Does a graft located in the canopy of a rubber tree affect the morphologies of cells in the adjacent wood?

Abstract

The objective of this study was to characterize the wood anatomical structure of a rubber tree clone, under the influence of two different canopy grafts. The following rubber trees were selected in the system of a double-grafted PB 311 + FX 2784 and PB 311 + MDF 180. For each tree, discs of wood were cut from the affected branch immediately below the insertion of clone at right angles to the axis, from which the regions corresponding to tension, in opposite and normal wood, were identified. The anatomical analyses were conducted in accordance with the standards established by the International Association of Wood Anatomy Committee. The Kruskal-Wallis nonparametric test was applied for multiple comparisons among the types of woods and radial positions studied, at 5% of significance. Still, multivariate associations were assessed among the anatomical characteristics of both double-grafted rubber trees, by means of a two-step cluster analysis. Quantitative morphological differences were observed in the wood cells of the double-grafted studied clones. The ray height and the vessels diameter were the most important morphologic characteristics for the distinction. The canopy clone exhibited the ability to modulate the quantitative anatomical characters of the panel clone, depending on the plant’s needs.

Full Article

Does a Graft Located in the Canopy of a Rubber Tree Affect the Morphologies of Cells in the Adjacent wood?

Glaycianne Christine Vieira dos Santos,^a,* João Vicente de Figueiredo Latorraca,^b Lívia Fernanda Lavrador Toniasso,^c Letícia Maria Alves Ramos,^a José Henrique Camargo Pace,^a Sabrina Mayer de Almeida,^d and Thayanne Caroline Castor Neto ^a

The objective of this study was to characterize the wood anatomical structure of a rubber tree clone, under the influence of two different canopy grafts. The following rubber trees were selected in the system of a double-grafted PB 311 + FX 2784 and PB 311 + MDF 180. For each tree, discs of wood were cut from the affected branch immediately below the insertion of clone at right angles to the axis, from which the regions corresponding to tension, in opposite and normal wood, were identified. The anatomical analyses were conducted in accordance with the standards established by the International Association of Wood Anatomy Committee. The Kruskal-Wallis nonparametric test was applied for multiple comparisons among the types of woods and radial positions studied, at 5% of significance. Still, multivariate associations were assessed among the anatomical characteristics of both double-grafted rubber trees, by means of a two-step cluster analysis. Quantitative morphological differences were observed in the wood cells of the double-grafted studied clones. The ray height and the vessels diameter were the most important morphologic characteristics for the distinction. The canopy clone exhibited the ability to modulate the quantitative anatomical characters of the panel clone, depending on the plant’s needs.

Keywords: Canopy graft; Tension wood; Wood anatomy; Rubber tree; Radial variation

Contact information: a: Programa de Pós-Graduação em Ciências Ambientais e Florestais, Instituto de Florestas, Universidade Federal Rural do Rio de Janeiro, Seropédica/RJ, Brazil; b: Departameto de Produtos Florestais, Instituto de Florestas, Universidade Federal Rural do Rio de Janeiro, Seropédica/RJ, Brazil; c: Plantações Michelin da Bahia LTDA, Igrapiúna/Ba, Brazil.; d: Graduação em Engenharia Florestal, Instituto de Florestas, Universidade Federal Rural do Rio de Janeiro, Seropédica/RJ, Brazil;

* Corresponding author: annechristine.santos@hotmail.com / ORCID 0000-0002-1722-4073

INTRODUCTION

The rubber tree (Hevea brasiliensis (Willd. ex Adr Jussieu) Muell. Arg.) is a native species of the Amazon rainforest, and its economic exploitation is based on the latex obtained, starting at 5 to 7 years and extending for 25 to 30 years (Rahman et al. 2013). The bleeding technique improvement and the use of clones has enabled the rubber tree planting to be successful in the market, guaranteeing the planting uniformity regarding the vigor, bark thickness, latex production and properties, nutrition, and tolerance to diseases (Gonçalves and Marques 2008).

