Lolium Temulentum Research Articles

Plant extract contact toxicities to various developmental stages of Colorado potato beetles (Coleoptera: Chrysomelidae)

AbstractThe contact toxicities of methanol extracts from the nine plant species Hedera helix, Artemisia vulgaris, Xanthium strumarium, Humulus lupulus, Sambucus nigra, Chenopodium album, Salvia officinalis, Lolium temulentum and Verbascum songaricum were tested on the developmental stages of Colorado potato beetle (CPB) (Leptinotarsa decemlineata). About 2 mL of plant extract, 40% (w/w), was applied to the first instar to fourth instar larvae and adult beetles using a Potter spray tower. Most of the tested plant extracts caused relatively low mortality in all the beetle instars. Among the plant extracts, H. lupulus extract was the most toxic to all stages of the insect, except for the adult beetles. Larval mortality ranged from 40% in the fourth instars to 84% in the third instars. In a second series of experiments, dose–response bioassays using H. lupulus extract produced lethal concentration 50 (LC50) values ranging from 10%, 12%, 17% to 46% (w/w) active ingredient (plant material) for instars 1–4, respectively. This increasing mortality trend, however, did not extend to the adult stage where even the maximum dose of 40% plant material did not provide sufficient mortality to allow estimation of a LC50. These results demonstrated that the extract from H. lupulus has potential as an active ingredient in biological pesticides developed to manage larval instars of the CPB. The potential uses of this plant extract may be in conventional and organic pest management or as part of a mixture of plant extracts or conventional insecticides. Before extracts can be considered as biological control agents, their impact on natural enemies should be assessed.

GA4 Is the Active Gibberellin in the Regulation ofLEAFYTranscription andArabidopsisFloral Initiation

Flower initiation in Arabidopsis thaliana under noninductive short-day conditions is dependent on the biosynthesis of the plant hormone gibberellin (GA). This dependency can be explained, at least partly, by GA regulation of the flower meristem identity gene LEAFY (LFY) and the flowering time gene SUPPRESSOR OF CONSTANS1. Although it is well established that GA(4) is the active GA in the regulation of Arabidopsis shoot elongation, the identity of the GA responsible for the regulation of Arabidopsis flowering has not been established. Through a combination of GA quantifications and sensitivity assays, we show that GA(4) is the active GA in the regulation of LFY transcription and Arabidopsis flowering time under short-day conditions. The levels of GA(4) and sucrose increase dramatically in the shoot apex shortly before floral initiation, and the regulation of genes involved in GA metabolism suggests that this increase is possibly due to transport of GAs and sucrose from outside sources to the shoot apex. Our results demonstrate that in the dicot Arabidopsis, in contrast with the monocot Lolium temulentum, GA(4) is the active GA in the regulation of both shoot elongation and flower initiation.

Evaluation of Lolium temulentum as a model grass species for the study of salinity stress by PCR-based subtractive suppression hybridization analysis

Soil salinity is one of the major abiotic stresses responsible for reduced persistence, yield and biomass accumulation in many crops including forage grass. Forage grass species are generally polymorphic, obligate out-crossers, that are self-incompatible. Because of their high genetic diversity, the mechanisms of salt tolerance are poorly understood. Consequently, the development of a useful model grass plant for the study of abiotic stresses is of great importance. We propose the use of Lolium temulentum L. (Darnel ryegrass), a diploid self-fertile species with a short life cycle (2–3 months), as a model system for the study of forage/turf grass species. To evaluate the utility of L. temulentum as a model grass species to study salt stress, a PCR-based subtractive suppression hybridization library was generated and sequenced. A total of 528 unique sequences were identified, among which 167 corresponded to orthologs of previously identified plant stress response genes. The expression patterns in leaf, crown and root tissues of selected genes were analyzed by Northern blot analysis, demonstrating salinity depended regulation of gene expression. These preliminary studies provide proof of concept supporting the use of L. temulentum as a model forage grass for molecular genetic analyses of salinity stress.

