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[Objective]To analyze the effects of different factors on embryoid formation using unpollinated ovaries of Luffa ssp. cultivar ‘Xulv No. 1’ as experimental material. [Method] An L16(215) orthogonal test was set up to analyze the effects of ovary collection time, 4 ℃ cold pretreatment, 35 ℃ dark heat shock treatment, TDZ hormone, 6-BA hormone, ABA hormone, NAA hormone and 2,4-D hormone on embryoid formation in vitro cultured Luffa ssp. ovaries. [Result]Different factors had different effects on the development process of ovary slices. Among them, collecting female flowers that opened on the same day and adding TDZ hormone to MS medium had a promoting effect on embryoid formation in ovary slices. However, 2,4-D hormone had an inhibitory effect on embryoid formation in Luffa ssp. ovaries but could promote rooting of ovary slices. In addition, the interaction between 4 ℃ cold pretreatment and TDZ hormone and between TDZ hormone and female flower collection time also had a significant effect on embryoid formation in Luffa ssp. . [Conclusion]The effective way to form embryoids from Luffa ssp. ovaries is: collecting ovaries on day of flowering+4 ℃ cold pretreatment+0. 04 mg / L TDZ hormone induction in vitro. [ABSTRACT FROM AUTHOR]

The article discusses a new genome editing system developed to enhance the efficiency of breeding inducer and haploid sorting in maize. By utilizing a Cas9 system with specific promoters and markers, inducer lines can be generated more efficiently, improving breeding outcomes. The system allows for visible sorting of haploid embryos at different stages of development, aiding in early identification and research on haploid formation mechanisms. Additionally, automated sorting equipment has significantly increased sorting efficiency, benefiting maize breeding programs. [Extracted from the article]

Cucumis melo ssp. agrestis is a vital crop in the Cucurbitaceae family. Traditional breeding has been the primary method, but prolonged artificial selection has led to a gradual decline in the genetic diversity of thin-skinned melon. Haploid induction system combined with distant hybridization, backcross transfer, gene editing and other breeding techniques can realize germplasm resource innovation, increase genetic diversity of thin-skinned melon, and help thin-skinned melon breeding. This work first established a regeneration system for unfertilized ovules in thin-skinned melons. Based on the regeneration system, experiments were conducted using unfertilized ovules of different genotypes. The results showed that genotype had an impact on the formation of callus tissue, green cells, and bud differentiation in unfertilized ovules. Through validation with eight genotypes of disease-resistant thin-skinned melon, this method exhibits a haploid induction rate of 12.26%, a diploid induction rate of 54.72%, and a tetraploid induction rate of 33.02%. After SSR and SNP molecular marker detection, the obtained haploid plants were resistant to powdery mildew, downy mildew, and frost mold.Key message: The development of an efficient haploid regeneration system for thin-skinned melons enhances genetic diversity and facilitates breeding for disease resistance in various genotypes. [ABSTRACT FROM AUTHOR]

Anther and microspore cultures are efficient methods for inducing haploids in plants. The microspore culture by chromosome-doubling method can produce double haploid lines, developing pure lines within the first or second generations. This study aimed to induce haploid plants in Platycodon grandiflorum using the shed-microspore culture method. P. grandiflorum floral buds (n = 1503) were cultured in six types of medium to induce haploids. Anthers were placed on a solid–liquid double-layer medium and cold pre-treated at 9 °C for one week, followed by incubation in the dark at 25 °C. Embryogenesis was observed after approximately 70 days of culture, producing haploid plants through regeneration. Of the 1503 floral buds, embryos developed in 120 buds, resulting in the induction of 402 individuals. Among the media used, Schenk and Hildebrandt (SH) and 1/2SH exhibited high efficiency, with embryogenesis ratios of 12% and 13.4%, respectively. Additionally, the highest embryogenesis ratio (15.3%) was observed in flower buds sized 10 mm or less. Therefore, we established shed-microspore culture conditions to induce haploids in P. grandiflorum. Using this method, haploids can be efficiently induced in P. grandiflorum, shortening the breeding period by enabling the rapid development of inbred lines. [ABSTRACT FROM AUTHOR]

