Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.

The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions. Cumulus-oocyte-complexes were in vitro matured and activated using Ca(2+)Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemProA (R) medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4. Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39 % and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker. Our data show that Ca2+ Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions.

Microcollinearity between autopolyploid sugarcane and diploid sorghum genomes

Abstract Background Sugarcane (Saccharum spp.) has become an increasingly important crop for its leading role in biofuel production. The high sugar content species S. officinarum is an octoploid without known diploid or tetraploid progenitors. Commercial sugarcane cultivars are hybrids between S. officinarum and wild species S. spontaneum with ploidy at ~12×. The complex autopolyploid sugarcane genome has not been characterized at the DNA sequence level. Results The microsynteny between sugarcane and sorghum was assessed by comparing 454 pyrosequences of 20 sugarcane bacterial artificial chromosomes (BACs) with sorghum sequences. These 20 BACs were selected by hybridization of 1961 single copy sorghum overgo probes to the sugarcane BAC library with one sugarcane BAC corresponding to each of the 20 sorghum chromosome arms. The genic regions of the sugarcane BACs shared an average of 95.2% sequence identity with sorghum, and the sorghum genome was used as a template to order sequence contigs covering 78.2% of the 20 BAC sequences. About 53.1% of the sugarcane BAC sequences are aligned with sorghum sequence. The unaligned regions contain non-coding and repetitive sequences. Within the aligned sequences, 209 genes were annotated in sugarcane and 202 in sorghum. Seventeen genes appeared to be sugarcane-specific and all validated by sugarcane ESTs, while 12 appeared sorghum-specific but only one validated by sorghum ESTs. Twelve of the 17 sugarcane-specific genes have no match in the non-redundant protein database in GenBank, perhaps encoding proteins for sugarcane-specific processes. The sorghum orthologous regions appeared to have expanded relative to sugarcane, mostly by the increase of retrotransposons. Conclusions The sugarcane and sorghum genomes are mostly collinear in the genic regions, and the sorghum genome can be used as a template for assembling much of the genic DNA of the autopolyploid sugarcane genome. The comparable gene density between sugarcane BACs and corresponding sorghum sequences defied the notion that polyploidy species might have faster pace of gene loss due to the redundancy of multiple alleles at each locus.

Genetic diversity and population structure of Musa accessions in ex situ conservation

Abstract Background Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa. Results From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis. Conclusions The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping.

Entstehung und Diversifizierung der mittel- und westeuropäischen Salicornia-Arten

