Cytogenetics of Lolium perenne

Summary A number of inbred lines of Lolium perenne have been developed as far as the fifth inbred generation and are being used for basic studies on the cytogenetics of the species. An analysis on variation in the pattern of chiasma frequency and distribution, including the parent plants and all generations down to the S5, reveals that the effect of inbreeding is to reduce chiasma frequency and to increase both the cell and bivalent variances. Evidence is presented for a genetic basis of polygenic control of chiasma formation and distribution, and a model is suggested for control over the three related components of variation which involves a two-stage level of regulation.     

Selection and stability of synthetic varieties of Lolium perenne

Summary The performance of three experimental cultivars of Lolium perenne selected for yield or water soluble carbohydrate content was monitored over four generations of seed multiplication under relaxed selection. In each variety the selected trait regressed towards that of the base population from which the selection line derived. This could be accounted for by residual genetic variation within the lines for the selected trait, and in some instances, by association of this variation with the fitness character, seed numbers produced. These results emphasize the need for practical breeding programmes to consider the nature of the gene action controlling the selected trait, if additive, directional selection should be effective in increasing the expression of the character. Where ambidirectional dominance and epistasis are important, consideration should be given to means of achieving reassortment of the controlling genes prior to selection.     

Genetic control of androgenetic response in Lolium perenne L.

In a study of androgenesis in 90 Norwegian genotypes of perennial ryegrass (Lolium perenne L.), heritabilities ranged from h _b ² =0.46 to 0.80. Very high or completely positive genotypic correlations were found between most characters of androgenetic response (e.g. embryo-like structures per 100 anthers, plants per 100 embryo-like structures, albino plants per 100 anthers, green plants per 100 anthers). Three genotypes, 2 Norwegian (7-5 and 9-5) and 1 Danish (245), which had significantly different androgenetic responses were selected to study the genetic control of the processes. Genotypes 7-5 and 9-5 were highly embryogenie, 7–5 and 245 were relatively high producers of green plants, while 9-5 was unable to produce green plants. The six possible reciprocal crosses between these three genotypes were made, and 10 or 11 F₁ plants from each cross were used for anther culture experiments. The cross 7-5 x 245 showed average superiority over both parents for total plant regeneration and green plant production, results not previously reported. The phenotypic correlations estimated among progenies from the crosses ranged from r=-0.99^*** to 0.81^***. These considerable changes, relative to the results of the screening experiment, are most likely the result of changed allele frequencies caused by the strong selection of parents in these crosses, and a relatively simple genetical control. This is also inferred from the large transgressive segregation observed.Abbreviations ANT anthers - ELS embryo-like structures - ALB albino plants - GRP green plants - DH doubled haploid plants     

Acclimation to freezing temperatures in perennial ryegrass (Lolium perenne)

The increasing demands being placed on natural grasslands in the era following the appearance of Bovine Spongiform Encephalitis require that forage crops provide a reliable extended season of growth, combined with good winter survival to ensure sward longevity. The ability to tolerate sub-zero temperatures is integral to the survival of perennial forages. Since the development of freezing tolerance is crucial to the survival and productivity of over-wintering crops, forage breeding programmes require an improved understanding of the individual characteristics that contribute to tolerance to sub-zero temperatures. Photosynthesis, carbohydrate content and changes in protein composition were investigated in two varieties of Lolium perenne which differ in their response to growth at low temperature.     

The effect of soil pH on rhizosphere carbon flow of Lolium perenne

Perennial rye-grass plants were grown at 15°C in microcosms containing soil sampled from field plots that had been maintained at constant pH for the last 30 years. Six soil pH values were tested in the experiment, with pH ranging from 4.3–6.5. After 3 weeks growth in the microcosms, plant shoots were exposed to a pulse of ¹⁴C-CO₂. The fate of this label was determined by monitoring ¹⁴C-CO₂ respired by the plant roots/soil and by the shoots. The ¹⁴C remaining in plant roots and shoots was determined when the plants were harvested 7 days after receiving the pulse label. The amount of ¹⁴C (expressed as a percentage of the total ¹⁴C fixed by the plant) lost from the plant roots increased from 12.3 to 30.6% with increasing soil pH from 4.3 to 6. Although a greater percentage of the fixed ¹⁴C was respired by the root/soil as soil pH increased, plant biomass was greater with increasing soil pH. Possible reasons for observed changes in the pattern of ¹⁴C distribution are discussed and, it is suggested that changes in the soil microbial biomass and in plant nitrogen nutrition may, in particular be key factors which led to increased loss of carbon from plant roots with increasing soil pH.     

