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Article

Leaf Surface Micromorphology in Hybrids of Wheat and ×Trititrigia × Elymus farctus

by
Alexander V. Babosha
1,*,
Pavla O. Loshakova
1,
Alina A. Pogost
1,
Margarita M. Gevorkyan
1,
Anastasia D. Alenicheva
1,
Galina I. Komarova
1,
Tatyana S. Wineshenker
2,
Irina N. Klimenkova
1 and
Vladimir P. Upelniek
1
1
N.V. Tsitsin Main Botanical Garden of Russian Academy of Sciences, Botanicheskaya 4, 127276 Moscow, Russia
2
Library for Natural Scieces of Russian Academy of Sciences, Malỳj Znamenskij per., 11/11, 119019 Moscow, Russia
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(11), 2490; https://doi.org/10.3390/agronomy14112490
Submission received: 26 September 2024 / Revised: 21 October 2024 / Accepted: 21 October 2024 / Published: 24 October 2024
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
Versions Notes

Abstract

:
The leaf surface micromorphology and the size of the stomatal complex of hybrids in the eighth seed generation from the crossing of ×Trititrigia cziczinii × Elymus farctus (f11814) on the wheat-like wheat-wheatgrass hybrid w107 were investigated by performing scanning electron microscopy of frozen samples (cryoSEM). The micromorphological characteristics of the paternal plants (w107) were dominant in the hybrid leaves. Costal long cells with silicified wavy walls, characteristic of w107 but absent in the mother plants f11814 and E. farctus, were observed in all hybrid samples examined. Conversely, shield-shaped prickles, a characteristic feature of E. farctus, were retained only in some hybrids. In addition, the maternal feature of Ω-shaped junctions of long epidermal cells in the intercostal zone was completely absent in hybrids. Quantitative parameters of the stomatal apparatus showed a weak correlation with micromorphological markers. Stomatal density on the adaxial side was inversely correlated with stomatal size, while variation in these parameters on the abaxial side occurred independently. The prevalence of paternal micromorphological traits in the hybrids seems to be a consequence of the elimination of genetic material from E. farctus, analogous to the loss of chromosomes from wild species observed in other distant crosses.

1. Introduction

Wild species of cereals have been successfully employed in wheat breeding to enhance its economic properties. Interspecific and intergeneric hybridization is a common phenomenon in the tribe Triticeae, particularly among the Elymus species [1,2,3]. It plays a pivotal role in the evolution of plants belonging to the wheat group [4]. Distant hybridization allows for the expansion of the trait set in the selection of new varieties of wheat and other cultivated cereals [5,6,7]. Wild relatives of wheat serve as donors of resistance to biotic [4,8,9] and abiotic stresses [10,11]. Additionally, these wild relatives are utilized in breeding programs for desirable baking qualities [12]. A breeding program is underway using a synthetic genus of ×Trititrigia plants (obtained by hybridization of wheat with wheatgrass) in an effort to integrate genes conferring resistance to powdery mildew and rust of select wheatgrass species into the wheat genome [13,14]. The introduction of genetic material from certain wheatgrass species has enabled the creation of new ×Trititrigia plants with enhanced grain quality at the N.V. Tsitsin Main Botanical Garden of the Russian Academy of Sciences [15,16].
The microstructure of the surface of cereal leaf laminae exhibits a high degree of diversity [17]. The variability in the composition of silica cells and trichomes (ranging from simple hairs to microspikes of varying size and shape) and their localization on the adaxial and abaxial sides of the leaf, as well as the peculiarities of the stomatal apparatus, provide useful characters for the taxonomy of the Poaceae family. It is to be expected that the appearance of the hereditary material of wild cereals in the wheat genome will result in an increase in micromorphological diversity among cultivated forms of this crop. Many features of leaf micromorphology, in particular, parameters of the stomatal apparatus and trichome density, play an important role in adaptation to environmental conditions and significantly affect plant productivity [18,19,20,21,22]. The high variability may provide an opportunity to utilize micromorphological markers for the distinction and control of varietal purity or as markers of economically valuable traits.
Hybrid forms may retain some features of the morphology of their wild parental forms [23,24,25,26]. The morphological traits of spikelets are mutually related and have high prognostic value for predicting wheat productivity [27], as well as for identifying foreign genetic material in wheat hybrids [23].
It is established that in amphidiploids derived from Aegilops kotschyi, Ae. biuncialis, and Secale cereale, the leaf surface characteristics of the wild-type parents are inherited, along with other traits, and can be manifested in the offspring [26,28]. However, thus far, research into surface microscale structures within the Poaceae has primarily been conducted for the purpose of classification. There has been little investigation into cultivated forms, particularly those resulting from distant hybridization.
In the 1930s, the hybridization of wheat and wheatgrass plants resulted in the creation of an intergeneric wheat-wheatgrass hybrid, which was subsequently designated as Triticum agropyrotriticum Cicin, or perennial wheat [29]. To date, the collection of soft wheat enriched with the genetic material of wild relatives from the Remote Hybridization Department of the N.V. Tsitsin Main Botanical Garden of the Russian Academy of Sciences has more than 250 accessions [15]. These plants possess fragments of the genome of Elytrigia intermedia (Host) Nevski and E. elongata (Host) Nevski, which exhibit 42 wheat chromosomes and 14 wheatgrass chromosomes, thereby representing a novel grain feed crop, ×Trititrigia. In the modern classification, they are designated as ×Trititrigia cziczinii Tsvel. and are included in the synthetic ×Trititrigia genus [30].
Additionally, the collection encompasses specimens of ×Trititrigia with genetic material from sand couch grass Elymus farctus [31]. This wild wheatgrass is a valuable donor of economic qualities, including resistance to diseases and salt tolerance [32,33]. Nevertheless, direct crossing with wheat is challenging. To facilitate the transfer of beneficial traits into the wheat genome, hybrids with ×Trititrigia were employed as an intermediate link. Additionally, the utilization of an intermediate (“bridge”) enables the production of hybrids with smaller segments of the wild species’ genetic material, thereby reducing the likelihood of undesirable wild traits.
The progeny of the 63-chromosomal hybrid ×Trititrigia × E. farctus [34], which exhibited a higher gluten fraction than standard varieties, represent a promising parental form for the creation of soft wheat with fragments of the genome of wild wheat relatives. It was found that these plants (in particular, f11814), which exhibited the characteristics of donors of high grain quality, were devoid of the negative traits that are characteristic of wheatgrass, such as the prolonged maturation of spikes.
Spring wheat-wheatgrass hybrid w107 exhibits basic properties similar to those of soft wheat while also displaying high grain quality [35]. However, w107, along with other wheat-wheatgrass hybrids, displays a notable disadvantage in terms of its poor resistance to head blight fungi. Hybrids f11814 × w107 exhibited resistance to head blight fungi until the seventh generation, which was attributed to the presence of wheatgrass genome fragments. In the seventh generation, only one sample with single affected grains was observed in this combination. While f11814 exhibits high grain quality, it is inferior to other more optimal soft wheat varieties in terms of thousand kernel weight and grain vitreousness [31]. Consequently, it is recommended that this sample not be utilized as a variety for baking purposes but rather as a donor of high grain quality. The hybrid combination f11814 × w107, in which both parents exhibit high grain quality, appears to be a highly promising candidate for the breeding of wheat accessions with high grain quality traits.
The objective of this study is to examine the micromorphological characteristics of maternal and paternal plants on the leaf surface in the F7-F8 progeny of crosses between the 63-chromosomal hybrid ×Trititrigia × E. farctus f11814 (maternal plants) and spring wheat-wheatgrass hybrid w107 (paternal plants).

