Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats

Concerns about genetically modified (GM) crops include transgene flow to compatible wild species and unintended ecological consequences of potential transgene introgression. However, there has been little empirical documentation of establishment and distribution of transgenic plants in wild populations. We present herein the first evidence for escape of transgenes into wild plant populations within the USA; glyphosate-resistant creeping bentgrass (Agrostis stolonifera L.) plants expressing CP4 EPSPS transgenes were found outside of cultivation area in central Oregon. Resident populations of three compatible Agrostis species were sampled in nonagronomic habitats outside the Oregon Department of Agriculture control area designated for test production of glyphosate-resistant creeping bentgrass. CP4 EPSPS protein and the corresponding transgene were found in nine A. stolonifera plants screened from 20,400 samples (0.04 +/- 0.01% SE). CP4 EPSPS-positive plants were located predominantly in mesic habitats downwind and up to 3.8 km beyond the control area perimeter; two plants were found within the USDA Crooked River National Grassland. Spatial distribution and parentage of transgenic plants (as confirmed by analyses of nuclear ITS and chloroplast matK gene trees) suggest that establishment resulted from both pollen-mediated intraspecific hybridizations and from crop seed dispersal. These results demonstrate that transgene flow from short-term production can result in establishment of transgenic plants at multi-kilometre distances from GM source fields or plants. Selective pressure from direct application or drift of glyphosate herbicide could enhance introgression of CP4 EPSPS transgenes and additional establishment. Obligatory outcrossing and vegetative spread could further contribute to persistence of CP4 EPSPS transgenes in wild Agrostis populations, both in the presence or absence of herbicide selection.     

Transgenic creeping bentgrass with delayed dollar spot symptoms

Creeping bentgrass (Agrostis palustris Huds) is animportant turfgrass used on golf course greens and fairways. It is susceptibleto a number of fungal pathogens and requires considerable fungicide use fordisease control. Transgenic approaches may be useful in improving the level ofdisease resistance. We have generated transgenic creeping bentgrass plantsexpressing PR5K from Arabidopsis thaliana (L.) Henyh. PR5Kis a receptor protein kinase whose extracellular domain is homologous to thePR5family of pathogenesis-related proteins. In a field test of plants inoculatedwith the fungal pathogen dollar spot (Sclerotiniahomoeocarpa F.T. Bennett) four of the eight transgenic lines showeddelays in disease expression of 29 to 45 days, relative to the control plants.     

Materials forPythium flora of Japan VIII Two species ofPythium:P. pyrilobum andP. oligandrum

Two species ofPythium (P. pyrilobum, P. oligandrum) having spherical sporangia with complex subglobose elements were isolated from the crown of creeping bentgrass [Agrostis palustris (cv. Penncross)] and from vegetable field soil, respectively. They are reported for the first time in Japan.     

Characterization of 215 simple sequence repeat markers in creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is a versatile, cross-pollinated, temperate and perennial turfgrass species. It occurs naturally in a wide variety of habitats and is also cultivated on golf courses, bowling greens and tennis courts worldwide. Isozymes and amplified fragment length polymorphisms (AFLPs) have been used to determine genetic diversity, and restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPDs) were used to construct a genetic linkage map of this species. In the current report, we developed and characterized 215 unique genomic simple sequence repeat (SSR) markers in creeping bentgrass. The SSRs reported here are the first available markers in creeping bentgrass to date. Eight hundred and eighteen alleles were amplified by 215 SSR loci, an average of 3.72 alleles per locus. Fifty-nine per cent of those alleles segregated in a 1:1 Mendelian fashion (P > 0.05). Twenty-two per cent had a distorted segregation ratio (P ≤ 0.05). These SSR markers will be useful for assessing genetic diversity in creeping bentgrass and will be important for the development of genetic linkage maps and identifying quantitative trait loci. These markers could enhance breeding programmes by improving the efficiency of selection techniques.     