The proliferation of the fungus causing the “South American leaf blight” (Microcyclus ulei (P. Henn) v. Arx) in the areas of naturally occurring rubber trees motivated the double-grafting development, that is, formed by three distinct genetic materials, by means of the double-grafting technique (Moraes et al. 2013). The double-grafting technique consists of initially performing a basal grafting using a high productivity latex clone (panel clone), from which bleeding panels (for latex extraction) will be established, and a material of broad genetic base, originating from seeds, as rootstock. Subsequently, between eight and twelve months after the first graft, a second graft is performed using a “South American leaf blight” resistant material (canopy clone) replacing the canopy of disease-susceptible clones. Thus, it was sought to gather in the same material, high productivity aspects of latex and the resistance/tolerance to the South American leaf blight. The research with double-grafted rubber trees were directed to the performance evaluation of these trees in terms of vigor, growth, and the grafting success, aiming to select clones compatible among themselves (Miranda 2000; Moraes 2000; Moraes and Moraes 2004; Moraes et al. 2011, 2013). However, the effect of canopy grafting on the anatomical structure of the panel clone has not been addressed in the literature, justifying the relevance of this study.

The wood cell morphology of tree species may be changed in the light of structural adjustments arising from the plant’s needs, environmental conditions, and silvicultural treatment (Naji et al. 2011, 2013a; Rita et al. 2015). This response sensitivity is responsible for inter- and intraspecific wood variation. Studies with different combinations of canopy/panel showed that the canopy graft can modify the latex characteristic of the panel clone (Moraes and Moraes 2004). This finding, associated with the structural plasticity of plant species, promoted the following question development “Do canopy grafts change the cell morphology of the panel clone wood?”.

In Brazil, at the end of the latex extraction period, the rubber tree wood is commonly intended to be used in the energy sectors due to, among other factors, to its low natural durability. However, with proper treatment, the same wood presents potential for use in the furniture industry. Nevertheless, the use of wood for nobler purposes can be affected due to the recurrent presence of reaction wood (Ratnasingam and Ma 2015), the development of which is associated with the cambium uneven growth, changes in the xylem cell morphology, and chemical and ultrastructural changes of the fiber’s secondary wall (Mellerowicz and Sundberg 2008). In hardwood, this wood is called tension wood, once it is formed on the side where the tension forces are requested in the wood (upper side to the stem inclination). The formation of this type of wood is associated with the mechanical and environmental tensions, such as the winds action, topography, and the canopy asymmetry (Ruelle 2014).

From macroscopic analysis, the reaction wood was observed by the pith displacement in relation to the geometric center of the log (Déjardin et al. 2010; Sultana et al. 2010) and the more widely spaced growth rings (Hillis et al. 2004). In freshly cut timber, the tension wood can be highlighted by a clear and bright coloration (Badia et al. 2005; Vidaurre et al. 2013).

In microscopic analysis, it is possible to observe changes in the vessel size and number, which are reduced in the tension wood region (Hiraiwa et al. 2007). However, for some species, the main anatomical differences are related to the fibers, which have an internal gelatinous layer (G layer) formed during the cell wall development of the fibers (Clair et al. 2011). This extra layer contains high levels of highly crystalline cellulose (approximately 10 to 20% more in comparison to normal wood), whose microfibrillar angle is oriented parallel to the fiber axis (in contrast with the helical provision in the secondary layers) (Patten et al. 2007).

The presence of tension wood can cause problems in wood processing and use. During the drying process, defects such as deformation, torsion, bending, and cracking are developed and are associated, in particular, with the contraction characteristics, which differ when compared to normal wood (Sultana and Rahman 2013). Identifying, understanding, and analyzing the characteristics of rubber tree wood and the mechanisms involved in the tension wood formation, can provide subsidies for the plantations planning, aiming the best use of the wood at the end of the latex extraction period.

The purpose of this study was to characterize the anatomical structure of the wood panel clone of Hevea brasiliensis (Willd. ex Adr Jussieu) Muell. Arg. (PB 311), under the influence of two different canopy grafts. The study was conducted on the basis of quantitative anatomical characters present in the panel clone wood, whose objectives were: (1) to characterize the radial variation (direction pith-cambium); (2) to analyze the variation in tension, for opposite and normal wood; and (3) to analyze the canopy graft influence on the rubber tree panel clone anatomy.

EXPERIMENTAL

Materials

The clones of Hevea brasiliensis used in this study were granted by the company Plantações Michelin da Bahia LTDA, in experimental plantations located in Igrapiúna (Bahia/Brazil (13°48’51″S, 39°8’54″W), with spacing 8 x 2.5m.