2. Materials and Methods

2.1. Origin of Hybrid Samples

The hybridization of line f11814 with w107 was performed in 2015 (f11814 × w107). Spring wheat-wheatgrass hybrid w107 served as pollinator. F1 plants were grown from seeds obtained in 2016. The hybrid combination f11814 × w107, studied in the collection of the Remote Hybridization Department of the N.V. Tsitsin Main Botanical Garden of the Russian Academy of Sciences, included 22 samples, which are the progeny of the original hybrid grains; 32 additional samples were selected in subsequent generations by sowing seeds from individual spikes. Four samples of the eighth seed generation of hybrids (F8) with large grains and high sedimentation and protein content (N4, N14, N16, and N23) were selected for leaf surface micromorphological studies. The growing period was between 90 and 110 days, depending on the sample.
The maternal line f11814 was selected from the 56-chromosomal progeny (f-hybrids) of the original 63-chromosomal F1 hybrid obtained by crossing ×Trititrigia cziczinii Tsvel. with sand couch grass Elymus farctus (Viv.) ex Melderis (Thinopyrum junceum (L.) Á.Löve). It contains 42 common wheat chromosomes and 14 wheatgrass chromosomes and is null6A-tetra 6D-somic. The line is a source of high grain quality and can also be assigned to ×Trititrigia [31,36]. Micromorphological data of f11814 were obtained from leaf samples in 2019 and 2023. Data from 2 other related f-hybrids (f9714 and f10405/16), as well as a sample (perennial) of sand couch grass E. farctus, were additionally used in statistical calculations in the mother plant group.
Spring wheat-wheatgrass hybrid w107 (paternal plants) was obtained by crossing a spring wheat-wheatgrass hybrid of wheat type 38/1 with winter wheat-wheatgrass hybrid 25 (w38/1 × w25). It also has high grain quality [34]. In statistical calculations, the group of paternal plants (WWH—wheat-wheatgrass hybrids) additionally included wheat-wheatgrass hybrids of wheat type w199 and w269 (samples were grown in 2022 and 2023).

2.2. Growing Conditions

Plants were grown in the experimental plot in Istra district (Moscow region). Each sample was sown with a cassette seeder in an area of 1.4 m2 at 0.5 m spacing. The soil of the plot is sod-podzolic and light and loamy. The zone is characterized by a cool, rather humid, and temperate continental climate. According to the weather station in New Jerusalem (Moscow region), the average temperature and precipitation from May to August were +16.3 °C and 275 mm in 2019, +16.8 °C and 152 mm in 2022, and +16.0 °C and 270 mm in 2023.

2.3. Determination of Spike Morphology and Grain Quality Parameters

Eight typical spikes were analyzed from each sample. Spike length, the number of spikelets in the spike, the number of flowers in the spikelet from the middle part of the spike, the number of grains in the spikelet, the number of grains in the spike, the weight of grains from the spike, and the weight of 1000 grains were determined.
The content of protein and gluten and grain vitreousness were determined according to the Russian state standards (FR.1.31.2009.06615) by spectroscopy in the near-infrared range using the analyzer InfraLUM FT-10 (Russia) [37]. The sedimentation index was determined by a modification of the Zeleny test with flour dispersion in 2% acetic acid [38]. The gluten deformation index was determined according to Russian state standards [39].

2.4. Scanning Electron Microscopy

The leaves of the middle layer of 1–5 samples of parental forms and hybrids in the earing or flowering stage (BBCH 61-71, June–July 2019–2023) were examined by scanning electron microscopy of frozen samples (cryoSEM). Fragments of the middle part of the leaf plate were fixed on a 2 × 4 cm copper plate, which was fixed on the table of the freezing attachment “Deben Cool stage” (UK) using computer thermal paste [40]. Good image quality is achieved by using thermal paste as an adhesive and a heat conducting compound for more efficient freezing. The samples were examined on a LEO-1430 VP scanning electron microscope (Carl Zeiss, Jena, Germany) using a QBSD backscattered electron detector under high vacuum conditions at −25 °C.
On the obtained micrographs, the presence or absence of different types of trichomes and silica cells in cell files in the costal and intercostal zones and additionally in the area of the middle vein on the abaxial and adaxial surfaces of the leaves were taken into account. The identification of micromorphological structures on the abaxial and adaxial surfaces of the leaf lamina was guided by the classification of microstructures according to Ellis (1979) [17]. The frequency of such structures was evaluated in scores as follows: 0—complete absence; 1—single structures; 2—the structure is typical for this sample; and 3—the sample is characterized by a particular abundance of these structures.
The sizes of microstructures were determined from digital images in the program Image J. Data from 20 to 30 microstructures were used to calculate the mean and error. For each micrograph, we calculated the values of the following micromorphological variables: AvRow (average cell row width, μm), L (stomatal length, mm), S (stomatal complex width, μm), L/S (ratio of stomatal complex length and width), St (stomatal density, units/mm2), StL (number of stomata per mm of stomatal cell row length, units/mm), and StRow (proportion of stomatal rows from all cell rows, %). The diameter of the base of shield-shaped prickles, the diameter of rounded short silica cells, the length of elongated silica bodies, and the length of apically directed prickles were also measured if appropriate micromorphological structures were present.

2.5. Statistical Analysis

Routine calculations were performed using the standard Microsoft Excel program. Data were analyzed using correlation analysis, principal component analysis, and exploratory factor analysis in JASP 0.9.2.0, PAST 4.03, and Jamovi 2.3.28 software (https://www.jamovi.org, accessed on 20 October 2024). One-way ANOVA and the Tukey test were performed using JASP 0.9.2.0 and online software [41].
Only data for the year with the most complete information are presented, except for micromorphology, where we can use multivariate analyses across years.

3. Results

3.1. Spike Morphology and Grain Quality

The spikes of samples from the hybrid combination f11814 × w107 are of the wheat-type white. The spikes may be spinous (Erythrospermum, N16 and 23) or spineless (Lutescens, N 4 and 14). The grains are red, oval, or oval-elongated. Threshing is characterized as good to excellent.
Plants from samples N16, 23, and 4 were medium-grown, close in height to the paternal form w107, and of the standard wheat variety Sudarynya for the Moscow region (Table A1, Figure A1). Sample N14 is tall, similar in height to the parental form f11814, and of the standard wheat variety Zlata used by us. The spike is mostly spindle-shaped. Plants of the maternal line f11814 had a higher number of spikelets per spike, flowers per spikelet, and total grains per spike, but the grains were smaller in size compared to w107 (Table 1). N14 was closer to f11814 in both the number of spikelets and height, while N23 was comparable to w107. The performance of hybrid plants was intermediate. Compared with the standard variety Sudarynya, the hybrid accessions were similar to it in spike length, number of spikelets in the spike, but had slightly lower values for the number of flowers in the spikelet and grains in the spike. Thus, hybrids generally occupy an intermediate position between the maternal and paternal forms in terms of plant height, spike length, and the number of spikelets in the spike.
Hybrid plants exceeded both f11814 and the standard variety Sudarynya in 1000 grain weight, which corresponded to the values of paternal plant w107. Indicators of spike and grain size from the 2022 season (Table A2) were slightly higher than those of 2023, but in both seasons, N4 values were at the maximum in the studied hybrid sample and N14 values were at the minimum.
Samples N4 and 16 are early maturing and samples N14 and 23 are medium maturing (Table 2). According to the sedimentation number, all samples (especially N4 and 23) have good baking properties (Table 2). The results of the rapid analysis carried out in 2022 show that all the hybrid samples have high grain vitreousness (above 53%), protein content (above 17%), and gluten content (above 32%) for soft wheat. Samples N4, 16, and 23 had yellow gluten, which is characteristic of many ×Trititrigia varieties. N14 has light gluten. The gluten content in 2023 was slightly lower (24.4, 36.2, 31.8, and 32.8% in samples N4, N14, N16, and N23, respectively), but even in this case, it corresponded to first-class wheat in this parameter for samples N14 and N23.

3.2. Micromorphology of Leaf Surface

The surface of the adaxial and abaxial sides of cereal leaves, including the samples studied by us, is formed by rows of long and shortened cells. Modifications of the latter are stomata, trichomes (prickles and macro-hairs), and various silica cells. The composition of cellular elements differs on the adaxial and abaxial side of the leaf. Different sets of trichomes and silica cells are arranged in cell rows on the veins above the conducting bundles (in the costal zone) and in the intercostal zone. Additionally, veins of different orders may exhibit specific micromorphological characteristics. For instance, the surface above the midvein is often prominent. Furthermore, cellular elements may occur at the leaf margin that are absent in cell rows in the middle of the leaf.
Upon examination of the microstructural elements of the leaf surface of parental forms and hybrid samples, a number of micromorphological markers were identified on the abaxial (B1–B8) and adaxial (D1–D4) sides of the leaf plate (Table 3, Figure 1, Figure 2 and Figure 3). This list does not include some surface microstructural elements that are common to the samples studied or that vary widely between years of observation, such as adaxial macro-hairs.

3.2.1. B1. Apically Directed Prickles in the Costal Zone on the Abaxial Leaf Side (Figure 1a, −♀−♂)

These prickles are absent in both parental forms and hybrid samples N23 and N4 but are evident in samples N14 and N16 on several veins near the leaf margin. The prickles are solitary or adjacent to an elongated basal cell. Samples N14 and N16 exhibit differences in the dimensions of the prickles of this type. In sample N14, the prickles are shorter in length (42 ± 1 μm), while in sample N16, they are larger (78 ± 7 μm).

