Hybrid Embryos Research Articles

Hybridization alters maternal and paternal genome contributions to early plant embryogenesis.

After fertilization, zygotic genome activation results in a transcriptionally competent embryo. Hybrid transcriptome experiments in Arabidopsis have concluded that the maternal and paternal genomes make equal contributions to zygotes and embryos, yet embryo defective (emb) mutants in the Columbia (Col) ecotype display early maternal effects. Here, we show that hybridization of Col with Landsberg erecta (Ler) or Cape Verde Islands (Cvi) ecotypes decreases the maternal effects of emb mutants. Reanalysis of Col/Ler and Col/Cvi transcriptomes confirmed equal parental contributions in Col/Cvi early embryos. By contrast, thousands of genes in Col/Ler zygotes and one-cell embryos were biallelic in one cross and monoallelic in the reciprocal cross, with analysis of intron reads pointing to active transcription as responsible for this parent-of-origin bias. Our analysis shows that, contrary to previous conclusions, the maternal and paternal genomes in Col/Ler zygotes are activated in an asymmetric manner. The decrease in maternal effects in hybrid embryos compared with those in isogenic Col along with differences in genome activation between Col/Cvi and Col/Ler suggest that neither of these hybrids accurately reflects the general trends of parent-of-origin regulation in Arabidopsis embryogenesis.

Genetic exchange with an outcrossing sister species causes severe genome-wide dysregulation in a selfing Caenorhabditis nematode.

Different modes of reproduction evolve rapidly, with important consequences for genome composition. Selfing species often occupy a similar niche as their outcrossing sister species with which they are able to mate and produce viable hybrid progeny, raising the question of how they maintain genomic identity. Here, we investigate this issue by using the nematode Caenorhabditis briggsae, which reproduces as a hermaphrodite, and its outcrossing sister species Caenorhabditis nigoni We hypothesize that selfing species might develop some barriers to prevent gene intrusions through gene regulation. We therefore examined gene regulation in the hybrid F2 embryos resulting from reciprocal backcrosses between F1 hybrid progeny and C. nigoni or C. briggsae F2 hybrid embryos with ∼75% of their genome derived from C. briggsae (termed as bB2) were inviable, whereas those with ∼75% of their genome derived from C. nigoni (termed as nB2) were viable. Misregulation of transposable elements, coding genes, and small regulatory RNAs was more widespread in the bB2 compared with the nB2 hybrids, which is a plausible explanation for the differential phenotypes between the two hybrids. Our results show that regulation of the C. briggsae genome is strongly affected by genetic exchanges with its outcrossing sister species, C. nigoni, whereas regulation of the C. nigoni genome is more robust on genetic exchange with C. briggsae The results provide new insights into how selfing species might maintain their identity despite genetic exchanges with closely related outcrossing species.

Induction of high triploid rate blunt snout bream (Megalobrama amblycephala) by hydrostatic pressure and analysis of gonadal sterility mechanism

At present, only cold shock method was used to induce the triploid of M. amblycephala, and because the triploid rate is not high enough, the triploid fish has not been applied in aquaculture on a large scale. This study aimed to obtain the best induction conditions of triploid by hydrostatic pressure, reveal the reproductive characteristics and mechanism of gonadal sterility of triploid M. amblycephala, provide an insights into the meiotic patterns of triploid fish. We successfully induced triploid in M. amblycephala using the hydrostatic pressure method. The highest triploid rate reached 85.92% at 55 MPa for 3 min after 3 min of fertilization. The relative DNA content of the triploid fish was 1.5 times of that of the diploid fish, and their chromosome karyotype was 3n = 72 = 27 m + 39sm + 6st. Both female and male triploids had dysplastic gonads, the testis of triploid males was able to produce 1.5n sperm without fertility because all the hybrid embryos of 2n♀ × 3n♂ died; however, the ovaries of triploid females belonged to complete sterility. The verification of gene expression patterns and interaction suggested that the hypothalamus-pituitary-gonad (HPG) axis process was disturbed and unsynapsed chromosome (UC) axis was blocked may jointly caused the complete sterility without egg production of female triploids. However, triploid males belonged to uncompletely sterility, which can produce a small number of sperm and complete fertilization, but the offspring cannot survive, may caused by the not completely blocked UC axis with low expression of hormad1 and iho1.

