Reproductive biology and pollen-pistil compatibility relationships in an Argentinian collection of Stevia rebaudiana Bertoni / Biología reproductiva y relaciones de compatibilidad polen-pistilo en una colección de Stevia rebaudiana Bertoni en Argentina

ABSTRACT Stevia rebaudiana Bertoni (Asteraceae) is a diploid species (2n=2x=22) with sexual and asexual reproduction. The sexual propagules are seeds produced by cross-pollination (allogamy) whereas the asexual propagules are either vegetative shoots or apomictic seeds. Various authors have reported that allogamy in this species is promoted by the expression of a sporophytic self-incompatibility (SSI) system. To introduce the cultivation of S. rebaudiana as a production alternative in Tucumán, Argentina, a germplasm collection of this species was established with accessions from four Argentinian provinces in the Famaillá Agropecuarian Experimental Station (EEA Famaillá), National Institute of Agropecuarian Technology (INTA). The reproductive biology of the collection was studied between 2014 and 2021 to develop strategies for breeding and conservation of these genetic resources. Fifty-six genotypes were analyzed, all of them were 2n=2x=22. Pollen viability was high (69.4 to 99.6%) in all the genotypes except in four of them, which exhibited low viability (36.0 to 51.5%) in 2015 and 2017. Forty-eight genotypic combinations were obtained by manual controlled crosses. In 12 of these combinations, one pollen tube was observed in the style zone and, in four of them, one pollen tube was observed in the embryo sac; these observations indicate, respectively, incompatible and compatible pollen-pistil relationships. Normal plump seeds were obtained in all compatible genotypic combinations. The observed incompatibility might be due to the functioning of the sporophytic homomorphic system and/or a cross-incompatibility system. The observed compatibility will allow the planification of controlled crosses within and between accessions of different geographical origins to generate genetically variable progenies for breeding purposes.

RESUMEN Stevia rebaudiana Bertoni (Asteraceae) es una especie diploide (2n=2x=22) con reproducción sexual y asexual. Los propágulos sexuales son semillas producidas por polinización cruzada (alogamia), mientras que los propágulos asexuales son brotes vegetativos y semillas apomםcticas. Varios autores han seסalado que la alogamia en esta especie se ve favorecida por la expresión de un sistema de autoincompatibilidad esporofםtica (SSI). Para introducir el cultivo de S. rebaudiana como alternativa productiva en Tucumán, Argentina, se estableció una colección de germoplasma de esta especie proveniente de cuatro provincias de la Argentina en la Estación Experimental Agropecuaria (EEA) Famaillá, Instituto Nacional de Tecnologםa Agropecuaria (INTA). Se estudió la biología reproductiva de la colección entre 2014 y 2021 para desarrollar estrategias de mejoramiento y conservación de estos recursos genéticos. Se analizaron 56 genotipos, que fueron 2n=2x=22. La viabilidad del polen fue alta (69,4 a 99,6%) excepto en cuatro de ellos que exhibieron baja viabilidad (36,0 a 51,5%) en 2015 y 2017. Se obtuvieron 48 combinaciones genotópicas mediante cruzamientos controlados manuales. En 12 de estas combinaciones, se observó un tubo polםnico en la zona estilar y, en cuatro de ellas, un tubo polםnico en el saco embrionario; estas observaciones indican, respectivamente, relaciones polen-pistilo incompatibles y compatibles. Se obtuvieron semillas rellenas normales en todas las combinaciones genotópicas compatibles. La incompatibilidad observada podría deberse al funcionamiento del sistema de autoincompatibilidad homomórfica esporofítica, un sistema de incompatibilidad cruzada, o ambos. La compatibilidad observada permitirá la planificación de cruzamientos controlados dentro y entre introducciones de diferentes orígenes geográficos para generar progenies genéticamente variables con fines de mejoramiento genético.

Chromosomal evolution in Cryptangieae Benth. (Cyperaceae): Evidence of holocentrism and pseudomonads.

