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The available methods for plant transformation and expansion beyond its limits remain especially critical for crop improvement. For grass species, this is even more critical, mainly due to drawbacks in in vitro regeneration. Despite the existence of many protocols in grasses to achieve genetic transformation through Agrobacterium or biolistic gene delivery, their efficiencies are genotype-dependent and still very low due to the recalcitrance of these species to in vitro regeneration. Many plant transformation facilities for cereals and other important crops may be found around the world in universities and enterprises, but this is not the case for apomictic species, many of which are C4 grasses. Moreover, apomixis (asexual reproduction by seeds) represents an additional constraint for breeding. However, the transformation of an apomictic clone is an attractive strategy, as the transgene is immediately fixed in a highly adapted genetic background, capable of large-scale clonal propagation. With the exception of some species like Brachiaria brizantha which is planted in approximately 100 M ha in Brazil, apomixis is almost non-present in economically important crops. However, as it is sometimes present in their wild relatives, the main goal is to transfer this trait to crops to fix heterosis. Until now this has been a difficult task, mainly because many aspects of apomixis are unknown. Over the last few years, many candidate genes have been identified and attempts have been made to characterize them functionally in Arabidopsis and rice. However, functional analysis in true apomictic species lags far behind, mainly due to the complexity of its genomes, of the trait itself, and the lack of efficient genetic transformation protocols. In this study, we review the current status of the in vitro culture and genetic transformation methods focusing on apomictic grasses, and the prospects for the application of new tools assayed in other related species, with two aims: to pave the way for discovering the molecular pathways involved in apomixis and to develop new capacities for breeding purposes because many of these grasses are important forage or biofuel resources. [ABSTRACT FROM AUTHOR]

Apomixis is the asexual production of seeds by plants and, in theory, would render low genetic diversity and even clonal lineages. However, recent studies have shown otherwise, although is not always clear where the genetic diversity of obligate apomicts comes from.We evaluated the genetic diversity among sister seedlings of M. albicans, an obligate apomictic species in Cerrado, Neotropical Savannas in Central Brazil. A total of 50 seedlings from five individuals were analysed using ISSR primers. We obtained 107 fragments, all with good resolution, consistently observed and replicable.The percentage of polymorphic loci ranged from 28.04% to 33.64% and Shannon's information index (I) averaged 0.173. The expected heterozygosity (He) averaged 0.117, similar to the observed for populations of M. albicans and other selfed species. Only two seedlings showed the same genotype (possible clones), but most differed at least for five loci. Most of variance was among progenies (62%), but we found that 38% was within progenies. Genetic distances separated the progenies in two groups, and analogous analyses between individuals reconstructed the original progenies clustering.The results confirmed a relatively high genetic diversity among sister seedling of this obligatory apomictic plant and clones were rare. This diversity can be generated during development, probably by restitutional meiosis or other recombination processes. These differences may accumulate into lineages and populations well adapted to heterogenous Cerrado environment. [ABSTRACT FROM AUTHOR]

Cenchrus spp. encompasses forage grasses that are especially important for drylands. Thus, information on their reproductive mechanism and genetic diversity is needed. The Active Germplasm Bank (AGB) of the Embrapa Semiárido (Tropical semiarid research center) has 115 accessions of Cenchrus spp. that were not molecularly characterized. Therefore, the objective of this work was to evaluate the genetic diversity of Cenchrus spp. accessions in the AGB, and identify their reproductive mechanisms using DNA markers. Specific SCAR markers Q8H, UGT197, and PCAB10, in addition to the SCAR marker 4HS* were used to identify genotypes with apomictic and sexual reproduction modes, respectively; the genetic diversity was characterized using the ISSR D12 (GA) 6CG and HB14 (CTC)3GC markers. The reproductive mechanism was analyzed using images for the presence or absence of the band in the expected amplicon size. For the analysis of genetic diversity, the gel images were exported to the BioNumerics Software (Applied Maths), in which polymorphism data were obtained and the dissimilarity dendrogram constructed by the UPGMA clustering method using the Dice coefficient. The markers related to the apomictic region were positive for all accessions. However, only the accessions CPATSA 102 and CPATSA 134 had positive markers using the primers for both reproduction strategies, indicating that these individuals can be facultative sexual. The cluster analysis discriminated two large groups and the accessions clustered mainly by the place of origin. It was also possible to identify duplicates within AGB. The identification and exclusion of duplicate accessions provides financial and labor savings in maintaining and managing the AGB. The two sexual accessions were selected for the breeding programs of Cenchrus spp. in Brazil. [ABSTRACT FROM AUTHOR]