Selection and wood samples collection of the rubber tree clones

Rubber trees were selected in a double-grafted system with variation in the canopy graft, aged 24 and 21 years, respectively: (1) root from seeds + clone PB 311 (panel) + clone FX 2784 (canopy); and (2) root from seeds + clone PB 311 (panel) + MDF 180 clone (canopy). The base and second grafts were performed at 8 to 9 months and 1.5 to 2 years of age (approximately 2.20m in height), respectively. Information on parental clones and country of origin of the respective clones are presented in table 1. For each situation, three trees were sampled. All the trees were stimulated with ethephon 4% (Ethrel ® 720, Bayer Crop Science, Research Triangle Park, NC, USA) aiming at the latex production. The choice of this double-grafted system was justified by the uneven diameter growth visual analysis among the clones of MDF 180 canopy and PB 311 panel (Fig. 1).

Table 1. Parental Clones and Country of Origin of the Hevea brasiliensis Clones Selected for the Present Study

MDF = Madre de Dios Firestone; FX = Ford Crossing; AVROS = Algemene Vereniging Rubber planters Oostkust Sumatra; PB = Prang Besar; RRIM = Rubber Research Institute of Malaysia. Source: Mattos et al. (2003).

From each tree, discs of wood were cut from the affected branch immediately below the insertion of canopy clone at right angles to the axis (Fig. 2). Samples of this material were deposited in the wood collection of the Forests Institute of the Federal University of Rio de Janeiro, with the following record numbers: 7714, 7715, 7716, 7717, 7718, and 7719. From the observation of the eccentric pith presence, specimens were observed in three radial regions covering the tension, of opposite and normal woods.

Fig. 1. Double-grafted trees of Hevea brasiliensis selected for this study. a: root from seed + PB 311 clone (panel) + FX 2784 clone (canopy). b: root from seed + PB 311 clone (panel) + MDF 180 clone (canopy). Plantações Michelin da Bahia LTDA, in plantations located in Igrapiúna, BA.

Fig. 2. Schematic drawing of obtaining wood discs and cross-sectional diagram of the trunk illustrating regions sampled for anatomical analysis. TW, OW, and NW = tension, for opposite and normal wood, respectively; PTW, POW, and PNW = pith of tension, for opposite and normal wood, respectively; MTW, MOW, and MNW = middle section of tension, for opposite and normal wood, respectively; CTW, COW, and CNW = cambium region of tension, for opposite and normal wood, respectively

Methods

Wood anatomy

For the histological cuts, transverse and longitudinal sections (radial and tangential) of 18 μm thickness were obtained in a microtome slide (MICRON HM 450) from the specimens. Then, the transverse sections were subjected to double staining with astra blue and safranin, in the proportion 9.5:0.5 (Bukatsch 1972). The lignified structures interact with safranin, acquiring red coloration, while the cellulosic structures react with the astra blue and, therefore, acquire blue coloration. This staining process allowed for the identification of the gelatinous fibers, rich in cellulose, often present in the tension wood. The longitudinal sections were subjected to safranin staining at 1% (Johansen 1940). Such cuts were used for the manufacturing of semi-permanent slides (Purvis et al. 1964). To assemble the permanent slides, the cuts after staining, were dehydrated in an alcohol series (20, 40, 60, 80, and 100%), treated with ethyl acetate, and set in resin.

For each specimen, wood fragments were selected in the direction of the fibers, for the tissue dissociation. The dissociation was performed according to methodology described by Franklin (1945), with changes in the temperature (70 ºC) and dissociation time (8 h). The fragments were stained with safranin at 1% and used for the semi-permanent slide manufacturing.

The histological cuts and dissociated tissues were employed in a quantitative microscopic study of the following anatomical characters: tangential diameter (µm), frequency (vessels/mm²) and length (µm) of the vessel elements; height (µm), width (µm), and frequency (radius/mm linear) of the rays; length (µm), total diameter (µm), and fiber wall thickness (µm).

All measurements were performed according to the standards established by the International Association of Wood Anatomy Committee (IAWA 1989). The image capturing was performed by means of a high-resolution camera coupled with an optical microscope Olympus CX40 connected to TSView software 6.2.4.5 (Tucsen Imaging Technology Co., Limited, Fujian, China). The images were analyzed in the software Image-Pro Plus^® 4.5.0.29.