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Whole genome duplications are implicated in genome instability and tumorigenesis. Human and yeast polyploids exhibit increased replication stress and chromosomal instability, both hallmarks of cancer. In this study, we investigate the transcriptional response of Schizosaccharomyces pombe to increased ploidy generally, and in response to treatment with the genotoxin methyl methanesulfonate (MMS). We find that treatment of MMS induces upregulation of genes involved in general response to genotoxins, in addition to cell cycle regulatory genes. Downregulated genes are enriched in transport and sexual reproductive pathways. We find that the diploid response to MMS is muted compared to the haploid response, although the enriched pathways remain largely the same. Overall, our data suggests that the global S. pombe transcriptome doubles in response to increased ploidy but undergoes modest transcriptional changes in both unperturbed and genotoxic stress conditions. [ABSTRACT FROM AUTHOR]

IVF laboratories routinely adopt morphological pronuclear assessment at the zygote stage to identify abnormally fertilized embryos deemed unsuitable for clinical use. In essence, this is a pseudo-genetic test for ploidy motivated by the notion that biparental diploidy is required for normal human life and abnormal ploidy will lead to either failed implantation, miscarriage, or significant pregnancy complications, including molar pregnancy and chorionic carcinoma. Here, we review the literature associated with ploidy assessment of human embryos derived from zygotes displaying a pronuclear configuration other than the canonical two, and the related pregnancy outcome following transfer. We highlight that pronuclear assessment, although associated with aberrant ploidy outcomes, has a low specificity in the prediction of abnormal ploidy status in the developing embryo, while embryos deemed abnormally fertilized can yield healthy pregnancies. Therefore, this universal strategy of pronuclear assessment invariably leads to incorrect classification of over 50% of blastocysts derived from atypically pronucleated zygotes, and the systematic disposal of potentially viable embryos in IVF. To overcome this limitation of current practice, we discuss the new preimplantation genetic testing technologies that enable accurate identification of the ploidy status of preimplantation embryos and suggest a progress from morphology-based checks to molecular fertilization check as the new gold standard. This alternative molecular fertilization checking represents a possible non-incremental and controversy-free improvement to live birth rates in IVF as it adds to the pool of viable embryos available for transfer. This is especially important for the purposes of 'family building' or for poor-prognosis IVF patients where embryo numbers are often limited. [ABSTRACT FROM AUTHOR]

Haploid induction is of great importance in the breeding of cross-pollinated crops such as maize, and it also expedites the development of new varieties in a shorter period in self-pollinated crops, i.e. soybean. The success rate of traditional techniques is almost negligible at less than 1%, and their applicability is dependent on external factors in soybean. There is a lack of standardized and reproducible systems, which makes it challenging to adopt the existing systems for haploid plant production. Therefore, there is a high demand for implementing innovative approaches for this crop due to the limitations of conventional methods. The BABY BOOM (BBM) gene, which generated haploidy at high rates (> 80%) when expressed in pearl millet or through ectopic expression in the egg cells of rice, maize, and tobacco, is a novel example with promising potential. In this study, we used the egg cell-specific promoter DD45, which was cloned from Arabidopsis, to ectopically express the native soybean BBM1 (GmBBM1) gene in soybean to observe the response to haploidy induction. Initially, the clone pDD45:GmBBM1 was successfully constructed and confirmed by PCR and Sanger sequencing. The construct was subsequently transformed into soybean via a half-seed approach. The expression of GmBBM1 in both flowers and leaves increased in the T0 transgenic soybean lines. The T0 plants and their seeds showed developmental abnormalities described by early senescence and flowering; however, the T1 plants exhibited normal growth characteristics. The ploidy levels of the T1 and T2 plants were determined by flow cytometry and chromosome counting. The flow cytometry histograms revealed haploidy in the T1 generation; however, further chromosome counting in T2 plants revealed changes in chromosome number, and aneuploidy, which may be due to spontaneous doubling. This system is especially important in legume crops, as no reports exist on the application of the BBM1 system in soybean. Our study will provide valuable insights for future research and advancing soybean breeding with haploid induction.Key message: The current investigation unveils the ectopic expression of native GmBBM1, which induces chromosomal aberrations in soybean. [ABSTRACT FROM AUTHOR]