Experimental Crossing Confirms Reproductive Isolation between Cryptic Species within Eulimnogammarus verrucosus (Crustacea: Amphipoda) from Lake Baikal.

Ancient lakes are known speciation hotspots. One of the most speciose groups in the ancient Lake Baikal are gammaroid amphipods (Crustacea: Amphipoda: Gammaroidea). There are over 350 morphological species and subspecies of amphipods in Baikal, but the extent of cryptic variation is still unclear. One of the most common species in the littoral zone of the lake, Eulimnogammarus verrucosus (Gerstfeldt, 1858), was recently found to comprise at least three (pseudo)cryptic species based on molecular data. Here, we further explored these species by analyzing their mitogenome-based phylogeny, genome sizes with flow cytometry, and their reproductive compatibility. We found divergent times of millions of years and different genome sizes in the three species (6.1, 6.9 and 8 pg), further confirming their genetic separation. Experimental crossing of the western and southern species, which are morphologically indistinguishable and have adjacent ranges, showed their separation with a post-zygotic reproductive barrier, as hybrid embryos stopped developing roughly at the onset of gastrulation. Thus, the previously applied barcoding approach effectively indicated the separate biological species within E. verrucosus. These results provide new data for investigating genome evolution and highlight the need for precise tracking of the sample origin in any studies in this morphospecies.

Induction of gynogenesis with ultra-violet irradiated Koi carp (Cyprinus carpio haematopterus) sperm demonstrates the XX/XY sex determination system in Opsariichthys bidens

Opsariichthys bidens (Chinese hooksnout carp) is a small stream fish with high economic value. As the males of this species grow faster than females, it follows that mono-sex breeding has significant potential for commercial production. However, little is known about the sex determination system in O. bidens, this is the key process that is used to develop mono-sex breeding in fish. In the present study, we induced gynogenesis by fertilizing O.bidens eggs with ultra-violet (UV)- inactivated Cyprinus carpio haematopterus (Koi carp) sperm followed by cold shock treatment. Results showed that the optimal induction conditions was 25 min duration of cold shock treatment at 2 min post fertilization (mpf), the highest fertilization rate and hatching rate for gynogenic embryos were 68.24 ± 3.75% and 12.54 ± 1.59%, respectively. Embryos derived from gynogenesis developed normally, but the hybrid embryos failed (created with non-UV-inactivated Koi carp sperm and O.bidens eggs). Genetic analysis demonstrated only maternal inheritance in the gynogenetic offspring. Histological analysis further showed that the gonads of all gynogenetic fish were ovaries, thus suggesting that the sex-determining system of O. bidens is male heterogametic (XX-XY). The present study is the first to establish a procedure for inducing gynogenesis, we also identified the sex determination system (SD) of O. bidens, that will be possible to produce only male.

Establishment of diploid hybrid strains derived from female Larimichthys crocea × male Larimichthys polyactis and transmission of parental mtDNA in hybrid progenies