Cryptangieae has recently been revised based on morphology and molecular phylogeny, but cytogenetic data is still scarce. We conducted this study with the aim of investigating the occurrence of holocentric chromosomes and pseudomonads, as well as understanding the mode of chromosomal evolution in the tribe. We performed analyses of meiotic behavior, chromosome counts, and reconstruction of the ancestral state for the haploid number. We present novel cytogenetic data for eight potentially holocentric species: Cryptangium verticillatum, Krenakia junciforme, K. minarum, Lagenocarpus bracteosus, L. griseus, L. inversus, L. rigidus, and L. tenuifolius. Meiotic abnormalities were observed, with parallel spindles being particularly noteworthy. Intra-specific variations in chromosome number were not found, which may indicate an efficient genetic control for the elimination of abnormal nuclei. The inferred ancestral haploid number was n = 16, with dysploidy being the main evolutionary mechanism. At least five chromosomal fissions occurred in Krenakia (n = 21), followed by a further ascending dysploidy event in Lagenocarpus (n = 17). As proposed for Cyperaceae, it is possible that cladogenesis events in Cryptangieae were marked by numerical and structural chromosomal changes.

Phenology, floral and reproductive biology of Dichorisandra rhizantha Aona (Commelinaceae), an endemic endangered species in an Atlantic Forest fragment.

Commelinaceae is an important component of the Atlantic Forest and its analysis can provide information on the conservation of that species. This study had the objective of analyzing the phenology and floral and reproductive biology of Dichorisandra rhizantha, growing in an Atlantic Forest fragment, to obtain data related to the form of reproduction and pollination mechanism of this species. We monitored the phenophases and reproductive biology of the D. rhizantha in the area studied using different methodologies and microscopy techniques (LM, SEM and FM). The flowering period occurs during the dry season. The species is andromonoecious and has daytime anthesis (4:30 a.m.-1:30 p.m.). It has purple zygomorphic flowers and rimose but functionally poricidal anthers. The pollen viability was 97.6% and the two floral morphs investigated contained a large quantity of pollen grains. The time of greatest stigma receptivity (anthesis) coincided with the pollen viability. Cross-pollination was the only efficient reproductive system of the species, with observation of gametophytic self-incompatibility and pollen tubes growth only as far as the middle or lower third of the pistil. We observed bee species of the genera Xylocopa and Euglossa visiting the flowers and acting as effective pollinators. The immature fruits were formed after about 3.25 ± 1 days, and the presence was noted of ants of the genus Cephalotes acting as dispersers of the seeds, with aril serving as the reward. Although the natural reproductive system is efficient, D. rhizantha is endangered, mainly due to fragmentation of habitat caused by deforestation and urban encroachment.

Adaptation of Interspecific Mesoamerican Common Bean Lines to Acid Soils and High Temperature in the Amazon Region of Colombia.

Knowledge of the physiological basis for improved genetic adaptation of common bean (Phaseolus vulgaris L.) lines to acid soils and high temperature conditions in the Amazon region of Colombia is limited. In this study, we evaluated the differences among 41 common bean lines in energy use, leaf cooling, photosynthate partitioning to pod formation and grain filling, and grain yield over two seasons under acid soil and high temperature stress in the Amazon region of Colombia. Common bean lines evaluated included medium and large seeded interspecific lines of Mesoamerican and Andean gene pools with different levels of adaptation to abiotic stress conditions and some lines are improved for iron and zinc (biofortified) concentration in seeds. We found three bean lines (GGR 147, SMG 21 and SMG 12) that were superior in their photosynthetic response, leaf cooling, photosynthate partitioning ability to pod formation and grain filling, resulting in grain yields exceeding 1900 kg ha-1 under acid soil and high temperature stress conditions. The superior photosynthetic performance was attributed to the efficient use of absorbed energy on the electron level in thylakoids, which is mainly oriented to a higher quantum yield of PSII (ΦII), lower energy dissipation in the form of heat (ΦNPQ), high linear electron flow (LEF) and high fraction of PSI centers in open state (PSIopen). We speculate that these photosynthetic and photosynthate partitioning responses of superior bean lines are part of the genetic adaptation to acidic soils and high temperature stress conditions. Among the evaluated bean lines, three lines (GGR 147, SMG 21 and SMG 12) combined the desirable attributes for genetic improvement of stress tolerance and biofortification. These lines can serve as parents to further improve traits (energy use efficiency and multiple stress resistance) that are important for bean production in the Amazon region.

The Effect of Temperature and Growth Regulators on Callus Induction in Watermelon Anthers

HIGHLIGHTS Callogenesis was induced from watermelon anthers The auxin 2,4-D at 2.0 and 5.0 μM concentrations induced callus formation. Anthers' responses to the pre-treatment at 4 °C varied according to the watermelon genotype.

