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.     

Development of a genomic microsatellite library in perennial ryegrass (Lolium perenne) and its use in trait mapping

Background and Aims: Perennial ryegrass (Lolium perenne) is one of the key forageand amenity grasses throughout the world. In the UK it accountsfor 70 % of all agricultural land use with an estimated farmgate value of £6 billion per annum. However, in termsof the genetic resources available, L. perenne has lagged behindother major crops in Poaceae. The aim of this project was thereforethe construction of a microsatellite-enriched genomic libraryfor L. perenne to increase the number of genetic markers availablefor both marker-assisted selection in breeding programmes andgene isolation. Methods: Primers for 229 non-redundant microsatellite markers were designedand used to screen two L. perenne genotypes, one amenity andone forage. Of the 229 microsatellites, 95 were found to showpolymorphism between amenity and forage genotypes. A selectionof microsatellite primers was selected from these 95 and usedto screen two mapping populations derived from intercrossingand backcrossing the two forage and amenity grass genotypes. Key Results and Conclusions: The utility of the resulting genetic maps for analysis of thegenetic control of target traits was demonstrated by the mappingof genes associated with heading date to linkage groups 4 and7.     

Partitioning of sugars in Lolium perenne (perennial ryegrass) during drought and on rewatering

Simulated swards of perennial ryegrass ( Lolium perenne ) growing in 1-m³ soil blocks in the glasshouse were either well watered or deprived of water for 57 d and then rewatered. The first aim was to measure effects of drought on sugar (water-soluble carbohydrate) composition of laminae and sheaths of mature laminae, and bases and laminae of young (growing) leaves. The second aim was to use pulse labelling with ¹⁴CO₂ to follow the partitioning of recently-fixed assimilates, and the assembly and consumption of reserve sugars (fructans). Over the last 7 d of drought growth almost stopped, old leaves died faster than they were replaced, and total sugar (which had doubled in concentration during drought) was rapidly consumed. Leaf laminae had lower content of total sugars and of large fructan (DP>5) than did growing bases and sheaths. Drought greatly reduced the rate at which sugar was exported from the laminae to the sheaths and growing leaf bases, and the rate at which it was converted to fructan. Nevertheless, fructan accumulated over the first 50 d of drought. Rewatering did not result in depolymerization and remobilization of sugars that had been formed during the last 7 d of drought, but stimulated their further assembly into high-DP fructans. Our hypothesis, that accumulation of neo-kestose (a DP-3 fructan) in droughted laminae was a symptom of sugar remobilization just before death, was disproved. It is concluded that sugar reserves contribute to drought resistance only under extreme conditions. The specific role of fructan in dry environments might be to improve regrowth when drought is relieved, rather than to enhance growth during drought.     

多年生黑麦草的逆境生理研究进展

在研究和借鉴国内外最新研究成果的基础上,系统论述和报道了多年生黑麦草在水分、温度以及重金属等逆境胁迫下的生理变化,展望了多年生黑麦草在逆境生理方面的研究趋势,指出多年生黑麦草的抗性机理以及相关分子生物学方面的研究将成为未来研究的重点和热点,为今后深入开展多年生黑麦草的研究和利用提供科学依据。     

Genotyping by RAD sequencing enables mapping of fatty acid composition traits in perennial ryegrass (Lolium perenne (L.))

Perennial ryegrass (Lolium perenne L.) is the most important forage crop in temperate livestock agriculture. Its nutritional quality has significant impact on the quality of meat and milk for human consumption. Evidence suggests that higher energy content in forage can assist in reducing greenhouse gas emissions from ruminants. Increasing the fatty acid content (especially α‐linolenic acid, an omega‐3 fatty acid) may thus contribute to better forage, but little is known about the genetic basis of variation for this trait. To this end, quantitative trait loci (QTLs) were identified associated with major fatty acid content in perennial ryegrass using a population derived from a cross between the heterozygous and outbreeding high‐sugar grass variety AberMagic and an older variety, Aurora. A genetic map with 434 restriction‐associated DNA (RAD) and SSR markers was generated. Significant QTLs for the content of palmitic (C16:0) on linkage groups (LGs) 2 and 7; stearic (C18:0) on LGs 3, 4 and 7; linoleic (C18:2n‐6) on LGs 2 and 5; and α‐linolenic acids (C18:3n‐3) on LG 1 were identified. Two candidate genes (a lipase and a beta‐ketoacyl CoA synthase), both associated with C16:0, and separately with C18:2n‐6 and C18:0 contents, were identified. The physical positions of these genes in rice and their genetic positions in perennial ryegrass were consistent with established syntenic relationships between these two species. Validation of these associations is required, but the utility of RAD markers for rapid generation of genetic maps and QTL analysis has been demonstrated for fatty acid composition in a global forage crop.     