In this study, a new type of diploid hybrid fish (LCP) was obtained successfully using interspecific hybridization of female Larimichthys crocea (LC, 2n = 48) and male Larimichthys polyactis (LP, 2n = 48). The embryonic development showed that the hatching rate of the hybrid embryos was the highest at 24 °C, with 35.75% of embryos hatching normally. A polymerase chain reaction (PCR) with specific primers (5S−F/−R) was used to distinguish LCP from their parents (LC and LP). Results indicate that LCP was hybrids with a hybrid genome that originated from original parents. Based on the chromosome number, LCP was a diploid hybrid fish with 48 chromosomes (2n = 48). Histological observations of the gonads revealed that both female and male LCP individuals developed normally and were potentially fertile. The analysis of mitochondrial genes showed that the late elimination and transcriptional silencing of paternal mitochondrial genome (mtDNA) may occur in hybrid progenies. The paternal mitochondrial gene of cox1 was detected at the blastula and tail bud stages, diminished at the heart-beating stage, then completely disappeared. This suggests that late elimination leads to mitochondrial heteroplasmy. The transcriptional status showed that cox1 mRNA was not detected at any of the stages examined in hybrid progenies, indicating that the paternal mtDNA may have been transcriptionally silenced and the influence on phenotype during embryonic development may have been excluded. This study provides basis for the establishment of diploid hybrid fish as a new germplasm resource, which in turn, can promote the formation of a new hybrid fish line and contribute in future fish genetic breeding.

Development of clubroot resistant cabbage line through introgressing six CR loci from Chinese cabbage via interspecific hybridization and embryo rescue

Multiple R-gene is required to achieve strong resistance against clubroot, a disease caused by Plasmodiophora brassicae in Cabbage (Brassica oleracea, 2n=18, CC). Most of the R-genes were identified in Chinese cabbage (B. rapa, 2n=20, AA), which could be transferred to cabbage via inter interspecific hybridization. We present a breeding scheme to transfer six major CR-loci namely, Crr1, Crr2, Crr3, CRa, CRb and CRc from Chinese cabbage line, LCR36 to cabbage line, Plimio. Approximately, 48% of the interspecific hybrid embryos were rescued, of which chromosomes of 48.15% colchiploid plants were successfully doubled. The hybridity of the colchiploid interspecific hybrids were confirmed by diagnostic sub-genome specific Conserved Ortholog Set (COS) markers, ploidy level, pollen fertility and meiotic chromosome number analyses. Backcross generations are characterized by very low seed set (0.57-1.33% success rate) in backcrossed generations (using Plimio as the recurrent parent). Marker assisted selection using the six CR-loci specific markers identified the resistant plants in F1 and backcross generations. MAS and bioassay test of BC2F2 plants revealed that plants having all six R-loci are resistant to clubroot. Development of subsequent backcross generations are in progress. More number of back-crosses and/or embryo rescue will be performed to ensure sufficient seed set. The success so far is the first report of successful introgression of multiple R-genes via interspecific hybridization in the initial backcrossed generations, indicating that an elite cabbage line having strong resistance to clubroot can be produced upon the completion of the breeding scheme.

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders.

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

Use of Biotechnological Methods to Support the Production of New Peach Hybrids

Peach [Prunus persica (L.) Batsch] is among the most demanded fruit crops in the world. Biotechnological methods help to originate new hybrid forms in order to increase the cultivar diversity and create new valuable genotypes. Cross combinations between the cultivars Clyde Wilson, Jerseyglo, Loadel, Summerglo and the promising cultivar ‘Nikitskiy Podarok’ have been done. The embryos of these hybrids germinated and formed plantlets after stratification at 4 °C for 45–60 days. The best regeneration rates in the hybrids ‘Loadel’ × ‘Nikitskiy Podarok’ and ‘Summerglo’ × ‘Nikitskiy Podarok’ (96.30% and 92.59%, respectively) were noted on hormone-free Monnier culture medium supplemented with 400.0 mg L−1 casein hydrolyzate. When the newly formed plantlets had necrosis of the shoot apex or immature roots, nodal shoot segments were used. At the same time, a high regeneration capacity was noted in the hybrids ‘Summerglo’ × ‘Nikitskiy Podarok’ and ‘Loadel’ × ‘Nikitskiy Podarok’ on B5 culture medium with 0.75 mg L−1 6–benzyl–aminopurine (BAP) + 0.1 mg L−1 indole–3–butyric acid (IBA). After the second subculture, the number of new adventitious shoots was 5.18 ± 0.18 and 4.95 ± 0.18 shoots per explant, respectively. The plants obtained from the hybrid embryos in a soil mixture soil: peat: sand (3:1:1) were adapted. The main morphological and anatomical features of the leaf blades in newly originated peach hybrids have been studied: the thickness of their tissues and the distribution of stomatal apparatus, as well as the physiological parameters of the photosystem II activity in regenerants cultured in vitro and during their in vivo acclimatization. The high capacity to post aseptic adaptation in the obtained hybrids has been shown.