HintergrundrnDie hygrohalophytische Gattung Salicornia ist in Mittel- und Westeuropa durch vier nah verwandte, sympatrisch vorkommende Arten vertreten. Es handelt sich um die zwei tetraploiden Arten S. procumbens und S. stricta und die diploiden Arten S. europaea und S. ramosissima. Morphologisch lassen sich die Arten zwar nur schwer voneinander unterscheiden, die morphologische Variation ist aber wiederum so hoch, dass mehrere distinkte Arten/Morphotypen unterschieden werden können. Bezüglich ihrer Verteilung im hochdynamischen Lebensraum Salzwiese findet man die verschiedenen Arten/Morphotypen in überlappenden Bereichen des Habitats. Ihr relativ vorhersagbares Auftreten entlang eines ökologischen Gradienten innerhalb ihres Lebensraumes scheint jedoch für eine ökologische Differenzierung der verschiedenen Arten/Morphotypen zu sprechen. Aufgrund des sympatrischen Vorkommens der scheinbar ökologisch und morphologisch differenzierten Morphotypen stellt sich die Frage, durch welche Prozesse diese entstanden sein könnten (genetische und ökologische Differenzierung) aber auch welche Prozesse die dauerhafte Koexistenz der Arten (reproduktive Isolationsmechanismen) aufrechterhalten.rnZielsetzungrnZiel dieser Arbeit war es, die Entstehung und Diversifizierung der mittel- und westeuropäischen Salicornia-Arten anhand von molekulargenetischen, ökologischen und reproduktionsbiologischen Methoden zu untersuchen.rnMethodenrnAnhand einer AFLP-Fragmentanalyse mit 89 Herkünften aus Großbritannien, Frankreich und Deutschland wurden molekulare Phylogenien erstellt sowie eine Hauptkomponenten- und Clusteranalyse durchgeführt. Um die ökologische Differenzierung und phänotypische Plastizität der vier Arten/Morphotypen zu untersuchen wurde ein reziprokes Transplantationsexperiment durchgeführt. Um die reproduktiven Isolationsmechanismen der Arten/Morphotypen zu untersuchen, wurden verschiedene Beobachtungen und Experimente durchgeführt.rnErgebnissernDie molekularen Analysen konnten zwar die beiden Artengruppen (Ploidiestufen) trennen, lieferten aber innerhalb dieser weder ein taxonomisches noch ein geographisches Signal. Akzessionen mit identischer Morphologie aus der gleichen Population verteilten sich in den Analysen in verschiedene genetische Cluster. Identische Morphotypen aus verschiedenen geographischen Regionen gruppieren teilweise zusammen. Das Transplantationsexperiment zeigte für die beiden tetraploiden Arten S. procumbens und S. stricta eine deutliche ökologische Differenzierung, bei S. procumbens in Form von verminderter Fitness und einer beschleunigten Phänologie, bei S. stricta nur in Form einer veränderten Phänologie. Bezüglich der Plastizität zeigten beide tetraploiden Arten eine konstante Morphologie. Die beiden diploiden Taxa S. europaea und S. ramosissima zeigten weder eine klare ökologische Differenzierung noch eine konstante Morphologie. Bezüglich der Reproduktionsbiologie konnte bestätigt werden, dass Selbstung bei allen Taxa der hauptsächliche Reproduktionsmodus ist. Bei den tetraploiden Taxa zeigte sich zwar ein geringes Maß an Fremdbefruchtung, bei den diploiden Taxa führen dagegen morphologische Besonderheiten zu hochgradiger Selbstung.rnRésumérnDie in Mittel- und Westeuropa vorkommenden Salicornia-Arten stellen keine evolutionären Einheiten dar. Die beiden tetraploiden Taxa sollten auf Grund ihrer parallelen Entstehung und ökologischen Differenzierung als Ökotypen angesprochen werden. Beide Ökotypen weisen ein hohes Ausbreitungspotential aus und persistieren als Inzuchtlinien mit geringem Anteil an Fremdbestäubung. Die diploiden Taxa sind weder ökologisch differenziert noch morphologisch stabil und sollten deshalb als nur ein morphologisch sehr variables, aus zahlreichen weitverbreiteten Inzuchtlinien bestehendes Taxon angesehen werden.

Multilocus analysis of genetic divergence between outcrossing Arabidopsis species: evidence of genome-wide admixture

P>Outcrossing Arabidopsis species that diverged from their inbreeding relative Arabidopsis thaliana 5 million yr ago and display a biogeographical pattern of interspecific sympatry vs intraspecific allopatry provides an ideal model for studying impacts of gene introgression and polyploidization on species diversification. Flow cytometry analyses detected ploidy polymorphisms of 2x and 4x in Arabidopsis lyrata ssp. kamchatica of Taiwan. Genomic divergence between species/subspecies was estimated based on 98 randomly chosen nuclear genes. Multilocus analyses revealed a mosaic genome in diploid A. l. kamchatica composed of Arabidopsis halleri-like and A. lyrata-like alleles. Coalescent analyses suggest that the segregation of ancestral polymorphisms alone cannot explain the high inconsistency between gene trees across loci, and that gene introgression via diploid A. l. kamchatica likely distorts the molecular phylogenies of Arabidopsis species. However, not all genes migrated across species freely. Gene ontology analyses suggested that some nonmigrating genes were constrained by natural selection. High levels of estimated ancestral polymorphisms between A. halleri and A. lyrata suggest that gene flow between these species has not completely ceased since their initial isolation. Polymorphism data of extant populations also imply recent gene flow between the species. Our study reveals that interspecific gene flow affects the genome evolution in Arabidopsis.