Crossing the divide: gene flow produces intergeneric hybrid in feral transgenic creeping bentgrass population

Gene flow is the most frequently expressed public concern related to the deregulation of transgenic events ( Snow 2002 ; Ellstrand 2003 ). However, assessing the potential for transgene escape is complex because it depends on the opportunities for unintended gene flow, and establishment and persistence of the transgene in the environment ( Warwick et al. 2008 ). Creeping bentgrass (Agrostis stolonifera L.), a turfgrass species widely used on golf courses, has been genetically engineered to be resistant to glyphosate, a nonselective herbicide. Outcrossing species, such as creeping bentgrass (CB), which have several compatible species, have greater chances for gene escape and spontaneous hybridization (i.e. natural, unassisted sexual reproduction between taxa in the field), which challenges transgene containment. Several authors have emphasized the need for evidence of spontaneous hybridization to infer the potential for gene flow ( Armstrong et al. 2005 ). Here we report that a transgenic intergeneric hybrid has been produced as result of spontaneous hybridization of a feral‐regulated transgenic pollen receptor (CB) and a nontransgenic pollen donor (rabbitfoot grass, RF, Polypogon monspeliensis (L.) Desf.). We identified an off‐type transgenic seedling and confirmed it to be CB × RF intergeneric hybrid. This first report of a transgenic intergeneric hybrid produced in situ with a regulated transgenic event demonstrates the importance of considering all possible avenues for transgene spread at the landscape level before planting a regulated transgenic crop in the field. Spontaneous hybridization adds a level of complexity to transgene monitoring, containment, mitigation and remediation programmes.     

Comparison of early development of three grasses: Lolium perenne, Agrostis stolonifera and Poa pratensis

BACKGROUND AND AIMS: To improve the management of grass communities, early plant development was compared in three species with contrasting growth forms, a caespitose (Lolium perenne), a rhizomatous (Poa pratensis) and a caespitose-stoloniferous species (Agrostis stolonifera). METHODS: Isolated seedlings were grown in a glasshouse without trophic constraints for 37 d (761 degrees Cd). The appearance of leaves and their location on tillers were recorded. Leaf appearance rate (LAR) on the tillers and site-filling were calculated. Tillering was modelled based on the assumption that tiller number increases with the number of leaves produced on the seedling main stem. Above- and below-ground parts were harvested to compare biomass. KEY RESULTS: Lolium perenne and A. stolonifera expressed similar bunch-type developments. However, root biomass was approx. 30 % lower in A. stolonifera than in L. perenne. Poa pratensis was rhizomatous. Nevertheless, the ratio of above-ground : below-ground biomass of P. pratensis was similar to that of L. perenne. LAR was approximately equal to 0.30 leaf d(-1) in L. perenne, and on the main stem and first primary tillers of A. stolonifera. LAR on the other tillers of A. stolonifera was 30 % higher than on L. perenne. For P. pratensis, LAR was 30 % lower than on L. perenne, but the interval between the appearance of two successive shoots from rhizomes was 30 % higher than the interval between two successive leaf stages on the main stem. Above-ground parts of P. pratensis first grew slower than in the other species to the benefit of the rhizomes, whose development enhanced tiller production. CONCLUSIONS: Lolium perenne had the fastest tiller production at the earliest stages of seedling development. Agrostis stolonifera and P. pratensis compensated almost completely for the delay due to higher LAR on tillers or ramets compared with L. perenne. This study provides a basis for modelling plant development.     

Assimilation and allocation of carbon and nitrogen of thermal and nonthermal Agrostis species in response to high soil temperature

We studied whether changes in the assimilation and allocation of carbon and nitrogen are associated with plant tolerance to high soil temperatures. Two Agrostis species, thermal Agrostis scabra, a species adapted to high-temperature soils in geothermal areas in Yellowstone National Park (USA), and two cultivars of a cool-season species, Agrostis stolonifera, L-93 and Penncross, were exposed to soil temperatures of 37 or 20 degrees C, while shoots were exposed to 20 degrees C. Net photosynthesis rate, photochemical efficiency, NO(3) (-)-assimilation rate and root viability decreased with increasing soil temperatures in both species. However, the decreases were less pronounced for A. scabra than for both A. stolonifera cultivars. Carbon investment in growth of plants exposed to 37 degrees C decreased more dramatically in both A. stolonifera cultivars than in A. scabra. Nitrogen allocation to shoots was greater in A. scabra than in both creeping bentgrass cultivars at 37 degrees C soil temperature. Our results demonstrate that plant tolerance to high soil temperature is related to efficient expenditure and adjustment of C- and N-allocation patterns between growth and respiration.     