The efficient doubled haploid (DH) plant production methods play a key role in accelerating the breeding of new varieties and hybrids in cultivated plants. Consequently, DH plant production methods are continuously improving for barley (Hordeum vulgare L.) breeding and research programs. Two plant regeneration (FHGR and K4NB) and three rooting media (MSr, N6I and ½N6I + Ca) were compared with four F1 barley cross-combinations to clarify the effect of medium on the regeneration of green and albino plantlets and acclimatization. The plant regeneration efficiency was higher using K4NB medium (74.53 green plantlets/100 anthers and 30.85 albino/100 anthers) compared to FHGR (55.77 green plantlets/100anthers and 21.32 albino/100 anthers). The percentage of acclimatization was highest when the K4NB regeneration medium was combined with the MSr rooting medium. Altogether, 61.83% of the anther culture-derived plantlets of 8 cross-combinations acclimatized to the greenhouse conditions, and 1403 acclimatized plantlets were produced from the F1 cross-combinations. Haploid (22.52%), diploid (69.37%) and tetraploid (8.11%) plantlets were identified among the 111 tested green plantlets by flow cytometric analyses. The tetraploid lines can be explored to offer new scopes for future barley research and breeding directions. Nearly one thousand DH plants have been integrated into our barley breeding program. [ABSTRACT FROM AUTHOR]

Centromeric nucleosomes are determined by the replacement of the canonical histone H3 with the centromere-specific histone H3 (CENH3) variant. Little is known about the centromere organization in allopolyploid species where different subgenome-specific CENH3s and subgenome-specific centromeric sequences coexist. Here, we analyzed the transcription and centromeric localization of subgenome-specific CENH3 variants in the allopolyploid species Arabidopsis suecica. Synthetic A. thaliana x A. arenosa hybrids were generated and analyzed to mimic the early evolution of A. suecica. Our expression analyses indicated that CENH3 has generally higher expression levels in A. arenosa compared to A. thaliana, and this pattern persists in the hybrids. We also demonstrated that despite a different centromere DNA composition, the centromeres of both subgenomes incorporate CENH3 encoded by both subgenomes, but with a positive bias towards the A. arenosa-type CENH3. The intermingled arrangement of both CENH3 variants demonstrates centromere plasticity and may be an evolutionary adaption to handle more than one CENH3 variant in the process of allopolyploidization. [ABSTRACT FROM AUTHOR]

Members of DOMAIN OF UNKNOWN FUNCTION 679 membrane proteins (DMPs) have the DUF679 domain, which plays an important role in the process of plant fertilization. In this study, bioinformatics methods were used to identify and analyze the DMP gene family in pepper. The location of the expression of the DMP gene family was explored according to the transient expression of Nicotiana benthamiana, and its expression patterns in different tissues and abiotic stress treatments were analyzed by qRT-PCR. A total of 17 CaDMP genes were identified from the three capsicum varieties, and sub-cellular localization prediction showed that CaDMPs were located on the cell membrane. Phylogenetic analysis showed that CaDMP5 in subgroup Ⅳ was highly homologous with haploid induction genes in Arabidopsis and maize, and its expression level in reproductive organs was significantly higher than that in other tissues, suggesting that CaDMP5 could be a candidate gene for haploid induction in pepper. The expression of CaDMPs increased to varying degrees after different stress treatments, indicating that the DMP gene plays an important role in plant growth and development. The CaDMP gene family was systematically analyzed in this study, which provided preliminary insights for the further research of Capsicum haploid breeding. [ABSTRACT FROM AUTHOR]

Cytological and chromosomal studies of ant-like spiders Myrmarachne melanocephala MacLeay, 1839, were undertaken with 12 males captured alive in the months of January to July 2023 from three habitats. The haploid count of male specimens was observed to be 13: 4 acrocentric (including X chromosome), 6 subtelocentric, 2 submetacentric, and 1 metacentric and showed X0 sex determination mechanisms, so the diploid count of the species was 25 (13+12). The single sex chromosome occupied roughly 4-6 per cent of the nuclear volume prominently in the Interphase -- Prophase I stage. The structure of X chromosome in interphase stage was circular-rectangular block. The peculiar shape of rod-shaped X chromosome was maintained from early pre-leptotene stage till the end of the division particularly the Prophase I. The synapsis started early from late leptotene and duration of zygotene was long enough to visualize the perfect ones in late zygotene. There were nine diplotene bivalents with interstitial chiasma. [ABSTRACT FROM AUTHOR]