Inheritance of Seed Shattering in Lolium temulentum and L. persicum Hybrids

Lolium temulentum and L. persicum are non-crop species found in wheat and barley fields. L. temulentum has non-shattering seeds like the associated grain crops, whereas L. persicum seeds shatter after maturity. We analyzed the inheritance mode of shattering tendency by comparing the F2 of L. temulentum and L. persicum hybrids. The selfed progeny of L. temulentum and L. persicum exhibited typical non-shattering (1.6% shattering) and shattering phenotypes (70.8%), respectively. F1 hybrids of L. temulentum×L. persicum and its reciprocals were of the shattering phenotype (71.4% and 63.8%, respectively), indicating that shattering is dominant to non-shattering. When the phenotype ratio was assumed to be 15 shattering: 1 non-shattering, the χ2 value for F2 segregation was not significant at the 5% level, and the reciprocal effect was not detected. This indicates that the non-shattering tendency is controlled by two recessive genes. The two-gene inheritance model of shattering tendency suggests that harvest is the selector for seed shattering in cultivated fields, thus the alternative tendency for non-shattering seeds of L. temulentum or shattering of L. persicum would be better adapted to cultivated fields.

Interspecific differentiation and phylogenetic relationships of poison ryegrass (Lolium temulentum L.) and Persian darnel (L. persicum Boiss. & Hohen. ex Boiss.)

To clarify the speciation and evolution of the inbreeding Lolium spp., poison ryegrass and Persian darnel, we analyzed genetic relationships using microsatellite and AFLP markers among accessions from Pakistan, where both species grow sympatrically or parapatrically. Dendrograms were constructed using the unweighted pair-group method with arithmetic averages (UPGMA), based on simple matching coefficient of similarity among 29 accessions of poison ryegrass and 16 accessions of Persian darnel. Most of the poison ryegrass and Persian darnel accessions were genetically divided into two clusters. One and two chloroplast RFLP (restriction fragment length polymorphism) haplotypes were identified in poison ryegrass and Persian darnel accessions, respectively, from Pakistan, which correspond to each cluster or subcluster of dendrograms. Some accessions, morphologically identified as Persian darnel, belong to neither poison ryegrass nor Persian darnel clusters and locate in other cluster between them. Because this intermediate group had the same haplotype as poison ryegrass, shared almost all alleles with poison ryegrass and/or Persian darnel, and was genetically closer to poison ryegrass than to Persian darnel, we hypothesize that the intermediate group was derived maternally from poison ryegrass via hybridization with Persian darnel. Key words: AFLP, chloroplast DNA-RFLP, Lolium persicum, Lolium temulentum, microsatellite, phylogenetic analysis

Use of tall fescue EST-SSR markers in phylogenetic analysis of cool-season forage grasses

Microsatellites or simple sequence repeats (SSRs) are highly useful molecular markers for plant improvement. Expressed sequence tag (EST)-SSR markers have a higher rate of transferability across species than genomic SSR markers and are thus well suited for application in cross-species phylogenetic studies. Our objectives were to examine the amplification of tall fescue EST-SSR markers in 12 grass species representing 8 genera of 4 tribes from 2 subfamilies of Poaceae and the applicability of these markers for phylogenetic analysis of grass species. About 43% of the 145 EST-SSR primer pairs produced PCR bands in all 12 grass species and had high levels of polymorphism in all forage grasses studied. Thus, these markers will be useful in a variety of forage grass species, including the ones tested in this study. SSR marker data were useful in grouping genotypes within each species. Lolium temulentum, a potential model species for cool-season forage grasses, showed a close relation with the major Festuca-Lolium species in the study. Tall wheat grass was found to be closely related to hexaploid wheat, thereby confirming the known taxonomic relations between these species. While clustering of closely related species was found, the effectiveness of such data in evaluating distantly related species needs further investigations. The phylogenetic trees based on DNA sequences of selected SSR bands were in agreement with the phylogenetic relations based on length polymorphism of SSRs markers. Tall fescue EST-SSR markers depicted phylogenetic relations among a wide range of cool-season forage grass species and thus are an important resource for researchers working with such grass species.

Relative efficiency of different Gramineae genera for haploid induction in triticale and triticale x wheat hybrids through the chromosome elimination technique