ABCtoolbox: a versatile toolkit for approximate Bayesian computations

Background The estimation of demographic parameters from genetic data often requires the computation of likelihoods. However, the likelihood function is computationally intractable for many realistic evolutionary models, and the use of Bayesian inference has therefore been limited to very simple models. The situation changed recently with the advent of Approximate Bayesian Computation (ABC) algorithms allowing one to obtain parameter posterior distributions based on simulations not requiring likelihood computations. Results Here we present ABCtoolbox, a series of open source programs to perform Approximate Bayesian Computations (ABC). It implements various ABC algorithms including rejection sampling, MCMC without likelihood, a Particle-based sampler and ABC-GLM. ABCtoolbox is bundled with, but not limited to, a program that allows parameter inference in a population genetics context and the simultaneous use of different types of markers with different ploidy levels. In addition, ABCtoolbox can also interact with most simulation and summary statistics computation programs. The usability of the ABCtoolbox is demonstrated by inferring the evolutionary history of two evolutionary lineages of Microtus arvalis. Using nuclear microsatellites and mitochondrial sequence data in the same estimation procedure enabled us to infer sex-specific population sizes and migration rates and to find that males show smaller population sizes but much higher levels of migration than females. Conclusion ABCtoolbox allows a user to perform all the necessary steps of a full ABC analysis, from parameter sampling from prior distributions, data simulations, computation of summary statistics, estimation of posterior distributions, model choice, validation of the estimation procedure, and visualization of the results.

Cytogenetics of pineoblastoma: four new cases and a literature review

Pineoblastoma represents a class of primitive neuroectodermal tumors (PNET) with poorly differentiated neuroepithelial cells that are histologically indistinguishable from medulloblastomas. It is a rare tumor, typically arising in childhood, and to date only a few cytogenetic cases have been published. We report four new cases in which conventional cytogenetics demonstrated the presence of an abnormal clone. The tumors showed a variety of ploidy levels, from hypodiploid to hypertetraploid. Both structural and numerical aberrations were frequent, and in three out of the four cases a large degree of cell-to-cell variation was observed. The most frequently involved chromosome in structural rearrangements was chromosome 1, observed in three of the four cases. The short arm was involved in two of the three cases; in the third case, the anomaly was in the long arm. Two cases showed unbalanced gain of chromosome 17q, one of them showing i(17)(q10). Together, the four cases illustrate the complex karyotypic nature of this tumor type and represent a step toward determining whether a nonrandom cytogenetic picture exists and how this may be related to other associated tumor types.

Niche differentiation between diploid and hexaploid Aster amellus

The maintenance of separated diploid and polyploid populations within a contact zone is possible due to both prezygotic and postzygotic isolation mechanisms. Niche differentiation between two cytotypes may be an important prezygotic isolating mechanism and can be studied using reciprocal transplant experiments. We investigated niche differentiation between diploid and hexaploid Aster amellus in their contact zone in the Czech Republic. Diploid populations are confined to habitats with low productivity, whereas hexaploid populations occur in habitats with both low and high productivity. Thus, we chose three diploid populations and six hexaploid populations, three in each of the two different habitat types. We analyzed habitat characteristics and carried out reciprocal transplant experiments in the field using both seeds and adult plants. Sites of diploid and hexaploid populations differed significantly in vegetation and soil properties. The mean number of juveniles was higher at sites of home ploidy level than at sites of foreign ploidy level, suggesting niche differentiation between the two cytotypes. On the other hand, transplanted adult plants survived at all sites and juvenile plants were able to establish at some sites of the foreign cytotype. Furthermore, the mean number of juveniles, survival, and flowering percentages were higher at home sites than at foreign sites, indicating local adaptation. We conclude that niche differentiation between the two cytotypes and local adaptation within each cytotype may contribute to the maintenance of diploid and hexaploid populations of A. amellus in their contact zone. Moreover, further factors, such as differences in flowering phenology and exclusion of minority cytotypes, should also be considered.

