**2.3 Genetic diversity of energy grass cultivar Szarvasi-1**

Besides studying the agronomical features of Szarvasi-1, it was important to reveal its genetic background, in order to ascertain its taxonomic position in the system of grasses (focusing on the Triticeae tribe), to assess genetic similarities among the closely related *Agropyron* and *Elymus* genera and to establish genetic relationships among native Hungarian populations of *E. elongatus* (the hypothesised ancestor of Szarvasi-1) and the cultivar.

As a first step the genetic background of Szarvasi-1 and its relatives was studied by RAPD (Randomly Amplified Polymorphic DNA) technique, which allowed the random study of the whole genome with no prior knowledge required. RAPDs can produce a large set of markers, which can be used for the evaluation of both between- and within-species genetic variation, more rapidly and easily than isozymes and microsatellites (Guadagnuolo et al., 2001). To determine the exact taxonomic position of the cultivar among its relatives, specific primers for sequencing specific DNA regions were used. The sequences were compared and phylogenetically analysed. Our results indicated a potential risk of gene flow, which is a possible disadvantage of planting Szarvasi-1 energy grass on large scale.

### **2.3.1 Interspecific study**

The interspecific variation of three *Elymus* and an *Agropyron* species together with the Szarvasi-1 cultivar was screened with 61 RAPD primers (Table 1.). The most informative 16

Tall wheatgrass is a Pontic-Mediterranean grass species. Its distribution ranges along the Mediterranean Basin from the Black Sea to the Iberian Peninsula. This vast area is covered by two, morphologically very different subspecies. The shorter and more fragile *E. elongatus* (Host) Runemark subsp. *elongatus* occurs in the western basin of the Pontus-Mediterranean area, while the taller and more robust *E. elongatus* (Host) Runemark subsp. *ponticus* (Podp.) Melderis occupies the Eastern Mediterranean Basin. The latter is also native to Hungary, reaching the north-westernmost part of its distribution in this area (Tutin et al., 1980). Szarvasi-1 energy grass was bred as an intra-specific hybrid of drought-tolerant and robust *E. elongatus* subsp. *ponticus* populations from Hungary and from different pontic areas (Janowsky & Janowszky, 2007). The 10-year-long breeding process was conducted in Szarvas (East Hungary) but more recently the new breed has been involved in extensive crop management studies in different parts of the country. The Szarvasi-1 tall wheatgrass cultivar was officially recognized by the Hungarian Central Agricultural Office in 2004.

Classification and nomenclature of wheatgrass species has been the subject of much taxonomic debate (Assadi Runemark, 1995; Mizianty et al., 1999; Murphy & Jones, 1999). Consequently, representatives of this genus are known by several scientific and vernacular names. Synonyms of *Elymus elongatus* (Host) Runemark (tall wheatgrass) include: *Agropyron elongatum* (Host) Beauv., *Elytrigia elongata* (Host) Nevski, *E. pontica* (Podp.) Holub, *Elymus varnensis* (Velen.) Runemark, *Lophopyrum elongatum* (Host) A. Löve and *Thinopyrum ponticum*

Besides studying the agronomical features of Szarvasi-1, it was important to reveal its genetic background, in order to ascertain its taxonomic position in the system of grasses (focusing on the Triticeae tribe), to assess genetic similarities among the closely related *Agropyron* and *Elymus* genera and to establish genetic relationships among native Hungarian populations of *E. elongatus* (the hypothesised ancestor of Szarvasi-1) and the

As a first step the genetic background of Szarvasi-1 and its relatives was studied by RAPD (Randomly Amplified Polymorphic DNA) technique, which allowed the random study of the whole genome with no prior knowledge required. RAPDs can produce a large set of markers, which can be used for the evaluation of both between- and within-species genetic variation, more rapidly and easily than isozymes and microsatellites (Guadagnuolo et al., 2001). To determine the exact taxonomic position of the cultivar among its relatives, specific primers for sequencing specific DNA regions were used. The sequences were compared and phylogenetically analysed. Our results indicated a potential risk of gene flow, which is a

The interspecific variation of three *Elymus* and an *Agropyron* species together with the Szarvasi-1 cultivar was screened with 61 RAPD primers (Table 1.). The most informative 16

possible disadvantage of planting Szarvasi-1 energy grass on large scale.