Abstract Callus induction is one of the pathways required for haploid plant regeneration through anther culture. Pollen viability, as well as the effect of growth regulators and cold pretreatment on anthers of two watermelon lines (Smile and Sugar Baby) to induce callus formation were herein evaluated. Pollen viability was estimated through the staining technique using 2% acetic carmine. Male flower buds were collected and disinfested to allow removal anthers. These anthers were placed on Murashige and Skoog medium, which was supplemented with 2,4-dichlorophenoxyacetic (2,4-D) at 0.0, 0.5, 1.0, 2.0 or 5.0 μM or with 6-benzylaminopurine at 0.0, 0.5, 1.0, 1.5, or 2.0 μM, in combination with 2.0 μM of 2,4-dichlorophenoxyacetic. Anthers were pretreated at 4 °C, for two days and then placed in vitro. Both watermelon lines provided high pollen viability rates (from 93 to 98%). The 2.0 and 5.0 μM concentrations of 2,4-D stimulated higher friable callus formation. The optimal concentration of 2,4-D was estimated at 3.78 μM and 4.17 μM, which had callus induction rates of 64% and 52%, respectively. The combination of 2.0 μM of 2,4-D and 6-benzylaminopurine did not lead to increased anther response to callus induction. The pre-treatment applied to flower buds at 4 °C enabled callus induction and the anther response to callus induction was genotype-dependent.

Influence of high temperature on the reproductive biology of dry edible bean (Phaseolus vulgaris L. )

The aim of this study was to investigate the effect of heat stress on 12 bean genotypes through the analysis of their reproductive biology in terms of flowering, pollen viability, meiotic behavior, and production. Plants were grown in a climate chamber at 25-20 °C (day and night) and at a high temperature treatment 37-26 °C (day and night) from the vegetative (V4) development stage to physiological maturity. The experimental design was 2 × 12 factorial arrangement with six replications and the factors consisted of heat treatments and genotypes. In three replications, the number of newly opened flowers was checked daily. At physiological maturity, the following traits were evaluated: percentage of pod set, number of pods, number of viable seeds, number of aborted seeds, 100 seed weight, and seed yield (g per plant). The other three replications were used to collect flowers to create slides to study viability of the pollen grain and analyze the meiotic behavior. The heat treatment factor significantly affected the following traits: total number of pollen grains, number of flowers, number of pods, pod set, number of viable seeds, 100 seed weight, and seed yield. The raised temperature reduced these variables, except for percentage of pod set, and increased meiotic irregularities. The mean values regarding seed yield were 16.39 g per plant for the control treatment and 7.46 g per plant under high temperature. IAC Imperador, FT Nobre, Pérola, BRS Estilo, and IAC Diplomata stood out for higher bean seed yield under increased temperature.(AU)

Influence of high temperature on the reproductive biology of dry edible bean (Phaseolus vulgaris L. )

The aim of this study was to investigate the effect of heat stress on 12 bean genotypes through the analysis of their reproductive biology in terms of flowering, pollen viability, meiotic behavior, and production. Plants were grown in a climate chamber at 25-20 °C (day and night) and at a high temperature treatment 37-26 °C (day and night) from the vegetative (V4) development stage to physiological maturity. The experimental design was 2 × 12 factorial arrangement with six replications and the factors consisted of heat treatments and genotypes. In three replications, the number of newly opened flowers was checked daily. At physiological maturity, the following traits were evaluated: percentage of pod set, number of pods, number of viable seeds, number of aborted seeds, 100 seed weight, and seed yield (g per plant). The other three replications were used to collect flowers to create slides to study viability of the pollen grain and analyze the meiotic behavior. The heat treatment factor significantly affected the following traits: total number of pollen grains, number of flowers, number of pods, pod set, number of viable seeds, 100 seed weight, and seed yield. The raised temperature reduced these variables, except for percentage of pod set, and increased meiotic irregularities. The mean values regarding seed yield were 16.39 g per plant for the control treatment and 7.46 g per plant under high temperature. IAC Imperador, FT Nobre, Pérola, BRS Estilo, and IAC Diplomata stood out for higher bean seed yield under increased temperature.

Intraspecific genetic divergence within Helianthus niveus and the status of two new morphotypes from Mexico.

