Interactions between Cellulose and (1,3;1,4)-ß-glucans and Arabinoxylans in the Regenerating Wall of Suspension Culture Cells of the Ryegrass Lolium multiflorum.

Plant cell walls (PCWs) form the outer barrier of cells that give the plant strength and directly interact with the environment and other cells in the plant. PCWs are composed of several polysaccharides, of which cellulose forms the main fibrillar network. Enmeshed between these fibrils of cellulose are non-cellulosic polysaccharides (NCPs), pectins, and proteins. This study investigates the sequence, timing, patterning, and architecture of cell wall polysaccharide regeneration in suspension culture cells (SCC) of the grass species Lolium multiflorum (Lolium). Confocal, superresolution, and electron microscopies were used in combination with cytochemical labeling to investigate polysaccharide deposition in SCC after protoplasting. Cellulose was the first polysaccharide observed, followed shortly thereafter by (1,3;1,4)-ß-glucan, which is also known as mixed-linkage glucan (MLG), arabinoxylan (AX), and callose. Cellulose formed fibrils with AX and produced a filamentous-like network, whereas MLG formed punctate patches. Using colocalization analysis, cellulose and AX were shown to interact during early stages of wall generation, but this interaction reduced over time as the wall matured. AX and MLG interactions increased slightly over time, but cellulose and MLG were not seen to interact. Callose initially formed patches that were randomly positioned on the protoplast surface. There was no consistency in size or location over time. The architecture observed via superresolution microscopy showed similarities to the biophysical maps produced using atomic force microscopy and can give insight into the role of polysaccharides in PCWs.

Relationship between epigenetic marks and the behavior of 45S rDNA sites in chromosomes and interphase nuclei of Lolium-Festuca complex.

The grasses of the Lolium-Festuca complex show a prominent role in world agricultural scenario. Several studies have demonstrated that the plasticity of 45S rDNA sites has been recently associated with the possible fragility of the loci. Often, these fragile sites were observed as extended sites and gaps in metaphases. This organization can be evaluated in relation to their transcriptional activity/accessibility through epigenetic changes. Thus, this study aimed to investigate the relationship of the 5-methylcytosine and histone H3 lysine-9 dimethylation in different conformations of 45S rDNA sites in interphase nuclei and in metaphase chromosomes of L. perenne, L. multiflorum and F. arundinacea. The FISH technique using 45S rDNA probes was performed sequentially after the immunolocalization. The sites showed predominantly the following characteristics in the interphase nuclei: intra- and perinucleolar position, decondensed or partially condensed and hypomethylated and hyper/hypomethylated status. Extranucleolar sites were mainly hypermethylated for both epigenetic marks. The 45S rDNA sites with gaps identified in metaphases were always hypomethylated, which justifies it decondensed and transcriptional state. The frequency of sites with hypermethylated gaps was very low. The structural differences observed in these sites are directly related to the assessed epigenetic marks, justifying the different conformations throughout the cell cycle.

B chromosomes are associated with redistribution of genetic recombination towards lower recombination chromosomal regions in perennial ryegrass.

Supernumerary 'B' chromosomes are non-essential components of the genome present in a range of plant and animal species-including many grasses. Within diploid and polyploid ryegrass and fescue species, including the forage grass perennial ryegrass (Lolium perenne L.), the presence of B chromosomes has been reported as influencing both chromosome pairing and chiasma frequencies. In this study, the effects of the presence/absence of B chromosomes on genetic recombination has been investigated through generating DArT (Diversity Arrays Technology) marker genetic maps for six perennial ryegrass diploid populations, the pollen parents of which contained either two B or zero B chromosomes. Through genetic and cytological analyses of these progeny and their parents, we have identified that, while overall cytological estimates of chiasma frequencies were significantly lower in pollen mother cells with two B chromosomes as compared with zero B chromosomes, the recombination frequencies within some marker intervals were actually increased, particularly for marker intervals in lower recombination regions of chromosomes, namely pericentromeric regions. Thus, in perennial ryegrass, the presence of two B chromosomes redistributed patterns of meiotic recombination in pollen mother cells in ways which could increase the range of allelic variation available to plant breeders.

Fragile sites of 45S rDNA of Lolium multiflorum are not hotspots for chromosomal breakages induced by X-ray.