**2. Description** 

(Podp.) Liu & Wang.

**2.3.1 Interspecific study** 

cultivar.

**2.1 Origin and distribution** 

**2.2 Taxonomy and nomenclature of** *Elymus elongatus* 

**2.3 Genetic diversity of energy grass cultivar Szarvasi-1** 

primers were retained, due to their ability to produce polymorphic, unambiguous and stable RAPD markers. Various banding patterns were revealed by different primers, but only polymorphic fragments of high intensity and moderate size (between 100 and 3000 bp) were used. About 98% (131 bands) of the total number of bands (136) were polymorphic. Though the high number of polymorphic bands allows the easy differentiation of analyzed samples using RAPD markers, it gives poor information regarding the relationships among the studied taxa.


Table 1. Hungarian *Agropyron* and *Elymus* taxa used in the interspecies study

Sequence analysis was performed for two DNA regions: the *rpo*A gene of the plastid genome including partial sequences of *pet*D and *rps*11 genes, which was successfully applied by Gitta Petersen and Ole Seberg (1997) to study the Triticeae tribe; and the intergenic spacers (ITS) of the rDNA, an extensively used marker in molecular phylogeny. These analyses resolved the exact taxonomic position of Szarvasi-1. Plant materials were collected from field and identified carefully using morphological characters. Total DNA was extracted from leaves, the targeted DNA loci were amplified in polymerase chain reactions (PCR) and sequenced. New DNA sequence data were deposited to GenBank. Cladistic analyses were performed with PAUP\* 4.0 software (Swofford, 2001) on Windows XP, using maximum parsimony, supplemented with additional public sequence data referring to the tribe. *Bromus inermis* was used as an outgroup. The analysis comprised 32 sequences representing 21 of the 24 monogenomic genera of the Triticeae. In the case of the *rpo*A data, the final matrix contained 1385 characters, of which 1276 (92%) were constant, 84 (6%) variable but uninformative and 25 (1.8%) informative. The analysis resulted in a 129-step-long parsimonious tree (Fig. 1.A) (consistency index including all characters = 0.9225, consistency index excluding uninformative characters = 0.7368, retention index = 0.9048). However, the results were based on only a small number of phylogenetically informative characters (1.8%) – concentrated mostly in the non-coding spacer regions. Therefore the study was completed by the analysis based on the nuclear ribosomal internal transcribed spacers (ITS) (Fig. 1.B). In the latter case the final matrix included 596 characters: 459 (77%) were constant, 53 (8.9%) variable but uninformative and 83 (13.4%) informative (tree length = 214 steps long, consistency index including all characters = 0.7617, consistency index excluding uninformative characters = 0.6731, retention index = 0.7475). In both cases, four sequences – *E. elongatus*, *E. elongatus* subsp. *ponticus* cv. Szarvasi-1, *E. hispidus*, and *A. cristatum* - were newly determined.

The phylogenetic relationships inferred from molecular data of both the *rpo*A gene and ITS regions supported the separation of the studied *Elymus* taxa from *A. cristatum –* formerly

Tall Wheatgrass Cultivar Szarvasi–1 (*Elymus elongatus* subsp. *ponticus* cv. Szarvasi–1)

utilization.

**energy grass** 

as a Potential Energy Crop for Semi-Arid Lands of Eastern Europe 273

homogenous population, the samples differed from each other by only 0.8%. The most heterogeneous population seemed to be the population of Hortobágy with 3.8% difference among individuals. According to the present state of our knowledge of the genetic relationships of Szarvasi-1 and other studied Hungarian tall wheatgrass populations we can claim that there is no genetic difference between the genotype of the Szarvasi-1 cultivar and that of the population of Hortobágy. This result suggests that the genetic material of the populations from pontic areas involved in the breeding process could disappear during the process. The ability to differentiate the tested populations by RAPD bands suggested that this technique may provide a rapid and inexpensive method for the identification of the three *Elymus* populations in Hungary and can be used to monitor the possible changes of energy grass genotype by outcrossing different, closely related *Elymus* taxa during their

**2.4 Morphology and anatomy of** *Elymus elongatus* **subsp.** *ponticus* **and Szarvasi-1** 

both their vegetative (Table 2.) and reproductive features (Table 3.).