The study was undertaken to evaluate the relative efficiency of different Gramineae genera for haploid induction in triticale (x Triticosecale) and triticale × wheat (Triticum aestivum) hybrids through the chromosome elimination (wheat × maize, Zea mays) system. Eight intergenotypic triticale and 15 triticale x wheat crosses were subjected to hybridization with nine different Gramineae genera viz., Z. mays, Sorghum bicolor, Pennisetum americanum, Setaria italica, Festuca arundinacea, Imperata cylindrica, Cynodon dactylon, Lolium temulentum and Phalaris minor in two separate experiments. This was followed by in vivo auxin treatment of the crossed spikes and subsequent rescue of the haploid embryos to regenerate green haploid plantlets. All the triticale and triticale x wheat crosses resulted in seed set in variable frequencies when hybridized with maize, I. cylindrica, pearl millet and sorghum. Seed set was also obtained with S. italica, F. arundinacea and P. minor in a few crosses in both groups. In general, all the triticale x wheat crosses, except for one in each case, resulted in embryo formation and green haploid plantlet regeneration when hybridizations were carried out with maize and I. cylindrica. However, the latter outperformed the former in embryo formation (25.48% vs. 20.0%) and regeneration (34.17% vs. 15.10%) frequencies, the differences being significant for regeneration frequencies. In the case of triticale hybrids, no significant differences between maize and I. cylindrica were observed for the three parameters of haploid induction. Embryo formation and regeneration were also observed in some of the triticale as well as triticale × wheat F1 hybrids when hybridized with sorghum and pearl millet.

Analysis of Lolium temulentum geographical differentiation by microsatellite and AFLP markers

SummaryThe genetic diversity and relationships of 48 Lolium temulentum accessions derived from eight countries (Morocco, Egypt, Tunisia, Italy, Ethiopia, Pakistan, Nepal and Japan) were analysed using seven microsatellite and 44 AFLP polymorphic loci to investigate the origin and distribution of genetic variation. Cluster analysis was performed using the unweighted pair‐group method with arithmetic averages (UPGMA) based on the simple matching coefficient of similarity. Nei's gene diversity (h) and the coefficient of gene differentiation (Gst) were calculated. The results from microsatellite analysis indicated that accessions from the same country did not always cluster together, probably because of the limited number of loci used. In contrast, AFLP clearly separated clusters between countries and/or regions; Pakistan–Nepal complex, the Mediterranean region, Ethiopia and Japan. The h value was much higher for microsatellite than for AFLP analysis, indicating that microsatellites are the more variable markers. For AFLP analyses, h values were highest in accessions from Pakistan–Nepal complex, and from the Mediterranean region. These are in agreement with proposals that the origin of L. temulentum lies between the South‐western Asia and the Mediterranean basin. The clear groupings of accessions from each country and/or region with high Gst (0.688) indicate that exchange of seeds between them is limited.

Genetic Diversity of Darnel (Lolium Temulentum L.) in Malo, Ethiopia Depends on Traditional Farming Systems

Darnel (Lolium temulentum L.) is a mimic weed associated with wheat and barley cultivation. Mimic weeds have evolved along with cultivated crops. Human impact on the genetic diversity of agricultural weeds was elucidated using darnel as a model. Three strains in each field in Gaitza village, Malo region, Ethiopia were randomly sampled, and the genetic composition of 120 samples was analyzed using RAPD analysis. Genetic diversity and distances were calculated using Nei's analysis, and cluster analysis was performed. These results indicated the occurrence of seed migration among the fields and a high genetic diversity of darnel in this region. Crop seed exchange and contamination of crop seeds with darnel seeds lead to the unintended artificial gene flow of darnel. Because darnel seeds and seedlings resemble their associated crop seeds and seedlings, traditional methods of crop cultivation have the unintended consequence of conserving the genetic diversity of darnel.

Awn of darnel (Lolium temulentum L.) as an anthropogenic dispersal organ: A case study in Malo, south‐western Ethiopia

Darnel (Lolium temulentum), a weed of wheat and barley, is either awned or awnless. The role of the darnel awn in dispersal was investigated in relation to human's agricultural practices in cereal cultivation in a Malo village, Gaytsa, south‐western Ethiopia. In 24 free‐threshing wheat fields and 11 emmer wheat fields, 129 and 88 spikes (one spike per plant), respectively, of darnel were randomly sampled. At the market in Gaytsa, 11 free‐threshing wheat grain samples and eight emmer wheat grain samples of 150–200 g were collected. For each grain sample, the number of wheat grains and awned and awnless grains of darnel were recorded. The relative percentage of the awned form in free‐threshing wheat fields was 8.53% and that in emmer wheat fields was 70.45%. The relative percentage of awned grains in free‐threshing wheat grains from the market collection was 3.55% and that in emmer wheat grain lots was 75.20%. The morphology of awned darnel grains resembles emmer wheat grains and awnless darnel grains resemble free‐threshing wheat grains. Thus, grain mimicry controls the close association of the two forms of darnel with the wheat species. The lack of an awn itself functions for darnel grain as a dispersal mechanism in free‐threshing wheat cultivation systems compared with other wild grass species.