Sites of 45S rDNA of Lolium are regions denominated fragile sites (FSs), constituting regions slightly stained with DAPI due to increased DNA unpacking in metaphasic chromosomes. Considered to be fragile regions in the genome, the FSs might be more responsive to induced breaks and result in chromosomal fragments and rearrangements, unless repairing mechanisms such as recombination or de novo telomere formation play a role at the break site of the DNA. Thus, this study aimed at investigating if SFs from Lolium are hotspots for the occurrence of breakages induced by X-ray and if they are regions favorable to synthesize new telomeres, using Hordeum vulgare as a comparative model. Lolium multiflorum and H. vulgare seedlings were irradiated with 20 and 50 Gy X-ray and evaluated one day following the irradiation and at 7-days intervals for a 28-days period, using FISH technique with 45S rDNA and Arabidopsis-type telomere probes in order to investigate the presence of chromosomal breakages and new telomere formation. H. vulgare did not survive after a few days of irradiation due to the increased rate of abnormalities. L. multiflorum also exhibited chromosomal abnormalities following the exposure, yet over the 28-days trial it had a decrease in the chromosomal damage rate and formation of de novo telomere has not been detected along this time. Despite being considered to be fragile regions in the genome, the 45S rDNA sites of Lolium are not hotspots to chromosomal breakages after the induction of breakages.

Mapping nano-scale mechanical heterogeneity of primary plant cell walls.

Nanoindentation experiments are performed using an atomic force microscope (AFM) to quantify the spatial distribution of mechanical properties of plant cell walls at nanometre length scales. At any specific location on the cell wall, a complex (non-linear) force-indentation response occurs that can be deconvoluted using a unique multiregime analysis (MRA). This allows an unambiguous evaluation of the local transverse elastic modulus of the wall. Nanomechanical measurements on suspension-cultured cells (SCCs), derived from Italian ryegrass (Lolium multiflorum) starchy endosperm, show three characteristic modes of deformation and a spatial distribution of elastic moduli across the surface. 'Soft' and 'hard' domains are found across length scales between 0.1 µm and 3 µm, which is well above a typical pore size of the polysaccharide mesh. The generality and wider applicability of this mechanical heterogeneity is verified through in planta characterization on leaf epidermal cells of Arabidopsis thaliana and L. multiflorum The outcomes of this research provide a basis for uncovering and quantifying the relationships between local wall composition, architecture, cell growth, and/or morphogenesis.

Stress memory induced transcriptional and metabolic changes of perennial ryegrass (Lolium perenne) in response to salt stress.

Preexposure to a stress could induce stable signals and reactions on plant physiology and gene expression during future encounters as a 'stress memory'. In this study, we found that two trainable genes, BPSP encoding putative brown plant hopper susceptibility protein and sucs encoding sucrose synthase displayed transcriptional memory for their considerably higher transcript levels during two or more subsequent stresses (S3, S4) relative to the initial stress (S0), and their expression returning to basal transcript levels (non-stressed) during the recovery states (R1, R2 and R3). Removing the repetitive stress/recovery exercise, activated transcriptional memory from two trainable genes persisted for at least 4 days in perennial ryegrass. The pretrainable genes with stress memory effort had higher response to the subsequent elevated NaCl concentration treatment than the non-trainable plants, which was confirmed by lower electrolyte leakage and minimum H2 O2 and O2 (-) accumulation. Salt stress elevated the content of 41 metabolites in perennial ryegrass leaves, and sugars and sugar alcohol accounted for more than 74.1% of the total metabolite content. The salt stress memory was associated with higher contents of 11 sugars and 1 sugar alcohol in the pretrainable grass leaves. Similarly, six sugars showed greater content in the pretrainable grass roots. These novel phenomena associated with transcriptional memory and metabolite profiles could lead to new insights into improving plant salinity acclimation process.

Functional repetitive sequences and fragile sites in chromosomes of Lolium perenne L.