Fig. 2. Fibrous root system of Szarvasi-1 energy grass (photo: Róbert W. Pál)

*Elymus* species are caespitose or rhizomatous perennials. The roots of *E. elongatus* are fibrous (Fig. 2.), and can reach lengths of 3.5 m. *E. elongatus* can grow to a height of 50-200 cm (Melderis, 1980; Barkworth, 2011), while Szarvasi-1 energy grass can reach 180-220 cm under optimal growing conditions. From the wheatgrass species that are native to Hungary, three members of the genus *Elymus* and a closely related *Agropyron* species were picked for morphological comparison with Szarvasi-1 energy grass (Fig. 3.), taking into consideration

also declared an *Elymus* species. All of the studied *Elymus* taxa form a well supported clade within *Pseudoroegneria*, *Lophopyrum*, and *Festucopsis,* corresponding with the results of Sha et al. (2010). Interestingly, the Hungarian *A. cristatum* is located far from the Danish accession of the species on the *rpo*A based phylogenetic tree. However, the *rpo*A sequence of *E.hispidus* and the ITS sequence of *E. repens* are very similar to those of the Szarvasi-1, suggesting the possibility of unwanted hybridization.

Fig. 1. Strict consensus tree based on phylogenetic analysis A: of the *rpo*A gene B: of the ITS sequence data. Numbers above and below branches indicate bootstrap support.

### **2.3.2 Interpopulational studies**

The interpopulation study compared 15 individuals from each population of *E. elongatus* and the Szarvasi-1 cultivar by RAPD markers. The samples originated from four different locations in Hungary: Hortobágy, Kunadacs, Tiszaalpár and Szarvasi-1 from Görcsöny. The method can be a valuable tool for populational studies (Reisch et al., 2003), though it has often been criticized for low reproducibility; in order to avoid this phenomenon, highly constant conditions were used and all reactions were repeated at least twice. The samples were screened with a total of 80 arbitrary 10-mer primers, out of which only 30 informative primers were retained. The total number of analyzed bands was 373. The percentage of polymorphic bands was 41.3% (154 bands). The RAPD data were used for calculation of pairwise genetic distances using the Simple matching coefficient. The distance matrix was used for cluster analysis using UPGMA (unweighted pair-group method with arithmetic averages). A dendrogram was generated using SYN-TAX 5.0 (Podani, 1993).

Consistent with other results (Díaz et al., 2000; Nieto-Lopez et al., 2000), RAPD analysis discriminated the studied populations. The samples from Kunadacs constituted the most

also declared an *Elymus* species. All of the studied *Elymus* taxa form a well supported clade within *Pseudoroegneria*, *Lophopyrum*, and *Festucopsis,* corresponding with the results of Sha et al. (2010). Interestingly, the Hungarian *A. cristatum* is located far from the Danish accession of the species on the *rpo*A based phylogenetic tree. However, the *rpo*A sequence of *E.hispidus* and the ITS sequence of *E. repens* are very similar to those of the Szarvasi-1, suggesting the

A. B. Fig. 1. Strict consensus tree based on phylogenetic analysis A: of the *rpo*A gene B: of the ITS

The interpopulation study compared 15 individuals from each population of *E. elongatus* and the Szarvasi-1 cultivar by RAPD markers. The samples originated from four different locations in Hungary: Hortobágy, Kunadacs, Tiszaalpár and Szarvasi-1 from Görcsöny. The method can be a valuable tool for populational studies (Reisch et al., 2003), though it has often been criticized for low reproducibility; in order to avoid this phenomenon, highly constant conditions were used and all reactions were repeated at least twice. The samples were screened with a total of 80 arbitrary 10-mer primers, out of which only 30 informative primers were retained. The total number of analyzed bands was 373. The percentage of polymorphic bands was 41.3% (154 bands). The RAPD data were used for calculation of pairwise genetic distances using the Simple matching coefficient. The distance matrix was used for cluster analysis using UPGMA (unweighted pair-group method with arithmetic

