Testing perennial ryegrass (Lolium perenne L.) as spaced plants in swards

Summary A method of testing spaced plants in swards of another grass crop is described. The procedure provides information on sward performance at each stage of the breeding scheme. A large number of entries may be included, together with whatever cutting frequency.When used for perennial ryegrass, the method favours the selection of productive and persistent plants.     

Introduction of a self-compatible gene of Lolium temulentum L. to perennial ryegrass (Lolium perenne L.) for the purpose of the production of inbred lines of perennial ryegrass

Interspecific hybrids between self-compatible species, Lolium temulentum L. and self-incompatible species, L. perenne L. were obtained using embryo rescue. Two cycles of backcrossing of interspecific hybrids with L. perenne were carried out. A 1: 1 segregation ratio of self comatibility and incompatibility was observed in backcross generations. These segregation data confirmed that self-compatibility of L. temulentum was controlled by a single gene. It suggested that the self-compatible gene of L. temulentum could be introduced to self-incompatible plants of L. perenne through interspecific hybridization. It appears that utilization of a self-compatible gene of L. temulentum would be useful for production of inbred lines of L. perenne. The possibility of breeding procedures of perennial ryegrass using a self-compatible gene was discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mitochondrial DNA analysis reveals cytoplasmic variation within a single cultivar of perennial ryegrass (Lolium perenne L.)

Summary The extent of mitochondrial DNA restriction fragment length polymorphisms among individual plant samples of perennial ryegrass was determined. A total of 72 plants from three cultivars Yorktown II, S23 and Riikka were surveyed using three restriction enzymes (BamHI,EcoRI andHindIII) and three mitochondrial gene probes (coxI, coxIII andnad9). Polymorphisms were noted within each of the two cultivars Yorktown II and S23, whereas in Riikka no variation was detected. It seems most likely that the mitochondrial genome diversity within the same cultivar has resulted from non-homogeneous ancestor cytoplasms. The hybridization-based assay employed is simple to perform, gives unambiguous results, and may thus be used in mass screening of perennial ryegrass populations for breeding purpose.     

Aneuploids of perennial ryegrass (Lolium perenne)

Summary Aneuploid plants of perennial ryegrass (Lolium perenne L.) with 2n=15 to 30 chromosomes were obtained by crossing a near-triploid (2n=3x+1=22) with a diploid or on open-pollination with diploids and tetraploids. Aneuploids occurred with a frequency of 83% in near triploid × diploid progeny and 92% on open-pollination with diploid and tetraploid plants. Aneuploid plants with 15 to 18 chromosomes resembled diploids in morphology and those with 19 to 30 chromosomes were akin to tetraploids. Meiotic studies suggested that most aneuploid plants resulted from transmission of aneuploid egg cells (n=8 to 23). Aneuploid plants with 2n=27 to 30 chromosomes in the progeny of 22×14 cross originated from unreduced egg cells. Plants with 19 to 21 chromosomes were recovered only by immature seed culture. Aneuploid plants with 26 to 30 chromosomes and triploids (2n=21) had higher pollen fertility and bigger seeds than plants with 15 to 22 chromosomes.     

Assessment of perennial ryegrass (Lolium perenne L.) for breeding. I Freezing tolerance

Summary Assessments of freezing tolerance, estimated in terms of temperature at which 50% mortality occurs (=LT₅₀), were obtained for 86 Lolium perenne accessions using a glycol bath test on naturally hardened field plants. Significant correlations were obtained between LT₅₀ and both heading dates of accessions and minimum temperature of the coldest month near their sites of origin. The effects of temperature and reproductive development on the hardening/dehardening of plants and their growth after freezing are discussed.     

Association of agronomic traits with isozyme loci in perennial ryegrass (Lolium perenne L.)

Summary Inbred lines possessing different genotypes at three isozyme loci, PGI-2, GOT-3 and ACP-1, were produced by two generations of selfing from two contrasting cultivars of perennial ryegrass. Four separate F₂ populations were obtained by selfing or crossing F₁ plants from crosses between cultivar inbred lines. F₂ plants were scored for genotype at the three isozyme loci and for eight agronomic traits. The three isozyme loci showed independent segregation confirming that they belonged to separate linkage groups. A consistent association was observed between water soluble carbohydrate content and genotype at the PGI-2 locus. Some association was also detected between genotype at the ACP-1 locus and heading date. The relevance of these observations to ryegrass breeding programmes is discussed.     

