Field performance of transgenic tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) plants and their progenies

Tall fescue (Festuca arundinacea Schreb.) is a hexaploid, outcrossing grass species widely used for forage and turf purposes. Transgenic tall fescue plants were generated by biolistic transformation of embryogenic cell suspension cultures that were derived from single genotypes of widely used cultivar Kentucky-31. Primary transgenics from two genotypes, their corresponding regenerants from the same genotypes and control seed-derived plants were transferred to the field and evaluated for 2 years. Progenies of these three classes of plants were obtained and evaluated together with seed-derived plants in a second field experiment. The agronomic characteristics evaluated were: heading date, anthesis date, height, growth habit, number of reproductive tillers, seed yield and biomass. The agronomic performance of the primary transgenics and regenerants was generally inferior to that of the seed-derived plants, with primary transgenics having fewer tillers and a lower seed yield. However, no major differences between the progenies of transgenics and the progenies of seed-derived plants were found for the agronomic traits evaluated. Primary transgenics and regenerants from the same genotype were more uniform than plants from seeds. Progenies of transgenics performed similarly to progenies of the regenerants. The addition of a selectable marker gene in the plant genome seems to have had little effect on the agronomic performance of the regenerated plants. No indication of weediness of the transgenic tall fescue plants was observed. Our results indicate that outcrossing grass plants generated through transgenic approaches can be incorporated into forage breeding programs.     

Inheritance of transgenes in transgenic tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> schreb.)

Summary Tall fescue (Festuca arundinacea Schreb.) is the most important forage species worldwide of the Festuca genus. Single genotype-derived embryogenic suspension cultures were established from tall fescue cultivar Kentucky-31, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric β-glucuronidase (gusA) gene was co-transformed with hph. Transgenic plants were recovered after microprojectile bombardment of suspension cells and subsequent selection in the presence of a high concentration of hygromycin. Fertile transgenic plants were obtained after vernalization under field conditions. T1 and T2 progenies were obtained after reciprocal crosses between transgenic and untransformed control plants. PCR and Southern hybridization analyses revealed a 1∶1 segregation ratio for both transgenes in the T1 and T2 generations. Southern hybridization patterns were identical for T0, T1, and T2 plants. The results demonstrated for the first time the stable meiotic transmission of transgenes following Mendelian rules in transgenic tall fescue.     

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.     

Reversible male sterility in transgenic tobacco carrying a dominant-negative mutated glutamine synthetase gene under the control of microspore-specific promoter

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.     

Pollen-mediated transgene flow in the wind-pollinated grass species tall fescue (Festuca arundinacea Schreb.)

Information regarding gene flow in wind-pollinated, outcrossing forage grasses is essential for any future releases of value-added transgenic cultivars. Experiments on pollen dispersal was carried out by growing transgenic tall fescue (Festuca arundinacea) in a central plot, surrounded by exclosures containing recipient plants up to a distance of 200 m from the central source plants in eight directions. The central transgenic tall fescue plants carried a chimeric hygromycin phosphotransferase gene (hph) and a chimeric -glucuronidase gene (gusA). Seeds were collected from the recipient plants and germinated seedlings were used for high throughput DNA isolation and polymerase chain reaction (PCR) analysis. More than 21,000 seedlings were PCR analyzed for the experiments conducted in three years. Transgenes were detected in recipient plants at up to 150 m from the central transgenic plot. The highest transgene frequencies, 5% at 50 m, 4.12% at 100 m and 0.96% at 150 m, were observed north of the central plot, the prevailing wind direction. Lower transgene frequencies were detected in other directions, particularly at 100 m and 150 m distances. No transgene was detected at 200 m distance in any direction. Transgene flow was less effective or ineffective when recipient plants were further away from the central donor plants. Southern blot hybridization analysis confirmed the transgenic nature of the PCR positive plants. A supplementary experiment demonstrated that transgene flow can be controlled by placing transgenic plantings downwind and long distances from non-transgenic seed increases, thus allowing tall fescue breeding and transgene development programs to be conducted concurrently at the same research station.     

Intergeneric somatic hybridization in Gramineae: somatic hybrid plants between tall fescue (Festuca arundinacea Schreb.) and Italian ryegrass (Lolium multiflorum Lam.)

Summary Tall fescue (Festuca arundinacea Schreb.) protoplasts, inactivated by iodoacetamide, and non-morphogenic Italian ryegrass (Lolium multiflorum Lam.) protoplasts, both derived from suspension cultures, were electrofused and putative somatic hybrid plants were recovered. Two different genotypic fusion combinations were carried out and several green plants were regenerated in one of them. With respect to plant habitus, leaf and inflorescence morphology, the regenerants had phenotypes intermediate between those of the parents. Southern hybridization analysis using a rice ribosomal DNA probe revealed that the regenerants contained both tall fescue- and Italian ryegrass-specific-DNA fragments. A cloned Italian ryegrass-specific interspersed DNA probe hybridized to total genomic DNA from Italian ryegrass and from the green regenerated somatic hybrid plants but not to tall fescue. Chromosome counts and zymograms of leaf esterases suggested nuclear genome instability of the somatic hybrid plants analyzed. Four mitochondrial probes and one chloroplast DNA probe were used in Southern hybridization experiments to analyze the organellar composition of the somatic hybrids obtained. The somatic hybrid plants analyzed showed tall fescue, additive or novel mtDNA patterns when hybridized with different mitochondrial gene-specific probes, while corresponding analysis using a chloroplast gene-specific probe revealed in all cases the tall fescue hybridization profile. Independently regenerated F. arundinacea (+) L. multiflorum somatic hybrid plants were successfully transferred to soil and grown to maturity, representing the first flowering intergeneric somatic hybrids recovered in Gramineae.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Festuca arundinacea</Emphasis> (Schreb.) and <Emphasis Type="Italic">Lolium multiflorum</Emphasis> (Lam.)