Geographic parthenogenesis, range expansion of apomictic plants after climate changes, has been described for Northern Hemisphere gametophytic apomicts. But similar trends have been observed for sporophytic apomicts of Cerrado, the savannas in Brazil. Eriotheca pubescens is a common Cerrado tree, an agamic complex of either hexaploid/polyembryonic apomicts or tetraploid/monoembryonic sexual individuals. Some populations have been described as a new species, Eriotheca estevesiae, all included in the Eriotheca Stellate Trichome Species Complex (ESTSC). Since breeding systems and ploidy are clearly associated with polyembryony and stomatal size, we used these ancillary features to map the reproductive and ploidy level traits of E. pubescens and E. estevesiae.Leaves and seeds were collected from individuals of 19 populations. Seeds were evaluated for the presence of polyembryony and leaves for stomatal measurements.Eight populations were monoembryonic while another eight were polyembryonic and for other three, the embryonic pattern was not readily verified. E. pubescens polyembryonic and hexaploid populations formed a homogeneous group, but monoembryonic plants were more variable. E. estevesiae populations were monoembryonic with smaller stomata. In contrast, some E. pubescens monoembryonic populations further south presented larger stomata. Despite these outliers, possibly mixed populations, stomatal size and embryonic pattern differed from northern to southern populations.Embryonic pattern and stomatal size indicated that northernmost populations of Eriotheca STSC (E. estevesiae) are diploid and sexual. Southernmost populations, mostly polyembryonic and with large stomata, are hexaploid and apomictic. This is in agreement with geographic parthenogenesis and range expansion of apomictic lineages to southern habitats available after the last glacial maximum. [ABSTRACT FROM AUTHOR]

Studies on the reproductive biology of Dorstenia species are scarce. We analyzed the sexuality, floral biology, and diaspore dispersal of Dorstenia arifolia and D. bonijesu, including their breeding systems and pollinators, in natural populations from Minas Gerais state, southeastern Brazil. We report here, for the first time, the occurrence of both unisexual (male) and bisexual coenanthia on the same individuals of Dorstenia arifolia, while only bisexual coenanthia were observed in D. bonijesu. In both species all coenanthia were long-lived and bisexual coenanthia were protogynous. We observed the occurrence of self-compatibility and myophily in Dorstenia arifolia, a species which was pollinated by small Lauxaniidae flies. Those insects not only used pollen as food resource but they also used coenanthia as oviposition sites, which is the first report to the studied species. Dorstenia bonijesu produced seeds, even having no floral visitors, suggesting an apomictic mechanism, which is a new breeding system reported to the genus. Both studied species are autochorous, but D. bonijesu also exhibited a peculiar type of hydrochory. The novelties reported herein indicate the need of additional studies to improve the understanding of the reproductive attributes of Dorstenia species. [ABSTRACT FROM AUTHOR]

The Asteraceae have a high diversity of reproductive mechanisms, as shown by studies conducted mainly with species from temperate regions. Aiming to contribute knowledge on the reproduction of tropical species, we compared the floral biology and breeding systems of the co-occurring speciesAdenostemma brasilianum,Bidens segetumandGrazielia intermedia, and additionally identified their pollinators. The study was conducted in a seasonal semideciduous forest fragment in southeastern Brazil. Florets opened in early morning and were protandrous. In the first day of anthesis, secondary pollen presentation occurred during the staminate phase, mainly in the morning. In the same day, the pistillate phase began, being long inA. brasilianumandB. segetum(up to 4 days) and short inG. intermedia(1 day). Pollen viability was high in all three species, except for twoG. intermediaindividuals which showed male sterility, thus characterising a gynodioecious population. Self-compatibility was observed inA. brasilianumand self-incompatibility inB. segetum. InG. intermedia, besides sexual reproduction, apomixis was also observed. This is the first report of apomixis in the genusGrazielia. Although the studied species show distinct reproductive mechanisms, all of them benefitted from cross-pollination, which was mostly done by butterflies. [ABSTRACT FROM AUTHOR]