Lolium perenne is considered a high-quality forage widely used in temperate regions to meet the shortage of forage during the winter. In this species, some peculiarities related to cytogenetic aspects have already been described, as the variability in number and position of 45S ribosomal DNA (rDNA) sites and the expression of fragile sites, which require further studies to support the understanding of their causes and consequences. In this way, this study aimed to evaluate the relationship between the expression of fragile sites and functional repetitive sequences (rDNA and telomeric) in chromosomes of diploid and polyploid cultivars of L. perenne. The techniques of FISH, Ag-NOR and fluorescence banding were used to assess the distribution of sites of 45S rDNA, 5S, telomeric sequences, and the transcriptional activity of the 45S ribosomal genes and the distribution of AT- and/or GC-rich sequences in L. perenne, respectively. There was variability in the number and location of 45S rDNA sites, which was not observed for 5S rDNA sites. One of the genotypes showed two 45S rDNA sites on the same chromosome, located in different chromosome arms. Breaks and gaps were found in 45S rDNA sites in most metaphases evaluated for both cultivars. Telomeric sequences were not detected at the end of the chromosomal fragments corresponding to the location of breaks at 45S sites. Apparently, the transcriptional activity was modified in fragile sites. Variation in the number and size of nucleoli, nucleolar fusions and dissociations were observed. All CMA(+) bands were colocalized with the 45S sites.

Deletion of the fungal gene soft disrupts mutualistic symbiosis between the grass endophyte Epichloë festucae and the host plant.

Hyphal anastomosis, or vegetative hyphal fusion, establishes the interconnection of individual hyphal strands into an integrated network of a fungal mycelium. In contrast to recent advances in the understanding of the molecular basis for hyphal anastomosis, knowledge of the physiological role of hyphal anastomosis in the natural habitats of filamentous fungi is still very limited. To investigate the role of hyphal anastomosis in fungal endophyte-plant interactions, we generated mutant strains lacking the Epichloë festucae soft (so) gene, an ortholog of the hyphal anastomosis gene so in the endophytic fungus E. festucae. The E. festucae Δso mutant strains grew similarly to the wild-type strain in culture but with reduced aerial hyphae and completely lacked hyphal anastomosis. The most striking phenotype of the E. festucae Δso mutant strain was that it failed to establish a mutualistic symbiosis with the tall fescue plant host (Lolium arundinaceum); instead, it killed the host plant within 2 months after the initial infection. Microscopic examination revealed that the death of the tall fescue plant host was associated with the distortion and disorganization of plant cells. This study suggests that hyphal anastomosis may have an important role in the establishment/maintenance of fungal endophyte-host plant mutualistic symbiosis.

Alien introgression in the grasses Lolium perenne (perennial ryegrass) and Festuca pratensis (meadow fescue): the development of seven monosomic substitution lines and their molecular and cytological characterization.

BACKGROUND AND AIMS: To address the issues associated with food security, environmental change and bioenergy in the context of crop plants, the production, identification and evaluation of novel plant phenotypes is fundamental. One of the major routes to this end will be wide hybridization and introgression breeding. The transfer of chromosomes and chromosome segments between related species (chromosome engineering or alien introgression) also provides an important resource for determining the genetic control of target traits. However, the realization of the full potential of chromosome engineering has previously been hampered by the inability to identify and characterize interspecific introgressions accurately. METHODS: Seven monosomic substitution lines have been generated comprising Festuca pratensis as the donor species and Lolium perenne as the recipient. Each of the seven lines has a different L. perenne chromosome replaced by the homoeologous F. pratensis chromosome (13 L. perenne + 1 F. pratensis chromosome). Molecular markers and genomic in situ hybridization (GISH) were used to assign the F. pratensis chromosomes introgressed in each of the monosomic substitutions to a specific linkage group. Cytological observations were also carried out on metaphase I of meiosis in each of the substitution lines. RESULTS: A significant level of synteny was found at the macro-level between L. perenne and F. pratensis. The observations at metaphase I revealed the presence of a low level of interspecific chromosomal translocations between these species. DISCUSSION: The isolation of the seven monosomic substitution lines provides a resource for dissecting the genetic control of important traits and for gene isolation. Parallels between the L. perenne/F. pratensis system and the Pooideae cereals such as wheat, barley, rye, oats and the model grass Brachypodium distachyon present opportunities for a comparison across the species in terms of genotype and phenotype.

Dark-mediated dormancy release in stratified Lolium rigidum seeds is associated with higher activities of cell wall-modifying enzymes and an apparent increase in gibberellin sensitivity.