Assessment of perennial ryegrass (Lolium perenne L.) for breeding. II. Components of winter hardiness

Summary When breeding for good winter hardiness a broad range of adaptations must be considered. Such a multitrait approach requires some understanding of physiological and genetical relationships between relevant traits.Fourteen winter related characters were measured on field grown plants from 86 perennial ryegrass accessions originating from a wide range of climates. Genetic components of variance and covariance were extracted from the data and a principal components analysis carried out. The first three independent components accounted for 80% of the variance represented by the genetic correlations and were broadly identified as winter growth, winter hardiness and early spring activity factors. None of the accessions exhibited a combination of extreme winter growth and winter hardiness although several with excellent winter hardiness showed better than average winter growth. Amounts of water soluble carbohydrate maintained through the winter appeared important for good survival.It was concluded that a selection index approach may prove useful in future breeding programmes as further information concerning physiological relationships becomes available.     

A relationship between heading date and in vitro tillering propensity in perennial ryegrass (Lolium perenne L.)

Summary In vitro tillers of over 1000 genotypes from nine contrasting Lolium perenne cultivars were cultured on various media containing the synthetic cytokinin, 6-benzylaminopurine (BAP), in order to determine the effect on tillering. The cultures were assessed after seven weeks for survival, tiller production, size and vigour. Added auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and -napthaleneacetic acid (NAA) had no consistent beneficial effects.A significant relationship was found between in vitro tiller production and cultivar heading date. Genotypes of early-heading, less persistent cultivars produced more tillers than genotypes from late heading more persistent cultivars. This was consistent with the generally higher in vitro tiller production in the biennial species Lolium multiflorum cultured on similar media.     

Spaced-plant traits related to seed yield in plots of perennial ryegrass (Lolium perenne L.)

Summary Seed yield in perennial ryegrass is low and selection criteria for high seed production are lacking. Indirect selection in spaced plants would be efficient, but spaced-plant traits need to be identified that correlate with seed yield in drilled plots. Spaced plants were studied of nine perennial ryegrass cultivars with a different seed production when grown in plots. Genotypic variation within cultivars for seed yield components and other traits was assessed in four environments in replicated clonal experiments. Extensive genotypic variation for most traits was present within each cultivar. Based on cultivar means of 25 or 50 genotypes, significant differences among cultivars were found for most traits. Cultivar-year interactions were found for most traits, but no significant cultivar-location interactions were generally found. Spaced-plant traits in general showed poor correlation to corresponding traits in drilled plots. Cultivars with contrasting seed yield in plots could not be distinguished on the basis of their spaced-plant traits and therefore no criteria for indirect selection for seed production in drilled plots could be identified in spaced plants.     

Suspension culture performance in commercial varieties of perennial ryegrass (Lolium perenne L.)

Summary Suspension culture performance in commercial varieties of perennial ryegrass was studied to assess the effect of variety on suspension culture response and plant regeneration. 179 suspension cultures were established from embryos of mature seeds of 21 varieties and one breeding population. Of these, 123 suspensions were morphogenic (21 varieties) and 66 suspensions (18 varieties) regenerated green plants. A number of suspension lines, originating from two different suspensions, retained the capacity for green plant formation for almost four years. Replicates performed with seed lots of different ages indicated that suspensions initiated from young seeds (1 year) were of better quality than suspensions initiated from older seeds (2–4 years). Varieties differed in their capacity to form morphogenic suspensions and suspensions capable of regenerating green plants, although the effect of variety was relatively small. It was concluded that responsive genotypes can be found within most varieties of Lolium perenne.     

Seed yield related to crop development and to yield components in nine cultivars of perennial ryegrass (Lolium perenne L.)

Summary We had previously found differences for seed yield among nine perennial ryegrass cultivars which were not associated with variation for seed weight. To detect the physiological basis of these genetic differences for seed yield, growth analyses were carried out. We related crop development and components of seed yield to seed yield during three years on clay and sandy soil. No significant differences occurred among cultivars for accumulation and partitioning of dry matter or the pattern of tiller production. Seed yield of the cultivars was not associated with ear number or total dry matter yield of the seed crop. Seed yield was more correlated with the number of seeds per unit area than with seed weight. The number of seeds as calculated after harvest from seed yield and seed weight was much lower than the number of seeds as estimated prior to harvest from seed yield components. The number of spikelets differed significantly among the cultivars, but the ranking was different from that for seed yield. The physiological basis of the genetic differences for seed yield is not clear. Implications for breeding perennial ryegrass are discussed.     

