Prezygotic Barriers Research Articles

Prezygotic barriers effectively limit hybridization in a rapid evolutionary radiation.

Hybridization is increasingly recognized as an important evolutionary process across the tree of life. In many clades, phylogenomic approaches have permitted unparalleled insight into the extent and frequency of hybridization. However, we continue to lack a deep understanding of the factors that limit and shape patterns of hybridization, especially in evolutionary radiations. In this study, we characterized patterns of introgression across Costus (Costaceae), a young evolutionary radiation of tropical understory plants that maintain widespread interfertility despite exhibiting strong prezygotic reproductive isolation. We analyzed a phylogenomic dataset of 756 genes from 54 Costus species using multiple complementary approaches - D-statistics, gene-tree-based tests, and phylogenetic network analyses - to detect and characterize introgression events throughout the evolutionary history of the radiation. Our results identified a moderate number of introgression events, including a particularly ancient, well-supported event spanning one of the deepest divergences in the clade. Most introgression events occurred between taxa or ancestral lineages that shared the same pollination syndrome (bee-pollinated or hummingbird-pollinated). These findings suggest that prezygotic barriers, including pollinator specialization, have been key to the balance between introgression and reproductive isolation in Costus.

Mismatch between pollen and pistil size causes asymmetric mechanical reproductive isolation across Phlox species.

Characterizing the mechanisms of reproductive isolation (RI) between lineages is key to determining how new species are formed and maintained. In flowering plants, interactions between the reproductive organs of the flower -the pollen and the pistil- serve as the last barrier to reproduction before fertilization. As such, these pollen-pistil interactions are both complex and important for determining a suitable mate. Here, we test whether differences in style length (a part of the pistil) generate a postmating prezygotic mechanical barrier between five species of perennial Phlox wildflowers with geographically overlapping distributions. We perform controlled pairwise reciprocal crosses between three species with long styles and two species with short styles to assess crossing success (seed set). We find that heterospecific seed set is broadly reduced compared to conspecific cross success and reveal a striking asymmetry in heterospecific crosses between species with different style lengths. To determine the mechanism underlying this asymmetric reproductive isolating barrier we assess pollen tube growth in vivo and in vitro. We demonstrate that pollen tubes of short-styled species do not grow long enough to reach the ovaries of long-styled species. We find that short-styled species also have smaller pollen and that both within and between species pollen diameter is highly correlated with pollen tube length. Our results support the hypothesis that the small pollen of short-styled species lacks resources to grow pollen tubes long enough to access the ovaries of the long-styled species, resulting in an asymmetrical, mechanical barrier to reproduction. Such reproductive isolating mechanisms, combined with additional pollen-pistil incompatibilities, may be particularly important for closely related species in geographic proximity that share pollinators.

The importance of reproductive isolation in driving diversification and speciation within Peruvian mimetic poison frogs (Dendrobatidae)

To explain how populations with distinct warning signals coexist in close parapatry, we experimentally assessed intrinsic mechanisms acting as reproductive barriers within three poison-frog species from the Peruvian Amazon belonging to a Müllerian mimetic ring (Ranitomeya variabilis, Ranitomeya imitator and Ranitomeya fantastica). We tested the role of prezygotic and postzygotic isolation barriers between phenotypically different ecotypes of each species, using no-choice mating experiments and offspring survival analysis. Our results show that prezygotic mating preference did not occur except for one specific ecotype of R. imitator, and that all three species were able to produce viable inter-population F1 hybrids. However, while R. variabilis and R. imitator hybrids were able to produce viable F2 generations, we found that for R. fantastica, every F1 hybrid males were sterile while females remained fertile. This unexpected result, echoing with Haldane’s rule of speciation, validated phylogenetic studies which tentatively diagnose these populations of R. fantastica as two different species. Our work suggests that postzygotic genetic barriers likely participate in the extraordinary phenotypic diversity observed within Müllerian mimetic Ranitomeya populations, by maintaining species boundaries.