Proportion of gelatinous fibers

For the determination of the proportion of gelatinous fibers, cross-sectional images containing such elements were analyzed in the Image-Pro Plus software using the “count/size” command (Kataria et al. 2012). The proportion was obtained by subtracting the blue component of the images (gelatinous fibers) from the red components (other lignified structures), manually demarcated (Purba et al. 2015).

Statistical analysis

After verifying the absence of normality in the residues (Shapiro-Wilk test, at the level of 95% confidence), the nonparametric Kruskal-Wallis test was performed, followed by the Bonferroni method for multiple comparisons among the types of woods and radial positions studied, both at 5% of significance. These analyses were performed using the statistical package Action Stat 3.2.60.1118. Multivariate associations were assessed among the anatomical characteristics of both double-grafted rubber trees, by a two-step cluster analysis. The grouping was performed according to the Bayesian Information Criterion (BIC), using for distance measured the log-likelihood. The significance of the variables within each cluster was determined by means of the Bonferroni test T-Test, at the level of 95% confidence. To do this, the statistical package IBM® SPSS® 20.0 was used.

RESULTS AND DISCUSSION

Wood Anatomy Features

No qualitative differences in the anatomy of the wood panel clone PB 311 was observed in the evaluated double-grafting systems, so the description below is valid for both cases.

Growth ring boundaries: These were slightly distinct and were possibly demarcated by fibrous areas.

Vessels: These showed that the wood was diffuse-porous. These were solitary vessels and were in radial multiples of 2 to 6, occasionally forming clusters, circular to oval section. There were simple perforation plates and the presence of appendages, with varying sizes, often at both ends. The inter-vessel pits were alternate; the vessel-ray pits had very reduced borders to apparently simple ones; the pits were rounded or angular. The tangential diameter was 71.24 µm to 371.39 µm. The vessel frequency (vessels/mm²) was 1 to 31. The vessel element length was from 536.52 µm to 1060.91 µm. There was a presence of common (Fig. 3A and 4B/D) and occasionally sclerotic tyloses in the region near the pith (Fig. 3E and 4C/E).

Fibers: These were non-septate, with thin-to-thick-walls, and with a length of 1102.40 µm to 1924.91 µm and simple pits. There was a presence of gelatinous fibers (Fig. 3A/B).

Axial parenchyma: This showed banded parenchyma reticulate.

Rays: The rays were numerous, ranging from 4 to 14 rays / linear mm. They were multi-serious with a width of 1 to 5 cells (Fig. 3C/D/E), They showed a heterogeneous cellular composition with procumbent body ray cells with mostly 2 to 4 rows of upright and/or square marginal cells (Fig. 3F). There was a presence of aggregate rays.

Mineral inclusions: There were calcium oxalate prismatic crystals present in the upright and/or square ray cells, in the axial parenchyma cells (occasionally forming short chains), and in the tyloses (Fig. 3D and 4D/E).

Both the double-grafted rubber trees presented vessels obstructed by common tyloses and occasionally sclerified in the region close to the pith. The obstruction can occur in a natural way, with the sapwood formation, or in response to biotic and abiotic stresses (attacks of pathogens, mechanical injuries, drought, or frost) (Feng et al. 2013; Dufraisse et al. 2017; Lesniewska et al. 2017; Pérez-de-Lis et al. 2018). The tyloses location in the material studied indicates that the formation happened naturally with the xylem aging. A wide variety of organic and mineral components may be contained in the tyloses formation process, among which stand out: gums, resins, starch, crystals, and phenolic compounds (De Micco et al. 2016). In this study, the presence of prismatic crystals of calcium oxalate associated with tyloses was identified.

Proportion of Gelatinous Fibers

The presence of gelatinous fibers in all the wood for both the studied double-grafted rubber trees was observed (Fig. 5). Among the double-grafted trees, the PB 311 clone under the influence of the FX 2784 canopy graft presented higher proportions of these elements in all radial positions, except in the region near the pith of the normal wood. The double-grafted PB 311 + FX 2784 showed a higher proportion of fibers in the normal and opposite wood, which were concentrated in the middle and cambium regions, respectively.