PREMISE: Collecting and characterizing the genetic diversity of wild relatives of crops can contribute importantly to sustainable crop production and food security. Wild sunflower, Helianthus niveus, occurs in arid regions in western North America and is partially cross-compatible with the cultivated sunflower (H. annuus). We assessed phylogenetic relationships and patterns of genetic divergence among three previously described subspecies (subsp. niveus, subsp. canescens, and subsp. tephrodes) as well as two new morphotypes of H. niveus recently discovered in extreme drought and dune habitats in Baja California, Mexico. METHODS: We measured 50 plants growing in a common garden for 27 morphological traits and conducted principal component analysis to assess patterns of phenotypic variation. Genome size of each accession was determined using flow cytometry. Pollen viability of first generation hybrids between taxa was tested to infer the strength of intrinsic postzygotic reproductive barriers. Finally, genotyping-by-sequencing data were used to investigate the genetic structure and phylogenetic relationships among the previously described subspecies and new morphotypes. RESULTS: The intraspecific genetic and phenotypic divergence of H. niveus populations closely tracks their geographical distribution. Subspecies niveus is phenotypically, genetically, and reproductively distinct from the other two subspecies and has a larger genome. Therefore, H. niveus as currently circumscribed should be considered to contain two distinct species, H. niveus and H. tephrodes. ABBA-BABA tests revealed substantial introgression between subsp. canescens and its sympatric congener H. petiolaris, which might contribute to their morphological similarities. The two new morphotypes collected in Mexico represent local ecotypes of subsp. niveus that occur in extreme drought and dune environments. Mantel tests showed a strong positive correlation between genetic and geographic distances. CONCLUSIONS: We conclude that geographic isolation is primarily responsible for intraspecific genomic divergence within H. niveus, while patterns of phenotypic variation appear to have been shaped by ecological selection and interspecific introgression.

The mitochondrial NAD+ transporter (NDT1) plays important roles in cellular NAD+ homeostasis in Arabidopsis thaliana.

Nicotinamide adenine dinucleotide (NAD+ ) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD+ biosynthesis is exclusively cytosolic. Hence, NAD+ must be imported into organelles to support their metabolic functions. Three NAD+ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T-DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD+ homeostasis for both metabolic and developmental processes in plants.

Pollen viability and meiotic irregularities in a collection of Stevia rebaudiana (Bertoni) Bertoni cultivated in Tucumán, Argentina / Viabilidad de polen e irregularidades meióticas en una colección de Stevia rebaudiana (Bertoni) Bertoni cultivada en Tucumán, Argentina

Stevia rebaudiana (Bertoni) Bertoni (2n=2x=22) (Asteraceae family) is a species of economic value due to the presence of steviol glycosides in leaves -mainly stevioside and rebaudioside A- which are non-caloric sugars. In 2013, a collection of plants (genotypes) from four contrasting environments was established in Tucuman, Argentina, for evaluation under local conditions and, eventually, breeding purposes. As a first step, pollen viability and meiosis was studied in a sample of 56 plants. The percentage of pollen viability varied from medium (69.4%) to high (99.6%) in 52 of them, but from low (36.5%) to relatively low (51.5%) in the remaining four. The latter four plants also presented pollen grains of heterogeneous size, which were classified as n (normal, the most frequent size in the sample),

Stevia rebaudiana (Bertoni) Bertoni (2n=2x=22) (familia Asteraceae) es una especie de valor economico debido a la presencia de glucosidos de esteviol en sus hojas, principalmente esteviosido y rebaudiosido A, que son azucares no caloricos. En 2013, se establecio una coleccion de plantas (genotipos) provenientes de cuatro ambientes contrastantes en Tucuman, Argentina, para su evaluacion en condiciones locales y, eventualmente, con fines de mejoramiento genetico. Como primer paso, se estudio la viabilidad del polen y la meiosis en una muestra de 56 plantas. El porcentaje de viabilidad del polen fue de medio (69,4%) a alto (99,6%) en 52 de ellas, pero de bajo (36,5%) a relativamente bajo (51,2%) en las cuatro restantes. Las ultimas cuatro plantas tambien presentaron granos de polen de tamano heterogeneo, que se clasificaron como n (normal, el tamano mas frecuente en la muestra),

Genetic and ecological niche modeling of Calydorea crocoides (Iridaceae): an endemic species of Subtropical Highland Grasslands