Assessing the temporal isolation barrier between two closely related species of the genus Fissurella off the southwestern coast of South America

ABSTRACT Knowledge of the reproductive isolation mechanisms that prevent gene flow between species is essential for understanding the process of speciation. In this study, we investigate the spawning periods in Fissurella cumingi and F. latimarginata, two closely related sympatric species of keyhole limpets with external fertilization, as a potential prezygotic barrier between them. The reproductive cycle was assessed in two distinct locations in northern Chile using the gonadosomatic index (GSI) and gonadal histology. Species, locality, and sex were designated as predictive variables, while average GSI values, as well as those of partial and total spawning, were considered dependent variables. Both species exhibit a continuous gametogenic cycle throughout the year, characterized by multiple spawning events. A generalized linear model (GLM) factorial ANOVA design revealed that neither species, locality nor sex were significant factors with respect to the GSI and partial spawning scores. However, sex was found to be the only significant factor related to total spawning. No significant interactions were observed among predictive factors. These findings suggest that reproductive periods may not serve as effective barriers to gamete encounters between species. Our study prompts questions about how closely-related externally-fertilizing species maintain reproductive isolation.

Ecological divergence despite common mating sites: Genotypes and symbiotypes shed light on cryptic diversity in the black bean aphid species complex.

Different host plants represent ecologically dissimilar environments for phytophagous insects. The resulting divergent selection can promote the evolution of specialized host races, provided that gene flow is reduced between populations feeding on different plants. In black bean aphids belonging to the Aphis fabae complex, several morphologically cryptic taxa have been described based on their distinct host plant preferences. However, host choice and mate choice are largely decoupled in these insects: they are host-alternating and migrate between specific summer host plants and shared winter hosts, with mating occurring on the shared hosts. This provides a yearly opportunity for gene flow among aphids using different summer hosts, and raises the question if and to what extent the ecologically defined taxa are reproductively isolated. Here, we analyzed a geographically and temporally structured dataset of microsatellite genotypes from A. fabae that were mostly collected from their main winter host Euonymus europaeus, and additionally from another winter host and fourteen summer hosts. The data reveals multiple, strongly differentiated genetic clusters, which differ in their association with different summer and winter hosts. The clusters also differ in the frequency of infection with two heritable, facultative endosymbionts, separately hinting at reproductive isolation and divergent ecological selection. Furthermore, we found evidence for occasional hybridization among genetic clusters, with putative hybrids collected more frequently in spring than in autumn. This suggests that similar to host races in other phytophagous insects, both prezygotic and postzygotic barriers including selection against hybrids maintain genetic differentiation among A. fabae taxa, despite a common mating habitat.

Reinstatement of the fission yeast species Schizosaccharomyces versatilis Wickerham et Duprat, a sibling species of Schizosaccharomyces japonicus.

Schizosaccharomyces japonicus Yukawa et Maki (1931) and Schizosaccharomyces versatilis Wickerham et Duprat (1945) have been treated as varieties of S. japonicus or as conspecific, based on various approaches including mating trials and nDNA/nDNA optical reassociation studies. However, the type strains of S. japonicus and S. versatilis differ by five substitutions (99.15% identity) and one 1-bp indel in the sequences of the D1/D2 domain of the 26S rRNA gene, and 23 substitutions (96.3% identity) and 31-bp indels in the sequences of internal transcribed spacer (ITS) of rRNA, suggesting that they may not be conspecific. To reassess their taxonomic status, we conducted mating trials and whole-genome analyses. Mating trials using the type strains showed a strong but incomplete prezygotic sterility barrier, yielding interspecies mating products at two orders of magnitude lower efficiency than intraspecies matings. These mating products, which were exclusively allodiploid hybrids, were unable to undergo the haplontic life cycle of the parents. We generated chromosome-level gap-less genome assemblies for both type strains. Whole genome sequences yielded an average nucleotide identity (ANI) of 86.4%, indicating clear separation of S. japonicus and S. versatilis. Based on these findings, we propose the reinstatement of S. versatilis as a distinct species (holotype strain: CBS 103T and ex-types: NRRL Y-1026, NBRC 1607, ATCC 9987, PYCC 7100; Mycobank no.: 847838).

Temperature and sex shape reproductive barriers in a climate change hotspot.

Climate change is altering species ranges and reproductive interactions in existing ranges, offering species new scope to mate and hybridize. The outcomes will depend on how environmental factors shape reproductive barriers across life stages, yet this is rarely assessed across the environments that species encounter in nature. We assess prezygotic and postzygotic barriers, and their dependence on temperature and parental sex, in species of a reef-building tubeworm (Galeolaria) from a fast-warming biodiversity hotspot in southern Australia. By replicating pure and reciprocal hybrid crosses across 5 temperatures spanning species' thermal ranges, we estimate thermal tolerance curves (defining niches) for crosses and reproductive isolation at each temperature. By also replicating crosses at 3 life stages, we partition the contributions of prezygotic barriers at fertilization, postzygotic barriers at embryogenesis, and postzygotic barriers at larval development to reproductive isolation. We show that barriers are weaker at fertilization and embryogenesis, but stronger and more temperature sensitive at larval development, as species diverge in thermal niche. Asymmetry of barriers between parental sexes, moreover, suggests a complex interplay between niche differentiation and maternal inheritance. Our findings point to a key role for temperature in reproductive isolation, but also challenges for predicting the fate of isolation in future climates.