Dormancy release in freshly matured, imbibed annual ryegrass (Lolium rigidum) seeds is inhibited by light and involves a decrease in seed sensitivity to abscisic acid. Other processes involved in dormancy release in the dark were investigated by measuring seed storage compound mobilisation and the activity of cell wall-degrading enzymes. Activities of endo-ß-mannanase and total peroxidase were higher in dark-stratified compared to light-stratified seeds, indicating that weakening of the structures constraining the embryo was accelerated in the dark. A dramatic degradation of storage proteins in light-stratified seeds, accompanied by induction of a high molecular mass protease, suggests that maintenance of storage(-like) proteins is also important in dark-mediated dormancy release. α-Amylase activity was induced in dark-stratified seeds at least 48 h prior to radicle emergence upon transfer to conditions permitting germination, or in light-stratified seeds supplied with exogenous gibberellin A(4). This suggests that (a) α-amylase is involved in stimulation of germination of non-dormant L. rigidum seeds, and (b) dark-stratified seeds have an increased sensitivity to gibberellins which permits the rapid induction of α-amylase activity upon exposure to germination conditions. Overall, it appears that a number of processes, although possibly minor in themselves, occur in concert during dark-stratification to contribute to dormancy release.

Bioavailability of heavy metals and their effects on the midgut cells of a phytopaghous insect inhabiting volcanic environments.

Organisms living in volcanic environments are chronically exposed to metals, either as particles or associated with gases, from volcanic emissions, being therefore potential sentinels of the effects derived from such exposure. Concentrations of Ca, Cd, Cu, Mg, Mn, Pb, Rb, and Zn were measured in soil, grass (Lolium perenne), and larvae of Pseudaletia uninpuncta captured in sites exposed and non-exposed to volcanic activity. The midgut epithelial cell morphometry and apoptosis of P. unipuncta larvae were also analyzed. Larvae from the site with volcanic activity showed higher levels of Cu, Mn, Rb and Zn. Metals such as Pb, Cd and Mg levels of P. unipuncta larvae were similar between sites. Apoptosis was higher in cells from digestive epithelium of larvae exposed to volcanic activity. Soils and grass not exposed to volcanic activity showed higher levels for most of the analyzed elements with the exception of Rb. Such result when compared with metal levels of larvae may reveal that bioavailability of elements differs between sites. The higher levels of Cd, Zn and Mg in soils and grass from the site with no volcanic activity are probably related to the severe artificial fertilization in the studied pastures. Such result, when compared with metal levels of larvae, suggest that the bioavailability of metals differs between sites.

Cytogenetics of Festulolium (Festuca x Lolium hybrids).

Grasses are the most important and widely cultivated crops. Among them, ryegrasses (Lolium spp.) and fescues (Festuca spp.) provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Species from the two genera display complementary agronomic characteristics and are often grown in mixtures. Breeding efforts to combine desired features in single entities culminated with the production of Festuca x Lolium hybrids. The so called Festuloliums enjoy a considerable commercial success with numerous cultivars registered all over the world. They are also very intriguing from a strictly cytogenetic point of view as the parental chromosomes recombine freely in hybrids. Until a decade ago this phenomenon was only known in general quantitative terms. The introduction of molecular cytogenetic tools such as FISH and GISH permitted detailed studies of intergeneric chromosome recombination and karyotyping of Festulolium cultivars. These tools were also invaluable in revealing the origin of polyploid fescues, and facilitated the development of chromosome substitution and introgression lines and physical mapping of traits of interest. Further progress in this area will require the development of a larger set of cytogenetic markers and high-resolution cytogenetic maps. It is expected that the Lolium-Festuca complex will continue providing opportunities for breeding superior grass cultivars and the complex will remain an attractive platform for fundamental research of the early steps of hybrid speciation and interaction of parental genomes, as well as the processes of chromosome pairing, elimination and recombination.

45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp.

BACKGROUND: In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported. METHODS AND RESULTS: During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region. CONCLUSIONS: The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.

Nondestructive analysis of senescence in mesophyll cells by spectral resolution of protein synthesis-dependent pigment metabolism.