Tracing of 2n egg occurrence in perennial ryegrass (Lolium perenne L.) using interploidy crosses

Several genotypes of Lolium perenne (2n = 2x = 14) were screenedfor the production of 2n eggs. In each cross a diploid genotype, used asfemale parent, was pollinated with a tetraploid genotype. All interploidycrosses were made in isolation cages. Flow cytometrical analysis of theprogeny of 154 diploid plants revealed 28 tetraploid descendants. Theseoffspring plants were harvested on 15 different diploid mother plants.Amplified Fragment Length Polymorphism (AFLP) markers were used tocheck the parenthood of these tetraploids. Two seedlings died, theremaining plants could, after AFLP fingerprinting, be identified as meiotictetraploids. The results obtained allowed to conclude that the tetraploidprogeny plants harvested on 15 diploid mother plants were the result ofthe fertilisation of an unreduced egg by a reduced pollen grain of thetetraploid plant.     

Components of resistance to ryegrass mosaic virus in a clone of Lolium perenne and their strain-specificity

Summary The extreme resistance to ryegrass mosaic virus (RMV) of a clone of Lolium perenne L. was due to a combination of two distinct types of resistance: resistance to infection and resistance to multiplication and movement of virus within the plant. Resistance to infection was quantitatively inherited and highly effective against three strains of RMV, while resistance to multiplication and movement was controlled by two complementary recessive genes and was effective against only two of the three RMV strains.     

Heritability estimates of spaced-plant traits in three perennial ryegrass (Lolium perenne L.) cultivars

Summary Seed yield in perennial ryegrass is low and unpredictable. Spaced-plant traits suitable for indirect selection for total seed yield in drilled plots would be very useful. The objectives of this investigation were to evaluate genetic variation for seed yield components and other traits among clones from three perennial ryegrass cultivars differing in seed yield and their open-pollinated progenies. Per cultivar, a random set of 50 genotypes was cloned and on each genotype seed was generated by open pollination. Clonal ramets of the parents were observed for 17 traits in 1986 at two locations. In 1987 and 1988, parents and progenies were observed as single plants in a randomized complete block design with two replications. There was little cultivar-environment interaction for most traits. The parents differed significantly for almost all traits. Half-sib (HS) families differed for only three to five traits. Broad-sense heritabilities (h² _b), based on variance components of the parents, were moderate to high; earliness had the highest h_b ². Narrow-sense heritabilities (h² _n), based on variance components among HS-families, were low to moderate and mostly not significant; for most traits h² _n estimates varied between years and cultivars. Flag leaf width and date of first anthesis showed the highest h² _n. Narrow-sense heritability estimates from parent-offspring regressions (h² _nPO) ranged from non-significant to high, depending on year and cultivar; they were generally higher than the corresponding h² _n estimates. Generally, h² _nPO was highest for earliness, flag leaf width, ear length and the number of spikelets per ear. Breeding methods that capitalize on additive genetic variance, such as mass selection, should result in improvement for these traits.     

Morphological variation in Dutch perennial ryegrass (Lolium perenne L.) populations,in relation to environmental factors

Summary Twenty-one Dutch Lolium perenne populations, fifteen European populations and six L. perenne cultivars were compared for morphological variation. Dutch populations clearly differed from the European populations and the cultivars. Dutch populations generally had reduced plant length and smaller leaves. For other characters, e.g. date of ear emergence, the Dutch populations showed as much variation as the European populations and cultivars they were compared with in this trial. Correlations between morphology and environmental factors at the site of origin were significant in several cases but were generally weak, and dependent on the set of populations studied. In order to maximize the phenotypic differences between the collected populations, soil type and management type appeared to be the most important factors for the choice of collection sites in the Netherlands. To determine suitable collection sites all over Europe, other factors like precipitation, latitude, altitude and temperature factors were also important. For subsampling of the locations, variation in management type within the location determined whether or not phenotypic different samples could be collected. The extensive use of cultivars in the Netherlands does not seem to have prevented the formation of distinct populations. Therefore in situ conservation of grassland seems a good alternative for genetic conservation of L. perenne in the Netherlands.Also associated with the WAU Department of Plant Taxonomy, P.O. Box 8010, 6700 ED, Wageningen, The Netherlands     

AFLP analysis of genetic diversity within and between populations of perennial ryegrass (Lolium perenne L.)