Testing for a role of postzygotic incompatibilities in rapidly speciated Lake Victoria cichlids.

Intrinsic postzygotic hybrid incompatibilities are usually due to negative epistatic interactions between alleles from different parental genomes. While such incompatibilities are thought to be uncommon in speciation with gene flow, they may be important if such speciation results from a hybrid population. Here we aimed to test this idea in the endemic cichlid fishes of Lake Victoria. Hundreds of species have evolved within the lake in <15k years from hybrid progenitors. While the importance of prezygotic barriers to gene flow is well established in this system, the possible relevance of postzygotic genetic incompatibilities is unknown. We inferred the presence of negative epistatic interactions from systematic patterns of genotype ratio distortions in experimental crosses and wild samples. We then compared the positions of putative incompatibility loci to regions of high genetic differentiation between sympatric sister species and between members of clades that may have arisen in the early history of this radiation, and further determined if the loci showed fixed differences between the closest living relatives of the lineages ancestral to the hybrid progenitors. Overall, we find little evidence for a major role of intrinsic postzygotic incompatibilities in the Lake Victoria radiation. However, we find putative incompatibility loci significantly more often coinciding with islands of genetic differentiation between species that separated early in the radiation than between the younger sister species, consistent with the hypothesis that such variants segregated in the hybrid swarm and were sorted between species in the early speciation events.

Arabidopsis leucine-rich repeat malectin receptor-like kinases regulate pollen-stigma interactions.

Flowering plants contain tightly controlled pollen-pistil interactions required for promoting intraspecific fertilization and preventing interspecific hybridizations. In Arabidopsis (Arabidopsis thaliana), several receptor kinases (RKs) are known to regulate the later stages of intraspecific pollen tube growth and ovular reception in the pistil, but less is known about RK regulation of the earlier stages. The Arabidopsis RECEPTOR-LIKE KINASE IN FLOWERS1 (RKF1)/RKF1-LIKE (RKFL) 1-3 cluster of 4 leucine-rich repeat malectin (LRR-MAL) RKs was previously found to function in the stigma to promote intraspecific pollen hydration. In this study, we tested additional combinations of up to 7 Arabidopsis LRR-MAL RK knockout mutants, including RKF1, RKFL1-3, LysM RLK1-INTERACTING KINASE1, REMORIN-INTERACTING RECEPTOR1, and NEMATODE-INDUCED LRR-RLK2. These LRR-MAL RKs were discovered to function in the female stigma to support intraspecific Arabidopsis pollen tube growth and to establish a prezygotic interspecific barrier against Capsella rubella pollen. Thus, this study uncovered additional biological functions for this poorly understood group of RKs in regulating the early stages of Arabidopsis sexual reproduction.

Ecological disruptive selection acting on quantitative loci can drive sympatric speciation

The process of speciation generates biodiversity. According to the null model of speciation, barriers between populations arise in allopatry, where, prior to biology, geography imposes barriers to gene flow. On the other hand, sympatric speciation requires that the process of speciation happen in the absence of a geographical barrier, where the members of the population have no spatial, temporal barriers. Several attempts have been made to theoretically identify the conditions in which speciation can occur in sympatry. However, these efforts suffer from several limitations. We propose a model for sympatric speciation based on adaptation for resource utilization. We use a genetics-based model to investigate the relative roles of prezygotic and postzygotic barriers, from the context of ecological disruptive selection, sexual selection, and genetic architecture, in causing and maintaining sympatric speciation. Our results show that sexual selection that acts on secondary sexual traits does not play any role in the process of speciation in sympatry and that assortative mating based on an ecologically relevant trait forces the population to show an adaptive response. We also demonstrate that understanding the genetic architecture of the trait under ecological selection is very important and that it is not required for the strength of ecological disruptive selection to be very high in order for speciation to occur in sympatry. Our results provide an insight into the kind of scenarios in which sympatric speciation can be demonstrated in the lab.

