Breeding of perennial ryegrass (Lolium perenne L.) for forage is mainly aimed at increase in herbage yield. However, abiotic stresses such as drought and winterkill threaten persistence and ability to produce stable aerial biomass of the plant. Field experiments, performed under natural conditions, rather than dissection of abiotic stress factors under artificial or semiartificial conditions, offers opportunity to evaluate the effect of complex of abiotic stresses on the plant performance. The aim of this study was to evaluate the relationship between dry matter yield and tolerance to winter kill and drought of perennial ryegrass ecotypes and cultivars differing in their ploidy level during a 2‐yr period. The field experiment was located in Akademija, Lithuania (55°40′ N, 23°87′ E) in 2015 and 2016. The germplasm consisted of 128 diploid and 25 tetraploid perennial ryegrass populations. Winterkill, spring growth and regrowth after cuts, drought damage, and dry matter yield were assessed. Short periods of lower than −10°C temperatures with slim snow cover determined low damages of the perennial ryegrass after winters. Medium correlation was estimated between drought damages and dry matter yield. Tetraploid genotypes of perennial ryegrass demonstrated higher tolerance to cold and drought stress conditions, better spring growth and regrowth after cuts, and higher dry matter yield.

Perennial ryegrass is an important agricultural crop, however, it is susceptible to winterkill. Freezing injury is caused primarily by ice formation. The LpIRI1 protein has the potential to inhibit ice recrystallization, thus minimize the damage. An association study was conducted using single nucleotide polymorphisms obtained through allele sequencing of the LpIRI1 gene and phenotypic data were collected using two phenotyping platforms in a perennial ryegrass association mapping population of 76 diverse genotypes. Winter survival (FWS) was evaluated under field conditions, while tiller survival (PTS) and electrolyte leakage (EL) at -8 and -12°C were determined under controlled-environment conditions. Proline content (PC) in cold-acclimated plants was measured prior to the freezing test. Significant variation in FWS, PTS, EL and PC was observed among genotypes in our panel. EL and PTS revealed significant negative correlations at -8°C (r s = -0.40) and -12°C (r s = -0.49). PC, however, did not show significant correlations with any of the measured traits, while FWS was correlated (r s = -0.48) with EL at -12°C. The LpIRI1 gene was found to be highly polymorphic with an average SNP frequency of 1 SNP per 16 bp. Association analysis revealed two non-synonymous SNPs being associated with increased EL, both located in the LpIRI1 leucinerich repeat. The results indicate that allelic variation in the LpIRI1 gene plays an important role in the cell membrane integrity of perennial ryegrass during freezing, and can be exploited for developing more freezing tolerant cultivars.

Drought is one of the critical abiotic stresses that significantly affect agricultural production, and current models predict an increase in its severity and intensity in the future. Generally, polyploidy has been found to improve the resistance of plants to abiotic stress. Understanding the role of ploidy in resistance to drought was achieved by comparing the response between diploids and their respective induced autotetraploids of Westerwolths ryegrass (Lolium multiflorum ssp. multiflorum). Field trials were carried out in the 2017 and 2018 growing seasons, and mild drought simulation experiments in controlled conditions were carried out to validate the effect of chromosome duplication. Results obtained from morphological traits in the field experiment revealed that the induced tetraploids were significantly (p < 0.05) taller, had longer inflorescences and larger flag leaf area than their diploid counterparts, especially in the year 2018 characterized by the prolonged drought. This study also revealed that the induced tetraploids produced more dry matter yield than their diploid progenitors, especially in drought periods. The induced tetraploids had significantly higher antiradical activity and phenolic content than the diploid progenitors in response to mild drought, and this significantly correlated with the plant performance in 2018 field trials, indicating that increased ploidy level plays an important role in conferring resistance to drought in Westerwolths ryegrass. Furthermore, the antiradical activity and total phenolic content proved to be a good tool to evaluate drought tolerance at the vegetative stage in Westerwolths ryegrass.

Perennial ryegrass (Lolium perenne L.) is a widely grown species in temperate regions as forage grass as well as for recreational and bioenergy production purposes. Information on perennial ryegrass genetic background facilitates breeding programs by providing an assessment of genetic diversity in exotic material. Genetic diversity of 104 genotypes of perennial ryegrass was evaluated using phenotypic drought traits and amplified fragment length polymorphism (AFLP) data. A high variation was observed for the drought tolerance traits. Chlorophyll fluorescence (F v /F m ) explained 88.8% of the whole variation observed in the collection, while re-growth accounted for 9.1% of all variation. A principal component analysis on the basis of phenotypic drought tolerance traits classified perennial ryegrass collection into three clusters. Three AFLP primer pairs produced a total of 210 fragments, 202 of which were polymorphic among all accessions. The genetic diversity of the collection was high with an average similarity coefficient of 0.46 and the average polymorphic information content of 0.29. The principal component analysis based on AFLP data did not cluster genotypes into any major group. A total of six AFLP fragments, identified as being prevalent in drought tolerant genotypes, together with the high degree of genetic homogeneity found, could provide a choice of selecting genotypes from this perennial ryegrass collection for a drought tolerance breeding program.

