E. Timms

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.

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.

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.

Analysis of the two nodulins, sucrose synthase and ENOD2, in transgenic Lotus plants

A partial sucrose synthase clone was isolated from a nodule cDNA library of Lotus japonicus, which had strong homology to sucrose synthase genes from Vicia faba and other plant species. The transcript was 2.7 kb in size and expression was enhanced in nodules compared to roots of L. japonicus. Two overlapping genomic clones containing sucrose synthase sequences were isolated from L. japonicus with a view to mapping and analysing the promoter of this gene.The early nodulin ENOD2 is a (hydroxy)proline-rich cell wall protein, which is expressed in the inner cortex surrounding the infected region of root nodules. In order to see if this nodulin is involved in the operation of the oxygen diffusion barrier, L. corniculatus was transformed with antisense ENOD2 gene constructs derived from the promoter and coding sequence of soybean. Two of the 20 antisense plant lines had significantly lower dry weight and nitrogenase activity as well as a different O2 profile through the nodules when compared to control plant lines, but there was no difference in ENOD2 transcriptional activity between the control and antisense plants. Possible reasons for this observation are discussed.

The effect of toxin-producing Rhizobium strains, on larvae of Sitona flavescens feeding on legume roots and nodules

Larvae of the weevil Sitona spp. specifically eat the root nodules formed on legume plants by the soil bacterium Rhizobium. This can adversely affect the nitrogen fixing activity in the root nodules and lead to decreases in yield. Transgenic rhizobia were used in a novel approach to the biological control of Sitona. Two transcriptional fusions were made in which the coding sequence of the insecticidal crystal protein gene (cryIIIA) from Bacillus thuringiensis subsp. tenebrionis was fused to either the promoter of the rhizosphere enhanced rhiA gene from R. leguminosarum biovar viciae, or the promoter of the nodule specific nifH gene from R. leguminosarum bv. trifolii. The two chimeric genes were transferred to R. leguminosarum bv. viciae and R. leguminosarum bv. trifolii, respectively. Bioassay systems which allowed easy inspection of the plant root systems were used to test the transgenic strains for their ability to protect their host plant from damage by larvae of Sitona flavescens. In both white clover and pea plants nodulated by the transgenic rhizobia a slightly smaller proportion of root nodules were damaged compared to the wild type control plants. In some of the bioassays this was accompanied by increased nitrogenase activity, but it was not reflected in increased plant growth, which was similar in both treatments. These results suggest that a toxin with much higher toxicity against Sitona larvae is necessary for this approach to biocontrol to succeed.

Effect of selectable gene to reporter gene ratio on the frequency of co-transformation and co-expression of uidA and hpt transgenes in protoplast-derived plants of tall fescue

Forty-six independent transformed plants were regenerated under hygromycin selection from cell-suspension derived protoplasts of Festuca arundinacea (Schreb.) after PEG-mediated transformation. Protoplasts were co-transformed with varying molar gene ratios (0.7:1–6:1) of a marker β-glucuronidase (uidA) gene and a selective hygromycin (hpt) resistance gene. Logistic regression analysis indicated that, as expected, the proportion of co-transformed plants tended to increase as the proportion of the marker gene was increased. However, although the proportion of plants co-expressing both genes tended to increase up to a molar ratio of 4:1, it appeared to fall at a molar ratio of 6:1. No statistically significant differences were found in the average copy number of the integrated uidA or hpt transgenes, either in GUS expressing, or in non-GUS expressing plants at the different molar ratios. When using naked-DNA gene transformation methods most authors use a molar ratio of 1:1; our data suggest that adding non-selected and selected transgenes at a higher Molar Gene Ratio would probably improve the proportion of plants regenerated which express both transgenes.