Optimization of isolation and storage of sperm cells from pollen of perennial ryegrass (Lolium perenne L.)

Summary A procedure for isolating sperm cells of perennial ryegrass (Lolium perenne L.) was developed. The sperm cells were released from the pollen grains by osmotic shock, with the right combination of pH and osmolality being important for optimal release. Various combinations of vitamins E, C and fetal calf serum were tested with the aim of improving yield and long-term viability, and their possible mode of action as important components for improvement of these two parameters is discussed. Under optimized conditions, a yield of 12% was established, and the storage time after which 50% of the sperm cells were still viable was improved to 60 h. Cytological observations demonstrated that sperm cells of perennial ryegrass are true protoplasts, which may allow future fusion experiments to be carried out.     

The isolation of viable egg cells of wheat (Triticum aestivum L.)

The isolation of viable egg cells of wheat has been achieved without enzymatic maceration of the ovules. 2,4-D applied to the stigmas resulted 3 to 7 days later in soft ovule tissues which disintegrated upon mechanical manipulation. The isolated egg cells were viable even 2 h after isolation. Their morphology corresponded to that of the in situ egg cells. The mean isolation frequency was 20% (two egg cells per ten ovules).     

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.     

Physicochemical characterization of cellulose from perennial ryegrass leaves (Lolium perenne)

In this study, we investigated the physicochemical properties of the cellulosic preparations obtained from both untreated perennial ryegrass leaves and de-juiced leaves. It was found that treatment at 22 degrees C with 18% NaOH and 18% KOH for 2h, and 10% NaOH and 10% KOH for 16 h yielded 28.2%, 28.8%, 22.7%, 23.4%, respectively, of 'cellulose' residue from untreated ryegrass leaves and 35.7%, 36.8%, 32.8% and 34.6%, respectively, from the de-juiced leaves. For each cellulosic fraction, the glucose content was 71.6%, 69.6%, 67.8%, 66.7%, 69.7%, 68.6%, 63.9% and 61.7%, respectively. The structure of the cellulose samples was examined using FTIR and CP/MAS (13)C NMR spectroscopy and X-ray diffraction. The cellulosic preparations were free of bound lignin except for noticeable amounts of residual hemicelluloses (28.4-38.3%), and had intrinsic viscosities between 275.1 and 361.0 mL/g, along with molecular weights from 144,130 to 194,930 g/mol. This study found that the cellulose samples isolated from both de-juiced ryegrass leaves and the untreated leaves had a much lower percent crystallinity (33.0-38.6%) than that from wood-based fibres (60-70%) and had much shorter fibres (0.35-0.49 mm) than those of either cereal straws, bagasse or wood. In addition, a partial disruption of the hydrogen bonds and microfibrils may occur during the de-juicing process by mechanical activity, which results in a decreased cellulose crystallinity and fibre length. These findings are significant in relation to hydrolysing ryegrass cellulose for bio-ethanol production.     

A comparative study of molecular and morphological methods of describing relationships between perennial ryegrass (Lolium perenne L.) varieties

A sample set of registered perennial ryegrass varieties was used to compare how morphological characterisation and AFLP^® (AFLP^® is a registered trademark of Keygene N.V.) and STS molecular markers described variety relationships. All the varieties were confirmed as morphologically distinct, and both the STS and AFLP markers exposed sufficient genetic diversity to differentiate these registered ryegrass varieties. Distances obtained by each of the approaches were compared, with special attention given to the coincidences and divergences between the methods. When correlations between morphological, AFLP and STS distances were calculated and the corresponding scatter-plots constructed, the variety relationships appeared to be rather inconsistent across the methods, especially between morphology and the molecular markers. However, some consistencies were found for closely related material. An implication could be that these molecular-marker techniques, while not yet suited to certain operations in the traditional registration of new varieties, could be suitable methods for investigating disputable distinctness situations or possible EDV (EDV= essentially derived variety. An EDV is a variety being clearly distinct from, but conforming in the expression of the essential characteristics of, an ’initial variety’ (IV) from which it is found to have been predominantly derived) relationships, subject to establishing standardised protocols and statistical techniques. Some suggestions for such a protocol, including a statistical test for distinctness, are given.     