Growth promotion of interspecific hybrid embryos between Cucumis anguria and Melon (C. melo) by fruit heating

Wild species of the genus Cucumis, such as C. anguria, are resistant to various melon (C. melo) diseases such as Fusarium wilt race 1.2 and are anticipated as genetic resources of melon. However, in the interspecific crosses between C. anguria and melon, the abortion of the embryos was observed in the early growth stage and could not be regenerated even when cultured. Therefore, for embryo rescue, it is necessary to promote embryo growth during abortion occurrence. This study investigated the effects of fruit heating on embryo growth in interspecific hybrids of C. anguria and melon. In the heating device, the minimum night temperature around the fruit was maintained above 21.5 °C. The ovule grain area of C. anguria × MR-1 with fruit heating was significantly enlarged compared to that without fruit-heating. It was also significantly enlarged compared with the ovule grain area of C. anguria self-pollination. Although, in C. anguria × MR-1 without fruit-heating, no embryo was observed until 10 days after pollination (DAP), and pre-embryos were observed at 13 DAP. Conversely, in C. anguria × MR-1 with fruit-heating, pre-embryos were even observed at 10 DAP. These results suggest that the possibility of embryo or ovule culture regeneration has increased due to embryo growth promotion.

Epigenetically mismatched parental centromeres trigger genome elimination in hybrids.

Wide crosses result in postzygotic elimination of one parental chromosome set, but the mechanisms that result in such differential fate are poorly understood. Here, we show that alterations of centromeric histone H3 (CENH3) lead to its selective removal from centromeres of mature Arabidopsis eggs and early zygotes, while wild-type CENH3 persists. In the hybrid zygotes and embryos, CENH3 and essential centromere proteins load preferentially on the CENH3-rich centromeres of the wild-type parent, while CENH3-depleted centromeres fail to reconstitute new CENH3-chromatin and the kinetochore and are frequently lost. Genome elimination is opposed by E3 ubiquitin ligase VIM1. We propose a model based on cooperative binding of CENH3 to chromatin to explain the differential CENH3 loading rates. Thus, parental CENH3 polymorphisms result in epigenetically distinct centromeres that instantiate a strong mating barrier and produce haploids.

Effects of small‐molecule compounds on fibroblast properties in golden snub‐nosed monkey (Rhinopithecus roxellana)

Golden snub-nosed monkey (Rhinopithecus roxellana) is an endangered primate species, whose molecular material for conservation purposes has not yet been maintained. Although small-molecule compounds (SMCs) have been reported to improve induced pluripotent stem cells (iPSCs), their efficiency in the interspecies-transferred nucleus is still unknown. We thus used the fibroblasts from the golden snub-nosed monkey treated with SMC as donor cells, injected into the enucleated oocytes of goats, to test such efficiency. Gene expression profiles in the cell-constructed embryos with and without SMCs were compared by qPCR. The results show that cell morphology undergoes remarkable changes (volume is smaller than normal cells, and many black spots in the cytoplasm were found); pluripotent genes (Oct4, Sox2, and Nanog) significantly increased with SMC treatment. This study demonstrates that SMCs alter the properties of donor cells and promote the expression of pluripotent genes in hybrid embryos.

Development and regeneration of wheat-rice hybrid zygotes produced by in vitro fertilization system.