* Over 6 d of dark-induced senescence, leaf segments of wild-type Lolium temulentum lost > 96% chlorophyll a + b; leaves from plants containing a staygreen mutation introgressed from Festuca pratensis, which has a lesion in the senescence-associated fragmentation of pigment-proteolipid complexes, retained over 43% of total chlorophyll over the same period. * Mutant segments preferentially retained thylakoid membrane proteins (exemplified by LHCP II) but lost other cellular proteins at the same rate as wild-type tissue. The protein synthesis inhibitor D-MDMP inhibited chlorophyll degradation and partially prevented protein loss in both genotypes, but tissues treated with the ineffective L-stereoisomer were indistinguishable from water controls. * Principal-components analysis of leaf reflectance spectra distinguished between genotypes, time points and D-MDMP treatments, showing the disruption of pigment metabolism during senescence brought about by the staygreen mutation, by inhibition of protein synthesis and by combinations of the two factors. * The build-up of oxidized, dephytylated and phaeo-derivatives of chl a during senescence of staygreen tissue was prevented by D-MDMP and associated with characteristic difference spectra when senescent mutant tissue was compared with wild-type or inhibitor-treated samples. The suitability of senescence as a subject for systems biology approaches is discussed.

Patterns of expression of a lolitrem biosynthetic gene in the Epichloë festucae-perennial ryegrass symbiosis.

Lolitrem B is synthesized by Epichloë festucae in associations with Pooid grasses. A complex cluster of at least 10 genes (ltm genes) is required for its synthesis. An early step in this pathway is catalyzed by ltmM, a symbiosis-expressed gene. PltmM-gusA reporter gene analysis was used to monitor ltmM gene expression patterns in planta. The minimum promoter length required for high-level gusA expression in infected seedlings is in the range of 480 to 782 bp. gusA was expressed by the endophyte in all infected vegetative plant tissues and in epiphyllous hyphae. Spikelets from reproductive tillers were analyzed at different developmental stages. During pre-anthesis, gusA expression was observed in all infected floral organs except the immature gynoecium. In post-anthesis florets, gene expression occurred almost exclusively in the gynoecium. Expression of gusA by the endophyte was observed in germinating seeds 24 h postimbibition and seedlings older than 6 days postimbibition in hyphae from the mesocotyl to the tip of the emerging first leaf. This work provides a detailed analysis of the spatial and temporal expression patterns of a symbiosis-expressed gene in planta.

Transcriptome analysis reveals season-specific rbcS gene expression profiles in diploid perennial ryegrass (Lolium perenne L.).

Perennial ryegrass (Lolium perenne L.) is a major grass species used for forage and turf throughout the world, and gains by conventional breeding have reached a plateau. Perennial ryegrass is an outcrossing, self-incompatible diploid (2n = 2x = 14) with a relatively large genome (4067 Mbp/diploid genome; Evans, G.M., Rees, H., Snell, C.L. and Sun, S. (1972) The relation between nuclear DNA amount and the duration of the mitotic cycle. Chrom. Today, 3, 24-31). Using tissues sourced from active pastures during the peak of the autumn, winter, spring and summer seasons, we analysed the ryegrass transcriptome employing a Serial Analysis of Gene Expression (SAGE) protocol, with the dual goals of understanding the seasonal changes in perennial ryegrass gene expression and enhancing our ability to select genes for genetic manipulation. A total of 159,002 14-mer SAGE tags was sequenced and mapped to the perennial ryegrass DNA database, comprising methyl-filtered (GeneThresher) and expressed sequence tag (EST) sequences. The analysis of 14,559 unique SAGE tags, which were present more than once in our SAGE library, revealed 964, 1331, 346 and 131 exclusive transcripts to autumn, winter, spring and summer, respectively. Intriguingly, our analysis of the SAGE tags revealed season-specific expression profiles for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco), LprbcS. The transcript level for LprbcS was highest in spring, and then decreased gradually between summer and winter. Five different copies of LprbcS were revealed in ryegrass, with one possibly producing splice variant transcripts. Two highly expressed LprbcS genes were reported, one of which was not active in autumn. Another LprbcS gene showed an inverse expression profile to the autumn inactive LprbcS in a manner to compensate the expression level.

Cell wall polysaccharides in differentiating anthers and pistils of Lolium perenne.