Senna rugosa (G.Don) H.S.Irwin & Barneby (Leguminosae) is a species from the Brazilian Cerrado commonly used for medicinal purposes. Chromosome variability (n = 14 and 28) described for this species indicates the occurrence of chromosome races and recent polyploidization in its evolution history. The aim of this study was to verify the relationship between ploidy level and occurrence of polyembryony in 5 accessions of S. rugosa. Cytogenetic analysis revealed the type of interphasic nuclei, chromosome number and morphology, and nuclear DNA content. Frequency of polyembryony was estimated by germination test. Pollen grain viability was estimated by different techniques. There was variability among the accessions of S. rugosa for interphasic nucleus type (semireticulate and nonreticulate), genome size (2.57 to 2.80 pg/2C), and polyembryony frequency (from 2 to 4 embryos). Pollen grain viability was 2.5% to 20%, depending on the technique used. Numeric chromosome variability was found among meristematic cells from each individual (2n = 28 to 2n = 112), with prevalence of 2n = 56. In mitotic metaphases with 2n = 56, all chromosomes were metacentric and of similar size. The data described, combined with previous descriptions of irregularities in chromosome pairing, suggest that S. rugosa is an autopolyploid facultative apomictic species, with interpopulation variability for level of apomixis expression. [ABSTRACT FROM AUTHOR]

Approximately 70% of the angiosperm species are polyploid, an important phenomenon in the evolution of those plants. But ploidy estimates have often been hindered because of the small size and large number of chromosomes in many tropical groups. Since polyploidy affects cell size, morphometric analyses of pollen grains and stomata have been used to infer ploidy level. Polyploidy is present in many species of the Cerrado, the Neotropical savanna region in Central Brazil, and has been linked to apomixis in some taxa. Eriotheca gracilipes and Eriotheca pubescens are common tree species in this region, and present cytotypes that form reproductive mosaics. Hexaploid individuals (2n = 6x = 276) are polyembryonic and apomictic, while tetraploid and diploid individuals (2n = 2x = 92, 2n = 4x = 184) are sexual and monoembryonic. We tested whether morphometric analysis can be used to estimate ploidy levels in E. gracilipes and E. pubescens individuals. Pollen material from diploid and hexaploid individuals of E. gracilipes, and tetraploid and hexaploid individuals of E. pubescens, were fixed in 50% FAA, and expanded leaves were dried in silica gel. Pollen grains and stomata of at least five individuals from each population were measured. The results demonstrate that all measures were significantly different among cytotypes. Individuals with higher levels of ploidy (hexaploid) all presented measurements that were higher than those with lower levels (diploid and tetraploid). There was no overlap between ploidy levels in each species at 95% confidence interval. Thus, the size of the pollen grains and stomata are effective parameters for analysis of ploidy levels in E. gracilipes and E. pubescens. [ABSTRACT FROM AUTHOR]

Apomixis and polyploidy have been important in the evolution of the angiosperms, and sporophytic apomixis has been associated with polyembryony and polyploidy in tropical floras. We studied the occurrence of polyembryony in populations of tetraploid Anemopaegma acutifolium, A. arvense and A. glaucum from the Brazilian cerrados, and histological features of sexual and apomictic processes were investigated in A. acutifolium. All populations and species were polyembryonic (68.9-98.4% of seeds). Normal double fertilization occurred in most ovules, with exceptions being that 3% of ovules were penetrated but not fertilized and in 4% of ovules both synergids were penetrated. The penetration of both synergids suggests a continuous attraction of pollen tubes and polyspermy. Adventitious embryo precursor cells ( AEPs) arose from nucellar and integumental cells of the ovule in pollinated and unpollinated A. acutifolium, indicating sporophytic apomixis. However, further embryo and endosperm development required pollination and fertilization. This pseudogamy also allows concurrent sexual embryo development. Similar polyembryony rates and polyploidy indicated that A. arvense and A. glaucum are also apomictic, forming an agamic complex similar to that observed for some species of confamilial, but not closely related Handroanthus. The co-occurrence of apomixis and polyploidy in different groups of Bignoniaceae indicates homoplasious origin of these agamic complexes. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 77-91. [ABSTRACT FROM AUTHOR]