Summary Hybridization plays a decisive role in the evolution and diversification of angiosperms. However, the mechanisms of wide hybridization remain open because pre‐ and post‐fertilization barriers limit the production and development of inter‐subfamily/intergeneric zygotes, respectively.We examined hybridization between wheat and rice using an in vitro fertilization (IVF) system to bypass these barriers. Several gamete combinations of allopolyploid wheat–rice hybrid zygotes were successfully produced, and the developmental profiles of hybrid zygotes were analyzed.Hybrid zygotes derived from one rice egg cell and one wheat sperm cell ceased at the multicellular embryo‐like structure stage. This developmental barrier was overcome by adding one wheat egg cell to the wheat–rice hybrid zygote. In the reciprocal combination, one wheat egg and one rice sperm cell, the resulting hybrid zygotes failed to divide. However, doubling the dosage of rice sperm cell allowed the hybrid zygotes to develop into plantlets. Rice chromosomes appeared to be progressively eliminated during the early developmental stage of these hybrid embryos, and c. 20% of regenerated plants showed abnormal morphology.These results suggest that hybrid breakdown can be overcome through optimization of gamete combinations, and the present hybrid will provide a new horizon for utilization of inter‐subfamily genetic resources.

Chrysanthemum embryo development is negatively affected by a novel ERF transcription factor, CmERF12

Embryo abortion often occurs during distant hybridization events. Apetala 2/ethylene-responsive factor (AP2/ERF) proteins are key transcription factor (TF) regulators of plant development and stress resistance, but their roles in hybrid embryo development are poorly understood. In this study, we isolated a novel AP2/ERF TF, CmERF12, from chrysanthemum and show that it adversely affects embryo development during distant hybridization. Transcriptome and real-time quantitative PCR demonstrate that CmERF12 is expressed at significantly higher levels in aborted ovaries compared with normal ones. CmERF12 localizes to the cell nucleus and contains a conserved EAR motif that mediates its transcription repressor function in yeast and plant cells. We generated artificial microRNA (amiR) CmERF12 transgenic lines of Chrysanthemum morifolium var. 'Yuhualuoying' and conducted distant hybridization with the wild-type tetraploid, Chrysanthemum nankingense, and found that CmERF12-knock down significantly promoted embryo development and increased the seed-setting rates during hybridization. The expression of various genes related to embryo development was up-regulated in developing ovaries from the cross between female amiR-CmERF12 C. morifolium var. 'Yuhualuoying'× male C. nankingense. Furthermore, CmERF12 directly interacted with CmSUF4, which is known to affect flower development and embryogenesis, and significantly reduced its ability to activate its target gene CmEC1 (EGG CELL1). Our study provides a novel method to overcome barriers to distant hybridization in plants and reveals the mechanism by which CmERF12 negatively affects chrysanthemum embryo development.

Optimization in vitro conditions for plum × apricot embryo rescue and modeling some critical factors by using artificial neural networks technology

The aim of this research was to overcome abortion postzygote incompatibility, and rescuing of hybrid embryos of early and mid-ripening plum cultivars, obtained from interspecific crossing between two plum cultivars (P. salicina L.) as female parents with four apricot cultivars (P. armeniaca L.) as male parents. The effects of cultivar, embryo developmental stage, plant growth regulators (PGRs), cold treatment, embryo size and removing seed coats were investigated on embryo germination at in vitro conditions. Results showed that the best conditions for embryo rescue were: harvesting embryos at 65 d after pollination (DAP) (91.33%), using completed MS medium with 0.5 mgL−1 6-benzyladenine (BA) and 1 mgL−1 indole-3-butyric acid (IBA), and embryo Stratification for 65 and 75 DAP- embryos at 4 °C for 30 d. Larger embryos testa removing treatment showed higher germination rates. Additionally, artificial neural networks (ANNs) was used to model two outputs which are the embryo generation percentage (GP) and complete plant percentage (CFP) based on three inputs including BA, IBA and embryo developmental stage (EDS). According to Multilayer 29 Perceptron (MLP), the best germination rate (100%) and the highest percentage of complete plant (100%) can be obtained by culturing the embryos at 65 DAP on MS medium containing 0.69 mgL−1 BA and 0.95 mgL−1 IBA, or at 75 DAP on MS medium containing 0.94 mgL−1 BA and 0.98 mgL−1 IBA. The MLP results showed a high correlation between the expected and the experimental values, which was 91.99%, 89.33% for GP and CFP, respectively. The results of the traditional statistical analysis by using Two Way ANOVA method were consistent with the results of the model obtained using MLP. Taking as a whole, this research developed an optimal protocol that may be useful in rescuing plum embryos in breeding programs.