3.2.2. B2. Basally Directed Prickles in the Costal and Midvein Zones on Abaxial Leaf Side (Figure 1b–d, −♀+♂)

These prickles are characteristic of the paternal plant w107 (Figure 2f) and are well represented in sample N4 (Figure 2a). The sizes of these prickles in w107 and N4 are close to 33–38 and 39–64 μm, respectively. Single prickles on the veins were observed in samples N14 and 16. The degree of appearance of the trait in the costal zone and on the middle vein differs among the different samples. In w107, the sizes of these barbs on the middle vein (50 ± 2 μm) exceed those on the veins of other orders (35 ± 1 μm).

3.2.3. B3. Basally Directed Long Thickened Macro-Hairs in the Costal and Midvein Zones (Figure 1c,j, −♀+♂)

The thickened hairs are specific to the paternal form and were observed in w107 samples in cell rows immediately adjacent to the veins (Figure 2a). Their length varies between 60 and 130 μm. Basally directed prickles (B2) can be observed in the same locations. They range in size from 38 to 70 µm and are sometimes similar to large basally directed hairs (e.g., Figure 2c). Neither hairs nor prickles are present in the parent form f11814 nor in samples N14, 16, and 23. In addition to w107, single hairs are observed on the middle vein of sample N4 (Figure 2a).

3.2.4. B4 and B5. Shield-Shaped Prickles (Shields) in the Costal (B4) and Midvein (B5) Zones on Abaxial Leaf Side (Figure 1a,c,d,f, +♀±♂)

The shields are prickles with a rounded base and a very short barb. An Ω-shaped connection to the anticlinal cell walls of the epidermal cells is evident along the base edge. In the absence of metal sputtering, cryoSEM reveals that the shields and silica cells are light in color. These structures occur singly and less frequently in pairs with a semicircular or elongated basal cell, alternating in a row with a long epidermal cell (Figure 1a,c,d). Along the leaf margin, the shields are abundant in all examined samples (Figure 2d). However, outside the leaf margin, they are abundant only on the middle vein and veins of the maternal form f11814 (Figure 2e).
The frequency of shields on the veins of the paternal form w107 exhibited considerable variation across different growing seasons. The number of these structures was particularly high during the 2023 season. However, they were notably absent in previous years. In contrast, the other wheat-wheatgrass hybrids, w199 and w269, exhibited a lack of shields. Conversely, shields were observed in other f-hybrids. These observations do not permit us to rule out the possibility that this trait was initially absent in the paternal plant and subsequently appeared in subsequent generations as a result of cross-pollination. In different hybrids, the occurrence of these structures varies. In samples N4, 14, and 16, shields are present on the middle vein, but less frequently (and not on every vein) on veins of higher orders. In sample N23, shields were not observed on the middle vein, but they did occur in isolated cases on veins in the vicinity of the leaf margin. Table 4 presents the dimensions of the base of the shields in the examined samples. In general, the diameter of the base is comparable or slightly larger than the width of the long cells of the cell row in which it is located. The size of the shield base was approximately the same on the midvein and veins of other orders. In all hybrid samples studied, they corresponded in size to the maternal form f11814 and were significantly smaller than the shields of the paternal plant w107.

3.2.5. B6. Rounded Short Silica Cells with Thin Sickle-Shaped and Darker Basal Cork Cells (Figure 1a–f, +♀+♂)

These microstructures are present in the costal and midvein zones on the abaxial side in all hybrids and both parental forms but are sometimes absent in samples w199 and w269. The rounded short silica cell, as well as other short cells, alternates with the long cell. In the intercostal zone, the rows with these cells are on either side of the stomatal rows. In some instances, these cells appear slightly more elongated and exhibit one or two thin longitudinal lines on the median vein (Figure 1c). In the majority of cases, the diameter of the rounded short silica cell aligns with the width of the cell row, excluding the thickness of the Ω-shaped junctions of the anticlinal cell walls on both sides. The mean diameter of the rounded short silica cell in the costal zone and on the middle vein was not statistically different between the examined samples. However, such cells adjacent to the stomatal rows exhibited a slight increase in diameter (p = 0.03). The mother form, f11814, demonstrated the lowest mean diameter, while the remaining samples exhibited slightly larger silica cells (Table 4).

3.2.6. B7. Oval or Rectangular Short Cells with Horizontally Elongated Silica Bodies with Sinuous Outline, Single or with Semicircular Basal Cell in the Costal Zone (Figure 1a,c, −♀+♂)

In the terminology proposed by Ellis (1979) [17], the horizontal direction is defined as the direction along the long axis of the leaf. Such cells are well represented on the veins of hybrid samples N14 (Figure 2b) and N23 (Figure 2d) and are completely absent in the parent form f11814. In the cell row, they alternate with a long epidermal cell, which is, however, shorter than similar long cells in rows with other structural elements, such as rounded silica cells. There may be one to four cell rows with costal long cells with silicified wavy cell walls adjacent to each other on different edges. The width of these cells is equivalent to the width of the cells in the row, taking into account the thickness of the Ω-shaped junction between neighboring anticlinal cell walls. N14 and N23 exhibit longer lengths than the remaining samples, with considerable variation (Table 4). Despite the pronounced visual differences between the samples, length exhibited statistically significant differences only in N14, likely due to the high variance observed in N23 (Table 4). In N14 and N23, silica cells exhibited an elongated shape and well-defined undulations at the junctions with the long cells of neighboring cell rows. N4, 16, and the paternal form w107 exhibit a greater prevalence of short silica cells with an oval shape, and the waviness along the edge is less pronounced. Additionally, numerous cells with a shorter length and a white longitudinal stripe are observed on the edge of the middle vein of all samples (except f11814) (Figure 1c).

3.2.7. B8. Ω-Shaped Junction of Horizontal Anticlinal Walls of Long Cells in the Intercostal Zone on the Abaxial Side (Figure 1f, +♀−♂)

The Ω-shaped junction of cells of different rows resembles a zipper and is a common feature observed in all plants studied, occurring in the costal zone, in the cell rows of the middle vein (Figure 1a–f), and near the leaf margin (Figure 2d). Additionally, in all hybrid samples and also in w107, this cell connection can be observed in the stomatal rows. However, in the intercostal cell rows between the stomatal rows, this marker is well expressed only in the mother form f11814 (Figure 1d,f and Figure 3e). In the leaves of these plants, the Ω-shaped junction of cells of different rows is present in cell rows that are not bordered by veins and stomata. This trait is also present in other f-hybrids. In the paternal form, the marker is present only in the 2023 samples. In earlier generations, it was observed only in the stomatal rows, as well as in all hybrid samples.
In instances where there is an Ω-shaped junction of cells situated between the stomata, the long cells that comprise these rows invariably alternate with a rounded silica cell that is characterized by a thin and dark basal cell on the abaxial side. Conversely, the adaxial side comprises rounded silica cells that are accompanied by a thin basal cell on the middle vein.

3.2.8. D1. Apically Directed Prickles in the Costal Zone on the Adaxial Leaf Side (+♀+♂)

All samples exhibit the presence of such prickles, although in f11814, the basal cell is semicircular (Figure 3e), while in the others, including the paternal form w107, it is elongated and wavy along the margin (Figure 3a,c,d,f). In some veins, apically directed prickles in the cell row alternate with a costal long cell with silicified wavy cell walls or several such cells that form a continuous line (Figure 1h and Figure 3b,c). The lengths of these prickles are presented in Table 4. Samples N4 and 14 exhibited small prickles that were comparable in size to similar microstructures of w107, while samples N23, N4 (2022), and N16 displayed larger prickles that were analogous to the parent form of f11814. Sample f11814 (2019) exhibited particularly large prickles (68.3 ± 5.0 μm), which were significantly different from those of all other samples, including the reseeding of this sample in 2023. While the basal cell shape in hybrid samples is consistently paternal (elongated with a wavy edge), some hybrids exhibited increased prickle size, which is more consistent with the mother plants.