Induction of cytoplasmic male-sterility into ryegrass (Lolium perenne)

Summary Data are presented which indicate that a cytoplasmic/genetic type male sterility has been induced into backcross progeny derived from intergeneric hybridization between Festuca pratensis (female parent) and Lolium perenne. Large numbers of male sterile genotypes have been obtained in all the backcross generations examined. The frequency and purity of maintainer genotypes is low and requires further breeding and selection. Analysis of data suggest that at least two loci are involved in fertility restoration. Conclusions regarding the genetic model are tentative and require further analyses.     

Rapid cycling of nitrogen and phosphorus from dying roots of Lolium perenne

Summary Previous experiments, using ³²P pulse labelling, showed that when roots of Lolium perenne were detached from the shoot, a substantial proportion of the phosphorus in the roots could within a few weeks be released and be captured by another, living plant. This paper describes experiments designed to confirm and further investigate this rapid nutrient transfer. Roots from plants grown with ample N and P were detached and placed in litter bags in soil. They lost up to 60% of their initial N and up to 70% of their P in three weeks. Even when roots were grown with deficient P supply, resulting in C:P ratios of 300–400, they lost 20–30% of their initial P. Time-courses of ³²P loss from roots suspended in solution gave results which agreed with these figures. The initially rapid rate of ³²P loss had declined greatly within three weeks. In a pot experiment small L. perenne plants showed a marked increase in their N and P content during 30 days after a neighbouring large plant's shoot was removed, supporting rapid capture of nutrients lost from the detached roots. To investigate P loss from roots while attached to the shoot, L. perenne shoots were clipped every four days and ³²P loss from the roots measured. After the third clip the rate of loss increased, eventually to more than four times that from the control plants.     

Neighbour specificity between Lolium perenne and Trifolium repens from a natural pasture

Summary Five genotypes of Trifolium repens and Lolium perenne were collected as neighbouring pairs along a fertility gradient in a natural pasture. After vegetative multiplication, the 25 possible combinations of Lolium genotype x Trifolium genotype were planted in the greenhouse in order to investigate competition between the genotypes. The comparison of the five combinations whose individual components had been neighbours with the combinations of genotypes that had not coexisted before disclosed no difference in total biomass production over 7 months. However, the yield of Trifolium increased when grown with the Lolium genotype which had been its natural neighbour, while the latter showed a decrease in yield. This neighbour specificity existed even when carryover effects from the sampling site had been eliminated (preconditioning period of 18 months) and when native Rhizobium strains were not present (inoculation with a non-native strain). The complex pattern of neighbour specificity with time indicated the importance of environmental conditions for its outcome. These results are a further confirmation of positive effects on the growth of Trifolium repens when grown together with its natural neighbour. They are discussed in the context of coexistence and coevolution     

Chromosome pairing in tetraploid hybrids between Lolium perenne and L. multiflorum

Summary Chromosome association at first meiotic metaphase in tetraploid hybrids between Lolium perenne and L. multiflorum was compared with that in autotetraploid L. perenne. The hybrids were found to have significantly higher levels of bivalent frequency, and lower levels of multivalent and chiasma frequency. A significant increase in multivalent frequency with increasing chiasma formation was found in both groups, but the increase was much less in the hybrids. These differences in chromosome associations between the two groups must therefore reflect differences in chiasma distribution and it is suggested that the results indicate a significant degree of preferential bivalent pairing in the hybrids.     

High yields of cytoplasts from protoplasts of Lolium perenne and Beta vulgaris using gradient centrifugation

Methods for the isolation of cytoplasts from suspension culture-derived protoplasts of the monocot Lolium perenne (perennial ryegrass) and the dicot Beta vulgaris (sugarbeet) have been determined. After comparing a range of gradients it was found that a discontinuous sucrose/mannitol gradient gave the highest cytoplast yields for both species tested: of the protoplasts loaded onto the gradient, for Beta >30% and for Lolium up to 45% could be recovered as cytoplasts. Sufficient protoplasts could be loaded onto the gradient to produce suitable numbers of cytoplasts for use in asymmetric somatic hybridisation experiments. Cytoplasts could be isolated from several suspension cultures of different ages. The cytoplast fraction was recovered from the upper part of the gradient in all cases and was only slightly contaminated (2–8%) with protoplasts. Lolium cytoplasts were small, evacuolate cells with granular cytoplasm. In contrast, Beta cytoplasts were larger and predominantly vacuolate. Both contained mitochondria as determined using fluorescence staining.Abbreviations 2,4-d 2,4 dichlorophenoxyacetic acid - M mannitol - S sucrose - P Percoll - S/M sucrose/mannitol gradient     

Associations between enzyme genotypes and dark respiration in perennial ryegrass,Lolium perenne L.

