels individually thus it is not possible to quantify these vascular connections. The low density tissues of the graft interface were also analyzed (Fig. 5C and F). In the GG, the pith is easily identifiable (Fig. 5C), whereas in the BG (Fig. 5F) it is not possible to separate, the pith and the necrotic tissues associated with the omega cut by greyscale level threshold. 4. Discussion 4.1. X-ray tomography as a method to study the 3D structure of the graft interface of grapevines: development on grafted vines of various ages High quality 3D images of the graft interface of grapevine can be produced using X-ray tomography especially for the vines studied 8 months after grafting. The parameter optimization used allowed us to visualize the internal tissue organization of the graft interface, the resolution was close to what classical histology can produce in terms of global tissue organization. Xylem, phloem, pith, ray and necrotic tissues, as well as the omega cut, could be identified in the different vines tested with this technique. We were able to distinguish the main tissues essential to describe a graft union. By contrast, the cambium was never visible. This result is not surprising as the cambium is thinner and very difficult to observe by classical histological methods (i.e. light microscopy) at the period of the year in which the graft interface samples were harvested (in October, the end of the grapevine growing season in Europe). It may also be noted that, at the resolution used here, the smallest vessels are not clearly visible with the X-ray tomography method used. It could be due to the resolution of the images taken or due to the limitations of computer power available. Depending on the size of the object analyzed and the resolution chosen, scans take between 7 and 50 min. As is frequently the case in imaging studies, a compromise must be found between the time necessary to acquire an image and its resolution. The resolution of the images of the graft interface 14 years after grafting had to be sacrificed because otherwise the number of data files produced would have been beyond the computing capacity available to the project. The large size of the graft interface of the grapevine 14 years after grafting required successive scans at 4 levels to capture the entire structure, with a 36 m resolution it took one and a half hours and produced 7 gigabytes of data. Concerning the second part of the study on the 8-month-old vines, a very good resolution was obtained for the scans (which took 48 min each). However, to analyze the tissue organization in 3D using the Imaris software the image resolution had to be reduced to 72 m. This resolution was insufficient to analyze vessels individually: they appear too close to each other. Thus, it was not possible to quantify vessel connectivity. It can be assumed Fig. 2. 3D Reconstruction of the graft union of an old grapevine plant (14 years after grafting) imaged using X-ray tomography at 36 m resolution (using Volocity software): (A) an external view of surface of the graft interface; (B) a full density virtual longitudinal section through the graft interface, the dense tissues are in white and the degraded tissues that has developed between the rootstock and scion are in dark grey; (C) a pseudocolor threshold density view of the graft interface, the
densest tissues are yellow and the least dense tissues are blue. Scale bare = 2.5 cm Gz, graft zone; Ro, rootstock; Sc, scion; Bu: Bud; pruning wounds (red stars), tissue degradation (red triangles) and huge xylem vessels (yellow arrows). Movie of the 3D vascular structure corresponding to varying angles of view can be seen in Supplementary Data 3 (Film 1).
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Fig. 3. Comparative visualization of a (A) “good graft” and a (B) “bad graft” 8 months after grafting: longitudinal sections of the graft union extracted from the 3D X-ray tomography reconstruction. Virtual longitudinal section obtained using ImageJ software. Scale bar = 1 mm. Gz, grafting zone; Ro, rootstock; Sc, scion; , omega; Xyv, xylem vessel; Ph, phloem; P, pith; Sa, sand.