Consistent with other results (Díaz et al., 2000; Nieto-Lopez et al., 2000), RAPD analysis discriminated the studied populations. The samples from Kunadacs constituted the most

sequence data. Numbers above and below branches indicate bootstrap support.

averages). A dendrogram was generated using SYN-TAX 5.0 (Podani, 1993).

possibility of unwanted hybridization.

**2.3.2 Interpopulational studies** 

homogenous population, the samples differed from each other by only 0.8%. The most heterogeneous population seemed to be the population of Hortobágy with 3.8% difference among individuals. According to the present state of our knowledge of the genetic relationships of Szarvasi-1 and other studied Hungarian tall wheatgrass populations we can claim that there is no genetic difference between the genotype of the Szarvasi-1 cultivar and that of the population of Hortobágy. This result suggests that the genetic material of the populations from pontic areas involved in the breeding process could disappear during the process. The ability to differentiate the tested populations by RAPD bands suggested that this technique may provide a rapid and inexpensive method for the identification of the three *Elymus* populations in Hungary and can be used to monitor the possible changes of energy grass genotype by outcrossing different, closely related *Elymus* taxa during their utilization.

### **2.4 Morphology and anatomy of** *Elymus elongatus* **subsp.** *ponticus* **and Szarvasi-1 energy grass**

*Elymus* species are caespitose or rhizomatous perennials. The roots of *E. elongatus* are fibrous (Fig. 2.), and can reach lengths of 3.5 m. *E. elongatus* can grow to a height of 50-200 cm (Melderis, 1980; Barkworth, 2011), while Szarvasi-1 energy grass can reach 180-220 cm under optimal growing conditions. From the wheatgrass species that are native to Hungary, three members of the genus *Elymus* and a closely related *Agropyron* species were picked for morphological comparison with Szarvasi-1 energy grass (Fig. 3.), taking into consideration both their vegetative (Table 2.) and reproductive features (Table 3.).

Fig. 2. Fibrous root system of Szarvasi-1 energy grass (photo: Róbert W. Pál)

Tall Wheatgrass Cultivar Szarvasi–1 (*Elymus elongatus* subsp. *ponticus* cv. Szarvasi–1)

fascicles in the case of the outer vascular bundles.

(photo: Ágnes Farkas)

Farkas)

as a Potential Energy Crop for Semi-Arid Lands of Eastern Europe 275

The stems of *E. elongatus* are robust and glabrous (Melderis, 1980). Our comparative histological studies, conducted on *E. hispidus*, *E. elongatus* and Szarvasi-1 energy grass, revealed that in Szarvasi-1 the epidermis of the stem is sclerenchymatous (Fig. 4.), covered with a thick cuticle, which suggests drought tolerance of the cultivar. Stomatal guard cells are in level with the epidermal cells (mesomorphic position), both in the stem (Fig. 4.) and the surrounding leaf sheath, which is typical in plants that require a moderate water supply. In the internodes collateral closed vascular bundles are arranged in two rings, embedded in the sclerenchymatous hypodermis and parenchyma. In the outermost cortical region of the culm in Szarvasi-1, clorenchyma alternates with sclerenchyma, or a continuous sclerenchymatous ring is formed. Third order vascular bundles are located in the hypodermis, while first and second order bundles can be found toward the centre of the stem (Fig. 5.). Vascular bundles are supported by a sclerenchymatous sheath and/or bundle cap, the latter often establishing direct contact with the hypodermal sclerenchymatous

Fig. 4. Sclerenchymatous epidermis with mesomorphic stoma in the stem of Szarvasi-1

Fig. 5. Vascular bundles of varying size in the stem of Szarvasi-1 energy grass (photo: Ágnes