Development of highly tissue culture responsive lines of Lolium temulentum by anther culture

Most of the important forage and turf grasses are outbreeders, require vernalization to flower, and in some cases are polyploid. With the development of gene sequencing information in grasses, there is an urgent need for a model system to perform large-scale functional analysis of candidate genes. We propose to use Lolium temulentum L. (Darnel ryegrass) as a model system for genetic manipulation studies in forage and turf grasses because L. temulentum has the following advantages: self-fertile, short life cycle (11 weeks), diploid, easy to grow, and closely related with other major grass species. In order to improve tissue culture response of L. temulentum, two relatively responsive lines were crossed and putative hybrid seeds obtained. Analysis of the F 1 plants by SSR markers confirmed that the F 1 plants were true hybrids. Anthers were dissected from F 2 plants of the cross, 3.0% of the cultured anthers responded with the formation of calluses. Green haploid plants were recovered from 48.9% of the anther-derived calluses. Seeds were harvested from doubled haploid plants and mature embryos were used as explants for comparing tissue culture responses with other lines. Besides anther culture, F 2 seeds of the cross were subjected to a cycle of selection for callus formation and plant regeneration, and F 4 seeds were obtained from the regenerated plants. Comparison of tissue culture response of different lines revealed that the doubled haploid plants had a much higher frequency of embryogenic callus formation than that of the parental lines and the F 4 seeds. This is the first report on the generation of green haploid and doubled haploid plants in L. temulentum. The technique was successfully used for the rapid production of homozygous diploid plants that are highly tissue culture responsive. The anther culture-derived new L. temulentum lines could be valuable material for functional test of genes in grasses.

Adjustment of Lateral Element Lengths in Hybrids of Lolium Temulentum x L. Multiflorum

Lateral element lengths and synaptic behaviour of meiotic chromosomes are compared in Lolium temulentum, L. multiflorum and their disomic and trisomic interspecific F1 hybrids. The 45 % difference in nuclear DNA amounts between the parents translates into a 66% difference in lateral element lengths at meiotic prophase. Despite the large length disparities, homoeologues form bivalents at meiotic prophase in the disomic hybrid, which are morphologically and functionally very similar to those of both parents. The relative lengths of the lateral elements of the trivalent of the trisomic hybrid do not conform to expectations. They are longer than expected compared with the rest of the complement, and cannot in many cases be identified as belonging to L. temulentum or L. multiflorum. The possible reasons for this anomaly are considered.

Absence of turnover and futile cycling of sucrose in leaves of Lolium temulentum L.: implications for metabolic compartmentation.

To study the interdependence of sucrose accumulation and its hydrolyzing enzyme, soluble acid invertase (AI; EC 3.2.1.26), in fructan-accumulating temperate grasses and cereals, experiments were performed in which sucrose synthesis was abolished in leaves of Lolium temulentum by four independent inhibitory factors, each having a distinct mechanism of action. Trials in the light with mannose or vanadate and in the dark with anoxia or cyanide showed that previously accumulated sucrose was stable in the tissue over a 5- to 6-h period. Conversely, putatively vacuolar AI activity in tissue homogenates was sufficient to completely convert endogenous sucrose to monosaccharide within the same period. Continuous invertase-mediated breakdown of sucrose was thus not a feature of this tissue. It is concluded that AI and sucrose were not in metabolic contact in vivo, implying differential compartmentation. In darkness, in uninhibited leaves, sucrose concentrations fell linearly with respect to time at a rate of -0.6 mg g(-1) FW h(-1), over a 5- to 6-h period. This value is equivalent to rates of dark respiration measured by gas exchange. Dark-utilisation of sucrose was not accompanied by monosaccharide accumulation in the tissue. The rate of sucrose loss was 3-fold lower than rates of extractable AI activity. Hence, if AI was involved in dark-utilisation, then this implies at least a partial differential localisation of enzyme and substrate. However, the dark-consumption of sucrose was completely abolished by anoxia and by cyanide. It follows that dark-mobilisation (unlike invertase hydrolysis per se) was respiration-dependent and did not result from a simple co-localisation of sucrose and invertase. Taken together, the results show that sucrose and invertase do not share the same metabolic compartment in grass leaves. It is possible that invertase has no role in the mobilisation of stored sucrose in leaves of the fructan-accumulating grasses.

Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum

Pigments, proteins and enzyme activity related to chlorophyll catabolism were analysed in senescing leaves of wild-type (WT) Lolium temulentum and compared with those of an introgression line carrying a mutant gene from stay-green (SG) Festuca pratensis. During senescence of WT leaves chlorophylls a and b were continuously catabolised to colourless products and no other derivatives were observed, whereas in SG leaves there was an accumulation of dephytylated and oxidised catabolites including chlorophyllide a, phaeophorbide a and 13 2 OH-chlorophyllide a. Dephytylated products were absent from SG leaf tissue senescing under a light-dark cycle. Retention of pigments in SG was accompanied by significant stabilisation of light harvesting chlorophyll-proteins compared with WT, but soluble proteins such as Rubisco were degraded during senescence at a similar rate in the two genotypes. The activity of phaeophorbide a oxygenase measured in SG tissue at 3d was less than 12% of that in WT tissue at the same time-point during senescence and of the same order as that in young pre-senescent WT leaves, indicating that the metabolic lesion in SG concerns a deficiency at the ring-opening step of the catabolic pathway. In senescent L. temulentum tissue two terminal chlorophyll catabolites were identified with chromatographic characteristics that suggest they may represent hitherto undescribed catabolite structures. These data are discussed in relation to current understanding of the genetic and metabolic control of chlorophyll catabolism in leaf senescence.

Development of microsatellite markers and their effectiveness in Lolium temulentum

SummaryScreening a genomic library of Lolium temulentum (darnel) with synthetic (GT)15 and (GA)15 oligonucleotides, we identified microsatellite‐containing clones. Based on the sequence data of the clones, specific primer pairs were synthesized and their effectiveness examined using 10 L. temulentum and two L. persicum accessions from Asia, Africa and Europe. Fifteen primer pairs amplified single fragments, and five of them showed polymorphism in polyacrylamide gel electrophoresis in the L. temulentum and L. persicum accessions. Fifteen primer pairs reported in L. perenne (perennial ryegrass) were also applied in this study. Nine of them amplified the microsatellite alleles, and two of the nine showed polymorphic alleles in L. temulentum and L. persicum. For the polymorphic markers, polymorphic information content values ranged from 0.60 to 0.86 with an average of 0.76 among 10 L. temulentum accessions. The microsatellite markers developed for L. temulentum and L. perenne should be informative and powerful in analysing the intraspecific genetic diversity and the phylogenetic relationships of L. temulentum and L. persicum.

Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L.

The invertases of Lolium temulentum have been characterized at the enzyme level. However, studies on the expression of the genes coding for these enzymes have been lacking. To elucidate the role of acid 'invertase-like' genes in sucrose metabolism and carbon partitioning in Gramineae further, three 'invertase-like' homologous clones were isolated from L. temulentum cDNA expression libraries based on leaf tissue, using maize soluble invertase probes. The effect of developmental stage and alterations in carbohydrate status on the expression and tissue distribution of these genes was investigated. The three highly homologous genes (Inv 1:2, Inv 1:4, and FT 2:2) show different patterns of expression and different tissue distribution. Inv 1:2 was predominantly expressed in root tissue. Expression increased during the dark in root and tiller base tissue. Minimal variations in gene expression were observed in leaf tissue following changes in carbohydrate status. Inv 1:4 was predominantly expressed in tiller bases, leaf sheath, and leaf base, with increased expression in tissue samples in the dark period. FT 2:2 was also predominantly expressed in tiller bases, leaf sheath, and leaf base. Higher expression was observed in leaf tissue following increases in carbohydrate content, in a manner that paralleled the regulation and spatial occurrence of fructan in the leaf tissue. Whilst invertases and fructosyltransferases are difficult to distinguish at the level of the whole sequence, analysis of 5' sequence and specific amino acids allows discrimination which correlates with patterns of expression within the tissue. Based on expression patterns and sequence characteristics, it is proposed that Inv 1:2 and Inv 1:4 code for soluble acid invertases, whilst FT 2:2 codes for a fructosyltransferase.

Gibberellin structure and function: biological activity and competitive inhibition of gibberellin 2- and 3-oxidases.