The transcription factor CmLEC1 positively regulates the seed-setting rate in hybridization breeding of chrysanthemum

Distant hybridization is widely used to develop crop cultivars, whereas the hybridization process of embryo abortion often severely reduces the sought-after breeding effect. The LEAFY COTYLEDON1 (LEC1) gene has been extensively investigated as a central regulator of seed development, but it is far less studied in crop hybridization breeding. Here we investigated the function and regulation mechanism of CmLEC1 from Chrysanthemum morifolium during its seed development in chrysanthemum hybridization. CmLEC1 encodes a nucleic protein and is specifically expressed in embryos. CmLEC1’s overexpression significantly promoted the seed-setting rate of the cross, while the rate was significantly decreased in the amiR-CmLEC1 transgenic chrysanthemum. The RNA-Seq analysis of the developing hybrid embryos revealed that regulatory genes involved in seed development, namely, CmLEA (late embryogenesis abundant protein), CmOLE (oleosin), CmSSP (seed storage protein), and CmEM (embryonic protein), were upregulated in the OE (overexpressing) lines but downregulated in the amiR lines vs. wild-type lines. Future analysis demonstrated that CmLEC1 directly activated CmLEA expression and interacted with CmC3H, and this CmLEC1–CmC3H interaction could enhance the transactivation ability of CmLEC1 for the expression of CmLEA. Further, CmLEC1 was able to induce several other key genes related to embryo development. Taken together, our results show that CmLEC1 plays a positive role in the hybrid embryo development of chrysanthemum plants, which might involve activating CmLEA’s expression and interacting with CmC3H. This may be a new pathway in the LEC1 regulatory network to promote seed development, one perhaps leading to a novel strategy to not only overcome embryo abortion during crop breeding but also increase the seed yield.

Endosperm-based incompatibilities in hybrid monkeyflowers.

Endosperm is an angiosperm innovation central to their reproduction whose development, and thus seed viability, is controlled by genomic imprinting, where expression from certain genes is parent-specific. Unsuccessful imprinting has been linked to failed inter-specific and inter-ploidy hybridization. Despite their importance in plant speciation, the underlying mechanisms behind these endosperm-based barriers remain poorly understood. Here, we describe one such barrier between diploid Mimulus guttatus and tetraploid Mimulus luteus. The two parents differ in endosperm DNA methylation, expression dynamics, and imprinted genes. Hybrid seeds suffer from underdeveloped endosperm, reducing viability, or arrested endosperm and seed abortion when M. guttatus or M. luteus is seed parent, respectively, and transgressive methylation and expression patterns emerge. The two inherited M. luteus subgenomes, genetically distinct but epigenetically similar, are expressionally dominant over the M. guttatus genome in hybrid embryos and especially their endosperm, where paternal imprints are perturbed. In aborted seeds, de novo methylation is inhibited, potentially owing to incompatible paternal instructions of imbalanced dosage from M. guttatus imprints. We suggest that diverged epigenetic/regulatory landscapes between parental genomes induce epigenetic repatterning and global shifts in expression, which, in endosperm, may uniquely facilitate incompatible interactions between divergent imprinting schemes, potentially driving rapid barriers.