We are presenting the pattern of distribution of several carbohydrate epitopes, which constitute an important component of cell walls, within the anthers and pistils of a monocot grass species, perennial ryegrass (Lolium perenne L.). The results of immunocytochemical studies revealed that the flower organs are rich in (1-->3, 1-->4)-beta-D-glucans and possess surprisingly high amounts of methylesterified pectic domains that bind JIM7 antibody and pectin side chains rich in (1-->4)-beta-D-galactose residues which react with LM5 antibody. The presence of arabinogalactan protein epitopes binding JIM13 is restricted to microspores and ovule integuments. The results are discussed in terms of possible functions of cell wall polysaccharides and arabinogalactan proteins in the differentiation of flower organs.

Polyploidy and cellular mechanisms changing leaf size: comparison of diploid and autotetraploid populations in two species of Lolium.

BACKGROUND AND AIMS: Growth and development of plant organs, including leaves, depend on cell division and expansion. Leaf size is increased by greater cell ploidy, but the mechanism of this effect is poorly understood. Therefore, in this study, the role of cell division and expansion in the increase of leaf size caused by polyploidy was examined by comparing various cell parameters of the mesophyll layer of developing leaves of diploid and autotetraploid cultivars of two grass species, Lolium perenne and L. multiflorum. METHODS: Three cultivars of each ploidy level of both species were grown under pot conditions in a controlled growth chamber, and leaf elongation rate and the cell length profile at the leaf base were measured on six plants in each cultivar. Cell parameters related to division and elongation activities were calculated by a kinematic method. KEY RESULTS: Tetraploid cultivars had faster leaf elongation rates than did diploid cultivars in both species, resulting in longer leaves, mainly due to their longer mature cells. Epidermal and mesophyll cells differed 20-fold in length, but were both greater in the tetraploid cultivars of both species. The increase in cell length of the tetraploid cultivars was caused by a faster cell elongation rate, not by a longer period of cell elongation. There were no significant differences between cell division parameters, such as cell production rate and cell cycle time, in the diploid and tetraploid cultivars. CONCLUSION: The results demonstrated clearly that polyploidy increases leaf size mainly by increasing the cell elongation rate, but not the duration of the period of elongation, and thus increases final cell size.

Enhanced post-thaw viability of cryopreserved cells by oxygenated perfluorocarbon or Pluronic F-68.

Viability and growth following cryopreservation have been examined following (i) culture of Oryza sativa cv. Taipei 309 (Japonica rice) in medium overlaying oxygenated perfluorocarbon (PFC) liquid (Flutec PP6), and (ii) culture of two cvs. of Japonica rice (Taipei 309, Tarom), and Lolium multiflorum in media with Pluronic F-68 (0.01-0.2% w/v). The mean viability of cv. Taipei 309 rice cells following 4 days in medium overlaying oxygenated Flutec (0.45 +/- 0.07; n = 20) was significantly (P < 0.05) greater than control (0.35 +/- 0.08). Cell viability of cv. Tarom was increased by 400% (P < 0.001) with 0.1% (w/v) Pluronic F-68 in the recovery medium. Mean cell viability was also elevated 3-fold (P < 0.001) and 2-fold (P < 0.05) with 0.2% (w/v) and 0.01% (w/v) Pluronic, respectively. A 2-fold increase (P < 0.001) in viability occurred for Taipei 309 cells exposed to 0.01% (w/v) Pluronic F-68; that of Lolium cells was increased by 31% (P < 0.01) under similar conditions. Pluronic F-68 (0.01% w/v) also stimulated biomass, by 29-32%, in both cvs. Taipei 309 and Lolium at 30 days post-thawing.

Genetic control of synapsis and recombination in Lolium amphidiploids.

Homologous bivalent formation in amphidiploids of Lolium is promoted during meiosis by diploidising genes carried by A-chromosomes, and by supernumerary B-chromosomes. The site and mode of action of these diploidising factors were investigated by comparing the relative frequencies of pairing configurations at meiotic prophase and metaphase I in several different hybrid genotypes. The results indicate that diploidising genes act predominantly by increasing the stringency of synapsis at early stages of meiotic prophase. By contrast, B-chromosomes appear to promote bivalent formation by ensuring that homoeologously paired chromosome segments within multivalents do not crossover. The results show that the additive effects of diploidising genes and B-chromosomes are to a certain extent separable in terms of their mode of action and timing during meiosis.