Observing the Structure Diversity of Historic Heirloom Apple Tree (Malus domestica Borkh.) Wood in Central Slovakia

Abstract

There is a vast diversity of historic heirloom apple varieties around the world. Apples of various colors, sizes, tastes, uses, and ripening times can be found. In Slovakia, there has recently been a rising interest to preserve these apple varieties. The life of a tree is partially influenced by the growing conditions of the environment where the tree grows. In this paper, macroscopic and microscopic characteristics of four selected heirloom apple trees growing in Slovakia’s Štiavnické vrchy were observed. Studying the microscopic structure of historical heirloom apple trees wood could reveal specific cell arrangements of tissues. This could help to specify the individual varieties.

1. Introduction

Apple trees of heirloom cultivars are valuable from a genetic aspect in the selection of new fruit tree cultivars [1]. They are one of the most important and favorite fruit trees cultivated in cold and temperate climates [2]. Every country has a great variety of local heirloom apple varieties (also called traditional apple cultivars). The number of currently known apple varieties in the world exceeds 10,000, although the number of cultivated varieties is significantly lower [2]. Each variety has specific characteristics, such as blooming onset, ripening, resilience to diseases and frost, size of the apples, their taste, smell, color, use, and preserving and storage time [3]. Some historic and local heirloom apple trees are rare, with grafts and samples precious to obtain.

Cultivating apple trees has an abundant history in Slovakia. In recent years, more and more people have searched for grafts of heirloom varieties to save them from disappearing [2]. Apple trees can tolerate various climates. The best yield and best quality of the fruit was grown in altitudes approximately 350 m above sea level (MASL) in the former Czechoslovakia [3]. In some locations and when choosing the right cultivar, good yields can also be grown in altitudes of 400–500 MASL [3]. Historic heirloom apple trees are usually undemanding, resistant to diseases and frost, and do not require the regular application of chemicals such as pesticides or insecticides. Climate change is adversely affecting newer commercial apple varieties, while heirloom diversity shows more resistance to the negative effects of weather, pests, and diseases [4]. The study [4] on climate change and apple diversity showed that growers observed an increased occurrence of spring freezes after a mild winter and warm spring that resulted in early-bloom onset as the greatest impact on apple growing.

Apple trees provide not only delicious, healthy fruit but also wood. Fruit trees must be pruned regularly to maintain a healthy growth [3]. Because of pruning, plenty of branch wood is produced yearly. There is a sizeable portion of waste wood after the winter cut of orchards and vineyards in production areas [5]. This waste wood has the potential to be used for various purposes.

According to research [5] on the energy potential of the wood biomass of orchards in the Czech Republic, apple tree wood and other fruit tree wood show a considerable energy potential. The net calorific value of the fruit tree wood mass is comparable to the net calorific value of spruce bark, softwood, or brown coal [5]. This research [6] investigated the suitability of using fruit tree branch wood in particleboard production. Results showed that the addition of chips from branches in the core of three-layer experimental particleboards resulted in up to 50% of the total amount of wood chips being considered successful, since no appreciable effects on the properties of the boards were observed [6].

In addition, cultivated tree species were employed to create common objects [7]. The study [7] examinated 701 samples from 337 historical farming tools from museum specimens. The team identified 19 wood species. Pear fruit tree wood (Pyrus species) was found as the teeth of a rake, head of an ash rake, and the beater of a flail. Freight sledge was found to be made from plum fruit tree wood (Prunus species), specifically, the runners of the sledge and pieces of the bearing construction [7].

To understand more about the physical and mechanical properties of wood, we must research its microscopic structure. The microscopic structure provides answers to many questions about wood properties and growth conditions of the tree. In this paper, we selected branch-wood samples from four heirloom apple trees and studied the cross-sections under a light microscope. According to [8], apple tree wood is diffuse porous with homocellular uniseriate to biseriate rays with a cell height of 12–18 cells.

2. Material and Methods

The altitudes, locations, and coordinates of each tree are listed in

Table 1. Locations of apple trees.