Genetic and historical relationships among geothermally adapted Agrostis (bentgrass) of North America and Kamchatka: evidence for a previously unrecognized, thermally adapted taxon

Agrostis species have been known to evolve ecotypes rapidly in response to unusual edaphic conditions. The geographic distribution of Agrostis taxa in Lassen Volcanic National Park (California) and Yellowstone National Park (Wyoming) in the United States and the Valley of the Geysers (Kamchatka Peninsula) in Russia suggests that Agrostis scabra might have independently evolved morphologically similar ecotypes several times. We used RAPDs to show that, contrary to expectation, the thermal populations are not independently evolved, but instead constitute a single taxon that currently has four names. A UPGMA including the four thermal and nine nonthermal Agrostis taxa showed that the thermal cluster divides into geographically distinct subclusters, but that two morphologically distinct thermal taxa do not cluster independently. Even though currently confused with the thermal populations, nonthermal A. scabra is not closely related. An analysis of molecular variance (AMOVA) showed significant differentiation between the thermal populations and the nonthermal species sampled in this study. Splitting a hypothesized thermal operational taxonomic unit (OTU) into its components (geographically separated populations) does not greatly affect the partitioning of variation among OTUs. All thermal populations therefore should be assigned to the same taxon, but its taxonomic rank cannot be determined at this time.     

Using empirical data to model transgene dispersal

One element of the current public debate about genetically modified crops is that gene flow from transgenic cultivars into surrounding weed populations will lead to more problematic weeds, particularly for traits such as herbicide resistance. Evolutionary biologists can inform this debate by providing accurate estimates of gene flow potential and subsequent ecological performance of resulting hybrids. We develop a model for gene flow incorporating exponential distance and directional effects to be applied to windpollinated species. This model is applied to previously published data on gene flow in experimental plots of Agrostis stolonifera L. (creeping bentgrass), which assessed gene flow from transgenic plants resistant to the herbicide glufosinate to surrounding non-transgenic plants. Our results show that although pollen dispersal can be limited in some sites, it may be extensive in others, depending on local conditions such as exposure to wind. Thus, hybridization under field conditions is likely to occur. Given the nature of the herbicide resistance trait, we regard this trait as unlikely to persist in the absence of herbicide, and suggest that the ecological consequences of such gene flow are likely to be minimal.     

Is genetic engineering ever going to take off in forage,turf and bioenergy crop breeding?

Background

Genetic engineering offers the opportunity to generate unique genetic variation that is either absent in the sexually compatible gene pool or has very low heritability. The generation of transgenic plants, coupled with breeding, has led to the production of widely used transgenic cultivars in several major cash crops, such as maize, soybean, cotton and canola. The process for regulatory approval of genetically engineered crops is slow and subject to extensive political interference. The situation in forage grasses and legumes is more complicated.

Scope

Most widely grown forage, turf and bioenergy species (e.g. tall fescue, perennial ryegrass, switchgrass, alfalfa, white clover) are highly self-incompatible and outcrossing. Compared with inbreeding species, they have a high potential to pass their genes to adjacent plants. A major biosafety concern in these species is pollen-mediated transgene flow. Because human consumption is indirect, risk assessment of transgenic forage, turf and bioenergy species has focused on their environmental or ecological impacts. Although significant progress has been made in genetic modification of these species, commercialization of transgenic cultivars is very limited because of the stringent and costly regulatory requirements. To date, the only transgenic forage crop deregulated in the US is ‘Roundup Ready’ (RR) alfalfa. The approval process for RR alfalfa was complicated, involving several rounds of regulation, deregulation and re-regulation. Nevertheless, commercialization of RR alfalfa is an important step forward in regulatory approval of a perennial outcrossing forage crop. As additional transgenic forage, turf and bioenergy crops are generated and tested, different strategies have been developed to meet regulatory requirements. Recent progress in risk assessment and deregulation of transgenic forage and turf species is summarized and discussed.     

Revealing constitutively expressed resistance genes in Agrostis species using PCR-based motif-directed RNA fingerprinting

Agrostis species are mainly used in athletic fields and golf courses. Their integrity is maintained by fungicides, which makes the development of disease-resistance varieties a high priority. However, there is a lack of knowledge about resistance (R) genes and their use for genetic improvement in Agrostis species. The objective of this study was to identify and clone constitutively expressed cDNAs encoding R gene-like (RGL) sequences from three Agrostis species (colonial bentgrass (A. capillaris L.), creeping bentgrass (A. stolonifera L.) and velvet bentgrass (A. canina L.)) by PCR-based motif-directed RNA fingerprinting towards relatively conserved nucleotide binding site (NBS) domains. Sixty-one constitutively expressed cDNA sequences were identified and characterized. Sequence analysis of ESTs and probable translation products revealed that RGLs are highly conserved among these three Agrostis species. Fifteen of them were shown to share conserved motifs found in other plant disease resistance genes such as MLA13, Xa1, YR6, YR23 and RPP5. The molecular evolutionary forces, analysed using the Ka/Ks ratio, reflected purifying selection both on NBS and leucine-rich repeat (LRR) intervening regions of discovered RGL sequences in these species. This study presents, for the first time, isolation and characterization of constitutively expressed RGL sequences from Agrostis species revealing the presence of TNL (TIR-NBS-LRR) type R genes in monocot plants. The characterized RGLs will further enhance knowledge on the molecular evolution of the R gene family in grasses.     

