No description available.

No description available.

An exceptionally effective transformation procedure has been established by using class I embryo-derived rice callus. Every treated callus clump yielded multiple independently transformed plants (average 40 plantlets). Analysis of genomic DNA blots and pollen expressing green fluorescent protein (GFP) from T0 plants revealed that 64% bore a single locus T-DNA insertion in which half had one T-DNA copy. Additive transgene expression was observed fromT0 plants with GFP driven by mUbi1 promoter. Transgenic plants could be rapidly characterized by analyzing GFP pollen from T0 plants without the need for further generations or genomic DNA blot analysis. Agrobacterium tumefaciens-mediated transformation of microspore-derived callus for generating large numbers of T-DNA haploid and doubled haploid(DH) plants has also been investigated. The established transformation procedure resulted in 100% transformation frequency for class I microspore-derived rice callus. Each callus typically yields multiple independent transgenic plants. Genomic DNA blot analysis suggested 98% of the transgenic plants are independent events. About half of the transgenic plants were identified as haploid plants, whereas half are DH hemizygous or homozygous transgenic plants. DH homozygous transgenic plants were obtained from T0plants and confirmed by pollen GFP expression and genomic blot analysis in T0transgenic DH plants. In this study, about 60% ofT0transgenic DH plants had a single locus T-DNA insertion of which 45% bore one T-DNA copy. Furthermore, in a population of over 2,000 haploid and doubled haploid T-DNA plants , about 25% showed phenotypic differences from non-transformed haploid plants. Approximately 5% were seriously phenotypically abnormal including lethal or semi-lethal mutants. This highly efficient transformation procedure using microspore-derived callus could be valuable in speeding up plant breeding and in new gene discovery. Diversification of the mUbi1 promoter led to a minimal promoter that has a similar function as the original mUbi1. Transient and stable transformation as measured from gene expression driven by the minimal promoter suggested that it has a similar function as the original wild type promoter.

No description available.

Molecular marker analysis of seven putative onion (Allium cepa) doubled haploid (DH) lines developed at Texas A&M University was conducted to verify genetic homozygosity. Analysis was also conducted on five equivalent conventional inbred lines, breeding lines developed from the same parental crosses as the DH lines, and the original parent lines. The markers have revealed polymorphisms within the parental lines and the conventional inbreds, but not in the DH lines. We can conclude therefore that these seven lines are true DH lines. Performance of these DH lines was tested in two field locations and compared to commercial check lines. Bulbs from the various crosses were evaluated for eight bulb traits: diameter, height, centers/bulb, ring thickness, number of rings/bulb, bulb weight, soluble solids content, and pungency. Some crosses were detected that yielded significantly greater bulb weight than the check lines. However, these lines also had significantly greater numbers of centers per bulb. To test how these lines would perform in a breeding program, two full diallel analyses were conducted according to Griffing’s Model I, Method 1. The first consisted of a four parent diallel cross using two red DH lines and two yellow DH lines. Bulbs from the various crosses were evaluated for the same eight bulb traits mentioned above. Significant variation was detected for genotypic, general combining ability (GCA), specific combining ability (SCA), reciprocal (REC), maternal (MAT), and nonmaternal (NMAT) effects for all traits except number of rings/bulb, soluble solids content, and pungency. Significant environmental effects were only detected with number of centers per bulb. The second diallel analysis, a four parent diallel with two DH lines and two inbred lines from the breeding program, showed significant variation for the same effects for all traits except soluble solids content. Generally, GCA effects were more important than SCA effects in explaining the variation observed between crosses. For all traits GCA and SCA were always larger than the reciprocal effects (divided into maternal and nonmaternal components).

Numerous factors can influence the evolutionary fate of mutations. Despite this, we tend to study strong evolutionary drivers, or evolution under simple contexts, in part because they are the conditions we have a means to study. My thesis evaluates novel computational approaches to advance detection, and study, of factors that influence a mutation’s evolutionary outcome. First, I present the novel computational tool AEGIS that I use to detect phylogenetic signals of correlated evolution followed by an experimental approach to evaluate the role of epistasis as a potential cause of correlated evolution among sites associated with antibiotic resistance in Pseudomonas aeruginosa. Second, I developed rSHAPE, a novel in silico approach for experimental evolution with asexual haploids, to complement empirical work by providing a common framework in which to test various evolutionary scenarios. After demonstrating that rSHAPE replicates the expected evolutionary dynamics of de novo mutations, I provide evidence that the common laboratory practice of serial passaging may increase stochasticity of evolutionary outcome. Through my work, I have demonstrated that a marriage of computational and experimental approaches will offer new opportunities to understand how the interaction of evolutionary factors influence the fate of mutations.