In the nodes of *Elymus* species, the bundles located in the outer region possess a welldeveloped sclerenchymatous bundle cap, which is kernel-shaped in *E. elongatus* (Fig. 6.) and ovate in *E. hispidus*. The phloem consists of sieve tubes and companion cells; the xylem comprises two large tracheas, tracheids and xylem parenchyma, accompanied by a

Fig. 3. Stem, leaf, inflorescence and spikelet of Szarvasi-1 energy grass (drawing: Emőke Oláh)


Table 2. Vegetative features of wheatgrass (*Elymus* and *Agropyron*) taxa (data are based on our own observations and some literature references, see Melderis, 1980 and Barkworth, 2011)

Fig. 3. Stem, leaf, inflorescence and spikelet of Szarvasi-1 energy grass (drawing: Emőke Oláh)

trichomes

long, sparse trichomes on adaxial side

prominent venation, surface and margin bearing spinules

prominent venation, surface and margin bearing spinules

Table 2. Vegetative features of wheatgrass (*Elymus* and *Agropyron*) taxa (data are based on our own observations and some literature references, see Melderis, 1980 and Barkworth, 2011)

**Leaf leaf blade ligule auricle** 

membranous,

membranous,

membranous,

membranous,

truncate -

truncate long

truncate medium

dentate medium

dentate long

**Stem height (cm)** 

*E. repens* rhizomatous 40-120 dense venation membranous,

*A. pectiniforme* fibrous 25-60 adaxial side with

**Taxon /** 

**Character Root system**

*E. hispidus* rhizomatous 40-150

*E. elongatus* fibrous 50-200

Szarvasi-1 fibrous 50-220

The stems of *E. elongatus* are robust and glabrous (Melderis, 1980). Our comparative histological studies, conducted on *E. hispidus*, *E. elongatus* and Szarvasi-1 energy grass, revealed that in Szarvasi-1 the epidermis of the stem is sclerenchymatous (Fig. 4.), covered with a thick cuticle, which suggests drought tolerance of the cultivar. Stomatal guard cells are in level with the epidermal cells (mesomorphic position), both in the stem (Fig. 4.) and the surrounding leaf sheath, which is typical in plants that require a moderate water supply. In the internodes collateral closed vascular bundles are arranged in two rings, embedded in the sclerenchymatous hypodermis and parenchyma. In the outermost cortical region of the culm in Szarvasi-1, clorenchyma alternates with sclerenchyma, or a continuous sclerenchymatous ring is formed. Third order vascular bundles are located in the hypodermis, while first and second order bundles can be found toward the centre of the stem (Fig. 5.). Vascular bundles are supported by a sclerenchymatous sheath and/or bundle cap, the latter often establishing direct contact with the hypodermal sclerenchymatous fascicles in the case of the outer vascular bundles.

Fig. 4. Sclerenchymatous epidermis with mesomorphic stoma in the stem of Szarvasi-1 (photo: Ágnes Farkas)

Fig. 5. Vascular bundles of varying size in the stem of Szarvasi-1 energy grass (photo: Ágnes Farkas)

In the nodes of *Elymus* species, the bundles located in the outer region possess a welldeveloped sclerenchymatous bundle cap, which is kernel-shaped in *E. elongatus* (Fig. 6.) and ovate in *E. hispidus*. The phloem consists of sieve tubes and companion cells; the xylem comprises two large tracheas, tracheids and xylem parenchyma, accompanied by a

Tall Wheatgrass Cultivar Szarvasi–1 (*Elymus elongatus* subsp. *ponticus* cv. Szarvasi–1)

Fig. 8. Non-glandular trichomes on the leaf of Szarvasi-1 (photo: Ágnes Farkas)

Fig. 9. Vascular bundles in the leaf of Szarvasi-1 (photo: Ágnes Farkas)

numbers of florets in each species (Table 3.).

