Anne-Sophie Spilmont*, Camille Carrere, Yosra Hmedi and Guillaume Mathieu
a Institut Français de la Vigne et du vin (IFV), Montpellier, France.
* Corresponding author: email@example.com
The production of grafted grapevine plant material is a complex process from grafting to final sorting in nurseries. To reach the market in France, grafted grapevines must meet different criteria including a manual graft union test entitled “thumb test” that is used to evaluate the mechanical resistance of the junction. This test depends on each person and is therefore subjective. The aim of this study was to evaluate the possibility of using internal criteria, quantitative and objective, to select good quality grafted grapevines instead of the current criteria.
The scanning parameters were first optimized to allow analyzing an important number of vines while maintaining sufficient resolution for imaging analysis. This study was done on two batches with different degrees of grafting. 60 to 80 grafted vines were randomly selected in these two combinations to be scanned before being assessed with the regulatory criteria, including the thumb test. Many variables concerning the graft union and the anatomy of both the scions and the rootstocks were measured for all the vines. The comparison was then made between batches of plants that pass or do not pass the “thumb test”. Two internal criteria related to the wood production and the volume of “air and necrosis” in the Omega interface appeared to be discriminatory and so relevant. This opens prospects for the use of these methods for graft quality assessment in the medium term.
Article extracted from the presentation held during Enoforum Web Conference (23-25 February 2021)
Grafting is currently used in almost all wine-growing countries. Grafting provides not only resistance to soil-borne pests but can also improve adaptability to various soils and environment, and as such is also used to improve fruit yield and quality (Ollat et al., 2016).
The production of grafted grapevine plant material is a complex process with many steps running over one year, from grafting to final sorting in nurseries. Even in scion/rootstock combinations which normally graft well, the development and longevity of grafts can vary, which suggests that there may have differences in terms of “quality” in the marketed plants. There is no clear definition of what constitutes a high-quality grafted grapevine plant. Waite et al. (2015) suggested that high quality grafted grapevines must be undamaged, without physical defects and present fully healed graft unions underlying the importance to have precise tools to evaluate it.
To reach the market in France, grafted grapevines must meet three criteria by law: resistance to a manual graft union test, a minimum number of three roots and a woody, lignified stem of at least 2 cm long (https://www.legifrance.gouv.fr/). Amongst these criteria, the one concerning the graft union solidity is the most subjective as this test is currently realized manually (by the “thumb test”) and can so vary greatly between different people and over time.
Successful grafting in plants requires the development of a functional vascular system between the scion and the rootstock. This is supposed to be evaluated by the defined criteria indicated above which are related to the junction and the good development of the new grafted vines from the point of view of both roots and shoots. Nevertheless, many questions remain unanswered such as how is really organised the junction? is the thumb test a good test to evaluate the connections between the partners? … Since several years, medical imaging, and especially X-Tomography, appeared to be of great interest to study anatomy and 3D organisation of the plants (Mc Elrone et al, 2013; Wason et al., 2017). This method was also used onto vines to characterize xylem vessel refilling and connections (Brodersen et al, 2011; Brodersen et al. 2018) and to describe the graft union of few vines (Milien et al, 2012). The objectives of this study were to i) improve knowledge of what distinguishes plants that pass or fail the test and ii) identify internal relevant criteria to select good quality grafted grapevines.
Materials and Methods
Two scion/rootstock combinations with different degrees of grafting success (i.e. producing different proportions of marketable plants after one year in the nursery) were used in this study. 200 vines per batch were bench omega grafted in March 2019 then sorted from the nursery in December 2019, to be analyzed. 60 to 80 grafted vines were randomly selected in these two combinations to be scanned by RX-microtomography before being assessed with the regulatory criteria, including the thumb test.
High resolution computed tomography acquisition and 3D reconstruction
Grafted vines were scanned at the Montpellier RIO Imaging Center (France) using EasyTom 150 RX Solution (Figure 1). The conditions were the same as described by Milien and al (2012) with some modifications to reduce the time of analyze and allow high-speed acquisitions.
The Electrons emitted from the source (sealed X-ray tube 40-150kV, spot size < 5um, 75W) were accelerated (50kV) and focused on an anode where they generate X-ray photons which are filtered with an aluminium filter (1mm of thickness) to reduce the X-Rays intensity and preserve the plant survival. Each projection was magnified through a series of lenses and relayed onto a 1758x1242 pixel plan captor camera. Then, algorithms were used to transform radiographies into virtual slices. Raw tomographic projection images were reconstructed into approximately 1250 sample TIF image slices using XAct software with an algorithm to create cross-section image stacks (8-bit or 16-bits series).
