Susan Dalton

Plant regeneration from cell suspension protoplasts of Festuca arundinacea Schreb. (tall fescue) and Lolium perenne L. (perennial ryegrass).

Summary Protoplasts were isolated from one Lolium perenne L. and three Festuca arundinacea Schreb. embryogenic cell suspensions 13 to 30 weeks after initiation from groups of chopped mature embryos. Colonies were formed by protoplasts of all four suspensions. When plated onto solidified medium the morphogenesis of protoplast colonies was similar to that of plated cell suspension colonies. Protoplast and cell suspension colonies of two Festuca lines regenerated green and albino shoots, the third Festuca line was unable to regenerate and the Lolium line formed virtually only albino shoots. These results were repeated in several protoplast isolations over a two month period. Over 100 Festuca protoplast derived cell colonies formed green shoots and over 200 colonies formed albino shoots. Almost 300 Lolium protoplast derived cell colonies formed albino shoots and two colonies formed green shoots. Green Festuca plandets derived from protoplasts were established in soil, but require vernalisation before crossing can be attempted.

Transgenic plants of Lolium multiflorum, Lolium perenne, Festuca arundinacea and Agrostis stolonifera by silicon carbide fibre-mediated transformation of cell suspension cultures

Seven Lolium multiflorum, one Lolium perenne, 12 Festuca arundinacea and six Agrostis stolonifera plants were regenerated following transformation with a hygromycin resistance gene and hygromycin selection, from cell suspension colonies treated with silicon–carbide whiskers. Transformation was confirmed by PCR and Southern blotting; the latter also showed that six of the L. multiflorum plants were independent transformants (insufficient molecular evidence was obtained for the seventh), nine of the 12 F. arundinacea plants were independent transformants, but that all the A. stolonifera plants were derived from a single transformation event. Most plants tested contained fewer than five integrated transgene copies. Transgene expression was confirmed by reverse transcriptase-PCR (RT-PCR). Of the one A. stolonifera and three L. multiflorum transformants regenerated after co-transformation with both the hygromycin resistance gene and the β-glucuronidase (gusA) gene, none were found to express GUS activity. L. multiflorum regenerants from older (14–16 week old) cell suspensions showed loss of female fertility, but analysis of the progeny from three plants showed that the transgenes were being inherited as a single dominant allele with a high frequency of transmission of hygromycin resistance.

Agrobacterium tumefaciens-mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.)

Abstract.Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and β-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24–68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.

Co-transformed, diploid Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass) and Lolium temulentum (darnel) plants produced by microprojectile bombardment

Abstract A total of 37 plants (30 Lolium multiflorum Lam., 6 L. perenne L., 1 L. temulentum L.) were regenerated from cell suspension colonies bombarded with plasmid DNAs encoding a hygromycin resistance gene (HYG) expressed under a CaMV35S promoter and a β-glucuronidase (GUS) gene expressed under a truncated rice actin1 promoter and first intron, or a maize ubiquitin promoter and first intron. Resistant plants were regenerated under hygromycin selection and transferred to soil. PCR analysis showed that the co-transformation frequency of the GUS gene varied from 33% to 78% of transformants, while histochemical staining of leaf tissue from soil-grown plants showed that the co-expression frequency varied from 37% to 50%. The transgenic nature of the plants was demonstrated by Southern hybridisation analysis, which also showed that the non-selected (GUS) gene was generally present at a higher copy number than the selected (HYG) gene.

Manipulating the phenolic acid content and digestibility of Italian ryegrass (Lolium multiflorum) by vacuolar targeted expression of a fungal ferulic acid esterase

In grass cell walls, ferulic acid esters linked to arabinosyl residues in arabinoxylans play a key role in crosslinking hemicellulose. Although such crosslinks have a number of important roles in the cell wall, they also hinder the rate and extent of cell wall degradation by ruminant microbes and by fungal glycohydrolyase enzymes. Ferulic acid esterase (FAE) can release both monomeric and dimeric ferulic acids from arabinoxylans making the cell wall more susceptible to further enzymatic attack. Transgenic plants of Lolium multiflorum expressing a ferulic acid esterase gene from Aspergillus niger, targeted to the vacuole under a constitutive rice actin promoter, have been produced following microprojectile bombardment of embryogenic cell cultures. The level of FAE activity was found to vary with leaf age and was highest in young leaves. FAE expression resulted in the release of monomeric and dimeric ferulic acids from cell walls on cell death and this was enhanced severalfold by the addition of exogenous β-1,4-endoxylanase. We also show that a number of plants expressing FAE had reduced levels of cell wall esterified monomeric and dimeric ferulates and increased in vitro dry-matter digestibility compared with nontransformed plants.

The effect of selection pressure on transformation frequency and copy number in transgenic plants of tall fescue (Festuca arundinacea Schreb.)