<p> The ɣ-radiosensitivity of haploid and diploid Oedogonium cardiacum cells was measured and compared to other cell lines. With the doubling of the chromosome complement, the Do value doubled, but the extrapolation number decreased four-fold. A general conclusion was drawn from the results that at all doses of ɣ-radiation, the diploids were more resistant than the haploids. A new radiation technique was used and compared to that used routinely in the laboratory. The further use of the technique was not recommended since the data obtained with the diploid line only was not as reliable as one would like.</p> <p> The degree of synchrony of Oe. cardiacum zoospore cultures was measured using cell division as the biological end-point and a mathematical expression, the percent phasing, as the index of synchrony. It was intended that this research problem be secondary to the radiation studies. The percent phasing values were determined for cells growing in two inorganic media and in the presence of an inhibitor, hydroxyurea. However the degree of synchrony was not improved beyond that of the routine laboratory procedure. Attempts to improve the size of the synchronous populations collected also proved unsuccessful.</p> / Thesis / Master of Science (MSc)

Plant breeding is focused on selection of new genotypes with improved traits. Conventional methods based on hybridization and those based on biotechnology (somatic hybridization, genetic transformation, ploidy manipulation, etc.) are used to create novel genetic variations. Biotechnology provides powerful tools for plant breeding, for instance, haploid technology allows achievement of homozygous lines from heterozygous parents in one step, which reduces significantly the time required by conventional methods. Concerning woody species, characterized by self-incompatibility, long juvenile period and high degree of heterozygosity, this technique is the only way to get homozygous lines. Haploid plants are of great interest for breeding and genomic studies, being used in mutation research, genetic analysis, genome mapping and gene transfer. Gametic embryogenesis, based on cellular totipotency, produces an embryo from an immature gamete, by switching its developmental pathway from gametophytic to sporophytic. This research is focused on inducing gametic embryogenesis in two important Mediterranean fruit crops: almond (Prunus dulcis Mill.) via in vitro anther culture and mandarin (Citrus reticulata Blanco) via isolation and microspore culture. Also ploidy manipulation was applied to loquat (Eriobotrya japonica (Thunb.) Lindl.) for getting genotypes whit different ploidy levels. The experiments were carried out through years 2014, 2015 and 2016 at the 'Università degli Studi di Palermo' (UNIPA) as well as at the 'Instituto Valenciano de Investigaciones Agrarias' (IVIA). Regarding the almond anther culture, formation of calli and production of embryos was achieved through the direct embryogenesis route. On then other hand, early embryo regeneration is reported, for the first time, from isolated microspore culture of mandarin, 'Mandarino Tardivo di Ciaculli'. Our results report the evidence of gametic embryogenesis and the production of homozygous regenerants in almond and mandarin, two species extremely recalcitrant to microspore embryogenesis. However, the results are affected by many factors that need further studies to better understand the embryogenic development and to increase the rate of embryo achievement. Moreover, another biotechnological tool (ploidy manipulation) was also applied for implementing the IVIA loquat breeding program. Polyploid plants are of great interest in this species, due to its potential for producing seedless genotypes via direct use of triploids or crosses between tetra and diploids. Aimed at obtaining new loquat genotypes, with different ploidy levels (polyploids), colchicine was applied to seeds before germination, to induce chromosome duplication. A total of three triploids (3x) and one tetraploid (4x) were obtained. / La mejora genética tiene como objetivo la selección de nuevos genotipos con mejores características. Los métodos de mejora convencional basados en hibridaciones y aquellos basados en Biotecnología (hibridación somática, transformación genética, manipulación de la ploidía, etc.) se utilizan para obtener nueva variación genética. La Biotecnología proporciona herramientas poderosas en mejora genética, por ejemplo, la obtención de haploides permite obtener líneas homocigotas en un solo paso, disminuyendo significativamente el tiempo requerido usando métodos convencionales. Respecto a especies leñosas, caracterizadas por autoincompatibilidad floral, largo período juvenil y alto grado de heterocigosidad, esta técnica es el único método de obtención de líneas homocigotas. Los genotipos haploides tienen un alto interés en estudios genómicos, siendo utilizados en estudios de mutaciones, análisis genéticos, mapeo genético y transferencia genética. Este estudio tiene