Sample	Town/Village	GPS Coordinates		Altitude (m)	Branch Age
		Latitude	Longitude
M1	Vyhne–Ovadovo	48°30′12.528″ N	18°49′1.9914″ E	555	12
M2		48°30′25.884″ N	18°48′34.307″ E	546	9
M3	Hliník and Hronom	48°32′10.737″ N	18°47′19.540″ E	244	3
M4	Repišťe	48°30′6.7314″ N	18°50′32.676″ E	610	4

. Pictures of all trees, their bark, apples, and leaves were taken and are shown in Figure 1. The apple trees are in the north-western part of Štiavnické vrchy (Štiavnické Mountains).

Exact varieties of the selected apple trees were researched in various pomology books [2,3,9,10]. Perfect matches were not found and all selected apple trees might be local heirloom varieties. The closest apple varieties are as follows: M1 could be a Gaesdonker Renette, M2 could be a “Londýnske” (literal translation is Londoner), and the M3 variety was White Transparent Apple. Growers of the M3 tree cut the branches and used the trunk as rootstock for M2 grafts (scion) in 2017. The area showing the graft on M3 is shown in Figure 1, picture 3b. M4 did not yield any apples this year, so it was not possible to determine an apple variety. M1 and M2 trees grow in a maintained orchard, M3 grows in a garden, and M4 grows in an abandoned orchard.

Only one tree per apple variety was used in this experiment, as there were not more trees to obtain samples from. Branch-wood samples were cut from the lower areas of the canopy. The branch-wood samples were obtained after pruning in early spring. One branch from one tree was cut. Collected branches were either 3–4 cm in diameter or had at least three growth rings. For the macroscopic observation, one 1 cm-thick block was cut and sanded until the surface was smooth to touch and suitable for macroscopic observation. Small blocks with dimensions of 3 × 3 × 7 mm were marked in each branch block and were cut out. One small block from each branch block was cut. The small blocks were embedded in epoxy resin. These resin-wood samples were cut into 15 μm-thick microsections on a sledge microtome (Reichert, Wien, Austria). The microsections were stained with three stains: Toluidine, Safranin, and a combination of Astra Blue and Safranin. These stains are among the most important for wood anatomists; they are widely used and call for minimum reagents and techniques [11]. After staining, the microsections were mounted in Euparal (BioQuip Products Inc., Rancho Dominguez, CA, USA).

3. Results and Discussion

The macroscopic observation showed interesting results (Figure 2). Significant macroscopic characteristics in bark and growth ring width were observed despite a small difference in the altitudes of the selected trees. The M3 altitude was the lowest among the selected trees, with the town where M3 grows located at the foot of Štiavnické vrchy, in Žiarska kotlina (Žiarska Basin). The villages where M1, M2, and M4 are located are in mountain valleys. Even though the M3 tree is grafted from the M2 tree, the differences are obvious. M3 had wider growth rings and thinner bark than M2. In comparison to M3, M2 and M1 had narrow growth rings, While M4 had the second-widest growth rings. Among the selected apple trees, M1 had the thickest bark, M2 and M4 had similarly thick bark, and M3 had the thinnest bark.

Microscopic observation of the selected heirloom apple trees also showed interesting results. The results are shown in Figure 3, with the numbers in the individual figures corresponding to the nomenclature in Table 1. Sample M1 had the least distinctive growth ring boundary (Figure 3, M1gr), even though the growth rings were macroscopically visible and distinct (Figure 2, M1). The other three samples had a thicker layer of libriform fibers at the terminal zone of the growth rings. The most distinctive growth rings were found in sample M4 (Figure 3, M4gr). In samples M2 and M4, the libriform fibers at the terminal zone of the growth ring have thicker cell walls than the libriform fibers at the initial zone of the growth ring. This is denoted by black arrowheads (libriform fibers with thin walls) and by white arrowheads with a black outline (libriform fibers with thick walls). Sample M3 had wide growth rings. The growth rings of the selected branch-wood sample were up to 8 mm thick. Hence, only the growth ring boundary is shown at a smaller magnification in Figure 3, M3gr.

Staining with the Astra Blue and Safranin combination revealed axial parenchyma in all samples. Figures M3ap-a, M2ap, and M3ap-b in Figure 3 depict the visibility of axial parenchyma in various stains. White arrows with a black outline point at the axial parenchyma in the respective figures. Because the branch-wood sample material was selected in early spring, at the end of March 2022, there is an abundance of nutrients in the axial parenchyma and ray cells parenchyma. The ray cell parenchyma had a blue-to-purple color in microsections stained with Astra Blue and Safranin (Figure 3, M2ap, pale yellow arrow with an outline, visible also in M4gr), whereas the axial parenchyma absorbed a blue color.