The inflorescence is an erect spike, which is long and slender in each wheatgrass species, except for *A. pectiniforme*, where it is short and dense, with numerous, overlapping spikelets. In *E. elongatus* the rachis is nearly flat on the side facing the spikelets, usually spinose-ciliate on the main angles (Melderis, 1980). Compared to *E. repens*, the spikelets are less overlapping and more loosely arranged in *E. elongatus*, sitting close to the rachis, and strongly compressed laterally; the rachilla is strigulose. The spikelet consists of varying

elements of the protoxylem.

as a Potential Energy Crop for Semi-Arid Lands of Eastern Europe 277

(xeromorphic position), when guard cells reach the bottom half of epidermal cells. Nonglandular trichomes (bristles) are present in large numbers (Fig. 8.), especially on the adaxial side of the leaf, enhancing the drought-tolerance of the plant by reducing water loss. The two epidermal layers sandwich a chlorenchymatous, homogenous mesophyll layer, consisting of spongy parenchyma. In Szarvasi-1 energy grass the basal leaf blade is strongly wavy in transverse section, due to the ridges formed by the longitudinally running veins that correspond to collateral closed bundles, arranged in a characteristic pattern formed by the alternation of smaller and larger bundles (Fig. 9.). The vascular bundles are surrounded by an inner sclerenchymatous and an outer parenchymatous bundle sheath. Bundles of first order are accompanied by hypodermal sclerenchyma. In both *E. elongatus* and Szarvasi-1 the sclerenchymatous bundle cap is more developed than in *E. hispidus*. In *E. elongatus* cell wall thickening also reaches a higher level in the sclerenchymatous tissues. In the adaxial part of the primary bundles we can often see rexigenous intercellular spaces, containing the broken

rexigenous intercellular space. The walls of vessels and tracheids are strengthened by annular or spiral thickening (Fig. 7.).

Fig. 6. Bundle cap in the stem of Szarvasi-1 (photo: Ágnes Farkas)

Fig. 7. Vessels with annular and spiral thickening in the stem of Szarvasi-1 (photo: Ágnes Farkas)

The leaves of *Elymus* species are flat or more or less convolute (Melderis, 1980). In *E. elongatus* they are convolute, however, in Szarvasi-1 this feature is not typical. The leaf blade is grey-green in *E. elongatus*, as opposed to the blue-grey colour of *E. hispidus* (Melderis, 1980). The leaf blade of *E. elongatus* is 2.5-5 mm wide, prominently and closely veined. Similarly to *E. hispidus*, one margin of the leaf sheath can bear trichomes in *E. elongatus* as well; sparse spinules, and sometimes also short setae can be observed on the surface and the edge of the leaf (Melderis, 1980). The ligule is short and membranous; the presence and length of the auricle varies with *Elymus* species (Table 2.) (Häfliger Scholz, 1980; Melderis, 1980; Barkworth, 2011).

The leaf epidermis in Szarvasi-1 is mostly sclerenchymatized, with thickened cell walls and a thick layer of cuticle, all of which highlight drought tolerance of the energy grass. In the intercostal region of the adaxial epidermis, a group of large bulliform cells can frequently be observed, which play a role in rolling up the leaf blade in the case of drought, thereby reducing transpiration. Both the adaxial and abaxial epidermis may contain stomata, however, they are more frequent on the abaxial side. Most stomatal guard cells are at the level of epidermal cells (mesomorphic position), however, in some cases guard cells may rise above the epidermis (hygromorphic position), or the stoma can become slightly sunken

rexigenous intercellular space. The walls of vessels and tracheids are strengthened by

Fig. 7. Vessels with annular and spiral thickening in the stem of Szarvasi-1 (photo: Ágnes

The leaves of *Elymus* species are flat or more or less convolute (Melderis, 1980). In *E. elongatus* they are convolute, however, in Szarvasi-1 this feature is not typical. The leaf blade is grey-green in *E. elongatus*, as opposed to the blue-grey colour of *E. hispidus* (Melderis, 1980). The leaf blade of *E. elongatus* is 2.5-5 mm wide, prominently and closely veined. Similarly to *E. hispidus*, one margin of the leaf sheath can bear trichomes in *E. elongatus* as well; sparse spinules, and sometimes also short setae can be observed on the surface and the edge of the leaf (Melderis, 1980). The ligule is short and membranous; the presence and length of the auricle varies with *Elymus* species (Table 2.) (Häfliger Scholz, 1980; Melderis,