{\it In vivo\/} induction of DNA changes in cervicovaginal epithelium by perinatal estrogen exposure

Epidemiological studies have associated estrogens with human neoplasm such as the endometrium, cervix, vagina, breast, and liver. Perinatal exposure to natural (17$\beta$-estradiol (17$\beta$-E$\sb2)\rbrack$ and synthetic (diethylstilbestrol (DES)) estrogens induces neoplastic changes in humans and rodents. Previous studies demonstrated that neonatal 17$\beta$-E$\sb2$ treatment increased the nuclear DNA content of mouse cervicovaginal epithelium that preceded histologically evident neoplasia. In order to determine whether this effect was specific to 17$\beta$-E$\sb2,$ associated with chromosomal changes, and relevant to the human, female BALB/c mice were treated neonatally with either 17$\alpha$-estradiol (17$\alpha$-E$\sb2)$ and 5$\beta$-dihydrotestosterone ($5\beta$-DHT), both inactive steroids in adult reproductive tissue, or 17$\beta$-E$\sb2.$ Ten-day-old mice received pellet implants of 17$\beta$-E$\sb2,$ 17$\alpha$-E$\sb2,$ $5\beta$-DHT, or cholesterol. Seventy-day-old cervicovaginal tracts were examined histologically and flow cytometrically. 17$\beta$-E$\sb2$-treated animals were evaluated by fluorescent in situ hybridization (FISH) using a probe specific for chromosome 1. Trisomy of chromosomes 1, 7, 11, and 17 was evaluated by FISH in cervicovaginal material from 19 DES-exposed and 19 control patients.^ $17\beta$-E$\sb2, 17\alpha$-E$\sb2$, and $5\beta$-DHT-induced dramatic developmental and histological changes in the cervicovaginal tract, including hypospadia, hyperplasia, and persistent cornification. The changes induced by 17$\alpha$-E$\sb2$ were equivalent to 17$\beta$-E$\sb2.$ Neonatal 17$\alpha$-E$\sb2$-induced adenosquamous cervicovaginal tumors at 24 months. 17$\alpha$-E$\sb2$ and $5\beta$-DHT significantly increased the nuclear DNA content over control animals, but at significantly lower levels than 17$\beta$-E$\sb2.$ DNA ploidy changes were highest (80%) in animals treated neonatally and secondarily with 17$\beta$-E$\sb2.$ Secondary 17$\alpha$-E$\sb2$ and $5\beta$-DHT administration, unlike 17$\beta$-E$\sb2,$ didn't significantly increase DNA content. Chromosome 1 trisomy incidence was 66% in neonatal 17$\beta$-E$\sb2$-treated animals. Trisomy was evident in 4 DES-exposed patients: one patient with trisomy of chromosomes 1, 7, and 11; one patient with chromosome 7 trisomy; and two patients with chromosome 1 trisomy. These data demonstrated the biological effects of 17$\alpha$-E$\sb2$ and $5\beta$-DHT were age-dependent, 17$\alpha$-E$\sb2$ was equivalent to 17$\beta$-E$\sb2$ and tumorigenic when administered neonatally, and histological changes were not steroid specific. Chromosomal changes were associated with increased nuclear DNA content and chromosomal changes may be an early event in the development of tumors in human DES-exposed tissues. ^

The prognostic significance of sialyl-Tn antigen in women treated with adjuvant chemotherapy for breast cancer