The axial parenchyma was saturated well with the blue color, while the rest of the wood structure was stained bright red. It was very easy to distinguish axial parenchyma from libriform fibers. Since the axial parenchyma was filled with nutrients, it was visible also in microsections stained with Toluidine and Safranin (Figure 3, M2ap and M3ap-b), even in an unstained microsection. In Toluidine staining, the axial parenchyma appeared as a darker blue or even purple color. A gradient in saturation of the blue staining of the axial parenchyma is visible in the microsections stained with Astra Blue and Safranin, which is visible in M1gr and M4gr of Figure 3. The blue staining is most saturated in the axial parenchyma close to the bark, with the saturation becoming lesser towards the pith (Figure 3, M1gr and M4gr).

According to the pronounced axial parenchyma staining, the following findings can be stated: The most intensive transport of nutrients in branch wood during spring happens in the proximity of the bark. The axial parenchyma in the selected apple branch wood is dominantly scanty paratracheal. Diffuse apotracheal axial parenchyma was also found.

Astra Blue has an affinity to cellulose. As a counterstain to Safranin O, it has been used to identify early stages of delignification in wood [11]. According to [12], Astra Blue stains cellulose blue only in the absence of lignin and safranin stains lignin regardless of whether cellulose is present. Ref. [13] studied lignification and cell wall thickening of ray parenchyma cells in Scots pine sapwood. Thanks to the staining with Safranin–Astra Blue, significant differences in lignification and cell wall thickening of ray parenchyma cells were observed in the outer sapwood of Scots pine [13].

In our experiment, the nutrients inside the axial and ray parenchyma absorbed the blue color from the Astra Blue stain. The cell walls of some axial parenchyma also seem to have absorbed the blue color. Blue staining of axial parenchyma in our experiment could signal lignification of the axial parenchyma. Ref. [14] used Safranin O and Astra Blue staining to distinguish cellulose from lignified cells in Gala apple cultivars. In the mentioned study, parenchyma cells absorbed the Astra Blue stain and the rest of the wood structure absorbed the Safranin O stain. A further study on the lignification of axial parenchyma in apple tree wood needs to be conducted.

If the apple trees do not have enough moisture in the soil during the first half of the year, limited ring growth results [3]. There are many other various influences on tree growth, growth ring, and bark width. The researches [15,16] found the influence of altitude on tree growth was an important explanatory factor. The latter also mentioned research focused on the correlation of altitude with climatic variables such as temperature and precipitation. Temperature had a strong influence and precipitation had a low influence on pine tree growth during the growth season [17]. Temperature, water, and nutrient availability were the most important factors for tree growth [18].

Bark thickness increases with stem diameter and decreases with distance from the stem base. It is species-specific and changes with tree size and height on stem [19]. Bark thickness traits were affected by temperature and precipitation, while soil factors had almost no effects on bark thickness traits [20].

Tree rings are one of the most important proxy data sources for reconstructing past climate variability [17]. When studying the above-mentioned influences on apple tree growth, if these influences result in changes in the microscopical structure of the wood, they should be studied in future research.

4. Conclusions

The objective of this work was to observe and compare the wood structure of selected historic heirloom apple varieties growing in Slovakia’s Štiavnické vrchy, given there has been a growing interest in historic heirloom apple varieties in recent years. Macroscopic and microscopic observations showed that the historic heirloom apple trees can adapt to various, often difficult environment and growing conditions.

Despite a small difference in the altitudes of the trees, there was a significant difference in bark and growth ring width. At a lower altitude, the growth rings are wide and the bark is thin. The trees growing in higher altitudes and a cold environment had a thick bark and narrow growth rings. In the microscopic observation, a difference in the libriform fibers layer thickness was found. Selected heirloom apples trees had a different libriform fiber layer to growth ring width ratio.

The results of this work could be expanded by studying the radial and tangential sections, which would provide an even deeper look into the wood structure of the selected historic heirloom apple varieties. It is important to also study the wood structure of historic heirloom species. Changes in the microscopic wood structure provide information about the lifecycle of the trees. This can be used as a guide for growers when choosing an apple variety and for apple breeders.