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-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.     

Collection and storage of pollen from Salix (Salicaceae)

Genetic improvement of willows through traditional breeding can be facilitated by pollen collection and storage so that female flower receptivity need not be synchronized with pollen shed for breeding. Two experiments were completed to test the effectiveness of various organic solvents for willow pollen collection. In the first experiment, seven pollen collection treatments and an untreated control were tested with two willow clones. The other experiment tested three treatments that showed promise in the initial experiment and an untreated control with eight willow clones. Toluene and carbon tetrachloride were effective for pollen extraction, with average pollen germination percentages that were >15%, but both chemicals reduced pollen viability by 10-20% compared with an untreated control based on in vitro germination tests. Pollen extracted with carbon tetrachloride or toluene was successfully used in controlled pollination, and >100 new families were produced with this technique. Pollen viability remained high after 18 mo of storage at -20°C. Based on our results, toluene is the preferred solvent for future willow pollen extractions because it is as effective as carbon tetrachloride, is not a known carcinogen, and is less expensive.     

农杆菌介导的高羊茅高效遗传转化和转基因植株再生

用带有质粒pDBA121(含hpt基因和bar基因)的农杆菌EHA 105转化高羊茅(Festucaarundinacea Schreb.)胚性悬浮细胞,建立了可重复的、高效的农杆菌介导的高羊茅遗传转化系统.商业用的除草剂Basta直接用于转化细胞的筛选.基因型、受体材料的类型、培养基成分和筛选剂影响农杆菌介导的转化频率.悬浮细胞的农杆菌转化效率为每克悬浮细胞再生2.85～10.9株转基因植株,大大高于基因枪法的高羊茅转化效率(2～5株).经PCR分析和Southern杂交检测表明,bar基因已整合进入高羊茅基因组,转基因植株Basta喷洒试验表明bar基因已成功地实现高水平的表达.此转化系统的建立为高效地将外源有用基因导入高羊茅并高效稳定地表达奠定了基础.     

Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

Three grass host species--tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.--each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.     

Preliminary note on potential use of forage crops for soil phytoremediation of dieldrin

The aim of this trial was to evaluate the feasibility of using tall fescue (Festuca arundinacea) and alfalfa (Medicago sativa) for phytoremediation of dieldrin. Experimental trial was carried out in greenhouse with temperature and light control. Each tested crop were seeded in individual pots (10 plants/pot) filled with contaminated soil (47 microg/kg dieldrin) and uncontaminated soil collected in sites located in the province of Latina (Italy). Samples of soil, root, and aerial part of plants were analysed at 3 and 6 months after seeding. The analytical determinations in soil and plant samples were carried out by GLC-ECD and confirmed by GLC-MS. After 6 months in the greenhouse, recoveries of dieldrin from soil planted with tall fescue and alfalfa were significantly lower than recoveries in unplanted control soil. Dieldrin residue values in root did not differ between the two different sampling times for each forage crop tested, but they were always higher in fescue than in alfalfa. Residue levels in aerial part were low (< 10 microg/kg) in the two forage crops. Preliminary results seem to confirm the ability of tested plants to enhance dissipation of dieldrin in soil at low level of contamination.     

Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea)

Tall fescue (Festuca arundinacea) is the predominant cool-season pasture grass in the USA. Embryogenic calluses were induced from seeds/caryopsis of elite tall fescue cultivars Jesup and Kentucky-31, and were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. Agrobacterium strains LBA4404 and EHA105 harboring pCAMBIA vectors or the super-binary vector pTOK233 were used to infect the embryogenic callus pieces. The number of hygromycin resistant calluses obtained per dish of infected callus pieces was in the range of 2.0-5.8, and the number of transgenic plants recovered per dish of infected callus pieces was in the range of 0.4-1.7. When transformation efficiency was calculated based on the number of transgenic plants recovered and the number of original intact calluses used, the transformation frequency was in the range of 1.9-8.7%. The use of the easily available pCAMBIA vectors could produce equivalent results as the superbinary vector pTOK233. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. Expression of the transgenes was confirmed by northern hybridization analysis, GUS staining, and detection of GFP signals. Fertile transgenic plants were obtained after vernalization in the greenhouse. Progeny analysis revealed Mendelian inheritance of the transgenes.     