Summary In this study, we determined whether relationships existed between dark respiration and genotype at five enzyme polymorphisms in perennial ryegrass, Lolium perenne L. Positive correlations were found between Q ₁₀ of dark respiration and genotype at the phosphoglucomutase (PGM) and 6-phosphogluconate dehydrogenase (6PGD) loci. Plants doubly homozygous for the common allele at these loci were found to have Q ₁₀ values 20% higher than those for double heterozygotes. In plants that were heat stressed for five consecutive days, Q ₁₀ was found to be negatively correlated with apparent vigor after stressing. Individuals homozygous for PGM and 6PGD (with higher Q ₁₀ values) exhibited more apparent damage following the stress than heterozygous individuals. Both PGM and 6PGD occupy positions in metabolism with regulatory potential. Although caution must be used in assigning causal relationships, the results suggest that specific forms of these enzymes are directly related to, or are correlated with, the determinants of respiratory efficiency in L. perenne.     

Effects of fungal endophytes on the seed and seedling biology of Lolium perenne and Festuca arundinacea

Summary Many grasses are infected by endophytic fungi that grow intercellularly in leaves, stems, and flowers and are transmitted maternally by hyphal growth into ovules and seeds. The seed biology and seedling growth of endophyte-infected and uninfected perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) were investigated under controlled environmental conditions. The percentage of filled seeds produced by infected tall fescue was over twice of uninfected tall fescue; infected and uninfected perennial reegrass had similar percentages. Weights of seeds from infected and uninfected plants were similar in both species. Seeds from infected plants of both species exhibited a higher rate of germination than seeds from uninfected plants. Shoot growth in the greenhouse was compared by making three sequential harvests of above-ground plant parts from infected and uninfected plants of both species. Infected perennial ryegrass plants produced significantly more biomass and tillers than uninfected plants after 6 and 10 weeks of growth and significantly more biomass after 14 weeks of growth. Infected tall fescue plants produced significantly more biomass and tillers than uninfected plants after 10 and 14 weeks of growth. The physiological mechanism of enhancement of growth is not known. The results of this study suggest that infected plants may have a selective advantage in populations with uninfected members.     

Characterization of a family of ice-active proteins from the Ryegrass, Lolium perenne

Five genes coding for ice-active proteins were identified from an expressed sequence tag database of Lolium perenne cDNA libraries. Each of the five genes were characterized by the presence of an N-terminal signal peptide, a region enriched in hydrophilic amino acids and a leucine-rich region in four of the five genes that is homologous with the receptor domain of receptor-like protein kinases of plants. The C-terminal region of all five genes contains sequence homologous with Lolium and Triticum ice-active proteins. Of the four ice-active proteins (IAP1, IAP2, IAP3 and IAP5) cloned, three could be expressed in Escherichia coli and recovered in a functional form in order to study their ice activity. All three ice-active proteins had recrystallization inhibition activity but showed no detectable antifreeze or ice nucleation activity at the concentration tested. IAP2 and IAP5 formed distinct hexagonal-shaped crystals in the nanolitre osmometer as compared to the weakly hexagonal crystals produced by IAP3.     

Effects of genotype and temperature on pollen tube growth in perennial ryegrass (Lolium perenne L.)

Summary Low yield in seed crops of perennial ryegrass is related to low fertilization efficiency and low temperature during anthesis. To study the effect of genotype and temperature on pollen performance, we conducted greenhouse experiments at controlled temperatures. Individual florets of four genotypes that are known to differ in seed production were hand pollinated at four temperatures (14°, 18°, 22°, 26° C) both in vivo and via a semiin-vitro method involving excised florets on agar. Pollen germination and tube growth were determined with UV-fluorescence microscopy and scored in six classes at 2 h after pollination in vitro and after 0.5, 2 and 5 h in vivo. In vitro, both genotype and temperature had a significant effect on the performance of self-pollen. Pollen tube growth increased with temperature. In cross-pollinations, the pistil parent had a significant effect on pollen tube growth, and there was also a significant pistil-by-temperature interaction. In vivo, genotype and temperature significantly affected pollen performance. The genotype-by-temperature interaction was only significant 5 h after pollination. One genotype with low seed yield was pseudoself-compatible and was a relatively poor mother after cross-pollination. The effects of genotype and temperature on the growth of self-pollen might be exploited in a breeding programme.A.G. Stephenson was on a sabbattical leave at SVP in 1987     

Genetic segregation in relation to chromosome pairing in tetraploid hybrids between Lolium perenne and L. multiflorum

Summary Segregation at one of the loci controlling tiller-base pigmentation was studied to determine the mode of inheritance in tetraploid hybrids between Lolium perenne and L. multiflorum. The results could be explained by tetrasomic inheritance and thus did not support previous reports of a degree of preferential chromosome pairing in this material. However, double reduction and aneuploidy may to some extent have masked any tendency to disomic segregation brought about by preferential pairing. Moreover, there was significant heterogeneity between families in the segregation ratios which may indicate genetically controlled differences in pairing behaviour. The results are related to previous cytological and genetic studies.     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.