Abstract Evolutionary analyses have been widely used for evaluation of genetic diversity of natural populations and correlate these data to the fitness of the species, especially in the case of threatened species. Calydorea crocoides occurs in a restricted area at altitudes from 800 to 1500 m in southern Brazil and is considered endangered. A study assessing genetic diversity, cytogenetic features and ecological niche was performed aiming to characterize C. crocoides by multidisciplinary approaches. Molecular data highlighted that most of the total variation (76%; p < 0.001) was found within populations and the parameters of genetic diversity were high at the species level (PPB = 98.97%; I = 0.4319; h = 0.2821). Gene flow (Nm) was estimated in 0.97 individuals per generation. Cytogenetically, C. crocoides presents a bimodal karyotype and low asymmetry. DAPI banding pattern was uniform, but the CMA-signal evidenced a pericentric inversion in the population ESC688. The species presents high pollen viability and two different morphologies of pollen grains. Our data showed high levels of polymorphism maintained in this species that could ensure conservationist practices in which the main goal is to preserve the evolutionary potential of the species through the maintenance of genetic diversity.

Cytological and genome size data analyzed in a phylogenetic frame: Evolutionary implications concerning Sisyrinchium taxa (Iridaceae: Iridoideae)

Abstract Sisyrinchium is the largest genus of Iridaceae in the Americas and has the greatest amount of cytological data available. This study aimed at investigating how genomes evolved in this genus. Chromosome number, genome size and altitude from species of sect. Viperella were analyzed in a phylogenetic context. Meiotic and pollen analyses were performed to assess reproductive success of natural populations, especially from those polyploid taxa. Character optimizations revealed that the common ancestor of sect. Viperella was probably diploid (2n = 2x =18) with two subsequent polyplodization events. Total DNA content (2C) varied considerably across the phylogeny with larger genomes detected mainly in polyploid species. Altitude also varied across the phylogeny, however no significant relationship was found between DNA content changes and altitude in our data set. All taxa presented regular meiosis and pollen viability (> 87%), except for S. sp. nov. aff. alatum (22.70%), suggesting a recent hybrid origin. Chromosome number is mostly constant within this section and polyploidy is the only source of modification. Although 2C varied considerably among the 20 taxa investigated, the diversity observed cannot be attributed only to polyploidy events because large variations of DNA content were also observed among diploids.

URS Campestre seedless orange: a new mutant with female sterility

ABSTRACT: Seedlessness in fruit is a trait that is much sought after by juice making industries. Close to the city of São Sebastião do Caí, in the state of Rio Grande do Sul (RS), Brazil, a new mutant orange originating from natural mutation was identified and selected as a seedless material. To determine the mechanisms involved in the absence of seeds, the reproductive structures of this new mutant by comparison with a Valencia sweet orange as control, a cultivar with a profusion of seeds, was analyzed in terms of meiotic behavior, meiotic index, pollen viability, in vitro germination, and ovule features to determine the grounds for seed absence. Other morphological analyzes allowed for visualizing the structures of normal appearance and size in both cultivars. Meiotic analysis identified chromosome normal pairing with a predominance of bivalents at diakinesis and metaphase 1. URS Campestre flowers at different developmental stages had anthers and ovaries whose dimensions are typical while pollen grain analysis pointed to a standard developmental pattern, normal meiosis, high viability (84 %) and elevated in vitro pollen tube germination rates (63 %). The cv. Valencia and URS Campestre ovules had a similar shape and morphology, sharing an anatropous orientation, and two integuments. In the internal ovule analyses of Valencia sweet oranges, normal embryo sac cells were identified: presence of one egg cell and two synergids, three antipodes and a bigger and central cell containing two polar nuclei. However, the analysis of ovules from URS Campestre reveals an apparent senescence or non-formation of an embryo sac, where only a few highly stained and collapsed cells could be identified. These results led to the conclusion that female sterility in URS Campestre, with a total absence of a female gametophyte, is the limiting factor for fertilization and seed production.(AU)