3.2.9. D2. Shield-Shaped Prickles, Single or with Semicircular Basal Cell (Figure 1g and Figure 3e, +♀−♂), and Rounded Short Silica Cells (Figure 1g–i and Figure 3e) in the Costal and Midvein Zones of the Adaxial Side

On the adaxial side of the leaf, shields are present on the veins and middle vein in the maternal form f11814 (as well as in other f-hybrids) and absent in the paternal form and in all hybrid samples studied. When the shield had a basal cell, it had a rounded or semicircular shape (Figure 1g). The same basal cells are also observed in f11814 in apically directed prickles. However, an exception is observed in w107 plants grown in 2023, in which single shields on individual veins were noted. In this case, the basal cells of these shields were elongated, similar to those observed in the prickles of w107 and hybrid samples. On veins f11814, the diameter of the shield on the adaxial leaf side (27.5 ± 1.1 μm) was found to be greater than that on the abaxial side (24.6 ± 1.0 μm). In the f11814 samples studied, the presence of the shield was always accompanied by the presence of round silica cells with enfolding crescentic cork cells on the veins (17.9 ± 0.8 μm), which were slightly larger than similar elements on the abaxial side. These features occur only on the veins or on the veins and midvein, usually together. They are absent in paternal w107 and hybrids.

3.2.10. D3. Basal Cells of the Prickles and Shield-Shaped Prickles Are Oval or Rectangular

Oval or rectangular short cells with horizontally elongated silica bodies with sinuous outlines exist in the costal and midvein zones. Long cells with silicified wavy cell walls in the costal zone are on the adaxial leaf side (Figure 1h–j and Figure 3a,b,d–f). It is a general observation that short and long silicified cells, exhibiting an Ω-shaped wavy edge on their veins, are frequently observed on the same specimens. However, the long cells may be absent on the middle vein. In some cases, costal long cells with silicified wavy cell walls are arranged in a consecutive manner without an interval, creating a distinct light line in the costal zone. The rows of long cells may contain prickles, indicating that such long cells are basal for them. Conversely, basal cells with a semicircular shape, which are characteristic of the maternal form, are absent.
Costal short and long cells with silicified wavy cell walls were observed in the paternal plant w107 (and other wheat-wheatgrass hybrids studied) and in all hybrid samples. However, they were absent in the maternal line f11814 and in other f-hybrids. Therefore, in our sample set, the D2 and D3 trait complexes are mutually exclusive.
The length of the short silica cells on the midvein, as well as the expression of Ω-shaped waviness, were observed to be markedly smaller than on the veins of other orders (Table 4). Among the hybrid samples, N4 was distinguished by shorter silica cells on the veins, exhibiting a greater degree of similarity to the silica cells of the main vein. In the set of examined samples, the presence of such silica cells was found to correlate with the presence of short hairs or prickles in the intercostals near the stomata on the abaxial side (Figure 1a,b,e and Figure 3a–d,f).

3.2.11. D4. Long Cells with Silicified Wavy Cell Walls in the Midvein Zone on the Adaxial Leaf Side (Figure 3f, −♀+♂)

These markers are characteristic of the paternal plants of w107 and the hybrid samples studied but are absent in the maternal line f11814. Additionally, the basal silica cells of the prickles and shields on the veins are also elongated in this case. In contrast to the D3 trait, the silicified cells on the main vein were absent not only in all f-hybrids but also in the other wheat-wheatgrass hybrids studied (w199 and w269). This trait is specific to w107 and its progeny.

3.2.12. Maternal and Paternal Markers

It can be observed that the characteristic features of maternal plants on the abaxial side are the presence of Ω-shaped anticlinal cell walls in the intercostal zone and the formation of shields on the middle vein. However, it should be noted that although both of these traits have not been previously observed in paternal w107 plants, they were observed in them in 2023. Therefore, it cannot be unequivocally considered that these traits are markers of maternal plants. Furthermore, the Ω-shaped anticlinal cell walls in the intercostal zone are absent in all hybrid samples. Similarly, the shields on the middle vein are also found in samples N14 and 16 (and in w107 of the 2023 reproduction year). However, in other locations (on the veins and along the leaf margin), they are present in almost all examined samples, although less frequently than in f11814. On the adaxial leaf side, a feature specific to the maternal form is the semicircular basal cell at the prickles and shields on the veins. In the paternal form w107, as well as in all hybrid plants, the basal cell is elongated with a wavy edge. The single shields on veins and midveins, as well as the rounded silica cells on the midveins, are well expressed in f11814 and w107 in 2023, but completely absent in hybrid samples.
Paternal plants exhibit basally directed prickles or thickened hairs on veins and the middle vein. These characteristics are absent in the mother plants of f11814 but are well represented in N4 and, to some extent, in other samples. Short hairs or prickles near the stomata (hooks), which are also characteristic only of paternal plants, are present in all samples except N23. Silica cells with an oval or rectangular shape and a wavy margin, which may be single or accompanied by a thin dark basal pair, are absent in the parent plants and present in all hybrid samples. On the adaxial side, costal long cells with silicified wavy cell walls are absent in f11814 and present in w107 and all hybrid lines. In some rows on the midveins, there is a lack of long cells but cells of a similar morphology but of a shorter length are present.

3.3. Principal Component Analysis of Micromorphologic Markers

A principal component analysis of the scatter of 12 micromorphological markers on both sides of the leaf allows us to identify two components, PCA1 and PCA2 (54.1 and 25.5% of the variability). The predominance of paternal traits in the hybrid samples is visualized in a scatter plot (Figure 4). The maternal form f11814, as well as other f-hybrids and E. farctus, form a separate group that differs from the hybrid samples in PCA1 values. Markers B3-B5, B7, B8, D2, and D3 contribute the most to this component. The hybrid samples form a compact group within the range occupied by wheat-couch grass hybrids. They are close to the paternal form w107 and differ from the other wheat-couch grass hybrids in PCA2 values. B2, B6, D1, and D4 markers contribute most to PCA2. PCA2 accumulates traits specific to w107 and absent in other wheat-couch grass hybrids. The exploratory factor analysis (Table 5) also shows that the investigated micromorphological markers can be grouped into two factors of a similar composition as PCA1 and PCA2, linked within each group by positive or negative correlations.

3.4. Quantitative Micromorphological Analysis of Stomatal Apparatus and Cell Row Width

Figure 5 shows the quantitative parameters of stomata and cell row width in hybrid samples and parental plants on the abaxial and adaxial leaf sides. In all tested samples, the number of stomata in a row (StL) and the proportion of stomatal rows themselves (StRow) are higher on the adaxial side of the leaf than on the abaxial side, which determines a similar relationship for the surface stomatal density (St). According to ANOVA, this difference is significant (p < 0.01) for St and StL. At the same time, both parental forms and individual hybrid samples had few differences among themselves in terms of stomatal parameters. The width of the cell rows in all hybrid samples corresponded approximately to the parameters of the paternal form and was significantly less than the width of the rows in f11814. The size of stomata and the ratio of stomatal length to width (Figure 6) were similar on both sides of the leaf. Sample N23 was characterized by shorter stomata, whereas the stomata of samples N14 and N16 were significantly longer than those of the paternal plants.
Table 6 shows the correlation matrix of dependence between the quantitative micromorphological variables. The exploratory factor analysis showed the presence of four factors (Table 5). Similarly to Figure 4, some samples related to paternal and maternal plants were added to calculate the parameters of Table 5 and Table 6. Factor RC1 is formed by the mutually correlated (p < 0.05–0.001) variables of cell row width and the proportion of stomatal rows on both sides of the leaf. The most significant in this group are the correlations between the width of cell rows on both sides of the leaf (R = 0.794, p < 0.001) and the width of cell rows and the proportion of rows with stomata on the adaxial side (R = 0.903, p < 0.001).
The parameters for stomatal length on both leaf sides and stomatal density on the adaxial side had the highest component loadings for the RC2 factor. Abaxial and adaxial stomatal length were correlated with each other (R = 0.766, p < 0.001), and each of these variables was inversely correlated with adaxial stomatal density (R = -0.562 and -0.498, respectively, p < 0.05).
For the RC3 factor, the adaxial width of the stomatal complex (SD) had the highest component loading. Stomatal density on the abaxial side of the leaf (StB, StLB) did not appear to be related to stomatal length on the same side nor to stomatal density on the opposite side of the leaf, forming a separate RC4 factor. Unexpectedly, there was a correlation between stomatal length and stomatal width only on the adaxial side (R = 0.664, p < 0.01). Stomatal length and width on the abaxial side were not correlated (R = −0.03). The variables PCA1 and PCA2, generated from the results of the PCA of the micromorphological markers (Figure 4), as well as the original markers B1-B8 and D1-D4 from Table 3, were weakly correlated with the parameters of the stomatal complex. The only quantitative parameter that could be calculated for all samples and that was not related to the stomatal apparatus, the diameter of the rounded short silica cells (RoundRibABA), also did not correlate with any of the stomatal apparatus parameters studied.
The principal component analysis of the stomatal complex and cell row width variables revealed four components explaining 28.3, 21.8, 17.5, and 13.4% of the variability, respectively. In PCA1 and PCA2 coordinates (Figure 7), groups of samples close to maternal (F) and paternal (WCH) plants overlapped strongly. The hybrid samples formed a compact group within the region occupied by WCH and did not overlap with the F group. An analysis of the scatterplot of samples in PCA1 and PCA2 coordinates in Figure 6, as well as similar plots with other principal components, indicates that the differences between hybrids and the parent form f11814 and other f-hybrids are due to PCA2, and the components PCA1, PCA3, and PCA4 reflect the variability of samples within each group. In turn, the RC1 and RC2 factor variables from Table 5 (stomatal length and width of cell rows on both sides and stomatal density on the abaxial side of the leaf) contribute approximately equally to PCA2.
The analysis of stomata and cell rows, as well as surface micromarkers, indicates a certain predominance of traits of paternal plants w107 in hybrid samples. Similarity with paternal plants is observed in such parameters as the width of cell rows and the proportion of stomatal rows. At the same time, some samples had significant differences from both paternal and maternal plants in terms of stomata length.

