Dalton, S. J., Robson, P. R. H., Buanafina, M. M., Bettany, A. J. E., Timms, E., Wiffen, D., Morris, P. (2005). Nylon mesh as an improved support for bombarded calli or cell suspensions. Molecular Breeding for the Genetic Improvement of Forage Crops and Turf. Proceedings of the 4th International Symposium on the Molecular Breeding of Forage and Turf. XXth International Grassland Congress, July 2005, Aberystwyth, Wales, Humphreys, M. O. (ed), p. 236

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.

. 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.

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.

Bettany, A. J. E., Dalton, S. J., Timms, E. J., Hauck, B., Michael, A. J., Walton, N. J., Morris, P. (2002). Modifying lignin and cell wall phenolics in forage grasses by altering precursor supply. Grassland science in Europe, Multifunctional Grassland: Quality Forages, Animal Products and Landscapes. Proceedings 19th General Meeting of the European Grassland Federation, La Rochelle, France, 27-30 May 2002, Durand, J-L.Emile, J-C.Huyghe, C.Lemaire, G. ISBN:2950411037, 7, 62-63

Robbins, M. P., Allison, G. G., Bettany, A. J. E., Dalton, S. J., Davies, T. E., Hauck, B., Hughes, J-W., Timms, E. J., Morris, P. (2002). Biochemical and molecular basis of plant composition determining the degradability of forage for ruminant nutrition. Pages 37-43 in: Durand, J-L., Emile, J-C., Huyghe, C., Lemaire, G. (Eds). Multifunctional Grassland: Quality Forages, Animal Products and Landscapes. Grassland Science in Europe, 7, ISBN:2950411037. Proceedings of the 19th General Meeting of the European Grassland Federation, La Rochelle, France, 27-30 May 2002.

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.

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 beta-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. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

The expression of a β-glucuronidase (GUS) transgene, under the control of the rice actin1 promoter and first intron, introduced via PEG-mediated transformation of protoplasts, has been followed through a number of generations of vegetative propagation (tillering) in the forage grass Festuca arundinacea. Gene expression was found to be particularly unstable during early generations of tillering, but more stable in the fourth or fifth tiller generation. Loss of transgene expression was not due to deletion of the transgene nor was it due to gross rearrangements in the transgene as demonstrated by Southern blot analysis of expressing and non-expressing tillers. Loss of transgene expression due to hypermethylation of the transgene promoter has been shown previously, but in this case it was not possible to detect differences in the methylation patterns of the coding and promoter regions of expressing and non-expressing tillers. The implications for screening of transgenic plants intended to be vegetatively propagated are discussed.

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.