Background and purpose. Sialyl-Tn(STn) represents an aberrantly glycosylated mucin epitope which is expressed in breast cancer and other adenocarcinomas and is an important target for the development of novel immunotherapeutic approaches. It is a marker of adverse prognosis in colon and ovarian cancer, but information about its prognostic impact in breast cancer is limited. The primary aim of the present study was to investigate the influence of STn expression on outcome of invasive breast cancer in 207 women who received anthracyline-containing adjuvant chemotherapy in a prospective clinical trial.^ Methods. Expression of STn was determined by an immunohistochemical procedure using the B72.3 monoclonal antibody. The extent of staining was determined by two observers using a 0 through 4 point scale, with 0 representing $<$5% of cells staining; 1: 5-25%; 2: 26-50%; 3: 51-75%; and 4: $>$75%. Intraobserver and interobserver agreement was.78-.92 (kappa). Kaplan-Meier and Cox proportional regression survival analyses were used to compare STn-negative and STn-positive patients.^ Results. Forty-eight (23%) of the 207 specimens demonstrated positive staining of STn. With a median follow-up of five years, STn-positivity was associated with a higher 5-year recurrence-free survival time than STn-negativity (67% vs. 80%, respectively; p = 0.03). STn expression was significantly associated with menopausal status (p = 0.04) but not other conventional prognostic markers. The risk of breast cancer recurrence and death was assessed by multivariate Cox regression analyses with adjustment for lymph node status, tumor size, menopausal status, hormone receptor status, nuclear grade, S-phase fraction and ploidy. In the final multivariate model for recurrence-free survival, the three factors that showed prognostic significance were: lymph node status (hazard ratio (HR) 3.04, 95% confidence interval (CI) 1.08-8.49), STn expression (HR 2.02, 95% CI 1.09-3.73), and tumor size (HR 1.96, 95% CI 1.05-3.64). STn was also associated with worse overall survival (HR 2.16, 95% CI 0.95-4.92) in multivariate analysis.^ Conclusion. STn antigen was shown to be a predictor of poor outcome in breast cancer. This tumor-associated antigen may be a valuable marker for identifying individuals at high risk of developing recurrent disease who may benefit from adjuvant therapy targeted at STn following definitive local therapy. Further study is needed to clarify the biologic and prognostic role of STn in breast cancer. ^

DNA hypomethylation and oxidative stress-mediated increase in genomic instability in equine sarcoid-derived fibroblasts

It is widely accepted that equine sarcoid disease, the most common skin associated neoplasm in equids, is induced by bovine papillomavirus (BPV-1). Although BPV-1 DNA has been found in almost all examined sarcoids so far, its detailed impact on the horse's host cell metabolism is largely unknown. We used equine fibroblast cell lines originating from sarcoid biopsies to study BPV-1-associated changes on DNA methylation status and oxidative stress parameters. Sarcoid-derived fibroblasts manifested increased proliferation in vitro, transcriptional rDNA activity (NORs expression) and DNA hypomethylation compared to control cells. Cells isolated from equine sarcoids suffered from oxidative stress: the expression of antioxidant enzymes was decreased and the superoxide production was increased. Moreover, increased ploidy, oxidative DNA damage and micronuclei formation was monitored in sarcoid cells. We postulate that both altered DNA methylation status and redox milieu may affect genomic stability in BPV-1-infected cells and in turn contribute to sarcoid pathology.