URS Campestre seedless orange: a new mutant with female sterility

ABSTRACT: Seedlessness in fruit is a trait that is much sought after by juice making industries. Close to the city of São Sebastião do Caí, in the state of Rio Grande do Sul (RS), Brazil, a new mutant orange originating from natural mutation was identified and selected as a seedless material. To determine the mechanisms involved in the absence of seeds, the reproductive structures of this new mutant by comparison with a Valencia sweet orange as control, a cultivar with a profusion of seeds, was analyzed in terms of meiotic behavior, meiotic index, pollen viability, in vitro germination, and ovule features to determine the grounds for seed absence. Other morphological analyzes allowed for visualizing the structures of normal appearance and size in both cultivars. Meiotic analysis identified chromosome normal pairing with a predominance of bivalents at diakinesis and metaphase 1. URS Campestre flowers at different developmental stages had anthers and ovaries whose dimensions are typical while pollen grain analysis pointed to a standard developmental pattern, normal meiosis, high viability (84 %) and elevated in vitro pollen tube germination rates (63 %). The cv. Valencia and URS Campestre ovules had a similar shape and morphology, sharing an anatropous orientation, and two integuments. In the internal ovule analyses of Valencia sweet oranges, normal embryo sac cells were identified: presence of one egg cell and two synergids, three antipodes and a bigger and central cell containing two polar nuclei. However, the analysis of ovules from URS Campestre reveals an apparent senescence or non-formation of an embryo sac, where only a few highly stained and collapsed cells could be identified. These results led to the conclusion that female sterility in URS Campestre, with a total absence of a female gametophyte, is the limiting factor for fertilization and seed production.

Apomixis does not affect visitation to flowers of Melastomataceae, but pollen sterility does.

Apomixis is an asexual seed reproduction mechanism thorough which embryos are originated from material tissues inside the ovules, without precedent fertilisation. It allows plants to colonise new habitats, even in places where flower visitors are scarce or where plants are isolate. Apomixis seems to be related to pollen sterility and, in species with flowers that offer pollen as a reward for pollinators, the amount or quality of the pollen offered by these species may influence the amount of the visits and specific composition of the visitors. In order to test this hypothesis, we studied breeding systems of 16 species of Melastomataceae and their flower visitors, evaluating composition and abundance of the visits to apomictic and sexual species. Apomictic plants with no viable pollen or with pollen with low viability did not receive visits from pollinators, and consequently probably produce strictly apomictic fruits. On the other hand, apomictic and sexual plants with high pollen viability do receive visits; in this case, apomictic plants may produce fruits and seeds through both sexual and apomictic methods. The species composition of insects visiting Melastomataceae with high pollen viability was similar, regardless of whether the plants were apomictic or not. It seems that pollen viability levels are important to determine visits to the flowers irrespective of breeding system.

Estimativa da viabilidade polínica de araçazeiro (Psidium cattleianum Sabine) através de distintos métodos de coloração / Estimation of pollen viability of strawberry guava (Psidium cattleianum Sabine) through distinct staining methods

RESUMO A análise histoquímica dos grãos de pólen através do uso de corantes permite a determinação de sua viabilidade polínica. O objetivo deste estudo foi comparar a eficiência dos corantes orceína acética 2% e reativo de Alexander modificado, para estimar a viabilidade polínica de vinte acessos de araçazeiro (Psidium cattleianum Sabine) – uma árvore de frutos muito apreciados que também é utilizada para fins medicinais. Botões florais na pré-antese foram coletados e fixados em etanol:ácido acético (3:1) por 24 horas, após os botões foram transferidos para etanol 70% e mantidos sob refrigeração. A técnica de esmagamento das anteras foi utilizada no preparo das lâminas. Para cada acesso foram preparadas 2 lâminas por corante e analisados 500 grãos de pólen por lâmina. A viabilidade polínica foi estimada através da porcentagem de grãos de pólen viáveis. De modo geral, observou-se que os grãos de pólen corados com orceína acética 2% apresentaram viabilidade alta, acima de 98,1%, e os acessos não diferiram entre si. A viabilidade polínica através da coloração com a solução de Alexander variou de 43% (acesso Silveira Martins 1) a 97% (acesso Candelária 1), mostrando que provavelmente exista variabilidade genética entre os acessos. Em apenas dois acessos não houve diferença significativa entre os valores de viabilidade encontrados através dos dois corantes testados. Conclui-se então que a orceína acética 2% superestimou a viabilidade polínica de P. cattleianum e que o reativo de Alexander é o mais preciso, devido a sua dupla coloração (verde de malaquita + fucsina ácida). Assim, indica-se o reativo de Alexander para análise de estimativa da viabilidade polínica de genótipos de araçá envolvidos em um programa de melhoramento genético.