Matrilineal (MTL) gene is known to induce haploidy because of the 4-bp insertion in the last exon of the gene in maize haploid inducer stock. Orthologue gene search in wheat genome identified TaPLA genes on wheat chromosomes 4A, 4B and 4D. The DNA sequence homology between the maize MTL gene and TaPLA gene orthologues in wheat was 86–88%. The present study involves the selection of EMS mutants with mutations in TaPLA genes from Cadenza (6X) and Kronos (4X) TILLING populations with an aim to identify/develop a haploid inducer stock in wheat. A total of 45 mutants including 24 for Cadenza and 21 for Kronos were selected with 18 having mutations in TaPLA.4A, 17 in TaPLA.4B and 10 in TaPLA.4D gene. On applying KASP assay using mutation-specific markers, 36 markers showed clear polymorphism among the parents and the mutants. On the basis of validated KASP assay and the mutations leading to the changes in the amino acid, their biochemical properties and nature of amino acids, a total of 17 Cadenza and 14 Kronos mutants have been selected to be further used for pyramiding of these mutations. The validated KASP assay will be used to check the true F1s and homozygosity status of the mutant alleles in F2s. The homozygous F2s will be further used to cross with hexaploid or tetraploid wheat to check the haploidy induction ability. [ABSTRACT FROM AUTHOR]

Simple Summary: The nutrient strategy is a significant part of Gephyrocapsa huxleyi's physiology. Unlike other studies mainly focusing on photosynthesis, we conducted a short-term incubation to test the difference in phagocytosis capacity between diploid calcified G. huxleyi RCC1266 and haploid non-calcified G. huxleyi PML B92/11 in different light regimes. The results suggested that a higher percentage has been found in the non-calcified strain as compared to the calcified strain, no matter whether it was under light or in the dark. Additionally, the non-calcified strain showed a greater percentage in the dark than under light. Coccolithophores play a significant role in marine calcium carbonate production and carbon cycles, attributing to their unique feature of producing calcareous plates, coccoliths. Coccolithophores also possess a haplo-diplontic life cycle, presenting distinct morphology types and calcification states. However, differences in nutrient acquisition strategies and mixotrophic behaviors of the two life phases remain unclear. In this study, we conducted a series of phagocytosis experiments of calcified diploid and non-calcified haploid strains of coccolithophore Gephyrocapsa huxleyi under light and dark conditions. The phagocytosis capability of each strain was examined based on characteristic fluorescent signals from ingested beads using flow cytometry and fluorescence microscopy. The results show a significantly higher phagocytosis percentage on fluorescent beads in the bacterial prey surrogates of the non-calcified haploid Gephyrocapsa huxleyi strain, than the calcified diploid strain with or without light. In addition, the non-calcified diploid cells seemingly to presented a much higher phagocytosis percentage in darkness than under light. The differential phagocytosis capacities between the calcified diploid and non-calcified haploid Gephyrocapsa huxleyi strains indicate potential distinct nutritional strategies at different coccolithophore life and calcifying stages, which may further shed light on the potential strategies that coccolithophore possesses in unfavorable environments such as twilight zones and the expanding coccolithophore niches in the natural marine environment under the climate change scenario. [ABSTRACT FROM AUTHOR]

Summary: In planta haploid induction (HI), which reduces the chromosome number in the progeny after fertilization, has garnered increasing attention for its significant potential in crop breeding and genetic research. Despite the identification of several natural and synthetic HI systems in different plant species, the molecular and cellular mechanisms underlying these HI systems remain largely unknown. This review synthesizes the current understanding of HI systems in plants (with a focus on genes and molecular mechanisms involved), including the molecular and cellular interactions which orchestrate the HI process. As most HI systems can function across taxonomic boundaries, we particularly discuss the evidence for conserved mechanisms underlying the process. These include mechanisms involved in preserving chromosomal integrity, centromere function, gamete communication and/or fusion, and maintenance of karyogamy. While significant discoveries and advances on haploid inducer systems have arisen over the past decades, we underscore gaps in understanding and deliberate on directions for further research for a more comprehensive understanding of in vivo HI processes in plants. [ABSTRACT FROM AUTHOR]