como objetivo la inducción de embriogénesis gamética en dos especies mediterráneas muy importantes: el almendro (Prunus dulcis Mill.) por medio de cultivo in vitro de anteras y el mandarino (Citrus reticulata Blanco) por medio de aislamiento de microesporas. Además, se ha estudiado la obtención de poliploides en níspero (Eriobotrya japonica (Thunb.) Lindl.) con el objetivo de obtener genotipos con diversos niveles de ploidía. Los experimentos se llevaron a cabo en los años 2014, 2015 y 2016 en la 'Università degli Studi di Palermo' (UNIPA) y en el 'Instituto Valenciano de Investigaciones Agrarias' (IVIA). Respecto al cultivo de anteras en almendro, la formación de callos y producción de embriones se obtuvo mediante embriogénesis directa. Por otro lado, se ha conseguido regenerar por primera vez embriones a partir de microesporas aisladas en el cultivar de mandarino 'Mandarino Tardivo di Ciaculli'. Los resultados obtenidos muestran que la embriogénesis gamética y la regeneración de embriones homocigótos en almendro y mandarino, dos especies extremadamente recalcitrantes para la embriogenésis a partir de microesporas, es posible. Sin embargo, los resultados se ven afectados por muchos factores que necesitan estudios adicionales para comprender mejor el desarrollo embriogénico y para aumentar la tasa de obtención del embriones. Además, otra herramienta biotecnológica (manipulación de la ploidía) se aplicó con el objetivo de implementar el programa de mejora de níspero del IVIA. Las plantas poliploides en esta especie tienen un alto interés, pues podrían permitir la obtención de frutos sin semilla, por medio de la obtención directa de triploides o mediante cruzamiento entre tetraploides y diploides. Con el objetivo de obtener nuevos genotipos de níspero con diferentes niveles de ploidía (poliploides), se aplicó colchicina a semillas sin germinar con el fin de inducir la duplicación cromosómica y se obtuvieron 3 triploides (3x) y un tetraploide (4x). / La millora genètica té com objectiu la selecció de nous genotips amb millors característiques. Els mètodes de millora convencional basats en hibridacions i aquells basats en Biotecnologia (hibridació somàtica, transformació genètica, manipulació de la ploïdia, etc.) s'utilitzen per aconseguir nova variació genètica. La Biotecnologia proporciona eines poderoses en millora genètica, per exemple, l'obtenció d'haploides permet obtenir línies homozigòtiques en un sol pas, disminuint significativament el temps requerit usant mètodes convencionals. Pel que fa a espècies llenyoses, caracteritzades per autoincompatibilitat floral, llarg període juvenil i alt grau d'heterozigosi, aquesta tècnica és l'únic mètode d'obtenció de línies homozigòtiques. Els genotips haploides tenen un alt interès en estudis genòmics, sent utilitzats en estudis de mutacions, anàlisis genètics, mapatge genètic i transferència genètica. Aquest estudi té com objectiu la inducció d'embriogènesi gamètica en dos espècies mediterrànies molt importants: l'ametller (Prunus dulcis Mill.) a través del cultiu in vitro d'anteres i el mandariner (Citrus reticulata Blanco) per mitjà d'aïllament de micròspores. A més a més, s'ha estudiat l'obtenció de poliploides en nespra (Eriobotrya japonica (Thunb.) Lindl.) Amb l'objectiu d'aconseguir genotips amb diversos nivells de ploïdia. Els experiments es van dur a terme en els anys 2014, 2015 i 2016 a la 'Università degli Studi di Palermo' (UNIPA) i a 'l'Institut Valencià d'Investigacions Agràries' (IVIA). Respecte al cultiu d'anteres en ametller, la formació de calls i producció d'embrions es va obtenir mitjançant embriogènesi directa. D'altra banda, s'ha aconseguit per primera vegada la regeneració d'embrions a partir de micròspores aïllades en el conrear de mandariner 'Mandarino Tardivo di Ciaculli'. Els resultats obtinguts mostren que l'embriogènesi gamètica i la regeneració d'embrions homozigotics en ametller i mandariner, dues espècies extremadament recalcitrants per l'embriogènesi a partir de micròspores, és possible. No obstant això, els resultats es veuen afectats per molts factors que necessiten estudis addicionals per entendre millor el desenvolupament embriogènic i per augmentar la taxa d'obtenció dels embrions. A més, una altra eina biotecnològica (manipulació de la ploïdia) es va aplicar amb l'objectiu d'implementar el programa de millora de nespra de l'IVIA. Les plantes poliploides en aquesta espècie tenen un alt interès, ja que podrien permetre l'obtenció de fruits sense llavor, per mitjà de l'obtenció directa de triploides o mitjançant encreuament entre tetraploides i diploides. Amb l'objectiu d'aconseguir nous genotips de nespra amb diferents nivells de ploïdia (poliploides), es va aplicar colquicina a llavors sense germinar per tal d'induir la duplicació cromosòmica i es van obtenir 3 triploides (3x) i un tetraploide (4x). / Cimò, G. (2017). Ploidy manipulation for genetic improvement in some Mediterranean fruit crops [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/79874