that with the use of a more powerful computer, not requiring a reduction in image resolution, the individual vessels may be studied. 4.2. Application to compare anatomy of a good and a bad grafted union The X-ray tomography method developed here to visualize in 3D the graft interface was applied to study grafting success in young grapevines. Our aim was to understand how grapevine tissues and structures organize in response to the grafting. Two genotypes displaying differing degrees of grafting success were selected for analysis. The first grapevine plant, termed GG, showed normal development of the scion and roots, whereas the second one, termed BG, had reduced scion development. The GG is characterized by a regular structure of large diameter consisting of well connected tissues, both in the wood and the phloem. The quantity of wood produced after grafting is an important indicator of cambial activity. Many starch granules are visible in the xylem rays and numerous continuous vessels between the scion and the rootstock were identified. By contrast, in the BG, the structures appear not so wellorganized and the tissues are not completely joined together. The density of the wood is heterogeneous with necrotic areas of lower density and only few continuous vessels were revealed in the BG. The irregular vessel orientation observed in the BG has also reported in cherries and apples grafted onto dwarfing rootstocks (Soumelidou et al., 1994; Olmstead et al., 2006). The xylem contains fewer vessels in the some examples of dwarfing rootstocks, which could be due to developments at the graft interface (Soumelidou et al., 1994; Olmstead et al., 2006). The union between the vascular elements of scion and rootstock is thought to be the critical event in the formation of a successful graft. The vessel connections were observed in the both BG and GG. Nevertheless there were fewer in the BG, which is coherent with its reduced scion development. The criteria defined here for the bad graft cannot be applied to all the cases of graft failure or
incompatibility; grafting failure is complex and could result from numerous factors. It is the first time, to our knowledge, that the junctions between scion and rootstock have been revealed in 3D. 4.3. Advantages and drawbacks of X-ray tomography compared to classical histology 4.3.1. Sample preparation One practical advantage of X-ray tomography is that no chemical fixation or sample preparation is needed, thus avoiding the complex and time-consuming process of obtaining good histological slides of woody material. In addition, artifacts such as cracks and cell wall damage are often formed during sample preparation for classical histology, especially on woody samples. It can so be used on material difficult or too precious to section for histological examination such as fossil plants (Smith et al., 2009). Unlike classical histology, X-ray tomography is not destructive or invasive so it can be used to image live plants. This method may also be used to follow the kinetics of tissue development at the graft interface or to detect potential flaws in the graft interface in plants used for commercial or scientific purposes. Although X-ray tomography is non-destructive, the potential impact of the ionizing effects of the X-rays on the living tissue has to be considered. Dhondt et al. (2010) observed a growth inhibition of Arabidopsis seedlings that were scanned with X-rays on a daily basis. This technique can also be applied on multiple specimens in identical conditions (image acquisition and analysis), which can allow some degree of automation (Brodersen et al., 2011). 4.3.2. Resolution and image quality of X-ray tomography images In this study the resolution of the images obtained with classic histology techniques was superior to those obtained with X-ray tomography, although they were comparable on a global tissue structure level. It is due to the limit of the scan resolution and the computer capacity for images analysis. The resolution used for the image analysis was not sufficient to study vessels individually.
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Fig. 4. Comparative visualization of a “good graft” (A–C) and a “bad graft” (D–F) 8 months after grafting: transverse sections of the graft union extracted from the 3D X-ray tomography reconstruction. Virtual transverse sections obtained using ImageJ software; sections were taken from the scion (A and D), the graft zone (B and E), the rootstock (C and F). Scale bar = 1 mm. On the insert, each virtual transverse section can be localised inside the volume, visualization with Imaris software (see arrows). Scale bar = 4 mm. Gz, grafting zone; Ro, rootstock; Sc, scion; Xy, xylem; Ph, phloem; S, starch; P, pith.
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Fig. 5. Comparative visualization of a “good graft” (A–C) and a “bad graft” (D–F) 8 months after grafting: 3D vasculature obtained after vessel segmentation. (A and D) External surface views. For tissue segmentation, higher density structures were classified and coloured according to their threshold and their volume: vascular elements are in green (B and E) pith in red and some isolated vascular elements in blue (C and F) (visualization with Imaris software). Scale bar = 4 mm. Animated 3D images of the vascular structure corresponding to varying angles of view for the “good graft” and “bad graft” are in Supplementary Data 6 and 7 respectively (films 2 and 3).