• Demonstrated the correlation between morphological parameters of flower bud and microspore developmental stages in onion for the first time. • This study indicated the positive correlation between flower bud length and diameter, and anther length and diameter and microspore developmental stages. • Combination of flower bud length, diameter, and the green colour intensity of anthers was found as easily applicable, rapid, and accurate criteria for identification of microspore developmental stages. • The most reliable approach for accurate visualization of microspore stages in onion involved diluting DAPI in PBS and fixing the anthers in carnoy's fixative. Correlations between the morphological characteristics of flower buds and the developmental stages of the male gametophyte have practical implications as a reliable indicator for selecting suitable plant material for androgenesis. However, the application of androgenesis in onions has been challenging, despite extensive research on anther culture. This research paper presents findings on the associations between flower bud length and diameter, anther length and diameter, and the developmental stages of the male gametophyte across ten onion genotypes. All the morphological parameters studied in this study were positively correlated with stages of microspore development. The most responsive stages for anther culture in most of the crops are the late uninucleate and early binucleate stages. Although the bud stage and microspore development stage among the genotypes varied but this stage was typically observed in flower buds with a length of 2.5 to 3.5 mm and a diameter of 2.0 to 2.7 mm. Likewise, the late uninucleate and early binucleate stages were found in anthers with length of 1.2 to 1.6 mm and diameter of 0.55 to 0.75 mm. Additionally, we examined the utility of the aforementioned morphological parameters in identifying microspore developmental stages. Based on our results, we propose a combination of flower bud length, diameter, and the green colour intensity of anthers as easily applicable, rapid, and accurate criteria for identifying microspore developmental stages. Different methods were employed to evaluate the efficiency of DAPI staining fluorescence for visualizing microspore stages. These methods included staining with or without anther fixation, diluting DAPI in PBS with or without anther fixation, and using a combination of DAPI, EDTA, Triton X100, and PBS buffer, with or without a fixative. The study concluded that the most reliable approach involved diluting DAPI in PBS and fixing the anthers in Carnoy's fixative, providing an accurate visualisation of microspore stages. [ABSTRACT FROM AUTHOR]

Maize is one of the most successful crops with regard to the utilization of heterosis. The haploid induction technique is one of the fastest methods to obtain pure maize material at the present stage. However, the molecular mechanism of haploid doubling is rarely reported. In this study, we treated B73 and ZNC442 haploid young shoots with colchicine for 0 h, 6.2 h, and 10 h, and analyzed the differentially expressed genes (DEGs). We found that colchicine treatment for 6.2 h and 10 h compared to 0 h resulted in a total of 4868 co-DEGs. GO enrichment analysis and KEGG metabolic pathway analysis found significantly enriched 282 GO terms and 31 significantly pathways, respectively. Additionally, The GO term and KEGG pathway genes of spindle, cytoskeleton, microtubules and nuclear division were selected for analysis, and three candidate genes were screened by taking intersections. Zm00001d033112, Zm00001d010525, and Zm00001d043386 were annotated as kinesin-associated protein 13, kinesin-like protein KIN-10C, and kinesin light-chain LC6, respectively. The real-time fluorescence quantification (RT-PCR) results revealed that Zm00001d033112, Zm00001d010525, and Zm00001d043386 had the same trends as RNA-seq. Interestingly, Zm00001d033112 is homologous gene AT3G20150 in Arabidopsis, which was involved in the regulation of chromosome movement and mitotic spindle assembly. Our study suggests that kinesin genes may play an important role in doubling chromosomes, thus providing valuable information for future studies on the molecular mechanisms of chromosome doubling in maize. [ABSTRACT FROM AUTHOR]

Haploid sporophytes of Anisocampium niponicum with 2n = 40, were produced artificially by induced apogamy in vitro. They were subsequently transplanted into pots and two of them have been cultivated for the investigation of sporogenesis and/or production of chimera for more than 20 years. Haploid A. niponicum is sterile, but an abnormal chimeric pinnule that developed spontaneously in a single frond produced sporangia with spores. Each sporangium bore approximately 32 spores that were almost uniform in size. Sowing of these spores resulted in 50 gametophytes. Of 20 gametophytes cultured individually, five produced sporophytes apogamously after eight months. Both the gametophytes and subsequent apogamous sporophytes showed a chromosome number of 2n = 40. Our study demonstrates that a haploid sporophyte offspring can be produced from a haploid mother sporophyte via haploid spores. Since asexual reproduction is a prominent evolutionary process in ferns, the reproduction of a haploid A. niponicum sporophyte by unreduced spore formation might help to elucidate how apogamous ferns occur and evolve. [ABSTRACT FROM AUTHOR]