Experimental models for p53, hepatitis B and aflatoxin hepatocarcinogenesis

The major risk factors for liver cancer in Southeast Asia: HBV infection, aflatoxin exposure and p53 expression/mutation, were examined in experimental models. Four groups were examined for development of hepatocellular carcinoma (HCC) with and without neonatal exposure to aflatoxin (AFB$\sb1)$: (Group I.) Transgenic HBsAg mice with one p53 allele. (Group II) Transgenic HBsAg mice with two p53 alleles. (Group III) Non-transgenic litter mates with one p53 allele. (Group IV) Non-transgenic litter mates with two p53 alleles. HCC developed in Group I animals exposed to aflatoxin at an earlier time and were of a higher grade than those seen later in other groups. These results provide an explanation for as to why p53 is a target for deletion and/or mutation in human HCC especially when found in high risk areas where HBV infection and Aflatoxin B1 food contamination is high, and nicely illustrates a synergistic interaction among these three factors. None of the tumors analyzed had loss or mutation in the p53 gene.^ To determine the significance of the specific p53ser249 mutation found in HBV/aflatoxin associated human hepatomas in an in-vivo experimental model using transgenic mice, a two-nucleotide change in the mouse p53 gene at amino acid position 246, which is equivalent to that of 249 in human p53, was introduced. Transgenic mice with mutant p53 controlled by the albumin promoter were generated and shown to express the p53ser246 mutant RNA and protein specifically in liver. Three groups were examined for development of HCC with and without neonatal exposure to aflatoxin: (Group V) Transgenic p53ser246 mice with two p53 alleles. (Group VI) Transgenic p53ser246 mice with one p53 allele. (Group VII) Double transgenic for p53ser246 and HBsAg with two p53 alleles. One hundred percent of male mice with the three risk factors injected with aflatoxin developed high grade liver tumors, compared to 66.6% from group VI and only 14.2% of group V suggesting synergistic interaction between HBsAg and this particular ser246 p53 mutation.^ In order to examine the growth properties of hepatocytes and correlation with p53 loss and/or mutation, cell proliferation and ploidy analysis of liver from normal heterozyous, homozygous null mice and from transgenic mutant p53ser246, mice were studied. Loss of wild-type p53 increased G1/G0 ratios of cells as well as proliferation and decreased cell ploidy. The mutant p53ser246 did not show a significant effect on cell ploidy or proliferation. However a striking 5-10X increase in G1/G0 ratio suggests that this specific mutation specifically induces G0 to G1 transition, which in turn further predisposes hepatocytes to the damaging effect of Aflatoxin. (Abstract shortened by UMI.) ^

Taxonomía y caracterización del arbusto forrajero Atriplex halimus L.