Synthesis and Scope of the Role of Postmating Prezygotic Isolation in Speciation.

How barriers to gene flow arise and are maintained are key questions in evolutionary biology. Speciation research has mainly focused on barriers that occur either before mating or after zygote formation. In comparison, postmating prezygotic (PMPZ) isolation-a barrier that acts after gamete release but before zygote formation-is less frequently investigated but may hold a unique role in generating biodiversity. Here we discuss the distinctive features of PMPZ isolation, including the primary drivers and molecular mechanisms underpinning PMPZ isolation. We then present the first comprehensive survey of PMPZ isolation research, revealing that it is a widespread form of prezygotic isolation across eukaryotes. The survey also exposes obstacles in studying PMPZ isolation, in part attributable to the challenges involved in directly measuring PMPZ isolation and uncovering its causal mechanisms. Finally, we identify outstanding knowledge gaps and provide recommendations for improving future research on PMPZ isolation. This will allow us to better understand the nature of this often-neglected reproductive barrier and its contribution to speciation.

FLORAL PHENOLOGY AND MORPHOMETRIC ANALYSIS OF THREE COMMERCIALLY GROWN COFFEA SPECIES

Coffea arabica, C. canephora, and C. liberica’s coexistence in one location or proximity areas could promote interspecific pollination. However, information related to floral phenology and flower structure of these three coffee species is insufficient. This research aimed to identify the divergence of flowering phenology and flower morphometrics among C. arabica, C. canephora, and C. liberica. Flowering phenology observations continued daily from July to October 2020, extending to two locations of parapatric populations of three coffee species. Flower morphometric measurements ensued at the peak period of flowering. The results showed some co-anthesis periods among the three coffee species, with C. liberica starting to flower early. Moreover, C. liberica var liberica had the broadest flower diameter and the highest petal number, whereas the tetraploid C. arabica had the lowest. Consequences of flowering phenology and flower structure on a pre-zygotic barrier of spontaneous, as well as, controlled interspecific hybridization among the three coffee species were the discussion focus.

Patterns of admixture and introgression in a mosaic Mytilus galloprovincialis and Mytilus edulis hybrid zone in SW England.

The study of hybrid zones offers important insights into speciation. Earlier studies on hybrid populations of the marine mussel species Mytilus edulis and Mytilus galloprovincialis in SW England provided evidence of admixture but were constrained by the limited number of molecular markers available. We use 57 ancestry-informative SNPs, most of which have been mapped genetically, to provide evidence of distinctive differences between admixed populations in SW England and asymmetrical introgression from M. edulis to M. galloprovincialis. We combine the genetic study with analysis of phenotypic traits of potential ecological and adaptive significance. We demonstrate that hybrid individuals have brown mantle edges unlike the white or purple in the parental species, suggesting allelic or non-allelic genomic interactions. We report differences in gonad development stage between the species consistent with a prezygotic barrier between the species. By incorporating results from publications dating back to 1980, we confirm the long-term stability of the hybrid zone despite higher viability of M. galloprovincialis. This stability coincides with a dramatic change in temperature of UK coastal waters and suggests that these hybrid populations might be resisting the effects of global warming. However, a single SNP locus associated with the Notch transmembrane signalling protein shows a markedly different pattern of variation to the others and might be associated with adaptation of M. galloprovincialis to colder northern temperatures.

Partial and asymmetrical reproductive isolation between two sympatric tropical shrub species: Cnidoscolus aconitifolius and C. souzae (Euphorbiaceae).

Reproductive isolation is conferred by several barriers that occur at different stages of reproduction. Comprehensive reviews on the topic have identified that barriers occurring prior to zygote formation are often stronger than those that occur afterward. However, the overrepresentation of temperate perennial herbs in the current literature precludes any generalization of this pattern to plants that present other life forms and patterns of distribution. Here, we assessed reproductive isolation barriers and their absolute contribution to reproductive isolation and asymmetry in Cnidoscolus aconitifolius and C. souzae, two closely related tropical shrub species that co-occur on the Yucatan peninsula. The reproductive barriers assessed were phenological mismatch, pollinator differentiation, pollen-pistil incompatibility (three pre-zygotic barriers), fruit set failure, and seed unviability (post-zygotic barriers). Reproductive isolation between the study species was found to be complete in the direction C. aconitifolius to C. souzae, but only partial in the opposite direction. One post-zygotic barrier was the strongest example. Most barriers, particularly the pre-zygotic examples, were asymmetrical and predicted the direction of heterospecific pollen flow and hybrid formation from C. souzae to C. aconitifolius. Both parental species, as well as the hybrids, were diploid and had a chromosome number 2n = 36. More studies with tropical woody perennials are required to fully determine whether this group of plants consistently shows stronger post-zygotic barriers.