The haploid and doubled haploid plants serve as valuable tools for breeders due to their ability to expedite the mapping of genes of agronomic importance, as well as accelerate the breeding cycle for generation of novel hybrids and improved homogenous varieties. Successful anther/microspore culture largely depends on the use of microspores at appropriate developmental stages at the time of culture, which can be specific for each plant species and genotype. In the present study, we described the visible morphological characteristics of flower buds and anthers at different developmental stages to identify the optimal microspore stage within the anther/buds of two pepper hybrids, Indra and Lakshmi. This information enabled us to predict the suitable microspore stage for successful haploid production. To enhance the visualization of nuclei in the pepper microspores, different concentrations of FeCl3 were employed as a mordant to Carnoy's fixative I, followed by DAPI staining. A clear and distinct nucleus was observed using DAPI staining procedures in the pepper microspores when fixed in Carnoy's solution containing ferric chloride (40–90 µl) as mordant. The use of mordant thus facilitated the efficient cytological analysis of the pepper microspores. Present results indicate that, to achieve efficient haploid production, flower buds with an average length of 4.4 to 5.02 mm for the hybrid Indra and 5.15 to 5.40 mm for the hybrid Lakshmi should be utilized. Additionally, these buds should have a calyx covering approximately 80–90% of the total bud length. We observed that in such buds, microspores are in the late-uninucleate and early binucleate stage which has been reported to be the most conducive stage for androgenesis induction in pepper. [ABSTRACT FROM AUTHOR]

This article discusses the successful generation of haploid plants in foxtail millet through the expression of the dandelion PARTHENOGENESIS (PAR) gene in egg cells. Haploid induction is a valuable technique in crop breeding, and this study demonstrates its feasibility in foxtail millet, a significant multigrain crop. The researchers used a heterologous expression system to introduce the PAR gene into foxtail millet and observed haploid induction in six out of eleven transgenic lines. The haploid plants exhibited distinct characteristics and complete loss of fertility. This research provides insights into the potential application of haploid induction in foxtail millet breeding programs. [Extracted from the article]

Anther and microspore cultures are efficient methods for inducing haploids in plants. The microspore culture by chromosome-doubling method can produce double haploid lines, developing pure lines within the first or second generations. This study aimed to induce haploid plants in Platycodon grandiflorum using the shed-microspore culture method. P. grandiflorum floral buds (n = 1503) were cultured in six types of medium to induce haploids. Anthers were placed on a solid–liquid double-layer medium and cold pre-treated at 9 °C for one week, followed by incubation in the dark at 25 °C. Embryogenesis was observed after approximately 70 days of culture, producing haploid plants through regeneration. Of the 1503 floral buds, embryos developed in 120 buds, resulting in the induction of 402 individuals. Among the media used, Schenk and Hildebrandt (SH) and 1/2SH exhibited high efficiency, with embryogenesis ratios of 12% and 13.4%, respectively. Additionally, the highest embryogenesis ratio (15.3%) was observed in flower buds sized 10 mm or less. Therefore, we established shed-microspore culture conditions to induce haploids in P. grandiflorum. Using this method, haploids can be efficiently induced in P. grandiflorum, shortening the breeding period by enabling the rapid development of inbred lines.

Haploidization technique is the modern and advanced breeding technique used to enhance economically essential crops, including cucumber, to meet consumers' preferences and needs worldwide. This study aimed to evaluate the effects of factors such as genotype, thermal pre-treatments, and nutrient medium combinations that are considered in haploid production via ovary culture in four commercial cucumber varieties. Four different induction media with four distinct maturation medium combinations and three F1 and one open-pollinated (Beith Alpha) cucumber varieties were used. All explants (cucumber ovaries' slices) were inoculated in induction media and remained at 35 °C/3 d in the dark, then were transferred at 25 ± 1 °C 16-/8-h light/dark photoperiod for a further 9 d. The results of the study demonstrated that the embryo-like structure (ELS) formation rate was high in induction medium 2 which was successful in all varieties of Ptk40 (20%), Botanik (20%), Beith Alpha (16%), and Sardes (13.3%), respectively, compared to other induction medium combinations used in this study. Callus formation rates obtained from regeneration of medium 2 supplemented with 2.0 mg L−1 6-benzylaminopurine (BAP) and 0.5 mg L−1 1-naphthaleneacetic acid (NAA) were Ptk40 (65%), Sardes (60%), and Botanik (40%), and no callus was formed in Beith Alpha. Due to the findings of this study, the nutrient medium containing KIN and 2,4-D seems to be successful during induction of haploid embryo formation in cucumbers. One plantlet germinated but perished at early stage. The plantlet regeneration was observed to be low. Further studies are required to optimize the embryo regeneration medium composition due to the cucumber growing seasons, genotypes, and nutrient medium. [ABSTRACT FROM AUTHOR]