4. Discussion

The dynamics of manifestation of macromorphological features of parental forms in seed generations of hybrid plants can have a rather complex character. Thus, when crossing wheat with wild species or breeding samples enriched with wheatgrass genes, the spike form of wild plants prevailed in hybrids of the first generations [42,43]. The micromorphological characteristics of the leaf surface of the wild species also prevailed in the first generations of hybrids (×Trititrigia cziczinii × ‘Botanicheskaya 3’) on Elymus farctus [44]. Later, a partial return of the wheat phenotype can be observed in the progeny. This is due to the elimination of a significant part of the chromosomes of wild species in hybrids. This effect has been repeatedly observed in interspecific and intergeneric crosses of cereals [45,46,47,48]. The elimination of part of the chromosomal material occurred already at the embryonic stage during seed formation [49,50], as well as in plants obtained by somatic hybridization [51].
A number of micromorphological markers of the leaf surface have been identified, which allows for the formalization of the features of the surface structure of parental forms and hybrid plants. The data obtained allow us to identify several sets of micromorphological markers that occur together in the studied sample of breeding samples and are specific for one of the parental forms. The f11814 hybrid and other f-hybrids are distinguished by the presence of shield-shaped prickles (shields) on the abaxial midvein (B5) and in the adaxial costal zone (D2), as well as an Ω-shaped junction in the intercostal zone on the abaxial side of long epidermal cells (B8) that alternate with a rounded short silica cell (Complex 1). These characteristics have been observed for E. farctus [31] and its crosses [44]. The Ω-shaped junction in the intercostal zone can be seen in ×Trititrigia cultivars such as Otrastayushchaya 55 and Mnogoletnyaya 4015 [52]. The presence of Complex 1 was exclusive to the maternal plants yet exhibited a markedly diminished expression in the hybrid samples studied, as well as in other wheat-wheatgrass hybrids. In hybrid samples N14 and 16, shields were observed solely on the middle vein. Conversely, shields on the abaxial veins of higher orders (B4) were evident in both parents and less frequently in hybrid samples.
Characteristic of the paternal plants of w107 and other wheat-wheatgrass hybrids studied by us were oval or rectangular cells with horizontally elongated silica bodies with sinuous outlines in the abaxial and long cells with silicified wavy cell walls in the adaxial costal zones (B7, D3), as well as basally directed long and thickened macro-hairs on veins (B3), which can be attributed to Complex 2. These features were present in some hybrid samples but were absent in the f-hybrids. Many features of the first and second complexes were mutually exclusive in the sample we examined. In particular, in F11814, even the basal cells of prickles and shields were rounded rather than elongated in the absence of silica cells with wavy edges. At the same time, in the ×Trititrigia samples mentioned above, elements of both complexes (shields, Ω-shaped junction, and elongated silica bodies with sinuous outlines) were present. Another group of features can be distinguished, namely basally directed prickles on abaxial veins (B2), apically directed prickles (D1), and long silicified cells on the adaxial side (D4). Within the sample studied, these characters were specific to w107 and its progeny, some of which may have been present in f11814 but were absent in other wheat-wheatgrass hybrids.
In a large number of plants from different taxonomic groups, there is an inverse relationship between stomatal size and stomatal density [53,54,55]. This dependence results from the geometric relationship between these parameters in the epidermis [55]. However, there may be other reasons for this; for example, it is thought that maximizing stomatal conductance by a denser packing of stomata on the leaf surface should not reduce the mechanical strength of the leaf [56]. Optimizing stomatal morphology during the evolution of covered seeds toward higher values of stomatal conductance resulted in plants with smaller but more frequently arranged stomata [53].
Obviously, the number and size of stomata should influence drought resistance and plant productivity. However, the inverse correlation between these parameters, as well as the presence of other influencing factors, leads to more complex and often contradictory experimental data. In one study, a drought-tolerant cultivar had small and numerous stomata [57]. However, it has also been shown that reducing stomatal density improves drought tolerance in wheat and rice [58,59,60]. Water exchange also depends on the stomatal opening rate, which decreases with increasing stomatal size and increases with increasing density [61]. Faster dynamic rates of small stomata may contribute to photosynthetic capacity and productivity and affect the transpiration rate and plant tolerance to water deficit.
The interdependence between yield and stomatal apparatus parameters in cereals may have a different sign in the works of different researchers. In a sample of 20 soft and durum wheat and landraces, grain yield was positively correlated with stomatal area but negatively correlated with stomatal frequency [62]. In rice, grain yield was also correlated with stomatal area but not with stomatal size and the number of stomata [63]. On the contrary, in another study, high-yielding rice varieties had higher stomatal density and slightly shorter stomatal length [64]. In a study [65], wheat yield correlated with stomatal length and width on both sides of the leaf and not with stomatal frequency, but only under drought conditions. Finally, no significant correlations between yield and stomatal traits were observed under field conditions, regardless of wheat moisture conditions [57].
The quantitative parameters of micromorphology that we studied concerned the structure of the stomatal apparatus, the width of the cell rows, and the size of some leaf surface elements. The stomatal density on the adaxial side of the leaf exceeded that on the abaxial side in all samples, which is in agreement with the results obtained in samples of some other cereals [66]. Factor analysis allowed us to identify four groups of quantitative variables that correlated with each other. At the same time, the relationship of the variables included in one of the selected factors reflected the known phenomenon of an inverse correlation between density and the size of stomata. This relationship is fulfilled in samples of Aegilops neglecta with different ploidy [67], as well as in samples of wheat species [68]. However, in our cereal sample, only stomatal density on the adaxial side of the leaf was inversely correlated with stomatal size, while variation in stomatal density on the abaxial side occurred independently of stomatal size. The relationship with stomatal size was only related to stomatal length, and stomatal width also varied according to other laws. It should also be noted that quantitative variables describing the stomatal apparatus were weakly correlated with the composition and size of micromorphological markers.

5. Conclusions

Thus, the micromorphology of the leaf surface of hybrids obtained by crossing ×Trititrigia cziczinii × Elymus farctus (f-hybrid f11814) on a wheat-couch grass hybrid of wheat-type (w107) was studied by scanning microscopy of native frozen preparations (cryoSEM). The method used allows for fresh plant tissue to be studied in a near-native state, without the artifacts caused by sample fixation and metallization commonly used in standard scanning electron microscopy. Micromorphological markers specific to maternal and paternal plants were identified. This allows for the monitoring of formogenesis during distant hybridization and the use of these markers as an additional tool for the identification of wheat varieties and hybrids.
Both parents originated from wild cereals. However, only maternal plants retained a number of features of E. farctus micromorphology (shield-shaped prickles on the abaxial midvein and adaxial costal zone and Ω-shaped junctions of long epidermal cells in the intercostal zone on the abaxial side). We studied rather late seed reproductions of hybrid plants. In the leaf surface micromorphology of these hybrids, there was a clear predominance of paternal traits. This was manifested by the fact that all hybrid samples had long cells with silicified wavy cell walls, characteristic of the paternal plant, but these were absent in E. farctus and f-hybrids. On the contrary, the shields on the middle vein, characteristic of E. farctus and f-hybrids, were observed only in part of the hybrid samples, and the Ω-shaped junction was completely lost.
These changes in the micromorphology of the leaf surface of hybrid plants have obvious similarities with the elimination of part of the genetic material of wild species in hybrids in the first seed generations during distant hybridization, which is repeatedly described in the literature. It can be assumed that the distribution of micromorphological characteristics observed in hybrid plants is the result of an additional elimination of genetic material of E. farctus, the trigger of which was the crossing of the f-hybrid enriched with the genetic material of wheatgrass with a wheat-type plant.