Implications of headwater contact zones for the riverine barrier hypothesis: a case study of the Blue-capped Manakin (Lepidothrix coronata).

Rivers frequently delimit the geographic ranges of species in the Amazon Basin. These rivers also define the boundaries between genetic clusters within many species, yet river boundaries have been documented to break down in headwater regions where rivers are narrower. To explore the evolutionary implications of headwater contact zones in Amazonia, we examined genetic variation in the Blue-capped Manakin (Lepidothrix coronata), a species previously shown to contain several genetically and phenotypically distinct populations across the western Amazon Basin. We collected restriction site-associated DNA sequence data (RADcap) for 706 individuals and found that spatial patterns of genetic structure indicate several rivers, particularly the Amazon and Ucayali, are dispersal barriers for L. coronata. We also found evidence that genetic connectivity is elevated across several headwater regions, highlighting the importance of headwater gene flow for models of Amazonian diversification. The headwater region of the Ucayali River provided a notable exception to findings of headwater gene flow by harboring non-admixed populations of L. coronata on opposite sides of a < 1-km-wide river channel with a known dynamic history, suggesting that additional prezygotic barriers may be limiting gene flow in this region.

Genetics and the Evolution of Prezygotic Isolation.

The significance of prezygotic isolation for speciation has been recognized at least since the Modern Synthesis. However, fundamental questions remain. For example, how are genetic associations between traits that contribute to prezygotic isolation maintained? What is the source of genetic variation underlying the evolution of these traits? And how do prezygotic barriers affect patterns of gene flow? We address these questions by reviewing genetic features shared across plants and animals that influence prezygotic isolation. Emerging technologies increasingly enable the identification and functional characterization of the genes involved, allowing us to test established theoretical expectations. Embedding these genes in their developmental context will allow further predictions about what constrains the evolution of prezygotic isolation. Ongoing improvements in statistical and computational tools will reveal how pre- and postzygotic isolation may differ in how they influence gene flow across the genome. Finally, we highlight opportunities for progress by combining theory with appropriate data.

Rapid evolution of pre-zygotic reproductive barriers in allopatric populations.

Adaptive divergence leading to speciation is the major evolutionary process generating diversity in life forms. The most commonly observed form of speciation is allopatric speciation which requires that gene flow be prevented between populations by physical or temporal barriers, as they adapt to their respective environments. Eventually, these adaptive responses lead to accumulation of mutations in different lines. The increased genetic distance between the lines is known to lead to the populations becoming reproductively isolated. A widely accepted theory is that speciation simply occurs as a by-product of adaptive response of the populations. Several examples of allopatric speciation from ecology and laboratory exist. However, we know little about the nature (pre- or post-zygotic) of barriers that arise first in this process. Understanding the first barriers that arise between populations is key to understanding how the process of speciation initiates. In recent years, fungi have been used as model organisms to answer questions related to the evolution of reproductive isolation. Here, we show rapid evolution of pre-zygotic barriers between allopatric yeast populations. We further demonstrate that these pre-zygotic barriers arise due to altered mating kinetics of the evolved population. Moreover, our non-adaptive evolution experiments with yeast under limited selection pressure also show rapid emergence of reproductive isolation. Overall, our results show that evolution of pre-zygotic reproductive barriers can occur as a result of natural selection or drift.IMPORTANCEA population diversifies into two or more species-such a process is known as speciation. In sexually reproducing microorganisms, which barriers arise first-pre-mating or post-mating? In this work, we quantify the relative strengths of these barriers and demonstrate that pre-mating barriers arise first in allopatrically evolving populations of yeast, Saccharomyces cerevisiae. These defects arise because of the altered kinetics of mating of the participating groups. Thus, our work provides an understanding of how adaptive changes can lead to diversification among microbial populations.