Strawberry (Fragaria x ananassa Duch.) which is one of the widely grown berry species in the world has economic and commercial importance. In commercial strawberry varieties, in order to increase yield and quality, it is necessary to obtain starting materials that are resistant/tolerant to biotic and abiotic stress factors. Biotechnological methods have an important place in strawberry breeding studies due to the long and costly process of classical breeding methods, the genetic expansion of seed production, high ploidy level and strong heterozygosity. Haploid plant production is an efficient breeding method that has been successfully applied to most plant species. However, due to the lack of sufficient haploid studies on strawberry and the fact that a specific protocol for this species has not yet been developed the necessary progress has not been made in this regard. In this study, the effectiveness of some factors determine the success in anther culture which has a significant place in obtaining haploid strawberries was investigated. For this reason, first, different sodium hypochlorite doses (NaOCl; 1%, 2%, 3%) and application durations (10, 15, 20 min) were used to determine the appropriate method for sterilisation, then cold pre-treatments (24, 36, 48, 72 hours at +4 °C) and different silver nitrate doses (AgNO3; 10, 20, 30, 40 mg l-1) were employed for callus induction in Festival strawberry variety. At the conclusion of the study it was observed that the lowest contamination rate (1%) was obtained by soaking in 1% sodium hypochlorite solution for 10 minutes. Cold pretreatment of flower buds at +4 °C for 36 hours produced the highest callus induction rate (96%). The evaluation of the effect of AgNO3 application at different doses on the callus induction rate revealed that the highest callus induction (82%) was obtained from 20 mg l-1 AgNO3 dosage. This study showed that anther culture practices in strawberry can be improved by using cold pre-treatment, appropriate sterilization method and silver nitrate addition to the medium. [ABSTRACT FROM AUTHOR]

Gynogenesis plays a crucial role in creating homozygous lines and accelerates the breeding process. This technique has been the subject of investigation in onion for several decades. But achieving desired efficiency levels, an effective diploidization protocol and a high survival rate of doubled haploids is still a limiting factor. Further, research on short-day onions, which occupy a major share in production, is still inadequate compared to long-day onions. This study investigated the gynogenic response of ten short-day onion genotypes, using whole flower buds, cultured on MS and B5 medium supplemented with different growth regulators and stress treatments. ‘Bhima Safed’ (9.52%) and ‘Bhima Dark Red’ (9.14%) recorded the highest gynogenesis efficiency. ‘Early Grano’ did not respond to gynogenesis indicating its recalcitrant nature. Among media, OGH8 (MS medium supplemented with 2 mg/l 2,4-D and 2 mg/l BAP) was found superior to OGH4 (B5 medium supplemented with 2 mg/l 2,4-D and 2 mg/l BAP) and 0MS (MS medium without growth regulators) in inducing gynogenesis. Cold pretreatment of flower buds for 2 days in Bhima Dark Red (7.33%) was superior to control (6.67%). Heat treatment of flower buds was not favourable for gynogenesis induction. The induced embryos were studied for root induction. RMS1 (16.86) and RMS2 (10.14) recorded the highest number of roots, while RMS5 (4.86) exhibited the highest propensity for shoot formation, ultimately resulting in the successful regeneration of whole plants. 51.39% of the induced plants were haploid, 26.39% spontaneous diploid, and 22.22% mixoploid. For diploidization, treatment of 500 μM colchicine for 4 h was superior with a 75% survival rate. Doubled haploid plants were cultured on the MS medium for bulbil induction. These bulbils were transferred under field conditions for flowering and seed production. This study presents a comprehensive strategy for gynogenesis induction and will be of benefit to onion researchers.Key message: This paper gives a comprehensive strategy of doubled haploid production from genotype selection, haploid induction, doubled haploids acclimatization to flowering, for the first time in short-day tropical Indian onions. [ABSTRACT FROM AUTHOR]