Author Contributions

Conceptualization, A.V.B., P.O.L. and V.P.U.; methodology, A.V.B. and P.O.L.; investigation, data curation, validation, and visualization, A.V.B., P.O.L., A.A.P., M.M.G., A.D.A., G.I.K., T.S.W. and I.N.K.; resources, P.O.L.; formal analysis, A.V.B.; writing—original draft preparation, A.V.B.; writing—review and editing, A.V.B., P.O.L., A.A.P., M.M.G., A.D.A., G.I.K., T.S.W., I.N.K. and V.P.U.; funding acquisition, A.V.B. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by the Russian Science Foundation, grant No. 24-26-00074, https://rscf.ru/project/24-26-00074/ (accessed on 23 October 2024).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Agronomy 14 02490 g0a1
Figure A1. Hybrid samples under study (BBCH59-73). From left to right: f11814 (maternal plant), N4, N14, N16, N23, w107 (paternal plant), cultivar Zlata (standard for Moscow region), cultivar Sudarynya (standard for Moscow region). Scale bar 1 m.
Figure A1. Hybrid samples under study (BBCH59-73). From left to right: f11814 (maternal plant), N4, N14, N16, N23, w107 (paternal plant), cultivar Zlata (standard for Moscow region), cultivar Sudarynya (standard for Moscow region). Scale bar 1 m.
Agronomy 14 02490 g0a1
Table A1. Height of the studied hybrids and parental forms. For comparison, data of standard varieties grown in Moscow region are presented.
Table A1. Height of the studied hybrids and parental forms. For comparison, data of standard varieties grown in Moscow region are presented.
Samplef11814 (♀)w107 (♂)N4N14N16N23ZlataSudarynya
Height, cm98.2 ± 1.6 a69.0 ± 4.0 bc68.2 ± 5.1 c98.2 ± 4.3 a82.0 ± 4.4 bc80.0 ± 2.9 bc84.4 ± 2.6 ab74.2 ± 1.2 bc
Values are average height of 5 typical plants ±SE (2024, F9). Averages in a row without a common superscript letter differ (p < 0.05), as analyzed by one-way ANOVA and the Tukey test.
Table A2. Main parameters of spike morphology of hybrid samples (growing season 2022).
Table A2. Main parameters of spike morphology of hybrid samples (growing season 2022).
ParameterSamples
N4N14N16N23
Spike length, cm10.0 ± 0.3 ab11.0 ± 0.4 a10.6 ± 0.3 ab9.4 ± 0.2 b
Number of spikelets in spike17.9 ± 0.2 b19.9 ± 0.5 a17.3 ± 0.4 b16.6 ± 0.3 b
Number of flowers in spikelet4.5 ± 0.23.8 ± 0.33.9 ± 0.14.3 ± 0.2
Number of grains in spike51.9 ± 2.6 a40.3 ± 2.6 b41.4 ± 2.1 b47.2 ± 2.0 ab
1000 grain weight, g51.3 ± 0.549.5 ± 1.848.1 ± 1.547.9 ± 2.7
Values are means of 8 replications ± SE. Averages in a row without a common superscript letter differ (p < 0.05), as analyzed by one-way ANOVA and the Tukey test.