Images were viewed and analyzed using ImageJ and Imaris softwares (Bitplane, France).
Figure 1: X-ray Tomograph EasyTom 150 used for the study; Montpellier RIO Imaging Center (Montpellier, France)
Identification of internal criteria to measure
The preliminary observations done onto grafted vines that pass or not the thumb test allowed us to define some criteria of interest. In Figure 2, two types of grafted vines are compared.
Figure 2: Virtual longitudinal section in the graft union extracted from 3D X-ray tomography reconstruction. Comparative view of a vine that pass (A) and does not pass (B) the thumb test. Air and necrosis part in the graft junction appear dark (not dense to the X-rays).
Some differences are visible in the graft junction where more “air and necrosis” are observed (in black) in the rejected vine (B: broken junction after thumb test). By contrast the diameter of the vine (indicated as <-->) appears larger in the vine that pass the thumb test (A).
Specific Macro tools were developed to measure i) the volume of the different tissues in the scion and in the rootstock and ii) the volume of the junction with the respective part of the “necrosis + air” area.
Development of specific methods for image processing
For the calculation of necrosis/air volume and healthy tissues in the omega zone, distances and volumes were calculated based on the x, y, and z coordinates of individual voxels using ImageJ software.
The stack was recroped to study a defined part of the scion and the rootstock. For both regions of interest, the volumes of the pith, the initial wood (xylem II) and the xylem produced post-grafting were calculated with the Macro specifically developed (Figure 3).
Figure 3: Xylem produced post grafting in the scion extracted from 3D X-ray Tomography reconstruction with Image J and Imaris softwares.
(A): Longitudinal and volume views: the volume of the new xylem II produced post grafting is represented in green.
(B): Transverse section with the different rings of wood (=xylem II).
Two other criteria concerning the grafting junction were also measured such as the global volume of the grafting area and the amount of residual air and necrosis part in this junction. A specific Macro was developed to delimit the junction area, then automatically calculate both the volume of the Omega Zone (as shown in Figure 4A) and the volume of “air and necrosis” in this area (in blue in Figure 4B).
Figure 4: Omega interface in the grafted vine
A: The Omega junction (in yellow) in positioned in the 3D view of the vine.
B: The limit of the Omega junction is represented in green and “air and necrosis” inside are represented in blue (3D view)
Two relevant criteria identified
After tomographic acquisition, each grafted vine was tested with the reglementary criteria including the thumb test. Then the macros were applied to obtain the measures on all the previously defined criteria.
The results concerning the quantity of xylem produced post-grafting in the scion is indicated in figure 5.
Figure 5: Quantity of xylem produced post-grafting related to the thumb test result, in the scion of the two combinations (OK: vines that pass the thumb test; C: vines that does not pass the thumb test)
The quantity of xylem produced post grafting in the scion is more important in the plants that pass the thumb test for both the combinations studied.
The results concerning the proportion of “air and necrosis” in the omega interface is indicated in figure 6.
Figure 6: Proportion of « air + necrosis » within the omega junction related to the thumb test result in the two combinations (OK: vines that pass the thumb test; C: vines that does not pass the thumb test)
For each combination we can observe a significant difference between the vines that pass or do not pass the thumb test. The grafts that pass the thumb test have significatively less “air and necrosis” in the junction area. Moreover, a threshold may be defined to separate these vines which may be of specific interest from a practical point of view.
Conclusions and perspectives
In this study we were able to characterize more precisely what distinguish plants that pass or fail the thumb test. Two inner criteria appeared discriminatory and so relevant to select good quality grafted grapevines. In medium term, X-ray Tomography might help or replace the manual sorting of the grafted vines. This opens prospects for improvement of graft quality assessment and, as a potential result, the durability of the vineyards.
From a more general point of view, imaging methods, as non-destructive tools can give very important information to undestand all the complex events during the grafting process. These innovative techniques open also new practical perspectives to help nursery men to identify the key points of the callusing process or to evaluate different grafting methods.
The authors would like to thank the “Plan National Dépérissement du Vignoble” for funding the ORIGINE project (FranceAgriMer-22001149-00001505). The authors wish a special thanks to the personnel of the Plant Material Department at the Espiguette Vineyard (Le Grau-du-Roi, France) for all the grafting and nursery work. 3D data acquisitions were performed using the µ-CT facilities of the MRI platform member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»), and of the Labex CEMEB (ANR-10-LABX-0004) and NUMEV (ANR-10-LABX-0020).
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