ABSTRACT The histochemical analysis of pollen grains through the use of stains makes it possible to determine their pollen viability. The purpose of this study was to compare the efficiency of 2% acetic orcein and Alexander’s stain modified, in estimating the pollen viability of twenty strawberry guava accessions (Psidium cattleianum Sabine) – a valued fruit tree which is used for medicinal purposes. Pre-anthesis flower buds were collected and fixed in ethanol:acetic acid (3:1 v/v) for 24 hours at room temperature, subsequently they were stored in 70% ethanol under refrigeration. The anther squash technique was performed for slide preparation. In each accession two slides per stain and 500 pollen grains were analyzed. The pollen viability was estimated according to the percentage of viable pollen grains. Overall, it was observed that pollen grains stained with 2% acetic orcein evinced a high viability, higher than 98,1%, and the accessions did not differ. The pollen viability estimated with Alexander’s stain varied from 43% (accession of Silveira Martins 1) to 97% (accession of Candelária 1), indicating that there is probably genetic variability among the accessions. There was no significant difference among the levels of viability found by means of the two stains in only two accessions. In conclusion, 2% acetic orcein overestimated the pollen viability of P. cattleianum, whereas the Alexander’s stain is in turn more accurate due to its two stains (green malachite and fuchsine). Thus, it is indicated Alexander’s stain for analysis estimation of pollen viability of strawberry guava genotypes involved in a genetic improvement program.

Successful crosses between fungal-resistant wild species of Arachis (section Arachis) and Arachis hypogaea.

Peanut (Arachis hypogaea) is the fifth most produced oil crop worldwide. Besides lack of water, fungal diseases are the most limiting factors for the crop. Several species of Arachis are resistant to certain pests and diseases. This study aimed to successfully cross the A-genome with B-K-A genome wild species previously selected for fungal disease resistance, but that are still untested. We also aimed to polyplodize the amphihaploid chromosomes; cross the synthetic amphidiploids and A. hypogaea to introgress disease resistance genes into the cultivated peanut; and analyze pollen viability and morphological descriptors for all progenies and their parents. We selected 12 A-genome accessions as male parents and three B-genome species, one K-genome species, and one A-genome species as female parents. Of the 26 distinct cross combinations, 13 different interspecific AB-genome and three AA-genome hybrids were obtained. These sterile hybrids were polyploidized and five combinations produced tetraploid flowers. Next, 16 combinations were crossed between A. hypogaea and the synthetic amphidiploids, resulting in 11 different hybrid combinations. Our results confirm that it is possible to introgress resistance genes from wild species into the peanut using artificial hybridization, and that more species than previously reported can be used, thus enhancing the genetic variability in peanut genetic improvement programs.

Short-term hybridisation activates Tnt1 and Tto1 Copia retrotransposons in wild tuber-bearing Solanum species.

Interspecific hybridisation in tuber-bearing species of Solanum is a common phenomenon and represents an important source of variability, crucial for adaptation and speciation of potato species. In this regard, the effects of interspecific hybridisation on retrotransposon families present in the genomes, and their consequent effects on generation of genetic variability in wild tuber-bearing Solanum species, are poorly characterised. The aim of this study was to analyse the activity of retrotransposons in inter- and intraspecific hybrids between S. kurtzianum and S. microdontum, obtained by controlled crosses, and the effects on morphological, genetic and epigenetic variability. For genetic and epigenetic analysis, S-SAP (sequence-specific amplification polymorphism) and TMD (transposon methylation display) techniques were used, respectively, with specific primers for Tnt1 and Tto1 retrotransposon families (Order LTR, Superfamily Copia). The results indicate that at morphological level, interspecific hybrid genotypes differ from their parental species, whereas derived intraspecific hybrids do not. In both cases, we observed significant reductions in pollen grain viability, and a negative correlation with Tnt1 mobility. Both retrotransposons, Tto1 and Tnt1, were mobilised in the genotypes analysed, with mobility ranging from 0 to 7.8%. Furthermore, at the epigenetic level, demethylation was detected in the vicinity of Tnt1 and Tto1 in the hybrids compared with the parental genotypes. These patterns were positively correlated with the activity of the retrotransposons. The results suggest a possible mechanism through which hybridisation events generate genetic variability in tuber-bearing species of Solanum through retrotranposon activation.