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Figure 1. Main micromorphological elements of abaxial (a–f) and adaxial (g–j) leaf surface. (a) Apically directed prickles (B1) and oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline (B7) in costal zone (N16); (b) basally directed prickles (B2), single and with basal round silica cell in costal zone. Rounded short silica cells with enfolding thin sickle-shaped or crescentic cork cells (B6), Ω-shaped anticlinal cell walls of costal long cells (N4); (c) basally directed prickles (B2) and oval silica cells (B7), almost round with white longitudinal stripe in midvein zone (N4); (d) single or with elongate basal cell shield-shaped prickles (B4) in costal zone (w107); (e) round silica cells (B6) with enfolding crescentic cork cells in costal and intercostal zones (N23); (f) round silica cells (B6) alternating with intercostal long cell with well-defined Ω-shaped (B8) anticlinal cell walls (f11814); (g) shield-shaped prickles (D2) and round silica cell (D3) paired with round basal cell (f11814); (h) oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline (D3) in costal zone and bulliform cells in intercostal zone on the adaxial leaf side (w107); (i) macro-hair in costal zone and small hairs or prickles (hooks) near stomata (f11814); (j) basally directed macro-hairs in costal and intercostal zones, bulliform cells in the intercostal zone between stomatal rows (w107). Scale bar 100 µm (a–f, h–j), 50 µm; (g) direction to leaf tip from bottom to top. bfc—bulliform cell; ccc—crescentic cork cell, enfolding the silica cell; cz—costal zone; ebc—elongated basal cell; esc—elongated or elliptical silica cell; iz—intercostal zone; lsc—long silica cell; mh—macro-hair; mvz—midvein zone; pr—prickle; rbc—round basal cell; rsc—round silica cell; sh—shield-shaped prickle; st—stomata; Ωcw—Ω-shaped anticlinal cell walls.
Figure 1. Main micromorphological elements of abaxial (a–f) and adaxial (g–j) leaf surface. (a) Apically directed prickles (B1) and oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline (B7) in costal zone (N16); (b) basally directed prickles (B2), single and with basal round silica cell in costal zone. Rounded short silica cells with enfolding thin sickle-shaped or crescentic cork cells (B6), Ω-shaped anticlinal cell walls of costal long cells (N4); (c) basally directed prickles (B2) and oval silica cells (B7), almost round with white longitudinal stripe in midvein zone (N4); (d) single or with elongate basal cell shield-shaped prickles (B4) in costal zone (w107); (e) round silica cells (B6) with enfolding crescentic cork cells in costal and intercostal zones (N23); (f) round silica cells (B6) alternating with intercostal long cell with well-defined Ω-shaped (B8) anticlinal cell walls (f11814); (g) shield-shaped prickles (D2) and round silica cell (D3) paired with round basal cell (f11814); (h) oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline (D3) in costal zone and bulliform cells in intercostal zone on the adaxial leaf side (w107); (i) macro-hair in costal zone and small hairs or prickles (hooks) near stomata (f11814); (j) basally directed macro-hairs in costal and intercostal zones, bulliform cells in the intercostal zone between stomatal rows (w107). Scale bar 100 µm (a–f, h–j), 50 µm; (g) direction to leaf tip from bottom to top. bfc—bulliform cell; ccc—crescentic cork cell, enfolding the silica cell; cz—costal zone; ebc—elongated basal cell; esc—elongated or elliptical silica cell; iz—intercostal zone; lsc—long silica cell; mh—macro-hair; mvz—midvein zone; pr—prickle; rbc—round basal cell; rsc—round silica cell; sh—shield-shaped prickle; st—stomata; Ωcw—Ω-shaped anticlinal cell walls.
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Figure 2. Abaxial leaf surface of hybrids and parents. (a) N4: midvein and costal zones with basally directed prickles, single shield-shaped prickles, and rounded short silica cells; (b) (N14) and (c) (N16): midvein zone with single shield-shaped prickles and rounded short silica cells, single elongate silica cells in costal zone on the right; (d) N23: single oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline in costal zone on the left and leaf margin with numerous shield-shaped prickles, rounded short silica cells, and well-expressed Ω-shaped anticlinal cell walls; (e) f11814 (mother): numerous shield-shaped prickles in costal zone, rounded short silica cells, and well-expressed Ω-shaped anticlinal cell walls in the intercostal zone; (f) w107 (father): basally directed prickles, single shield-shaped prickle, and rounded short silica cells similar to hybrids in midvein and costal zones. Scale bar 100 µm. Direction to leaf tip from bottom to top.
Figure 2. Abaxial leaf surface of hybrids and parents. (a) N4: midvein and costal zones with basally directed prickles, single shield-shaped prickles, and rounded short silica cells; (b) (N14) and (c) (N16): midvein zone with single shield-shaped prickles and rounded short silica cells, single elongate silica cells in costal zone on the right; (d) N23: single oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline in costal zone on the left and leaf margin with numerous shield-shaped prickles, rounded short silica cells, and well-expressed Ω-shaped anticlinal cell walls; (e) f11814 (mother): numerous shield-shaped prickles in costal zone, rounded short silica cells, and well-expressed Ω-shaped anticlinal cell walls in the intercostal zone; (f) w107 (father): basally directed prickles, single shield-shaped prickle, and rounded short silica cells similar to hybrids in midvein and costal zones. Scale bar 100 µm. Direction to leaf tip from bottom to top.
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Figure 3. Adaxial leaf surface of hybrids and parents. (a) N4: midvein and costal zones with apically directed prickles and oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline, basal cells of prickle are elongated or long; (b) (N14), (c) (N16), and (d) (N23): costal zone with apically directed prickles and oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline, basal cells of prickle are oval or rectangular; (e) f11814 (mother): apically directed prickles and shield-shaped prickles in costal zone, round basal cells in prickles of both types; (f) w107 (father): midvein and costal zones with apically directed prickles and oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline, basal cells of prickle are oval or rectangular, similar to hybrids. Scale bar 100 µm. Direction to leaf tip from bottom to top.
Figure 3. Adaxial leaf surface of hybrids and parents. (a) N4: midvein and costal zones with apically directed prickles and oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline, basal cells of prickle are elongated or long; (b) (N14), (c) (N16), and (d) (N23): costal zone with apically directed prickles and oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline, basal cells of prickle are oval or rectangular; (e) f11814 (mother): apically directed prickles and shield-shaped prickles in costal zone, round basal cells in prickles of both types; (f) w107 (father): midvein and costal zones with apically directed prickles and oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline, basal cells of prickle are oval or rectangular, similar to hybrids. Scale bar 100 µm. Direction to leaf tip from bottom to top.
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Figure 4. Scatter plot of samples in coordinates of the first and second principal components. Principal component analysis (PCA) was used to analyze the presence and degree of expression of 8 micromorphological markers on the abaxial side of the leaf and 4 markers on the adaxial side of the leaf. In the insets: (a) “Biplot” projection of the original variables onto the scattergram; (b) plot of eigenvalues. The eigenvalues expected under a random model (broken stick) are indicated by the red dashed line (plot of actual versus randomly generated eigenvalues). The dots represent the examined samples in 3 groups. Hybrids—hybrid samples obtained by crossing f11814 × w107; WWH—wheat-wheatgrass hybrids of wheat type, including paternal plants of w107; F—f-hybrids, progeny of crossing ×Trititrigia cziczinii Tsvel. with Elymus farctus, including mother plants f11814 and E. farctus specimens.
Figure 4. Scatter plot of samples in coordinates of the first and second principal components. Principal component analysis (PCA) was used to analyze the presence and degree of expression of 8 micromorphological markers on the abaxial side of the leaf and 4 markers on the adaxial side of the leaf. In the insets: (a) “Biplot” projection of the original variables onto the scattergram; (b) plot of eigenvalues. The eigenvalues expected under a random model (broken stick) are indicated by the red dashed line (plot of actual versus randomly generated eigenvalues). The dots represent the examined samples in 3 groups. Hybrids—hybrid samples obtained by crossing f11814 × w107; WWH—wheat-wheatgrass hybrids of wheat type, including paternal plants of w107; F—f-hybrids, progeny of crossing ×Trititrigia cziczinii Tsvel. with Elymus farctus, including mother plants f11814 and E. farctus specimens.
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Figure 5. Stomatal density and width of cell rows in hybrid samples and parental plants on abaxial and adaxial leaf sides. St—stomatal density; StRow—proportion of stomatal rows; AvRow—average width of cell rows. Values are mean ± SE. Means of the variables on the same leaf side without a common superscript letter are different (p < 0.05), as analyzed by one-way ANOVA and the Tukey test.
Figure 5. Stomatal density and width of cell rows in hybrid samples and parental plants on abaxial and adaxial leaf sides. St—stomatal density; StRow—proportion of stomatal rows; AvRow—average width of cell rows. Values are mean ± SE. Means of the variables on the same leaf side without a common superscript letter are different (p < 0.05), as analyzed by one-way ANOVA and the Tukey test.
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Figure 6. Parameters of stomata of hybrid samples and parental plants on abaxial and adaxial sides of the leaf. L—stomatal length; S—stomatal complex width; L/S—ratio of stomatal length and stomatal complex width. Values are mean ± SEM. Means of the variables on the same leaf side without a common superscript letter are different (p < 0.05), as analyzed by one-way ANOVA and the Tukey test.
Figure 6. Parameters of stomata of hybrid samples and parental plants on abaxial and adaxial sides of the leaf. L—stomatal length; S—stomatal complex width; L/S—ratio of stomatal length and stomatal complex width. Values are mean ± SEM. Means of the variables on the same leaf side without a common superscript letter are different (p < 0.05), as analyzed by one-way ANOVA and the Tukey test.
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Figure 7. Scatter plot of the studied samples in coordinates of the first and second principal components obtained by principal component analysis (PCA) of stomatal apparatus and cell row width. Insets: (a) “Biplot” projection of the original variables on the scattergram: AvRow—average cell row width; L—average stomatal length; S—average width of stomatal complex; L/S—ratio of stomatal length to stomatal complex width; St—stomatal density; StL—number of stomata per unit length of stomatal row; StRow—proportion of stomatal rows from all cell rows (parameters with indices B and D refer to the abaxial and adaxial side of the leaf, respectively); RoundRibABA—average for the sample diameter of rounded silica cells in the abaxial costal zone; (b) plot of eigenvalues. The eigenvalues expected under a random model (broken stick) are shown by the red dashed line (plot of actual versus randomly generated eigenvalues). The dots represent the studied samples in 3 groups. Hybrids—hybrid samples obtained by crossing f11814 × w107; WWH—wheat-wheatgrass hybrids of wheat type, including paternal plants of w107; F—f-hybrids, progeny of crossing ×Trititrigia cziczinii Tsvel. and Elymus farctus, including mother plants f11814 and a sample of E. farctus.
Figure 7. Scatter plot of the studied samples in coordinates of the first and second principal components obtained by principal component analysis (PCA) of stomatal apparatus and cell row width. Insets: (a) “Biplot” projection of the original variables on the scattergram: AvRow—average cell row width; L—average stomatal length; S—average width of stomatal complex; L/S—ratio of stomatal length to stomatal complex width; St—stomatal density; StL—number of stomata per unit length of stomatal row; StRow—proportion of stomatal rows from all cell rows (parameters with indices B and D refer to the abaxial and adaxial side of the leaf, respectively); RoundRibABA—average for the sample diameter of rounded silica cells in the abaxial costal zone; (b) plot of eigenvalues. The eigenvalues expected under a random model (broken stick) are shown by the red dashed line (plot of actual versus randomly generated eigenvalues). The dots represent the studied samples in 3 groups. Hybrids—hybrid samples obtained by crossing f11814 × w107; WWH—wheat-wheatgrass hybrids of wheat type, including paternal plants of w107; F—f-hybrids, progeny of crossing ×Trititrigia cziczinii Tsvel. and Elymus farctus, including mother plants f11814 and a sample of E. farctus.
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Table 1. Main parameters of spike morphology of hybrid samples and parental forms (growing season 2023).
Table 1. Main parameters of spike morphology of hybrid samples and parental forms (growing season 2023).
ParameterSamples
f11814 (♀)w107 (♂)N4N14N16N23Sudarynya
Spike length, cm14.7 ± 0.4 a8.4 ± 0.5 c9.6 ± 0.4 bc10.8 ± 0.4 b10.3 ± 0.3 b9.9 ± 0.4 bc10.0 ± 0.4 bc
Number of spikelets in spike18.4 ± 0.7 ab14.6 ± 0.7 c17.0 ± 0.5 ac19.4 ± 0.6 a17.0 ± 0.6 ac16.6 ± 0.6 bc17.3 ± 0.6 ac
Number of flowers in spikelet5.4 ± 0.2 a4.2 ± 0.2 bc4.4 ± 0.2 bc3.6 ± 0.2 c3.9 ± 0.2 c4.0 ± 0.2 bc4.8 ± 0.2 ab
Number of grains in spike47.4 ± 3.1 a30.4 ± 3.5 b36.4 ± 2.5 ab32.2 ± 2.5 b36.5 ± 2.5 ab35.2 ± 2.5 ab46.5 ± 2.9 a
1000 grain weight, g34.8 ± 1.8 b49.8 ± 1.8 a45.3 ± 1.5 a46.6 ± 1.5 a44.5 ± 1.5 a43.9 ± 1.5 a28.8 ± 1.7 b
Values are means ± SE. Means in a row without a common superscript letter differ (p < 0.05), as analyzed by one-way ANOVA and the Tukey test.
Table 2. Main grain quality parameters of 4 hybrid samples (2022, F7).
Table 2. Main grain quality parameters of 4 hybrid samples (2022, F7).
ParameterSample
N4N14N16N23
Vegetation, days9010590100
Sedimentation number (Zeleny test)55414155
Gluten, %30.74032.939
Grain vitreousness, %56.361.453.855.3
Protein, %17.219.617.418.9
Table 3. Main micromorphological markers on the leaf surface of hybrid samples (in scores from 0 to 3).
Table 3. Main micromorphological markers on the leaf surface of hybrid samples (in scores from 0 to 3).
Leaf SideNMarkerF11814
(♀)		W107
(♂)		N4N14N16N23
abaxialB1Apically directed prickles in the costal zone, solitary or adjacent to an elongated basal cell000110
B2Basally directed prickles in the costal and midvein zones022110
B3Basally directed long macro-hairs on midvein01-21000
B4Shield-shaped prickles, single or with semicircular basal cell in the costal zone21-20-1111
B5Shield-shaped prickles, single or with semicircular basal cell in the midvein zone30-20220
B6Rounded short silica cells with thin basal cork cell in the costal and midvein zones222122
B7Oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline, single or with semicircular basal cell in the costal zone00-21222
B8Ω-shaped junction of horizontal anticlinal walls of long cells in the intercostal zone20-20000
adaxialD1Apically directed prickles in the costal zone1-222222
D2Shield-shaped prickles, single or with semicircular basal cell, and also rounded short silica cells in the costal and midvein zones200000
D3Basal cells of the prickles and shield-shaped prickles are oval or rectangular. Oval or rectangular short cells with horizontally elongated silica bodies with sinuous outline in the costal and midvein zones. Long cells with silicified wavy cell walls in the costal zone (but may be absent on the midvein)022222
D4Long cells with silicified wavy cell walls in the midvein zone022222
The frequency of micromorphological structures is estimated in points: 0—complete absence; 1—single structures; 2—the structure is typical for this sample; 3—the sample is characterized by a special abundance of such structures.
Table 4. Dimensions of selected microstructures in hybrid samples and parental plants (2023).
Table 4. Dimensions of selected microstructures in hybrid samples and parental plants (2023).
Markerf11814 (♀)w107 (♂)N4N14N16N23
Diameter of the base of shield-shaped prickles in the costal and midvein zones on abaxial leaf side (B4 and B5)24.6 ± 1.0 b30.1 ± 1.3 a24.4 ± 0.9 b22.5 ± 0.3 b24.2 ± 0.7 b24.8 ± 1.8 b
Diameter of rounded short silica cells (abaxial intercostal zone) (B6)14.9 ± 0.4 b16.9 ± 0.2 ab16.0 ± 0.4 ab16.3 ± 0.4 ab17.7 ± 0.6 a17.2 ± 0.9 ab
Diameter of rounded short silica cells (abaxial costal and midvein zones) (B6, RoundRibABA)15.8 ± 0.716.2 ± 0.715.3 ± 0.416 ± 0.616.9 ± 0.716.4 ± 0.7
Length of elongated silica bodies in the abaxial costal zone (B7) -33.5 ± 2.6 ab29.4 ± 1.2 b37.1 ± 1.7 a30 ± 1.7 ab36.7 ± 3.1 ab
Length of elongated silica bodies in the abaxial midvein zone (B7) -24.6 ± 1.325.6 ± 1.124.4 ± 0.823.2 ± 0.826.1 ± 2.4
Length of apically directed prickles in the adaxial costal zone (D1) 49.9 ± 3.6 ab44.4 ± 1.8 bc39.4 ± 0.8 c43.9 ± 1.3 bc54.7 ± 2.1 a47.2 ± 2.2 ab
Values are means ± SEM. Means of the variable on the same leaf side without a common superscript letter differ (p < 0.05), as analyzed by one-way ANOVA and the Tukey test.
Table 5. Exploratory factor analysis with oblimin rotation of the parameters of stomatal complex, width of cell rows, and diameter of silica cells on the abaxial and adaxial leaf surfaces of the studied samples.
Table 5. Exploratory factor analysis with oblimin rotation of the parameters of stomatal complex, width of cell rows, and diameter of silica cells on the abaxial and adaxial leaf surfaces of the studied samples.
ParametersComponent Loadings
RC1RC2RC3RC4Uniqueness
StB 0.8500.2348
StRowB0.913 0.0988
AvRowB0.899 0.1698
LB 0.789 0.3366
SB−0.320 0.569 0.4032
StLB−0.365 0.3120.7720.1081
L/SB 0.689−0.379 0.3169
StD −0.751 0.3298
StRowD0.7770.323 0.1947
AvRowD0.947 0.0639
LD 0.7030.535 0.0716
SD 0.922 0.1125
StLD −0.891 0.1910
L/SD 0.450−0.751 0.1513
RoundRibABA 0.4260.495
PCA 1 * 0.5390.5591
PCA 2 −0.3050.7531
* Principal components PCA1 and PCA2 obtained by PCA of micromorphologic markers (Figure 4) are additionally included in the analysis. The values of component loadings less than 0.3 are not shown.
Table 6. Correlation matrix of quantitative micromorphologic parameters (n = 17).
Table 6. Correlation matrix of quantitative micromorphologic parameters (n = 17).
StBStRowBAvRowBLBSBStLBL/SBStDStRowDAvRowDLDSDStLDL/SD
StB
StRowB0.247
AvRowB−0.0410.902 ***
LB−0.123−0.121−0.165
SB−0.372−0.430−0.207−0.030
StLB0.570 *−0.299−0.231−0.2120.278
L/SB0.2980.208−0.0280.544 *−0.784 ***−0.278
StD−0.150−0.284−0.133−0.566 *0.1680.211−0.546 *
StRowD−0.0490.682 **0.564 *0.155−0.343−0.552 *0.298−0.456
AvRowD−0.0900.767 ***0.788 ***0.013−0.260−0.3470.172−0.4190.874 ***
LD−0.337−0.190−0.1850.765 ***0.242−0.2840.222−0.4620.2280.109—
SD−0.229−0.220−0.1070.2590.492 *0.143−0.155−0.1780.0420.1660.664 **—
StLD−0.174−0.0340.249−0.613 **0.1790.363−0.539 *0.734 ***−0.476−0.119−0.546 *−0.051—
L/SD0.0860.037−0.1820.387−0.366−0.4210.444−0.2430.150−0.1900.047−0.671 **−0.576 *—
RoundRib
ABA		0.3170.1410.1410.062−0.0040.387−0.004−0.3270.2390.3360.1290.295−0.130−0.352
AvRow—average cell row width; L—average stomatal length; S—average width of stomatal complex; L/S—ratio of stomatal length to stomatal complex width; St—stomatal density; StL—number of stomata per unit length of stomatal row; StRow—proportion of stomatal rows from all cell rows (parameters with indices B and D refer to the abaxial and adaxial side of the leaf, respectively); RoundRibABA—average for the sample diameter of rounded silica cells in the abaxial costal zone. The correlation coefficient is significant at the following levels: * p < 0.05, ** p < 0.01, *** p < 0.001.
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Babosha, A.V.; Loshakova, P.O.; Pogost, A.A.; Gevorkyan, M.M.; Alenicheva, A.D.; Komarova, G.I.; Wineshenker, T.S.; Klimenkova, I.N.; Upelniek, V.P. Leaf Surface Micromorphology in Hybrids of Wheat and ×Trititrigia × Elymus farctus. Agronomy 2024, 14, 2490. https://doi.org/10.3390/agronomy14112490