Embryo Culture of New Hybrid of Pecan (Carya Illinoensis) with Walnut (J. Regia and J. Nigra)

Present study was conducted to achieve intergeneric hybridization in Juglandaceae for the first time. Due to embryo abortion possibility, the embryo rescue technique was conducted via callus induction from immature hybrid embryo culture. For this reason, embryos were sampled at 20, 30, 40 and 60 days after pollination and cultured on two enrichment media (WPM and DKW). The developmental stage of immature embryo, kind of medium and addition of growth regulators to the induction medium played the role in promoting better callus and plantlet formation. The best quality and quantity of callus induction were achieved in 60 days’ explant. Maximum induction was in WPM and there was no callus induction in PGRs free medium. Browning was the most obstacle to conquer in callus proliferation. Germination of remained mature hybrid embryo until fruit harvest time was induced on three media (MS, WPM, and DKW). There were no significant differences between medium and male parents effect on germination parameters. But, MS and WPM performed better for hybrid embryo germination. The first germination was 7 days in MS and WPM and about 10 days for DKW after culture. In all medium the roots were robust and more developed than the leaves. The microplant transferred to soil after hardening.

Aggregation of Leopardus geoffroyi hybrid embryos with domestic cat tetraploid blastomeres.

Heterospecific embryo transfer of an endangered species has been carried out using recipients from related domestic females. Aggregation of an embryo from an endangered species with a tetraploid embryo from the species to be transferred could improve the development of pregnancy to term. The main objective of the present study was to analyze embryo aggregation in domestic cat model using hybrid embryos. For this purpose, we compared in vitro development of synchronic (Sync) or asynchronic (Async) and asynchronic with a tetraploid (Async4n) aggregation of domestic cat IVF embryos. Furthermore, aggregated blastocyst quality was analyzed by evaluation of the total cell number, cell allocation by mitotrackers staining of embryonic cells, expression of Oct4, Nanog, Sox2, Cdx2 genes, number of OCT4+ nuclei, and presence of DNA fragmentation. Additionally, the developmental rates of Async4n aggregation of domestic cat with Leopardus geoffroyi hybrid (hLg) embryos were evaluated. Async aggregation increased blastocyst cell number and the number of OCT4+ nuclei as compared to non-aggregated diploid (2n) and tetraploid (4n) embryos. Moreover, blastocysts produced by Async4n aggregation showed reduced rates of fragmented DNA. No differences were found in the expression of the pluripotent genes, with exception of the Cdx2 expression, which was higher in 4n and aggregated embryos as compared to the control group. Interestingly, hybrids embryos derived by Async4n aggregation with domestic cat embryos had similar rates of blastocysts development as the control. Altogether, the findings support the use of two-cell-fused embryos to generate tetraploid blastomeres and demonstrate that Async4n aggregation generates good quality embryos.

UTILIZACIÓN DE PLANTAS PUENTE EN EL MEJORAMIENTO DEL COMPLEJO Calibrachoa – Petunia

The hybridization technique through bridging plants uses indirect or intermediate crosses as an instrument to cross parents with different levels of ploidy. The objective of this work is to obtain hybrids that can be used as bridges in crosses with ornamental species of the genus Petunia and/or Calibrachoa. This can overcome the incompatibility barriers that arise when hybridization is direct between species. To obtain hybrids, directed crosses were carried out between: P. integrifolia, P. inflata, P. interior and P. axillaris subspecies axillaris; with C. linoides, C. ovalifolia, C. thymifolia, C. caesia, C. x hybrida var. Kabloom blue and C. x hybrida var. Kabloom yellow. The first step was the detection of putative combinations. In order to do so, the fruiting rate, which was defined as the formation of fruits every 10 intergeneric crosses, was compared, and variable results were found for each combination. A hybrid plant was obtained between P. inflata x C. x hybrida ‘kabloom yellow’, where morphological, pollen viability and meiotic studies confirm its nature. With the bridge hybrid obtained, reciprocal crosses were carried out with all of the Petunia and Calibrachoa species involved, obtaining 31 hybrid plants sharing morphological characteristics of both genera in combinations with P. inflata and P. integrifolia. The identification of intergenetic crosses with a high fruiting rate opens doors for future studies on the development of hybrid embryos. This would allow determining the optimum time to apply embryo rescue techniques in order to increase the number of hybrid bridging plants.