Effect of leaf age and nitrogen fertilisation on sporulation of crown rust (Puccinia coronata var. lolii) on perennial ryegrass (Lolium perenne L.)

Individual leaves of perennial ryegrass cv. Aberystwyth S23 of two leaf ages and at two levels of nitrogen fertilisation were point inoculated with Puccinia coronata f.sp. coronata in a growth chamber. In general, there was no significant difference in the lifespan of inoculated versus control leaves. However, the higher rate of nitrogen extended leaf lifespan more markedly in rusted than in control leaves. Uredospore production varied according to leaf age: colonies on juvenile leaves produced three times as many spores as those on mature leaves.     

Generation of large numbers of independently transformed fertile perennial ryegrass (Lolium perenne L.) plants of forage- and turf-type cultivars

Perennial ryegrass (Lolium perenne L.) is the most important grass species in areas with a temperate climate. Biolistic transfer of a ubiquitin promoter driven nptII expression cassette into mature or immature tissue derived calli of perennial ryegrass followed by paromomycin selection, resulted in the rapid and efficient production of fertile transgenic ryegrass plants. Transformation efficiencies after paromomycin selection in combination with the nptII selectable marker compared favourably with hygromycin selection in combination with the hph selectable marker. In total 83 independent nptII expressing plants were produced. Transformation frequency was highly affected by genotype, explant, selection regime and the duration of the callus induction period. The optimised transformation protocol for mature embryo derived calli of turf-type or forage-type cultivars resulted in an average transformation efficiency of 5.2% or 6.6% respectively. This converts into 1.7 or 2.2 independent transgenic plants per bombardment. Immature inflorescence- and immature embryo-derived calli were also successfully used as target for the gene transfer, resulting in transformation efficiencies of up to 3.7% or 11.42% respectively. Transgenic plants were transferred to soil 12 or 9 weeks after excision of mature and immature embryos or inflorescences respectively. Transgene integration and expression were confirmed by PCR and ELISA or western blot analysis. Southern blot analysis confirmed the independent nature of the transgenic lines. The majority of lines showed the integration of two to six transgene copies, while 21% of the analysed lines had a single copy insert. A short tissue culture period in comparison to recently published reports seems to be beneficial for the production of normal and fertile transgenic ryegrass plants. Consequently we report for the first time molecular evidence for sexual transgene transmission in fertile transgenic perennial ryegrass.     

干旱胁迫下内生真菌感染对黑麦草叶内几种同工酶的影响

以内生真菌感染(endophyte-infected,EI)与不感染(endophyte-free,EF)的黑麦草(Lolium perenne L．)种子建立实验种群,分别对其施加长时间不同强度的干旱胁迫,通过比较黑麦草体内过氧化物酶(POD)、超氧化物歧化酶(SOD)、多酚氧化酶(PPO)活性及其同工酶谱的变化以探讨保护酶系统在内生真菌——植物共生体的抗旱性方面所作的贡献。研究结果表明,水分胁迫和内生真菌对黑麦草3种酶的影响不仅表现在总量上而且表现在同工酶的酶谱及各区带的酶活力上。就总酶活力而言,EI和EF植株中POD、SOD和PPO的活性均随着干旱胁迫强度的增加而增加,进一步将EI和EF植株的酶活力进行比较,发现与EF植株相比,EI植株中POD和PPO的活性相对较低,而SOD的活性相对较高。从同工酶的谱带数量和强弱来看,POD同工酶各区带活力均随干旱胁迫强度的增加而增加,EI植株叶片增加的幅度高于EF叶片,而且EI叶片在重度胁迫下出现了1条新带SOD同工酶各区带活力在EI叶片中有随干旱胁迫增加而增加的趋势,而在EF叶片中有些区带酶活力增强,有些区带酶活力减弱,且EI叶片在中度胁迫下出现了1条新带;PPO同工酶随干旱胁迫的增强,EI和EF叶片均表现为有些区带酶活力增强,有些区带酶活力减弱。总之,内生真菌的感染虽然没有显著提高宿主植物黑麦草POD、SOD和PPO的活性,但使宿主黑麦草对干旱胁迫的反应更为迅速,其中既包括POD、SOD等酶活力的迅速升高,也包括新酶带的产生。     

Ultrastructural features of a Stagonospora species (Ascomycotina) in senescent tissues of perennial ryegrass (Lolium perenne L.)