Some gibberellin (GA) analogues, especially with C-16,17 modifications of GA(5), can inhibit growth of plants apparently by acting as competitors with the endogenous substrate of GA biosynthetic enzymes. Here, we directly confirm the competitive action of GA derivatives but also show that some analogues may retain significant bioactivity. A recombinant 3-oxidase from pea, which converts GA(20) to bioactive GA(1), was inhibited by GA(5), and 16,17-dihydro-GA(5) derivatives, especially if the C-17 alkyl chain length was increased by up to three carbons or if the C-13 hydroxyl was acetylated. Genetic confirmation that GA(5) analogues target 3-oxidases in vivo was provided by comparing the growth response of a WT (LE) pea with a 3-oxidase mutant (le-1). Two pea 2-oxidases that inactivate bioactive GAs, were inhibited by GA(1) and GA(3) but were generally insensitive to GA(5) analogues. alpha-Amylase production by barley half-seeds in response to GA analogues provided a method to study their action when effects on GA biosynthesis were excluded. This bioactivity assay showed that 16,17-dihydro GA(5) analogues have some inherent activity but mostly less than for GA(5) (5-50-fold), which in turn was 100-fold less active than GA(1) and GA(3). However, although C-17 alkyl derivatives with one or two added carbons showed little bioactivity and were purely 3-oxidase inhibitors, adding a third carbon (the 17-n-propyl-16,17-dihydro GA(5) analogue) restored bioactivity to that of GA(5). Furthermore, this analogue has lost its capacity to inhibit stem elongation of Lolium temulentum (Mander et al., Phytochemistry 49:1509-1515, 1998a), although it strongly inhibits the 3-oxidase. Thus, the effectiveness of a GA derivative as a growth retardant will reflect the balance between its bioactivity and its capacity to inhibit the terminal enzyme of GA biosynthesis. The weaker growth inhibition in dicots including pea (approximately 10%) than in monocots such as L. temulentum (>35%) is suggestive of taxonomic differences in the bioactivity of GAs and/or their effects on GA biosynthesis.

Gibberellins and flowering of grasses and cereals: prizing open the lid of the "florigen" black box.

Comprehensive studies in grasses show that gibberellins (GAs) play a role as a florigen. For Lolium temulentum, which flowers in response to a single long day (LD), GAs are a transmitted signal, their content increasing in the leaf early in the LD and then, hours later, at the shoot apex. There is a continuous trail of evidence of hormonal action of these GAs for L. temulentum and support for a similar role in the flowering of other LD-responsive temperate grasses and cereals. A characteristic of the initial flowering responses of grasses and cereals is their limited stem elongation. Interestingly, it is GAs with low effectiveness for stem elongation, GA5 and GA6, that reach the shoot apex and, structurally, are probably not degraded by 2-oxidase enzymes. By contrast, GA1 and GA4 cause stem elongation, may be inactive for floral evocation, and do not reach the vegetative shoot apex apparently because of susceptibility to degradation. However, GA4 can be florally active if protected against 2-oxidases either structurally or by using a 2-oxidase inhibitor. Later in inflorescence development, GA1 and GA4 can be detected at the shoot apex and are florally active if applied. The 2-oxidase restricting accessibility to the apex has probably declined at this time so there is a second florigenic, LD-regulated GA action. A growing body of molecular evidence supporting these actions of GA may provide a future basis for manipulating flowering of grasses and cereals.

Synthesis of gibberellin GA 6 and its role in flowering of Lolium temulentum

The induction of flowering by one long day (LD) in the grass Lolium temulentum is most closely mimicked by application of the gibberellins (GAs) GA 5 or GA 6, both of which occur naturally. These gibberellins promote floral development but have little effect on stem elongation. Endogenous GA 5 and GA 6 contents in the shoot apex double on the day after the LD and, for GA 5 (and we presume for GA 6 as well) reach a concentration known to be inductive for the excised shoot apex in vitro. They are, therefore, strong candidates as LD floral stimuli in this grass. The synthesis of GA 6 and an examination of its florigenic properties in L. temulentum are described.

Plant regeneration from embryogenic cell suspension cultures of Lolium temulentum

Lolium temulentum L. (Darnel ryegrass) is a self-fertile and diploid grass species with a relatively short life cycle. We propose to use L. temulentum as a model system for genetic manipulation studies in forage and turf grasses, since most of the important grasses are outcrossing, require vernalization to flower, and in some cases are polyploid. As the first step to develop an efficient regeneration and transformation system, we performed a large-scale genotype screening for tissue culture responses using 46 L. temulentum accessions. Embryogenic callus formation frequency ranged from <1% to 11% across all accessions tested. Embryogenic calluses of a few responsive accessions were used to establish cell suspension cultures. The regeneration frequency of green plantlets from the established cell suspension ranged from 15% to 39%. After transferring the regenerants to the greenhouse, fertile plants were readily obtained without any vernalization treatment. This efficient plant regeneration system is being used for genetic transformation studies. With the development of genomics approaches for the improvement of forage and turf grasses, L. temulentum could serve as a model system for testing gene functions.