RESUMEN
Bahiagrass (Paspalum notatum Flüggé) is the predominant forage grass in the southeastern US. The commercially important bahiagrass cultivar 'Argentine' is preferred for genetic transformation over sexual diploid cytotypes, since it produces uniform seed progeny through apomixis. Pseudogamous apomictic seed production in Argentine bahiagrass may contribute to transgene confinement. It is characterized by embryo development which is independent of fertilization of the egg cell, but requires fertilization with compatible pollen to produce the endosperm. Pollen-mediated gene transfer from transgenic, glufosinate-resistant apomictic bahiagrass as pollen donor at close proximity (0.5-3.5 m) with non-transgenic sexual or apomictic bahiagrass cultivars as pollen receptors was evaluated under field conditions. Hybridization frequency was evaluated by glufosinate herbicide resistance in >23,300 seedlings derived from open-pollinated (OP) pollen receptor plants. Average gene transfer between transgenic apomictic, tetraploid and sexual diploid bahiagrass was 0.03%. Herbicide-resistant hybrids confirmed by immuno-chromatographic detection of the PAT protein displayed a single copy bar gene identical to the pollen parent. Hybrids resulting from diploid pollen receptors were confirmed as triploids or aneu-triploids with significantly reduced vigor and seed set as compared to the parents. Transmission of transgenes to sexual bahiagrass is severely restricted by the ploidy difference between tetraploid apomicts and diploid sexual bahiagrass. Average gene transfer between transgenic apomictic tetraploid and non-transgenic, apomictic tetraploid bahiagrass was 0.17%, confirming a very low frequency of amphimixis in apomictic bahiagrass cultivars. While not providing complete transgene containment, gene transfer between transgenic apomictic and non-transgenic bahiagrass occurs at a much lower frequency than reported for other cross-pollinating or facultative apomictic grasses.
Asunto(s)
Flujo Génico/genética , Partenogénesis/genética , Paspalum/crecimiento & desarrollo , Paspalum/genética , Ploidias , Polen/genética , Cromosomas de las Plantas/genética , Citometría de Flujo , Resistencia a los Herbicidas/genética , Hibridación Genética , Fenotipo , Plantas Modificadas Genéticamente , Polimorfismo de Longitud del Fragmento de Restricción , Semillas/genética , Tiempo (Meteorología)RESUMEN
Pollen-mediated gene transfer from stress tolerant or herbicide-resistant transgenic plants may cause environmental or agronomic problems. Apomictic seed production found in some bahiagrass cultivars may serve as a natural transgene containment system. Under greenhouse conditions, the average gene transfer frequency from an herbicide-resistant apomictic tetraploid to a population of sexual diploid bahiagrass genotypes or apomictic tetraploid bahiagrass was 0.16% when the transgenic pollen donor was placed at 0.5-1.5 m distance from the non-transgenic pollen receptors. The herbicide-resistant hybrids were characterized for transgene integration, expression and ploidy, by Southern blot analysis, immuno-chromatography and flow cytometry, respectively. Hybrids resulting from open pollination of non-transgenic diploid female plants with transgenic tetraploid male plants were triploids or near-triploids, with 2n = 26-34. These hybrids displayed a wide range of phenotypic variability, including some non-persistent or non-flowering dwarf-type hybrids with good vigor, or hybrids with vegetative growth similar to non-transgenic plants, but with significantly reduced seed set. Non-flowering aneu-triploids with good vigor/field performance will provide the highest level of transgene containment. Embryo sac analysis of pollinated spikelets confirmed a high proportion of aborted ovules. An apospory-linked RFLP marker was detected in 13 of the 15 near-triploid hybrids. All flowering aneuploid hybrids displayed significantly reduced seed set, and none of the sexual near-triploid hybrids produced any seeds. All tetraploid gene transfer events carried the apospory-linked RFLP marker, suggesting that despite the presence of the aposporus locus, a low degree of sexuality co-exists in apomictic tetraploid cultivars. Thus, tetraploid apomictic bahiagrass does not provide complete transgene containment, although intra-specific gene transfer is drastically reduced compared to sexually reproducing perennial grasses.
Asunto(s)
Hibridación Genética , Paspalum/genética , Poliploidía , Flores/genética , Flores/crecimiento & desarrollo , Flujo Génico , Transferencia de Gen Horizontal , Vigor Híbrido , Paspalum/crecimiento & desarrollo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Polen/genética , Polimorfismo de Longitud del Fragmento de Restricción , TransgenesRESUMEN
Bahiagrass (Paspalum notatum Flugge) is an important turf and forage grass in the southeastern United States and other subtropical regions. Biolistic co-transfer of two unlinked, minimal, linear transgene expression cassettes (MCs) into the apomictic bahiagrass cv. Argentine was carried out to evaluate co-integration, quantify co-expression and analyze inheritance to apomictic seed progeny. Gold projectiles were coated with minimal unlinked nptII and bar expression cassettes in a 1:2 molar ratio. Complexity of transgene loci correlated with the amount of DNA used during gene transfer. Transgenic plants displayed a simple nptII integration pattern with 1-4 hybridization signals compared to the non-selected bar gene with 2 to more than 5 hybridization signals per transgenic line. Co-expression of unlinked nptII and bar genes occurred in 19 of the 20 co-transformed lines (95% co-expression frequency). Protein quantification revealed that several lines with complex integration patterns displayed a higher transgene expression than lines with simple transgene integration patterns. Several transgenic lines displayed hybridization signals indicative of concatemerization. Concatemers were confirmed following PCR amplification and sequence analysis of transgene loci. The obligate apomictic bahiagrass cv. Argentine produced uniform seed progeny without segregation of simple or complex transgene loci. NPTII- and PAT-ELISA, as well as herbicide application, confirmed stable expression of the nptII and bar gene at levels similar to the primary transformants. These results demonstrate that biolistic transfer of MCs support stable and high level co-expression of transgenes in bahiagrass.
Asunto(s)
Paspalum/genética , Transgenes , Secuencia de Bases , Southern Blotting , ADN de Plantas , Ensayo de Inmunoadsorción Enzimática , Genes de Plantas , Datos de Secuencia Molecular , Paspalum/fisiología , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Regeneración , Transformación GenéticaRESUMEN
To investigate whether knowledge and perceptions of antithrombotic therapy differ between ethnic groups in the UK, we conducted a cross-sectional questionnaire survey of patients attending anticoagulation clinics in three Birmingham teaching hospitals. 180 consecutive patients were recruited-135 white European, 29 Indo-Asian, 16 Afro-Caribbean. The average knowledge score was 5.5 out of 9, with no significant differences between the groups. Indo-Asians were significantly less likely than the other groups to know the name of the anticoagulant they were taking (warfarin) and Afro-Caribbeans to know the condition for which they were being anticoagulated. Few patients of any group were able to specify more than one side-effect of warfarin or the dose they were on. In logistic regression analysis the factors associated with a low score were age >61 years, having been born outside the UK, and the perception of difficulty in comprehension. Nearly half the Indo-Asians felt unable to understand what was said to them in the clinic, and 62% expressed a preference for a doctor of the same ethnic group. Although there were no significant between-group differences, this study points to gaps in the knowledge of patients from ethnic minorities and to deficiencies in the provision of information. In patient education, these groups should receive special attention.