Recent advances in X-ray tomography could overcome this limitation, for example Steppe et al. (2004) used high resolution X-ray tomography to study the anatomy of the wood of beech and oak trees, and Dhondt et al. (2010) studied Arabidopsis hypocotyls with a resolution of 0.85 m that allowed the visualization of individual cells. The use of the synchrotron radiation X-ray tomographic microscopy in modern and fossil plants provides high-quality anatomical detail, up to the cell resolution, comparable to traditional histology (Cloetens et al., 2006; Smith et al., 2009). In the X-ray tomography study performed here, we were able to identify the phloem, the xylem vessels and the pith, but the cambium and the parenchyma were not easily distinguishable. This observation was also made by Brodersen et al. (2011) on both hydrated and dehydrated grapevine stems. The cambium was
not visible here because of the period of sampling. The phloem appears somewhat brighter than the xylem, as these tissues are spatially separated it does not affect our interpretation of the images. Nevertheless, use of some contrasting agents could improve their identification. Dhondt et al. (2010) reported the use of a contrasting agent (such as iodine) to increase the contrast within the soft Arabidopsis hypocotyl tissues, however, as the samples must be fixed and dehydrated it limits the usefulness of the X-ray tomography technique. 4.4. Imaging the 3D structure The great advantages of X-ray tomography are (1) the ability to obtain digital sections in multiple planes, (2) the possibility of
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virtually dissecting a plant to visualize specific tissues and study the internal structure, and (3) the 3D visualization (which is very difficult to obtain by histological techniques). This technique appears much more easy and rapid than the techniques of classical histology. The 3D imaging after reconstruction has been done previously for different woody tissue types (Steppe et al., 2004; Brodersen et al., 2011). 3D data can also be acquired using MRI (magnetic resonance imaging). MRI has been used to study the graft interface in apples, grapevine and in pine trees, but the resolution was very low (Warmund et al., 1993; Leszczynski et al., 2000; Bahar et al., 2010). The small cells of cambium appear bright on a MRI image due to their high water density (due to the absence of air and the absence of a thick cell wall); therefore MRI gives a good indication of cambium development at the graft interface. In addition, MRI is well adapted to the study of sap flows in xylem and phloem vessels (as reviewed by Dhondt et al., 2010) and can be considered as complementary to X-ray tomography as it can provide information about sap flow around and across the graft interface. The development of multimodal imaging, combining X-ray tomography and MRI, would allow us to study not only the 3D structure of the graft interface but also the functioning of the vasculature by examining the saps flows. 5. Conclusion: future potential applications New scion varieties and new rootstock genotypes are currently being selected for and field tested (particularly in terms of their resistance to fungal diseases and challenging climates); however, it is also essential that all new genotypes can be grafted successfully. Understanding the spatial organization of the grafting events is of paramount interest. The potential of X-ray tomography to study the complexity of the graft interface of grapevine in 3D was evaluated. The main important tissues in a graft union (the pith, xylem vessels, phloem and necrotic tissues) and even some compounds such as starch were identified. The main interest of X-ray tomography method is associated with the image analysis; we present a non-destructive 3D visualization of the graft interface that could provide new insights into the spatial tissue organization of the graft interface in grapevine. It is the first time, to our knowledge, that 3D imaging of the graft interface and vascular connections has been reported. This method could open new avenues to study graft quality assessment in woody plants. As this technique is non-destructive and non invasive, it may be of great interest to image live plants and study the kinetics of tissue development on the same graft plant overtime. While the X-ray tomography will not replace systematically classical histology, it does offer an improved technique with the ability to combine 2D analysis in multiple planes and 3D reconstruction. Thus it appears as an ideal tool to understand morphological developments at the graft interface and it could explain grafting success or failure and/or incompatibility in grapevines, but also for many other woody plants. Acknowledgements This work and the position of MM were funded by the ‘Consortium Inter-Régional sur le Dépérissement de la Syrah’ which included the Région Languedoc-Roussillon, the Région Provence Alpes-Côte d’Azur, the Région Rhône-Alpes, FranceAgriMer, InterRhône and the Centre du Rosé. The authors wish to thank the Montpellier RIO Imaging Center and specifically Renaud Lebrun for his helpful advice on Skyscan 1076 use