The pycnidium of a Stagonospora sp. observed in degenerating leaf sheath and blade tissues of Lolium perenne L. contained concentric bodies similar to those found in the mycobiont of several lichens and a few other free-living ascomycetes. These bodies occurred in vegetative hyphae, hyphae of the pycnidial wall, in conidiogenous cells and in conidia and comprised a central electron-translucent core surrounded by a granular zone and an outer radially orientated fibrillar or lamellar zone. In some cases the entire body was surrounded by a translucent halo. A second feature of the interwoven hyphae of the pycnidial wall was the occurrence of pores connecting adjacent hyphae. These lateral pores appeared identical to pores in septa between cells of individual hyphae, and like them, were associated with Woronin bodies.     

Sink control of nitrogen uptake and assimilation during regrowth after-cutting of ryegrass (Lolium perenne L.)

Abstract. The ¹⁵N isotope was used to compare the uptake and the assimilation of NH₄⁺ and NO₃ nitrogen in ryegrass ( Lolium perenne L.) during regrowth after cutting. Uptake of nitrate-N, expressed per plant, was at all times greater than ammonium-N uptake and assimilation decreased in roots and stubble while its assimilation was maintained at a high level in leaves. It has been suggested that ammonium assimilation is directly related to the availability of carbohydrates in the sink organ (leaves) resulting from their remobilization from the source organs (roots and stubble). Nitrate reduction decreased in all organs, while the uptake of NO₃ was still high. After this first period of regrowth, nitrogen assimilation both from nitrate and ammonium increased in all the plants. Nitrate reduction capacity (expressed in μg NO₃-N reduced per g D.W. per d) is 7.5 and 22.5 times greater in leaves than in stubble and roots, respectively. Therefore, nitrogen assimilation in stubble and particularly in roots was mainly dependent on ammonium nitrogen.     

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.     

Dissecting the regulation of fructan metabolism in perennial ryegrass (Lolium perenne) with quantitative trait locus mapping

Quantitative trait locus (QTL) mapping, which can be a useful tool for dissecting complex traits, has been used here to study the regulation of fructan metabolism in temperate forage grasses. An F2 mapping family, derived from a high water-soluble carbohydrate (WSC) x low WSC cross, was used to map fructans and the other components of WSC (sucrose, glucose and fructose) in leaves and tiller bases of perennial ryegrass (Lolium perenne) in spring and autumn. To characterize regions of the genome that control basic carbohydrate metabolism, a strategy to minimize the impact of genotype (G) x environment (E), and E-effects on the characterization of G-effects, was adopted. Most traits were highly variable within the family. There was also considerable year-to-year environmental variation. However, significant genetic effects were detected, and several traits had high broad-sense heritability. QTL were identified on chromosomes 1, 2, 5 and 6. Leaf and tiller base QTL did not coincide. Individual QTL explained between 8 and 59% of the total phenotypic variation in the traits. Fructan turnover, metabolism and their genetic control, and the effect of environment, are discussed in the context of the results.     

Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene

Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat‐growing areas in China. Because it is vectored by the fungus‐like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co‐transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12‐1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12‐1 showed broad‐spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild‐type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus‐derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad‐spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.     

Isolation of viable egg cells of perennial ryegrass (Lolium perenne L.)

Summary Isolation of viable egg cells of perennial ryegrass (Lolium perenne L.) has been accomplished. After an enzyme incubation, ovules disintegrated into loose cells upon mechanical manipulation. The egg cells could be identified between the bulk of sporophytic cells derived from the macerated ovules. The morphology of the isolated egg cell corresponds to the morphology of the egg cell in situ and is comparable to the morphology of egg cells of other monocotyledons and angiosperms. Two hours after isolation the egg cells were still viable. The protocol proved reproducible and the yield was determined at 10%.     

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.