Potato haploids have been recent components in protoplast fusion research, strategies to combine wild and cultivated potato germplasm and the generation of economically valuable mutant phenotypes. Additionally, most major genetic mapping and QTL analyses in potato have utilized haploid germplasm to simplify linkage-mapping computations. The accuracy of genetic assumptions concerning the randomness and genetic purity of haploid genomes may directly affect the statistical validity of many results in current potato research. In the present study, AFLP analysis was conducted on two sibling S. phureja "BARD 1-3" monoploid populations derived by androgenesis in anther culture, and gynogenesis through the use of a haploid-inducing pollinator, S. phureja "IVP 101." Little indication of somaclonal variation and haploid-inducer gene introgression was found in the monoploid band data suggesting genomic stability. Segregation of marker alleles that were heterozygous in the parent was distorted from the expected 1:1 ratio in both populations, ranging from 35% in the gynogenic monoploids (GM) to 46% in the androgenic monoploids (AM). Genetic diversity appeared more random among the monoploid populations after skewed marker data was removed from phylogenetic analyses. Bilateral and unilateral marker skewness in the monoploid populations may respectively indicate common and unique segregation distorting loci (SDL) present in the AM and GM genomes. Representatives of both SDL types were located on a partial linkage map created using androgenic monoploid data. / Master of Science

Monopoloid potato (2n = 1x = 12) can be derived either paternally through anther/microspore culture or maternally through crossing with a haploid-inducing pollinator. Evidence from other genera suggests that androgenic and gynogenic haploid populations derived from the same parent may differ due to gametic selection and/or epigenetic factors. Our objectives were to derive androgenic and gynogenic monoploid populations from each of two diploid (2n = 2x = 24) Solanum phureja clones and compare their phenotypic characteristics in a greenhouse study. A haploid-inducing pollinator, S. phureja IVP101, was crossed to two selections of S. phureja (PP5 and BARD1-3). A total of 185 fruit was obtained from PP5 and 398 from BARD1-3, resulting from 85% and 65% fruit set, respectively. Seed lacking the dominant embryo spot marker carried by IVP101 were selected and germinated in vitro. From 29,300 PP5 x IVP101 seeds, 278 were spotless, resulting in 27 monoploids. Approximately 37 monoploids were obtained from the 99,500 BARD1-3 x IVP101 seeds of which 500 were spotless. In anther culture, PP5 and BARD1-3 yielded 0.16 and 1.67 embryos per anther respectively of which 51% and 44% of the regenerants were monoploid. A total of 32 anther-derived monoploids has been obtained from PP5 and 130 from BARD1-3. Rooted cuttings of 21 androgenic and 21 gynogenic PP5 monoploids were established in a greenhouse in each of three randomized complete blocks. Although the anther-derived monoploids appeared more vigorous, none of the differences between the two populations were significant. Flow cytometry revealed that anther derived progeny of PP5 and BARD1-3 had 3-8% greater nuclear DNA content than gynogenic monoploids derived from the same parental clones. / Master of Science