Abstract Protoplasts of Festuca arundinacea (Schreb.) were transformed with plasmids containing a hygromycin resistance gene, and plants were regenerated under a variety of selection regimes. Plant regeneration was highest under discontinuous selection, but resulted in the production of significant numbers of escapes. Continuous selection at low concentrations of hygromycin produced the highest numbers of transgenic plants without escapes. Transgene copy numbers for 83 plants were estimated and related to the selection regime under which the plants were regenerated. Discontinuous application of hygromycin produced a high proportion of plants containing two or fewer inserted copies of the transgene (87%), as did low levels (50 mg·l−1) of continuous selection on hygromycin (88%). Continuous selection at high concentrations of hygromycin biased selection in favour of multiple-copy containing plants. Using a plasmid encoding a hygromycin phosphotransferase enzyme of higher specific activity (pROB5) resulted in the production of significantly more transgenic plants.

Targeting expression of a fungal ferulic acid esterase to the apoplast, endoplasmic reticulum or golgi can disrupt feruloylation of the growing cell wall and increase the biodegradability of tall fescue (Festuca arundinacea).

In the cell walls of grasses, ferulic acid is esterified to arabinoxylans and undergoes oxidative reactions to form ferulates dimers, trimers and oligomers. Feruloylation of arabinoxylan is considered important not only because it leads to cross-linked xylans but also because ferulates may act as a nucleating site for the formation of lignin and hence link arabinoxylans to lignin by forming a lignin-ferulate-arabinoxylan complex. Such cross-linking is among the main factors inhibiting the release of fermentable carbohydrates from grasses either for ruminant nutrition or for biofuel production. We have found that significant reductions in the levels of monomeric and dimeric phenolics can be achieved in the growing cell walls during plant development in leaves of Festuca arundinacea by constitutive intracellular targeted expression of Aspergillus niger ferulic acid esterase (FAEA). We propose that this occurred by directly disrupting ester bonds linking phenolics to cell wall polysaccharides by apoplast targeting or by preventing excessive feruloylation of cell wall carbohydrates prior to their incorporation into the cell wall, by targeting to the Golgi membrane system. Plants with lower cell wall ferulate levels, which showed increased digestibility and increased rates of cellulase-mediated release of fermentable sugars, were identified. Targeting FAE to the Golgi was found to be more effective than targeting to the ER, which supports the current theories of the Golgi as the site of feruloylation of arabinoxylans. It is concluded that targeting FAEA expression to the Golgi or apoplast is likely to be an effective strategy for improving wall digestibility in grass species used for fodder or cellulosic ethanol production.

Plant Regeneration from Cell Suspension Protoplasts of Festuca Arundinacea Schreb., Lolium Perenne L. and L. Multiflorum Lam

A prerequisite for the use of many genetic manipulation techniques is the ability to regenerate plants from protoplasts, but in the cereals and grasses, plants have been regenerated in this way in only six species (1). Of these, only Oryza (2,3,4,5,6) and Saccharum (7) have been successfully transferred to soil. In vitro plants of three forage grasses: Festuca arundinacea (tall fescue), Loliurn perenne (perennial ryegrass) and L.multiflorum (Italian ryegrass), have now been regenerated from protoplast-derived cell colonies and green Festuca and L.multiflorum plants established in soil (8).

Regeneration of fertile transgenic tall fescue plants with a stable highly expressed foreign gene

One hundred and seventeen green tall fescue plants and 37 albino plants were regenerated from a glufosinate ammonium resistant callus clone co-transformed with the bar gene and the gusgene, both driven by the rice actin 1 promoter. The gus gene was not detectable in regenerated plants but the presence of the bar gene in these plants was detected by the polymerase chain reaction and integration of the bar gene into the genome by Southern blot hybridization. A high and stable expression of the bar gene was evident from the assay for phosphinothricin-N-acetyltransferase activity and from spraying plants with glufosinate ammonium herbicide. There was no detectable variation with respect to the level of bar gene expression among these plants. However, no inheritance of the bar gene was found in two populations of outcrossed progenies.

Genetic transformation of Dichanthium annulatum (Forssk)—an apomictic tropical forage grass

Eleven Dichanthium annulatum (Forssk) plants were regenerated from embryogenic callus co-transformed with two plasmids encoding either the hygromycin phosphotransferase gene (hph) or the β-glucuronidase(GUS) gene (uidA). Analysis of these putative transformants showed that three plants were transformed with the hph gene, showed the presence of the hph transcript and expressed hygromycin resistance after transfer to soil. Two of these also contained the uidA gene but did not express GUS and were shown to be the same transformation event. All three of the transformants set seed. Hygromycin resistance varied from 68–100% in the progeny of the three transformants. Transgene transmission appeared to have been mainly through apomixis.