Doubling of chromosome set directly affects plant performance through increase of organ size, higher feeding value and increased resistance to adverse environmental factors. Therefore efficient methods of polyploid induction are needed in order to develop new varieties of naturally diploid fodder grass species. The efficiency of antimitotic agents as colchicine, amiprophos-methyl, trifluralin and oryzalin was compared in a series of tetraploid induction experiments in Lolium multiflorum, L. perenne and Festuca pratensis, while newly developed tetraploid plants were compared to standard tetraploid varieties in the field trials. Colchicine treatment proved to be the most efficient method for in vitro cultured embryos in comparison with the other agents. Induced tetraploids of F. pratensis produced higher dry matter and seed yield and could be used for the development of new varieties. Induced tetraploid plants of Lolium spp. were equal to the standard varieties in field trials, therefore they could be used as parental genotypes in crosses. Induced tetraploids of F. pratensis produced higher dry matter and seed yield and could be used for development of new variety.

The current study was set out to establish the efficacy of different mitosis inhibitors (colchicine, amiprophos-methyl, trifluralin and oryzalin) used in the production of red clover (Trifolium pratense L.) and alsike clover (Trifolium hybridum L.) polyploids and to assess the agrobiological traits of tetraploid populations. Four cultivars and five wild populations of T. pratense, one cultivar and three wild populations of T. hybridum were used for polyploidization. The effect of conventional soaking of germinating seeds in a colchicine solution was compared with that of colchicine treatment of embryos in an in vitro culture. Colchicine treatment of T. pratense embryos resulted in 3.3 times higher production of tetraploids (teraploid yield 55.0%) compared with colchicine treatment of seedlings, and the production of chimeric plants was 1.9 times lower. The different mitosis inhibitors were characterised by a diverse polyploidization effect. Tetraploid yield depended not only on the mitosis inhibitor used but also on the plant species. Treatment of T. pratense embryos with colchicine, amiprophosmethyl and oryzalin yielded similar results in terms of tetraploid production (31.3-40.7%) and chimeras (14.3-22.4%). The concentration of trifluralin used for the production of T. pratense tetraploids was ineffective. Colchicine solution was more efficient for the development of T. hybridum tetraploids whose yield was 2.5 times as high as that obtained having treated the embryos with amiprophos-methyl. Induced tetraploid populations were compared to reference cultivars in field trials. The two T. pratense populations 'Radviliai 4n' and 'Arimaičiai 4n' stood out in this respect -their plants accumulated higher dry matter contents, produced more inflorescences than those of the reference cultivar and exhibited prolific seed yield capacity. In relation to the agrobiological traits, the tetraploid populations of T. hybridum did not surpass the reference cultivar.

Global warming is predicted to impact many agricultural areas, which will suffer from reduced water availability. Due to precipitation changes, mild summer droughts are expected to become more frequent, even in temperate regions. For perennial ryegrass (Lolium perenne L.), an important forage grass of the Poaceae family, leaf growth is a crucial factor determining biomass accumulation and hence forage yield. Although leaf elongation has been shown to be temperature-dependent under normal conditions, the genetic regulation of leaf growth under water deficit in perennial ryegrass is poorly understood. Herein, we evaluated the response to water deprivation in a diverse panel of perennial ryegrass genotypes, employing a high-precision phenotyping platform. The study revealed phenotypic variation for growth-related traits and significant (P &lt; 0.05) differences in leaf growth under normal conditions within the subgroups of turf and forage type cultivars. The phenotypic data was combined with genotypic variants identified using genotyping-by-sequencing to conduct a genome-wide association study (GWAS). Using GWAS, we identified DNA polymorphisms significantly associated with leaf growth reduction under water deprivation. These polymorphisms were adjacent to genes predicted to encode for phytochrome B and a MYB41 transcription factor. The result obtained in the present study will increase our understanding on the complex molecular mechanisms involved in plant growth under water deficit. Moreover, the single nucleotide polymorphism (SNP) markers identified will serve as a valuable resource in future breeding programs to select for enhanced biomass formation under mild summer drought conditions.

Some attempts have been made recently to expand the range of perennial grasses tailored to the requirements of modern farming. Attention has been drawn to the forage plant species that have not been widely used so far. Apart from the common cocksfoot (Dactylis glomerata L.), which is traditionally used as a forage, another cocksfoot species -slender cocksfoot (Dactylis polygama H.) can be found in natural habitats in Lithuania. The plants of this species are characterised by some attractive traits: late maturity, better and more stable indicators of feeding value, and lesser aggressiveness (do not form tussocks). The genus Dactylis includes diploid (2n = 2x = 14) and tetraploid (2n = 4x = 28) species. The tetraploid D. glomerata varieties exhibit better agromorphological and quality characteristics. The current work was aimed to develop more productive tetraploid populations of slender cocksfoot and to assess them for agromorphological traits and compare them with diploid populations. The most effective method of tetraploid populations' development was proved to be colchicine treatment of meristems of young inflorescences by maintaining them on a nutrient medium supplemented with 0.2% colchicine for 96 h. Depending on the genotype, the tetraploid yield ranged from 16.7% to 100%. A comparison of averaged biomass qualitative and quantitative indicators of diploid and tetraploid populations showed the tetraploid populations of D. polygama to be more, albeit negligibly, productive and of better quality.