Short-day and Low-temperature Control of Floral Induction in Festuca

The conditions necessary for floral induction to occur in tallfescue (Festuca arundinacea), meadow fescue (Festuca pratensis),and red fescue (Festuca rubra), have been investigated. Onlya Tunisian ecotype of tall fescue produced inflorescences undershort-day conditions when air temperatures were above 8 °C.Under short days with low temperatures nearly all plants ofS. 170 tall fescue and S. 215 meadow fescue produced inflorescencesafter 15 weeks' exposure, but S. 59 red fescue showed only asmall response. Evidence was obtained for the existence in bothtall fescue and meadow fescue of a juvenile stage during whichplants showed a reduced response to inductive conditions. Avariation of 35 days in the required length of exposure to inductiveconditions was demonstrated between families within the S. 170variety of tall fescue, indicating the possibility of selectingfor larger or smaller inductive requirements. A second generationof seed was produced within a 12-month period from inflorescenceswhich had developed in a heated glasshouse during the wintermonths.     

Viability of cotton and canola pollen on the proboscis of Helicoverpa armigera: implications for spread of transgenes and pollination ecology

Abstract.  1. Pollen can be transported thousands of kilometres by insects but its viability after long-distance transport is not known. Knowing the potential for this mechanism to cause outcrossing of transgenes from genetically modified (GM) plants is important for risk assessments.
2. The viability of pollen from cotton ( Gossypium hirsutum L.) and canola ( Brassica napus L.) was determined after placing it on the proboscis of Helicoverpa armigera moths for intervals of up to 32 h. Viability of both cotton and canola pollen declined at a much greater rate when in contact with the moth proboscis. Most was non-viable by 8 h compared with 16 h for control cotton pollen or 32 h for canola pollen.
3. There was no significant difference in the rate of decline of pollen viability between the five conventional cotton varieties, or between these and the one GM cotton variety used in these experiments.
4. The number of canola pollen grains remaining on the proboscis declined over time. Very few cotton pollen grains were retained on the proboscis.
5. The reduction in pollen viability during contact with the proboscis might indicate partial ingestion of the pollen via the proboscis.
6. The points above suggest that pollen is unlikely to remain attached or remain viable when carried over large distances by H. armigera . The implications for spread of pollen from transgenic plants and for pollination ecology in general are discussed.     

Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross‐pollinating crops

We have developed a novel hybridization platform that utilizes nuclear male sterility to produce hybrids in maize and other cross‐pollinating crops. A key component of this platform is a process termed Seed Production Technology (SPT). This process incorporates a transgenic SPT maintainer line capable of propagating nontransgenic nuclear male‐sterile lines for use as female parents in hybrid production. The maize SPT maintainer line is a homozygous recessive male sterile transformed with a SPT construct containing (i) a complementary wild‐type male fertility gene to restore fertility, (ii) an α‐amylase gene to disrupt pollination and (iii) a seed colour marker gene. The sporophytic wild‐type allele complements the recessive mutation, enabling the development of pollen grains, all of which carry the recessive allele but with only half carrying the SPT transgenes. Pollen grains with the SPT transgenes exhibit starch depletion resulting from expression of α‐amylase and are unable to germinate. Pollen grains that do not carry the SPT transgenes are nontransgenic and are able to fertilize homozygous mutant plants, resulting in nontransgenic male‐sterile progeny for use as female parents. Because transgenic SPT maintainer seeds express a red fluorescent protein, they can be detected and efficiently separated from seeds that do not contain the SPT transgenes by mechanical colour sorting. The SPT process has the potential to replace current approaches to pollen control in commercial maize hybrid seed production. It also has important applications for other cross‐pollinating crops where it can unlock the potential for greater hybrid productivity through expanding the parental germplasm pool.     

Comparison of tall fescue (Cyperales: Gramineae) to other cool-season turfgrasses for tolerance to European chafer (Coleoptera: Scarabaeidae)

Three cultivars of tall fescue, Festuca arundinacea Schreb., were compared with three cultivars each of fine fescue (Festuca spp.), Kentucky bluegrass (Poa pratensis L.), and perennial ryegrass (Lolium perenne L.) to evaluate tolerance to root-feeding by European chafer grubs, Rhizotrogus majalis (Razoumowsky). Potted turfgrasses were infested with initial densities equivalent to 33 or 66 grubs per 0.1 m2 on 19 August 2000. More grubs were added in late September and October, bringing the total to 66 or 143 grubs per 0.1 m2. Plant growth, root loss, weight gain, and survival of grubs were measured. The experiment was repeated in fall of 2001 with an initial density of 66 grubs per 0.1 m2. The proportion of root mass lost as a result of grub feeding was a function of turf species, root growth, grub survival, and grub growth during the test. Grubs gained the most weight and consumed the most roots when feeding on fine fescue. Fine fescue suffered the greatest percentage of root loss in 2000, despite having the most rapid root growth and largest mass in control pots. Cultivars of tall fescue appeared to be the most tolerant of grub feeding, having the smallest reduction in root mass in both years. Data from fine fescue, Kentucky bluegrass, and perennial ryegrass cultivars were not as consistent as tall fescue, because for some cultivars root growth and grub survival were different between years. We also found that grubs increased in mass by 20% when the mass of available roots was doubled.