Linkage map construction in allotetraploid creeping bentgrass (Agrostis stolonifera L.)

Creeping bentgrass (Agrostis stolonifera L.) is one of the most adapted bentgrass species for use on golf course fairways and putting greens because of its high tolerance to low mowing height. It is a highly outcrossing allotetraploid species (2n=4x=28, A₂ and A₃ subgenomes). The first linkage map in this species is reported herein, and it was constructed based on a population derived from a cross between two heterozygous clones using 169 RAPD, 180 AFLP, and 39 heterologous cereal and 36 homologous bentgrass cDNA RFLP markers. The linkage map consists of 424 mapped loci covering 1,110 cM in 14 linkage groups, of which seven pairs of homoeologous chromosomes were identified based on duplicated loci. The numbering of all seven linkage groups in the bentgrass map was assigned according to common markers mapped on syntenous chromosomes of ryegrass and wheat. The number of markers linked in coupling and repulsion phase was in a 1:1 ratio, indicating disomic inheritance. This supports a strict allotetraploid inheritance in creeping bentgrass, as suggested by previous work based on chromosomal pairing and isozymes. This linkage map will assist in the tagging and eventually in marker-assisted breeding of economically important quantitative traits like disease resistance to dollar spot (Sclerotinia homoeocarpa F.T. Bennett) and brown patch (Rhizoctonia solani Kuhn).     

Transgenic Agrostis mongolica Roshev. with enhanced tolerance to drought and heat stresses obtained from Agrobacterium-mediated transformation

Efficient procedures for regeneration and Agrobacterium-mediated transformation were established for Agrostis mongolica Roshev. and generated transgenic plants tolerant to drought and heat stresses using a regulatory gene from Arabidopsis, ABF3, which controls the ABA-dependent adaptive responses. The identification and selection of regenerable and reproducible callus type was a key factor for successful transformation. The transformation efficiency was 49.2% and gfp expression was detected in hygromycin-resistant calli and stem of putative transgenic plants. The result of Southern blot analysis showed that the ABF3 transgene was stably integrated into the genome of transgenic plants. Of the five transgenic lines analyzed, single transgene integration was observed in two lines and two copy integration was observed in three transgenic lines. Northern blot analysis confirmed that ubi::ABF3 was expressed in all transgenic lines. Transgenic plants exhibited neither growth inhibition nor visible vegetative phenotypic alternations. However, both transgenic and wild-type plants were highly sterile and did not flower during 3 years of growth period in the open field under subtropical Jeju Island climate. The stomata of the transgenic plants opened less than did stomata of the wild-type plants, and water content of the transgenic leaves remained about 3–4 fold higher than observed for wild-type leaves under drought stress. The transgenic plants showed about 2 fold higher survival rates under drought stress and about 3 fold higher survival rates under heat stress when compared to wild-type plants. Thus, overexpression of the Arabidopsis ABF3 gene results in enhancement of both drought and heat stress tolerance in Agrostis mongolica Roshev.     

Change in isolation frequency ofTyphula ishikariensis from turfgrass under snow cover on golf courses

The change in isolation frequency ofTyphula ishikariensis from bentgrass leaves under snor cover on golf courses near Sapporo, Hokkaido was investigated over three consecutive winters.Agrostis palustris Huds. cv. Penncross growing at these sites was severely infested withT. ishikariensis biotype B. Isolation tests showed thatT. ishikariensis biotype B was first isolated 9 to 34 d after snow cover in December–January, and isolation frequency peaked in February–March. The fungus was never isolated from leaves after snowmelt. The behavior ofT. ishikariensis on bentgrass was discussed in terms of winter hardiness of plants and its epidemiology.     