Amplified fragment length polymorphism (AFLP) analysis has been used to measure genetic diversity in perennial ryegrass (Lolium perenne L.) and to relate intra- and interpopulation variation to breeding history. Cluster analysis of AFLP data from contrasting populations showed features consistent with the origins of these varieties. Significant differences in intrapopulation diversity were detected and partial separation of different cultivars was observed. Restricted base cultivars, derived from small numbers of foundation clones, were suitable for this type of study, allowing near complete discrimination of closely related cultivars. Analysis of bulked samples was based on the pooling of genomic DNA from 20 individuals from 6 selected populations. Cluster analysis of AFLP data from bulked samples produced a phenogram showing relationships consistent with the results of individual analysis. AFLP profiling provides an important tool for the detection and quantification of genetic variation in perennial ryegrass. This revised version was published online in August 2006 with corrections to the Cover Date.     

Variation in digestibility in nine populations of perennial ryegrass (Lolium perenne) and its genetic basis

Summary Thirty five individuals from each of nine perennial ryegrass populations were sampled from set-stocked and zero-grazed swards and from an old permanent pasture. The swards were 6, 18, 30 months and 11 years old when sampled. The permanent pasture was at least 40 years old.Dry matter digestibility (DMD) and organic matter digestibility (OMD) were determined for green leaf material harvested 4 days after ear emergence. Significant differences were found between the populations, but genotypic differences were found only for OMD. The largest proportion of the total variation was due to differences between blocks. In the zero-grazed populations, DMD and OMD declined significantly with increasing sward age. This was not found for the set-stocked swards. Two sets of pair crosses, from which mid-parent, progeny regressions could be determined, were carried out.The regressions of offspring on mid-parent for set-stocked and zero-grazed swards were used to assess the presence of additive genetic variation and to estimate narrow sense heritability of DMD. The coefficients of correlation, 0.122 and 0.226 respectively, were non-significant, indicating an absence of additive genetic variation.     

Colchicine-induced heritable variation in cell size and chloroplast number in the leaf cells of inbred ryegrasses (Lolium perenne,L. multiflorum)

Summary A 3 hr period of treatment with a 0.2% aqueous solution of colchicine was given to one week old seedlings of 3 inbred lines of perennial ryegrass (Lolium perenne) and 4 lines of Italian ryegrass (L. multiflorum). The surviving plants developed as mixoploids and were self-pollinated. In the following generation (CT1) the seeds were grown without further treatment, and were screened cytologically at the seedling stage to separate out diploids from tetraploids. Only the diploids were kept, and in this CT1 generation comparisons were then made between controls (2x) and colchicine-treated diploids (C2x) for leaf mesophyll cell size and chloroplast number variation. Heritable differences were found such that the cell plan areas and/or the numbers of chloroplasts per cell were significantly greater in the C2x compared with the 2x treatment in 3 and 5 out of the 7 lines. In each case there was enhancement of chloroplast numbers independently of the variation in cell size.     

A database for a more efficient perennial ryegrass breeding programme

Summary The development and use of a computer database specifically designed to be used to store data from a plant breeding programme on perennial ryegrass (Lolium perenne) is described. Setting up a database requires data analysis in order to establish a conceptual dat model and a conceptual processing data. Every model has been written with MERISE method. With the models, a database has been built using ORACLE relational database management system. It is tailored to meet our own requirements which may be common to other database users.     

Stability of esterases and total proteins during seed storage of perennial ryegrass (Lolium perenne L.) and their use for cultivar discrimination

Polymorphisms in electrophoretic patterns obtained by isoelectric focusing (IEF) were examined to evaluate their suitability for cultivar identification in perennial ryegrass (Lolium perenne L.). It was possible to discriminate 64 (94%) of 68 cultivars by combining results from esterase and total protein analysis. Discrimination was based on quantitative differences (relative band intensity) rather than on qualitative differences (presence or absence of bands). Esterase patterns from different recent (fresh to seven years old) seed lots of the same perennial ryegrass cultivars were very stable. Occasionally, minor differences in band intensity were observed between recent and old (up to 30 years old) seed lots of a cultivar. Storage of meal samples up to two years at −20 ^°C had no effect on the total protein patterns. No correlation was found between esterase patterns and ploidy level, cultivar type (pasture or turf), heading date or breeding company. Esterase patterns appeared to be unsuitable markers for the selection of reference cultivars for distinctiveness, uniformity, and stability (DUS) testing, because no correlation was found between cultivars on the basis of esterase banding patterns and morphological characteristics. This revised version was published online in July 2006 with corrections to the Cover Date.