Effects of sheath tube length on leaf development in perennial ryegrass (Lolium perenne L.)

Position in and contribution of leaf laminae to the canopy of forage grasses are important both in determining herbage growth rates and intake rate by grazing animals. These canopy characteristics are controlled by the way dry matter is apportioned between sheath and lamina in growing leaves. The objective of this work was to determine how the development of individual leaves is affected by altering the effective length of the psuedostem tube, on the assumption that the light environment within the tube varied. The development of a leaf from initiation at the apex to maturity was followed by successive destructive dissections of tillers. Vertical incisions were made in the pseudostem of each tiller to three different depths. The three treatments imposed were — no incision (control), moderate and severe incision of the sheath length. Destructive harvests of tillers followed 3, 6, 12 and 24 days after imposition of treatments. Incision resulted in the length of the monitored leaf being reduced significantly at all harvests, and differentiation of the sheath beginning earlier. The length reduction reflected a reduction in both cell size and cell number and the effects were evident at the earliest harvest. The data support the theory that leaf size and timing of onset of sheath development are influenced by the environment of the developing leaf. The present results indicate that sheath tube length affects leaf development and suggests that the effects are substantially explained by a direct light effect on the location and depth of the elongation zone.     

Effect of perennial ryegrass (Lolium perenne L.) cultivars on the milk yield of grazing dairy cows

The objective of this experiment was to investigate the effect of four perennial ryegrass cultivars: Bealey, Astonenergy, Spelga and AberMagic on the milk yield and milk composition of grazing dairy cows. Two 4 × 4 latin square experiments were completed, one during the reproductive and the other during the vegetative growth phase of the cultivars. Thirty-two Holstein–Friesian dairy cows were divided into four groups, with each group assigned 17 days on each cultivar during both experiments. Within each observation period, milk yield and milk composition, sward morphology and pasture chemical composition were measured. During the reproductive growth phase, organic matter digestibility (OMD) was greater for Bealey and Astonenergy (P < 0.001; +1.6%). AberMagic contained a higher stem proportion (P < 0.01; +0.06) and a longer sheath height (P < 0.001; +1.9 cm). Consequently, cows grazing AberMagic recorded a lower milk yield (P < 0.001; −1.5 kg/day) and a lower milk solids yield (P < 0.001; −0.13 kg/day). During the vegetative growth phase, OMD was greater (P < 0.001; +1.1%) for Bealey, whereas the differences between the cultivars in terms of sward structure were smaller and did not appear to influence animal performance. As a result, cows grazing Bealey recorded a higher milk yield (P < 0.001; +0.9 kg/day) and a higher milk solids yield (P < 0.01; +0.08 kg/day). It was concluded that grass cultivar did influence milk yield due to variations in sward structure and chemical composition.     

Effects of Nitrogen fertilisation and regrowth period on fatty acid concentrations in perennial ryegrass (Lolium perenne L.)

Information on lipids in forages is scarce, and effects of nitrogen (N) application level and regrowth period on the fatty acid (FA) concentration and composition of perennial ryegrass (Lolium perenne L.) were studied. N was applied at 0, 45 and 100 kg ha^?1, and swards were cut after various regrowth periods, resulting in six treatments designed as randomised blocks with three replicates. The stages of development ranged from vegetative to elongating swards, with herbage yield levels from 1.9 to 4.2 t dry matter (DM) ha^?1. Concentrations of individual FA were determined by gas chromatography, and canopy characteristics and herbage quality were assessed. The treatments resulted in canopies with contrasting DM yields and protein concentrations. Five FAs, representing 98% of total FAs, were studied in detail. On an average, the concentration of these major FAs in fresh grass was 15.1 g kg^?1 DM, and 69% of the major FAs consisted of C18:3. Regrowth period affected (P < 0.05) the total FA concentration, and significantly (P < 0.01) lower concentrations of C18:3 and C16:1 were found after a longer period of regrowth. N application resulted in higher (P < 0.001) concentrations of all FAs. The FA composition was not affected by N application, but a longer regrowth period significantly (P < 0.001) decreased the proportion of C18:3 and increased those of C18:2 and C16:0. A strong, positive overall linear relation was found between the concentrations of total FAs and C18:3 with the crude protein concentration in the herbage. These studies demonstrate opportunities to affect the FA concentration and composition of FA in herbage through management strategies, which could affect milk FA composition.     