Cytochrome and alternative pathway activity in roots of thermal and non-thermal Agrostis species in response to high soil temperature

Partitioning of respiration between the cytochrome pathway (CP) and the alternative pathway (AP) may play an important role in plant adaptation to extreme environments. We examined changes in partitioning between CP and AP, and viability of roots associated with plant exposure to high soil temperature for two Agrostis species: Agrostis scabra Willd., a species adapted to high-temperature soils in geothermal areas in Yellowstone National Park, and Agrostis stolonifera L. (cv. Penncross) a heat-sensitive grass widely used in cool-climate regions. Roots of A. scabra and A. stolonifera were exposed to soil temperature of 37 or 20°C, while shoots were exposed to 20°C for 28 days. Root viability decreased, and total root respiration increased for both species at 37°C. The decline in root viability and the increase in respiration rates were less pronounced for A. scabra than for A. stolonifera . A larger proportion of total root respiration was attributed to the AP in A. scabra compared with that in A. stolonifera when both species were exposed to 37°C. At 7 and 14 days at 37°C, the relative proportion of respiration passing through AP increased by 12 and 10%, respectively, in A. scabra , whereas in A. stolonifera , AP increased by 4 and 1%, respectively. Our results suggest that maintaining a higher proportion of AP at a high soil temperature may contribute to root thermo-tolerance in A. scabra in comparison with A. stolonifera , and alternative respiration may play an important role in plant adaptation to high soil temperature.     

Ectopic expression of a cyanobacterial flavodoxin in creeping bentgrass impacts plant development and confers broad abiotic stress tolerance

Flavodoxin (Fld) plays a pivotal role in photosynthetic microorganisms as an alternative electron carrier flavoprotein under adverse environmental conditions. Cyanobacterial Fld has been demonstrated to be able to substitute ferredoxin of higher plants in most electron transfer processes under stressful conditions. We have explored the potential of Fld for use in improving plant stress response in creeping bentgrass (Agrostis stolonifera L.). Overexpression of Fld altered plant growth and development. Most significantly, transgenic plants exhibited drastically enhanced performance under oxidative, drought and heat stress as well as nitrogen (N) starvation, which was associated with higher water retention and cell membrane integrity than wild‐type controls, modified expression of heat‐shock protein genes, production of more reduced thioredoxin, elevated N accumulation and total chlorophyll content as well as up‐regulated expression of nitrite reductase and N transporter genes. Further analysis revealed that the expression of other stress‐related genes was also impacted in Fld‐expressing transgenics. Our data establish a key role of Fld in modulating plant growth and development and plant response to multiple sources of adverse environmental conditions in crop species. This demonstrates the feasibility of manipulating Fld in crop species for genetic engineering of plant stress tolerance.     

New Taxa of the Genus Agrostis from Yunnan

In the paper two species and one variety of the genus Agrostis L. are described as new from Yunnan Province, China. They are Agrostis lushuiensis,A. kunmingensis and A. myriantha Hook. f. var. yangbiensis.     

After grazing exclusion,is there any modification of strategy for two guerrilla species: <Emphasis Type="Italic">Elymus repens</Emphasis> (L.) Gould and <Emphasis Type="Italic">Agrostis stolonifera</Emphasis> (L.)?

Elymus repens (L.) Gould and Agrostis stolonifera (L.), are competitive grasses with guerrilla strategy that invade grasslands with a low stocking rate. In this work, we tested the hypotheses that grazing exclusion facilitates vegetative development of rhizomes and stolons of these clonal grasses and that such change is a key mechanism for their abundance in set-aside grasslands. The competitive capacities of these two guerrilla species were characterised by samples in plant community (species richness and biomass) and on the level of individual species (morphometric measurements on stolons and rhizomes) during a growing season. Compared to grasslands where grazing was excluded for three years, species richness was higher in grazed site and the plant community structure differed. Indeed, with grazing exclusion, a shift from annual species with a diversified growth-form to perennial species with a tall tussock and graminoid growth-form was monitored. In ungrazed situation, Elymus repens and Agrostis stolonifera were the dominant grasses, and the standing biomass for the lowland community showed a significant increase compared to the grazed site. Vegetative development increased competitive capacities of these two guerrilla species and led by phenomenon of competitive exclusion to the disappearance of annuals species. With grazing cessation, Elymus repens was found to increase the size of aerial traits (shoot length and the number of leaves per shoot) and this may both be propitious for achieving dominance within plant communities and also maintaining its competitive local advantage. By contrast, Agrostis stolonifera showed an increase in a root trait, i.e. rhizome length, in the fenced site, which provide good ability for spatial propagation and then to explore adjacent patches. We concluded that Elymus repens presented a morphological capacity to change its colonising strategy from a guerrilla strategy to a phalanx strategy, by morphological variability of aerial organs, when it was submitted to competitive stress and environment modifications. Agrostis stolonifera showed a capacity to escape aerial competition resulting from grazing cessation, than to increase underground propagation capacity. The present study highlighted the capacities of Elymus repens to respond in an adaptative way to competitive pressure.