AMA Style

Babosha AV, Loshakova PO, Pogost AA, Gevorkyan MM, Alenicheva AD, Komarova GI, Wineshenker TS, Klimenkova IN, Upelniek VP. Leaf Surface Micromorphology in Hybrids of Wheat and ×Trititrigia × Elymus farctus. Agronomy. 2024; 14(11):2490. https://doi.org/10.3390/agronomy14112490

Chicago/Turabian Style

Babosha, Alexander V., Pavla O. Loshakova, Alina A. Pogost, Margarita M. Gevorkyan, Anastasia D. Alenicheva, Galina I. Komarova, Tatyana S. Wineshenker, Irina N. Klimenkova, and Vladimir P. Upelniek. 2024. "Leaf Surface Micromorphology in Hybrids of Wheat and ×Trititrigia × Elymus farctus" Agronomy 14, no. 11: 2490. https://doi.org/10.3390/agronomy14112490

APA Style

Babosha, A. V., Loshakova, P. O., Pogost, A. A., Gevorkyan, M. M., Alenicheva, A. D., Komarova, G. I., Wineshenker, T. S., Klimenkova, I. N., & Upelniek, V. P. (2024). Leaf Surface Micromorphology in Hybrids of Wheat and ×Trititrigia × Elymus farctus. Agronomy, 14(11), 2490. https://doi.org/10.3390/agronomy14112490

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