SummaryPerennial 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 (2 n = 2 x = 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.

BackgroundPyrrolizidine alkaloids (PAs) are a class of secondary metabolites that function as feeding deterrents in a range of different plant species. In perennial ryegrass (Lolium perenne L.) the only PAs that have been identified are the thesinine-rhamnoside group, which displays significant genetic variation. Homospermidine synthase (HSS) has evolved from deoxyhypusine synthase (DHS) and catalyses the first step in the PA pathway, making it a key candidate for the investigation of genes influencing observed PA trait variation.ResultsDuring PCR amplification and sequence analysis of DHS we identified two putative HSS genes in perennial ryegrass. One of the genes (LpHSS1) was absent in some perennial ryegrass plants. Thesinine-rhamnoside levels were measured using liquid chromatography coupled with mass spectrometry in a diverse association mapping population, consisting of 693 plants free of fungal endophytic symbionts. Association tests that accounted for population structure identified a significant association of absence of the LpHSS1 gene with lower levels of thesinine-rhamnoside PAs. HSS-like gene sequences were identified for other grass species of the Poaceae, including tall fescue, wheat, maize and sorghum.ConclusionHSS is situated at the crucial first step in the PA pathway making it an important candidate gene for investigation of involvement in PA phenotypic variation. In this study, PA level in perennial ryegrass was strongly associated with the presence or absence of the LpHSS1 gene. A genetic marker, developed for the presence/absence of LpHSS1, may be used for marker-assisted breeding to either lower or increase PAs in breeding populations of perennial or Italian ryegrass to investigate a potential role in the deterrence of herbivore pests. The presence of HSS-like genes in several other Poaceae species suggests that PA biosynthesis may occur in plant family members beyond perennial ryegrass and tall fescue and identifies a potential route for manipulating PA levels.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1269-6) contains supplementary material, which is available to authorized users.

This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of microcolonies with plating efficiencies 3-10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.

Lolium perenne (ryegrass) is the most widely sown pasture grass in New Zealand providing forage for millions of sheep and cattle. Genetic improvement of Lolium species through conventional breeding has been particularly difficult because of its outbreeding and heterozygous nature. Biotechnology techniques that are not based on genetic modification technology, such as protoplast fusion, could help in the breeding of L. perenne as it enables the introgression of DNA (and associated desirable traits) from other species of plants that are otherwise unobtainable by conventional means. We intend to adapt this technology for L. perenne and report on our progress with the isolation of protoplasts, micro colony and callus formation, and shoot regeneration. Friable callus was induced from L. perenne seeds and used for isolation of protoplasts. After testing several combinations of enzyme mixtures at different molarities, a high yield of protoplasts (1.1 × 107 g-1 FW) with a viability of 82% has been obtained. Callus colonies were formed from protoplasts cultured on a nitrocellulose membrane over a feeder layer. Plating efficiencies of 0.1-0.2% were obtained. Regeneration of plantlets from these colonies were obtained on Linsmaier and Skoog medium supplemented with growth hormones. All the plants regenerated to date have an albino appearance.