Antagonistic RALF peptides control an intergeneric hybridization barrier on Brassicaceae stigmas

Pollen-pistil interactions establish interspecific/intergeneric pre-zygotic hybridization barriers in plants. The rejection of undesired pollen at the stigma is crucial to avoid outcrossing but can be overcome with the support of mentor pollen. The mechanisms underlying this hybridization barrier are largely unknown. Here, in Arabidopsis, we demonstrate that receptor-like kinases FERONIA/CURVY1/ANJEA/HERCULES RECEPTOR KINASE 1 and cell wall proteins LRX3/4/5 interact on papilla cell surfaces with autocrine stigmatic RALF1/22/23/33 peptide ligands (sRALFs) to establish a lock that blocks the penetration of undesired pollen tubes. Compatible pollen-derived RALF10/11/12/13/25/26/30 peptides (pRALFs) act as a key, outcompeting sRALFs and enabling pollen tube penetration. By treating Arabidopsis stigmas with synthetic pRALFs, we unlock the barrier, facilitating pollen tube penetration from distantly related Brassicaceae species and resulting in interspecific/intergeneric hybrid embryo formation. Therefore, we uncover a "lock-and-key" system governing the hybridization breadth of interspecific/intergeneric crosses in Brassicaceae. Manipulating this system holds promise for facilitating broad hybridization in crops.

Acoustic innovations in courtship sounds generated by hybridization in cichlid fish

Abstract During courtship, prezygotic barriers are thought to maintain the diversity of species since differences in phenotypic traits and reproductive behaviours are likely to be involved in maintaining species boundaries. However, introgressive hybridization has been reported in many different taxa and the resulting hybrids usually possess phenotypic features, including behaviours, that are intermediate between those of the parents. The outcome of hybrids may depend on the interactions between the intrinsic viability of the hybrids, the ecological environment of the species, and the fitness of hybrids in that ecological context. In cichlids, the mechanism of hybridization is increasingly documented as an important factor in their evolutionary radiation since it may provide novel gene combinations that promote speciation and adaptive radiation by generating new transgressive phenotypes. Cichlid species are separated mostly by pre-mating isolating mechanisms. Like colour patterns, acoustic communication is involved in sexual selection, but this pattern has been much less studied and the outcome of hybridization on acoustic features has never been examined. To gain novel insights into the evolution of their acoustic communication behaviour, we performed a hybrid cross between Chindongo saulosi and Maylandia estherae. Qualitative examination of courtship showed that the three groups possess the same sequence of behaviours. However, sounds produced during courtship showed specific signatures since all three groups differ at the level of sound duration and pulse period, with both features being intermediate in hybrids in comparison to the two parent species. Colour patterns in hybrids were also different from those of the parent species. These results show that hybridization can affect at least two kinds of prezygotic barriers, which may be important in the cichlid evolutionary process. However, playback experiments are required to test the strength of these features in sexual selection.

Embryo Rescue in Plant Breeding.

Embryo rescue (ER) techniques are among the oldest and most successful in vitro tissue culture protocols used with plant species. ER refers to a series of methods that promote the development of an immature or lethal embryo into a viable plant. Intraspecific, interspecific, or intergeneric crosses allow the introgression of important alleles of agricultural interest from wild species, such as resistance or tolerance to abiotic and biotic stresses or morphological traits in crops. However, pre-zygotic and post-zygotic reproductive barriers often present challenges in achieving successful hybridization. Pre-zygotic barriers manifest as incompatibility reactions that hinder pollen germination, pollen tube growth, or penetration into the ovule occurring in various tissues, such as the stigma, style, or ovary. To overcome these barriers, several strategies are employed, including cut-style or graft-on-style techniques, the utilization of mixed pollen from distinct species, placenta pollination, and in vitro ovule pollination. On the other hand, post-zygotic barriers act at different tissues and stages ranging from early embryo development to the subsequent growth and reproduction of the offspring. Many crosses among different genera result in embryo abortion due to the failure of endosperm development. In such cases, ER techniques are needed to rescue these hybrids. ER holds great promise for not only facilitating successful crosses but also for obtaining haploids, doubled haploids, and manipulating the ploidy levels for chromosome engineering by monosomic and disomic addition as well substitution lines. Furthermore, ER can be used to shorten the reproductive cycle and for the propagation of rare plants. Additionally, it has been repeatedly used to study the stages of embryonic development, especially in embryo-lethal mutants. The most widely used ER procedure is the culture of immature embryos taken and placed directly on culture media. In certain cases, the in vitro culture of ovule, ovaries or placentas enables the successful development of young embryos from the zygote stage to maturity.