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gene-associated single nucleotide polymorphism discovery in perennial ryegrass (Lolium perenne L.)

Molecular genetic marker development in perennial ryegrass has largely been dependent on anonymous sequence variation. The availability of a large-scale EST resource permits the development of functionally-associated genetic markers based on SNP variation in candidate genes. Genic SNP loci and associated haplotypes are suitable for implementation in molecular breeding of outbreeding forage species. Strategies for in vitro SNP discovery through amplicon cloning and sequencing have been designed and implemented. Putative SNPs were identified within and between the parents of the F₁(NA₆ × AU₆) genetic mapping family and were validated among progeny individuals. Proof-of-concept for the process was obtained using the drought tolerance-associated LpASRa2 gene. SNP haplotype structures were determined and correlated with predicted amino acid changes. Gene-length LD was evaluated across diverse germplasm collections. A survey of SNP variation across 100 candidate genes revealed a high frequency of SNP incidence (c. 1 per 54 bp), with similar proportions in exons and introns. A proportion (c. 50%) of the validated genic SNPs were assigned to the F₁(NA₆ × AU₆) genetic map, showing high levels of coincidence with previously mapped RFLP loci. The perennial ryegrass SNP resource will enable genetic map integration, detailed LD studies and selection of superior allele content during varietal development.     

Callus Formation from Somatic Hybridization of Wheat (Triticum aestivum L.) and Ryegrass (Lolium perenne L.) by Electrofusion

Somatic hybrid calli were recovered following electrofusion of protoplasts from a chloroplast-containing cell suspension culture of wheat (Triticum aestivum L.) and a cell suspension culture of ryegrass (Lolium perenne L.). The protoplasts of wheat were inactivated by iodoacetamide; in addition morphology and colour were used as markers to aid selection of putative hybrid calli. For isozyme analysis of putative hybrids, nine isozymes were tested for differences in bands between the parental lines. Of these, three showed differences (ADH, GOT, SDH). Analysis of ADH bands of calli indicated that six lines were hybrids. These lines were analysed with the ,ther isozymes, and at the DNA level by Southern hybridisation with a wheat ribosomal DNA probe. The overall results indicated that one line was an almost complete combination of the genomes of the parental lines, but the other 5 lines were probably partial hybrids. In the latter, some loss of the wheat genome had probably occurred.     

Fractional and structural characterization of hemicelluloses from perennial ryegrass (Lolium perenne) and cocksfoot grass (Dactylis glomerata)

Sequential three-stage treatments with 80% EtOH containing 0.2% NaOH, 2.5% H2O2-0.2% EDTA containing 1.5% NaOH and 2.5% H2O2-0.2% TAED containing 1.0% NaOH at 75 degrees C for 3h released 8.0% and 10.4%, 79.1% and 77.0% and 12.9% and 12.5% of the original hemicelluloses from perennial grass and cocksfoot grass, respectively. It was found that the four alkaline peroxide-soluble hemicellulosic fractions contained higher amounts of xylose (33.4-38.2%), uronic acids (9.3-15.3%) and rhamnose (3.0-3.9%), but were lower in glucose (25.1-28.3%), galactose (13.3-15.3%) and mannose (0.4-1.5%) than those of the two alkaline EtOH-soluble hemicellulosic fractions in which glucose (32.9-36.0%), xylose (20.1-22.6%), arabinose (14.1-21.4%), galactose (16.6-19.9%), mannose (4.1-9.9%) and uronic acids (3.4-7.4%) were the major sugar components. 13C NMR spectroscopy confirmed that all the six hemicellulosic fractions were composed of galactoarabinoxylans, 4-O-methylglucuronoarabinoxylans and beta-glucan. In addition, the studies showed that the four alkaline peroxide-soluble hemicellulosic fractions were more linear and acidic and had larger molecular weights (Mw, 28,400-38,650 g mol(-1)) than those of the two alkaline EtOH-soluble hemicellulosic fractions (Mw, 16,460-17,420 g mol(-1)).