The leaf epidermis in Szarvasi-1 is mostly sclerenchymatized, with thickened cell walls and a thick layer of cuticle, all of which highlight drought tolerance of the energy grass. In the intercostal region of the adaxial epidermis, a group of large bulliform cells can frequently be observed, which play a role in rolling up the leaf blade in the case of drought, thereby reducing transpiration. Both the adaxial and abaxial epidermis may contain stomata, however, they are more frequent on the abaxial side. Most stomatal guard cells are at the level of epidermal cells (mesomorphic position), however, in some cases guard cells may rise above the epidermis (hygromorphic position), or the stoma can become slightly sunken

Fig. 6. Bundle cap in the stem of Szarvasi-1 (photo: Ágnes Farkas)

annular or spiral thickening (Fig. 7.).

Farkas)

1980; Barkworth, 2011).

(xeromorphic position), when guard cells reach the bottom half of epidermal cells. Nonglandular trichomes (bristles) are present in large numbers (Fig. 8.), especially on the adaxial side of the leaf, enhancing the drought-tolerance of the plant by reducing water loss. The two epidermal layers sandwich a chlorenchymatous, homogenous mesophyll layer, consisting of spongy parenchyma. In Szarvasi-1 energy grass the basal leaf blade is strongly wavy in transverse section, due to the ridges formed by the longitudinally running veins that correspond to collateral closed bundles, arranged in a characteristic pattern formed by the alternation of smaller and larger bundles (Fig. 9.). The vascular bundles are surrounded by an inner sclerenchymatous and an outer parenchymatous bundle sheath. Bundles of first order are accompanied by hypodermal sclerenchyma. In both *E. elongatus* and Szarvasi-1 the sclerenchymatous bundle cap is more developed than in *E. hispidus*. In *E. elongatus* cell wall thickening also reaches a higher level in the sclerenchymatous tissues. In the adaxial part of the primary bundles we can often see rexigenous intercellular spaces, containing the broken elements of the protoxylem.

Fig. 8. Non-glandular trichomes on the leaf of Szarvasi-1 (photo: Ágnes Farkas)

Fig. 9. Vascular bundles in the leaf of Szarvasi-1 (photo: Ágnes Farkas)

The inflorescence is an erect spike, which is long and slender in each wheatgrass species, except for *A. pectiniforme*, where it is short and dense, with numerous, overlapping spikelets. In *E. elongatus* the rachis is nearly flat on the side facing the spikelets, usually spinose-ciliate on the main angles (Melderis, 1980). Compared to *E. repens*, the spikelets are less overlapping and more loosely arranged in *E. elongatus*, sitting close to the rachis, and strongly compressed laterally; the rachilla is strigulose. The spikelet consists of varying numbers of florets in each species (Table 3.).

Tall Wheatgrass Cultivar Szarvasi–1 (*Elymus elongatus* subsp. *ponticus* cv. Szarvasi–1)

source for cultivation the durability of the energy grass crops can be much shorter.

play an important role in the recycling of saline drainage waters for irrigation.

temperature sinks below – 35 °C.

**3.2 Gas exchange behaviour** 

humidity and carbon-dioxide.

as a Potential Energy Crop for Semi-Arid Lands of Eastern Europe 279

The life span of Szarvasi-1 energy grass cultivation can be 10-15 years long, but the temporal change of biomass production during this time has not yet been monitored sufficiently. We have only one complete data series monitoring the yields of an energy grass field on solonec alkaline soil for more than 10 years. According to this study it takes two years for energy grass cultivation to reach maximal biomass production, which can then be maintained for at least 7 years. At around the tenth year energy grass cultivation starts decrease in yearly biomass production. In semi-arid climates without a ground water table serving as water