El objetivo de esta Tesis Doctoral ha sido la revisión de la taxonomía infraespecífica de la especie Atriplex halimus L., la delimitación del área geográfica que ocupan sus taxones y su caracterización morfológica. En base a las citas bibliográficas y material de herbario consultado se ha trazado un mapa de distribución de la especie, que delimita su distribución como planta espontánea a la Cuenca Mediterránea, desierto de Siria, Macizo de Hoggar y Sahara Occidental. El resto debe considerarse poblaciones naturalizadas o cultivadas. La taxonomía actual, basada en relaciones filogenéticas, nos indica que lo adecuado es separar las poblaciones de A. halimus en dos clados que se caracterizan por tener diferente nivel de ploidía. Para definir el área de distribución de los clados diploide y tetraploide se ha determinado la ploidía de 19 poblaciones en el espacio comprendido entre las dos áreas de distribución conocidas. Y como resultado el área de distribución de ambos clados puede definirse trazando una recta que une el estrecho de Gibraltar con Estambul, las poblaciones al sur de esta línea son tetraploides a las que habría que añadir las poblaciones del este y sur de la isla de Cerdeña que son también tetraploides. Se han determinado cuales son los parámetros morfológicos que permiten caracterizar ambos clados, para lo cual se han estudiado 52 poblaciones que comprenden toda el área de distribución de la especie. Se han evaluado caracteres cualitativos y cuantitativos de valvas fructíferas, plántulas cultivadas en cámara y arbustos de 2 a 4 años situados en la misma parcela. El único elemento fiable para determinar la pertenencia al clado diploide o tetraploide es la medición del nivel de ploidía. Ninguno de los caracteres morfológicos estudiados es plenamente fiable para determinar si los individuos de una población pertenecen al clado diploide o tetraploide a pesar de realizarse las mediciones sobre poblaciones cultivadas en igualdad de condiciones de suelo y clima, y en condiciones muy favorables para la expresión de caracteres cualitativos y cuantitativos. Los caracteres cualitativos observados que guardan una mayor correlación con el nivel de ploidía son: la base de las hojas de las plántulas, el porte del arbusto, la ramificación, el ángulo de inserción de las ramas y la longitud de la inflorescencia. Respecto a los datos cuantitativos los más relevantes han sido, la longitud de los cotiledones y la longitud del limbo de la 1ª, 3ª o 5ª hoja de la plántula o la razón entre este valor y la anchura del limbo. ABSTRACT The aim of this Thesis has been in the revision of the infraspecies taxa of Atriplex halimus L., the delimitation of their natural growing area and their morphological characterization. Using references to relevant literature and consultation of herbariums, it has been possible to map the distribution of the species as spontaneous plant in the Mediterranean Basin, Desert of Syria, Massif of Hoggar and Western Sahara. The remaining populations must be considered as naturalized or cultivated. The current taxonomy, based on phylogenetic relationships, indicates that A.halimus should be divided in two clades. These clades are characterized by their different ploidy level. Samples of nineteen populations collected from localizations between the defined distribution area of both clades have been characterized for their ploidy level. As a result, it can be seen that the line of clade separation can be trazed from Istambul tothe Strait of Gibraltar, where the populations north to this line are classified as diploid and those south of the line are clsassified as tetraploid. The only exception of this rule is in Sardinia, hwre populations to the West and South of the Island are also classified as tetraploid. The morphological parameters, which include the characterization of both clades, have been defined, and the study of fifty-two populations from the entire distribution area according to these parameters have been analysed. The qualitative and quantitative characteristics of the fruit valves have been evaluated, with seedlings grown in a culture chamber, and 2-4 years old shrubs grown in the same orchard. None of the morphological characters under study are fully reliable to determine whether individuals in a population belong to clade diploid or tetraploid. This is despite measurements being made on populations grown under the same conditions of soil and climate, and in very favorable conditions for the expression of qualitative and quantitative traits. The qualitative characteristics that show a higher correlation with the ploidy level are: the base of the leaves of the seedlings, the bearing bush, the branch arrangement, the angle of insertion of the branches and the inflorescence length. With regards to the quantitative data the most relevant were the length of the cotyledons and the length of the blade of the 1st, 3rd or 5th leaves of the seedling, and the ratio of any of this values and the leaf blade width. The only reliable way to determine the diploid or tetraploid clade membership is the measurement of ploidy level.

Endosperm balance number manipulation for direct in vivo germplasm introgression to potato from a sexually isolated relative (Solanum commersonii Dun.)

Diploid (2n = 2x = 24) Solanum species with endosperm balance number (EBN) = 1 are sexually isolated from diploid 2EBN species and both tetraploid (2n = 4x = 48, 4EBN) and haploid (2n = 2x = 24, 2EBN) S. tuberosum Group Tuberosum. To sexually overcome these crossing barriers in the diploid species S. commersonii (1EBN), the manipulation of the EBN was accomplished by scaling up and down ploidy levels. Triploid F1 hybrids between an in vitro-doubled clone of S. commersonii (2n = 4x = 48, 2EBN) and diploid 2EBN clones were successfully used in 3x × 4x crosses with S. tuberosum Group Tuberosum, resulting in pentaploid/near pentaploid BC1 progenies. This provided evidence of 2n (3x) egg formation in the triploid female parents. Two selected BC1 pentaploid hybrids were successfully backcrossed both as male and as female parents with S. tuberosum Group Tuberosum. The somatic chromosome number varied greatly among the resulting BC2 progenies, which included hyperaneuploids, but also a number (4.8%) of 48-chromosome plants. The introgression of S. commersonii genomes was confirmed by the presence of S. commersonii-specific randomly amplified polymorphic DNA markers in the BC2 population analyzed. The results clearly demonstrate the feasibility of germplasm introgression from sexually isolated diploid 1EBN species into the 4x (4EBN) gene pool of the cultivated potato using sexual hybridization. Based on the amount and type of genetic variation generated, cumbersomeness, general applicability, costs, and other factors, it would be interesting to compare the approach reported here with other in vitro or in vivo, direct or indirect, approaches previously reported.