Polyembryony has been commonly associated with apomixis in the angiosperms and seems to be more common than expected, even in biomes where sexual reproduction processes are predominant. Recent studies in Cerrado, the Neotropical savannas of Central Brazil, showed high frequencies of apomixis and polyembryony and indicated these processes as reproductive and evolutionary alternatives for plants in these areas. In this sense, we investigated the occurrence of polyembryony and its relationships with ecological (season and type of dispersal, ploidy, species distribution and breeding system) and taxonomic (tribe) factors in the Melastomataceae, a mostly tropical family already known for its high frequency of apomixis and very common in Cerrado. We collected seeds from 69 populations of 53 species, which were sown in germination chambers. After seed germination, the presence and number of seedlings per seed were evaluated as a method to estimate polyembryony. We encountered 18 species (33.96%) with polyembryony (more than one seedling, or gemellar seedlings, originated per seed) concentrated in species of the tribe Miconieae (64%) and Microlicieae (16.67%), but absent in Melastomeae. Monoembryony was present only in sexual species, while all apomictic species were polyembryonic. In Miconia, the polyembryony was correlated with polyploidy, and monoembryony with diploid species. Polyembryony was more common among species with wide distribution in the Cerrado region, which indicates that the presence of gemellar seedlings is important for establishment and survival of the group in the Cerrado biome. [ABSTRACT FROM AUTHOR]

Difficulties in obtaining new breeding lines of Brachiaria (Trin.) Griseb., an important forage grass in Brazil, are mostly related to differences in ploidy among the accessions, and to apomixis, an asexual mode of reproduction. Usually, sexual accessions are diploid while apomicts are polyploid. Induced tetraploids of Brachiaria brizantha (A. Rich.) Stapf have been successfully obtained and this paper presents the results of a study of their reproductive modes and fertility. Despite frequent meiotic aberrations during microspore development, the induced tetraploids produced viable pollen and produced progeny after controlled self-pollination. Similarly to the original diploid sexual progenitor, embryo sacs of the Polygonum type with confirmed meiotic origin were present in the induced tetraploids suggesting chromosome doubling did not alter the reproductive mode. The embryo sac of the Polygonum type was also observed in progenies obtained after self and open pollination. Nevertheless, embryo sacs of the Polygonum and the Panicum types within the same ovule were observed in some progenies obtained after open pollination, probably having resulted from hybridization with tetraploid, apomictic plants. Indeed, the compatibility of the progeny with tetraploid, apomictic B. brizantha was confirmed by the formation of mature caryopses after controlled pollination. Evidence is presented that the induced tetraploids and their progeny are sexual plants and that they are compatible with natural tetraploid B. brizantha. The induced tetraploids will be useful for analyses of apomictic inheritance as well as in the development of sexual tetraploid lines in Brachiaria breeding programs. [ABSTRACT FROM AUTHOR]

The production of triploids and apomictic reproduction are important processes for polyploid establishment and cytotype coexistence, but we know little about the interaction between triploids and facultatively apomictic plants. To bridge this gap, we studied the pollen-dependent, facultatively apomictic orchid Zygopetalum mackayi from high-elevation outcrops of southeast Brazil. We described the nature of the contact between Z. mackayi cytotypes and patterns of genetic diversity and structure based on eight microsatellite markers and 155 individuals of pure tetraploid, pure diploid and mixed cytotype populations. Our results revealed high values of genetic and genotypic diversity within all populations of Z. mackayi. Each cytotype emerged as a genetic distinct cluster, combining individuals from different populations. Triploids clustered in an intermediate position between diploids and tetraploids. Most genetic variance is associated with individuals within populations and genetic differentiation is high among populations. Mixed cytotype populations of Z. mackayi originate from secondary contact. Triploids are hybrids between diploids and tetraploids and likely act as a bridge. Our results point to the predominance of sexual reproduction in all populations but do not corroborate previous basic chromosome number for this species. Polyploidy rather than facultative apomixis may explain the larger geographic distribution of tetraploids of Z. mackayi., (© 2020 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)