The flood tolerance of energy grass is relatively good, especially when the cultivation is at least two or three years old and the tussocks of the individuals are well developed. However, in the first year, the short and weak stems of the juveniles cannot tolerate permanent water cover and die out. Hence, the cultivated energy grass stand opens, the density of the stems declines and the establishment of the grass cultivation remains incomplete. In such a condition, weeds can gain multiple chances to invade and to establish. High salt concentrations of the soil can be tolerated by Szarvasi-1 energy grass, but only in wet habitats, where a several weeks long seasonal high water table can occur every year. Because of the high salt resistance, Szarvasi-1 energy grass can be used as salt-tolerant forage and can

Since *Elymus elongatus* subsp. *ponticus* is a native species of the continental and subcontinental climate in Eastern Europe, it tolerates well the summer high temperatures exceeding daily means of even 30-35 °C, and can also resist cold winter days when the

Tall wheatgrass is classified as C3 plant with cool season characteristics and seasonally different water use efficiency in moderately saline habitats (Bleby et al., 1997; Johnson, 1991). Several cultivars have previously been developed based on adaptability to different environmental conditions in Europe and Asia, but not from ecophysiological perspective. Szarvasi-1 energy grass was developed from a native population of tall wheatgrass (*Elymus elongatus* subsp. *ponticus*) that was adapted to slightly salty habitats. Therefore it was expected that *E. elongatus* cv. Szarvasi-1 will be a good candidate for biomass crop status because it produces large amounts of organic matter with relatively broad tolerance spectra and a high adaptability to different environments. Here we review the current knowledge on environmental gas exchange responses of Szarvasi-1 energy grass under greenhouse and field conditions to different environmental parameters such as temperature, light, air

We used the following photosynthetic parameters: *assimilation* as the measure of carbondioxide fixation, *transpiration* as the measure of water loss and *photosynthetic water use efficiency* as the ratio of carbon-dioxide input to water output. All of these parameters depend on stomatal regulation and the abiotic environment. In this section capacities and threshold limits of Szarvasi-1 energy grass gas exchange performance will be presented for a better knowledge of its abiotic environmental requirements (Fig. 10.). To define and to compare gas exchange capacities, growing pots were installed using three soil types (sandy soil, Alfisol-Mollisol, Aquic Mollisol) in the Botanic Garden of the University of Pécs with permanent irrigation. In addition, field experiments were established on three soil types (Alfisol, Alfisol-Mollisol, Aquic Mollisol) in South


Table 3. Reproductive features of wheatgrass (*Elymus* and *Agropyron*) taxa

The glumes of *Elymus* species are indurate-coriaceous, obtuse or truncate, with 1-11 veins, possessing a short awn or no awn at all. The glume can reach half or two thirds of the spikelet in *A. pectiniforme*, two thirds of the spikelet in *E. repens*, and one third of the spikelet in *E. hispidus*, *E. elongatus* and Szarvasi-1 (Fig. 3.). The glumes are 1-3-veined in *A. pectiniforme*, and 3-7-veined in the other taxa. The lemma of *E. elongatus* is obtuse, glabrous, unawned and 5-veined; the palea is two-keeled (Melderis, 1980; Barkworth, 2011). Similarly to other representatives of the Poaceae family, the stigma is feather-like in the *Elymus* genus, where stigmatic secretion is absent even in the mature stage of the stigma, and the receptive surface is discontinuous (Heslop-Harrison Shivanna, 1977). The fruit is a caryopsis.

The evaluated anatomical features allow the differentiation of *E. elongatus* and Szarvasi-1 energy grass from the other investigated members of the *Agropyron*-*Elymus* complex. Szarvasi-1 shows several anatomical traits that enhance drought tolerance, such as a sclerenchymatized epidermis covered by a thick cuticle and dense coverage by nonglandular hairs. On the other hand, the mesomorphic position of stoma guard cells is characteristic of an intermediate water requirement. This dual nature of the habitat tolerance of *Elymus elongatus* cv. Szarvasi-1 has to be taken into account when the new cropfields of this energy grass are planned.