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and Marc Lartaud for his help on the Volocity and Imaris software.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.scienta.2012.06.045.
References Bahar, E., Korkutal, I., Carbonneau, A., Akcay, G., 2010. Using magnetic resonance imaging technique (MRI) to investigate graft connection and its relation to reddening discoloration in grape leaves. J. Food Agric. Environ. 8, 293–297. Brodersen, C.R., Lee, E.F., Choat, B., Jansen, S., Phillips, R.J., Shackel, K.A., McElrone, A.J., Matthews, M.A., 2011. Automated analysis of three-dimensional xylem networks using high-resolution computed tomography. New Phytol. 191, 1168–1179. Buffard-Morel, J., Verdeil, J., Pannetier, C., 1992. Embryogenèses somatique du cocotier (Cocos nucifera L.) à partir de tissus foliaires: étude histologique. Can. J. Bot. 70, 735–741. Cloetens, P., Mache, R., Schlenker, M., Lerbs-Mache, S., 2006. Quantitative phase tomography of Arabidopsis seeds reveals intercellular void network. PNAS 103, 14626–14630. Dhondt, S., Vanhaeren, H., Van Loo, D., Cnudde, V., Inze, D., 2010. Plant structure visualization by high-resolution X-ray computed tomography. Trends Plant Sci. 15, 419–422. Ermel, F.F., Poessel, J.L., Faurobert, M., Catesson, A.M., 1997. Early scion/stock junction in compatible and incompatible pear/pear and pear/quince grafts: a histo-cytological study. Ann. Bot. 79, 505–515. Estrada-Luna, A.A., Lopez-Peralta, C., Cardenas-Soriano, E., 2002. In vitro micrografting and the histology of graft union formation of selected species of prickly pear cactus (Opuntia spp.). Sci. Hortic. 92, 317–327. Fisher, D., 1968. Protein staining of ribboned epon sections for light microscopy. Histochemie 16, 92–96. Fromm, J.H., Sautter, I., Matthies, D., Kremer, J., Schumacher, P., Ganter, C., 2001. Xylem water content and wood density in spruce and oak trees detected by high-resolution computed tomography. Plant Physiol. 127, 416–425. Kollmann, R., Glockmann, C., 1991. Studies on graft unions. 3. On the mechanism of secondary formation of plasmodesmata at the graft interface. Protoplasma 165, 71–85. Larabell, C.A., Nugent, K.A., 2010. Imaging cellular architecture with X-rays. Curr. Opin. Struct. Bio. 20, 623–631. Lee, J.M., Kubota, C., Tsao, S.J., Bie, Z., Echevarria, P.H., Morra, L., Oda, M., 2010. Current status of vegetable grafting: diffusion, grafting techniques, automation. Sci. Hortic. 127, 93–105. Leszczynski, R., Byczkowski, B., Jurga, S., Korszun, S., 2000. NMR microimaging studies of the union between stock and scion. Appl. Magn. Reson. 18, 147–153. Longuetaud, F., Saint-Andre, L., Leban, J.M., 2005. Automatic detection of annual growth units on Picea abies logs using optical and X-ray techniques. J. Nondestruct. Eval. 24, 29–43. Martinez-Ballesta, M.C., Alcaraz-Lopez, C., Muries, B., Mota-Cadenas, C., Carvajal, M., 2010. Physiological aspects of rootstock–scion interactions. Sci. Hortic. 127, 112–118. May, P., 1994. Using Grapevine Rootstocks – The Australian Perspective. Grape and Wine Research Corporation, Adelaide. Olmstead, M.A., Lang, N.S., Ewers, F.W., Owens, S.A., 2006. Xylem vessel anatomy of sweet cherries grafted onto dwarfing and non-dwarfing rootstocks. J. Am. Soc. Hortic. Sci. 131, 577–585. Pierret, A., Capowiez, Y., Moran, C.J., Kretzschmar, A., 1999. X-ray computed tomography to quantify tree rooting spatial distributions. Geoderma 90, 307–326. Pina, A., Errea, P., 2005. A review of new advances in mechanism of graft compatibility–incompatibility. Sci. Hortic. 106, 1–11. Pina, A., Errea, P., Schulz, A., Martens, H.E., 2009. Cell-to-cell transport through plasmodesmata in tree callus cultures. Tree Physiol. 29, 809–818. Pouget, R., 1990. Histoire de la lutte contre le phylloxera de la vigne en France: 1968–1895. In: INRA, co-publisher Office International de la Vigne et du Vin, Paris, France, p. 158. Ravaz, L., 1930. De l’influence spécifique réciproque du sujet et du greffon – effets de la greffe. Extrait des Annales de l’Ecole nationale d’Agriculture de Montpelllier 20, 1–15. Schöning, U., Kollmann, R., 1997. Phloem translocation in regenerating in vitro heterografts of different compatibility. J. Exp. Bot. 48, 289–295. Smith, S.Y., Collinson, M.E., Rudall, P.J., Simpson, D.A., Marone, F., Stampanoni, M., 2009. Virtual taphonomy using synchrotron tomographic microscopy reveals cryptic features and internal structure of modern and fossil plants. PNAS 106, 12013–12018.
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Soumelidou, K., Battey, N.H., John, P., Barnett, J.R., 1994. The anatomy of the developing bud union and its relationship to dwarfing in apple. Ann. Bot. 74, 605–611. Steppe, K., Cnudde, V., Girard, C., Lemeur, R., Cnudde, J.P., Jacobs, P., 2004. Use of X-ray computed microtomography for non-invasive determination of wood anatomical characteristics. J. Struct. Biol. 148, 11–21.
Warmund, M., Barritt, B., Brown, J., Schaffer, L., Jeong, B., 1993. Detection of vascular discontinuity in bud unions of ‘Jonagold’ apple on mark rootstock with magnetic resonance imaging. J. Amer. Soc. Hort. Sci. 118 (1), 92–96. Weatherhead, I., Barnett, J.R., 1986. Development and structure of unusual xylem elements during graft union formation in Picea sitchensis L. Ann. Bot. 57, 593–598.