Abnormal behavior of spindle during microsporogenesis of passiflora (passifloraceae) / Comportamento anormal de fuso durante a microsporogênese de passiflora (passifloraceae)ien

Passiflora genus, Passifloraceae family, has more than 500 species and 120 of them are native species of Brazil. All species produce fruits that are used as food, medicine and decoration. Floral buttons of five species were collected and fixed in a mixture of ethanol and acetic acid (3:1). The slides were prepared by squashing and staining with 1% propionic carmine. Results showed that during microsporogenesis there were few irregularities, mostly frequently related to chromosome irregular segregation as: precocious migration to poles in metaphase I and II, non-oriented bivalent chromosomes at metaphase I and II, and laggard chromosomes in anaphase I and II, forming micronuclei in telophases I and II and tetrad with microcyte. Another observed irregularity is related to the organization of spindle fibers in meiosis II as they organize themselves in T and V shapes and in sequential spindle. However, in the V-shaped spindle configuration, there was fusion between two nuclei that were close, forming triads instead of tetrads. Irregular chromosome segregation, abnormal spindles and irregularities in the cytokinesis process were responsible for the formation of monads, dyads, triads and polyads. However, the pollen grain viability was not harmed, presenting an 83.98% to 98.59% fertility variation.

O gênero Passiflora, família Passifloraceae, apresentam mais de 500 espécies, havendo no Brasil aproximadamente 120 espécies nativas. Todas as espécies produzem frutos que são utilizados como produtos alimentícios, medicinais e ornamentais. Botões florais de cinco espécies foram coletados e fixados em etanol/acido acético (3:1). As lâminas foram preparadas utilizando a técnica de esmagamento e coradas com carmim propiônico a 1%. Como resultado, observou-se que durante a microsporogênese poucas irregularidades foram encontradas, as mais frequentes estão relacionadas à segregação irregular dos cromossomos, tais como: migração precoce para os pólos em metáfase I e II, bivalente não orientado em metáfase I e II, e cromossomos retardatários em anáfase I e II, levando a formação de micronúcleos em telófases I e II, e micrócito em tétrades. Outra irregularidade observada esta relacionada a organização das fibras dos fusos em meiose II, que se organizam na forma em T, em V e fuso sequencial. Na configuração de fuso na forma de V ocorreu fusão entre dois núcleos que estavam próximos, formando tríade ao invés de tétrade. A segregação irregular dos cromossomos, a formação de fusos anormais e as irregularidades no processo de citocinese foram responsáveis pela formação de mônades, díades, tríades e políades como produtos final da meiose. Porém, a viabilidade dos grãos de pólen não foi comprometida, apresentado uma variação de 83,98% a 98,59% de fertilidade.

Botanical aspects of Heteropterys umbellata (Malpighiaceae): a cytological and palynological approach

The genus Heteropterys is one of the major genera in Malpighiaceae. However, few cytological and palynological studies were reported. The present work described for the first time the chromosome number, heterochromatin pattern, meiotic behavior, pollen viability and palynological aspects of Heteropterys ubellata, a very spread species. One large Brazilian population was evaluated using conventional techniques for meiotic studies and acetolyse to access the pollen morphology. The species showed 2n = 20 chromosomes, normal meiotic development and viable pollens. Great blocks of heterochromatin were observed around the centromers. DAPI staining was positive for centroeric heterochroatin, hile CMA3 ark as observed just at terinal regions of one pair of hoologues chromosomes. This result and the presence of one chromosome pair attached to the nucleoli during the pachytene and diakinesis suggested the presence of only one pair of NORs. Palynological analysis revealed that pollen grains are apolar, 6 porate and with colpoids associated to all pores. The pollen content was positive for the starch test, and the exine was rugulate with little psilate regions.

O gênero Heteropterys é um dos maiores gêneros entre as Malpighiaceae. Entretanto, poucos estudos citológicos e palinológicos foram relatados. O presente trabalho descreveu pela primeira vez o número cromossômico, o padrão heterocromático, o comportamento meiótico, a viabilidade polínica e aspectos palinológicos de Heteropterys umbellata, uma espécie muito disseminada. Uma grande população brasileira foi estudada utilizando técnicas convencionais para o estudo de cromossomos meióticos e acetólise para acessar a morfologia polínica. A espécie apresentou 2n = 20 cromossomos, desenvolvimento meiótico normal e pólens viáveis. Grandes blocos de heterocromatina foram observados ao redor dos cen-trômeros. A coloração com DAPI foi positiva para a hetero-cromatina centromérica, enquanto marcas com CMA3 foram observadas somente em um sítio terminal de um par de cromossomos homólogos. Este resultado e a presença de um par de cromossomos associados a um nucléolo durante o paquíteno e diacinese sugerem a presença de somente um par de RONs. A análise palinológica revelou que os grãos de pólen são apolar, com 6 poros e colpóides associados a todos eles. O conteúdo polínico foi positivo para o teste de amido e a exina mostrou-se rugulada com poucas regiões psiladas.