Allium tuncelianum (Kollman) Özhatay, Matthew and Şiraneci forms a single-cloved edible white bulb with a mild garlic (A. sativum) odor and taste. Its ability to form seeds makes it suitable for genetic improvement via classical and modern approaches. A detailed study was carried out to determine the gynogenic and somatic plant regeneration potential of two A. tuncelianum accessions (AT1 and AT2). Unopened flower buds of A. tuncelianum accessions were cultured in various BDS (Dunstan and Short, Physiol Plant 41:70-72, 1977) and MS (Murashige and Skoog, Plant Physiol 15:473-497, 1962)-based induction media. Accessions showed slight differences in their responses to gynogenesis and somatic shoot induction cultures. Gynogenic plantlets were obtained in six induction media (T2, T3, T6, T9, T12, and T15). Three of these (T2, T3, and T9) did not contain plant growth regulators (PGRs). Seventeen (0.09%) gynogenic plantlets were obtained from approximately 20,000 AT1 and AT2 flower buds used in gynogenesis induction experiments. The highest gynogenic plantlet production frequency (0.34%) in AT1 was achieved with buds cultured in T12 [MS-based medium with 1.0 mg L−1 α-naphthalene acetic acid, 8.0 mg L−1 isopentenyl adenine, and 100.0 g L−1 sucrose]. Flower buds of AT2 showed the best gynogenic response (0.44%) in T2 (PGR-free BDS–based medium with 50.0 g l−1 sucrose). Somatic regeneration was observed in six media (T5, T6, T11, T12, T14, and T15) containing at least 50.0 g L−1 sucrose and auxin- and cytokinin-type PGRs. Two hundred and thirty-three (1.18%) somatic plantlets were obtained from the cultured flower buds of AT1 and AT2. The highest somatic plantlet production frequencies for AT1 (4.44%) and AT2 (3.89%) were obtained from flower buds cultured in T5 [BDS-based medium with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid, 2.0 mg L−1 6-benzylaminopurine, and 50.0 g L−1 sucrose]. Three induction media (T6, T12, and T15) provided both gynogenic and somatic regeneration in A. tuncelianum. Eight of 17 (46.06%) gynogenic plantlets and 33 of 233 (14.16%) somatic plantlets continued to grow, resulting in healthy plants with green leaves and well-established root systems. The remaining gynogenic plantlets had abnormal development. Flow cytometric analysis of the well-developed gynogenic plants showed that two were haploid (25%), four were diploid (50%), and two were mixoploid (25%) containing both haploid and diploid cells. The nine abnormal gynogenic plantlets were diploid. Results obtained from this study suggest that doubled haploid technology can be used in the production of homozygous A. tuncelianum inbred lines in breeding programs. [ABSTRACT FROM AUTHOR]

Introduction Given the economic importance of growing flowers and plants in the world, the use of new technologies and methods in the improvement of ornamental plants in order to market them can play a significant role in marketing of these products and their trade in the international markets. Marigold (Tagetes spp.), is an annual and essential plant that is cultivated as an ornamental plant in many parts of the world (Neher, 1968). But in recent years, marigolds have been used as a commercial source of essential oils, biological compounds, pigments and cosmetics (Anonymous, 1976), control of malaria mosquitoes (Wanzala and Ogoma, 2013), antihypertensive, anti-inflammatory, antidepressant, antimicrobial, antimicrobial (Senatore and Feo, 1999), and control of nematode (Prasad et al., 1992). Anther or microspore culture is known to be an effective method for producing haploid plants (Henry and De Baizer, 1980). Hybrid seed production requires pure line (as a parent), and the double haploid method can reduce the production period of pure lines to 5-6 years. Production of hybrid seeds in this valuable plant is of great importance, and the double haploid method can be important in this regard. Materials and Methods In the anther culture of marigold, a culture medium containing 0.2 mg/l of Naphthalene acetic acid and 1 mg /l of 6-Benzylaminopurine was used. In this study, the effect of genotype, bud size and mannitol on androgenesis induction of marigold anthers was evaluated during two separate experiments. A factorial experiment was conducted in a completely randomized design with two factors including genotype at 5 levels (T1, T2, T3, T4, T5) and bud size at three levels (3-5, 5-10 and 10-15 mm) for first experiment. The second experiment was also performed as a factorial in a completely randomized design with 3 replications. In the recent experiment, the first factor included the method of mannitol application and the second factor included the different concentrations of mannitol (0.1, 0.2, 0.3, 0.4 and 0.5 M). The factor of application method was in 2 levels: 1: adding different concentrations of mannitol to the solid culture medium and 2: pre-treating the anthers with the liquid culture medium containing different concentrations of mannitol for 24 hours. The T4 genotype was used in the second experiment. Results In the first experiment, the effect of different bud sizes and 5 different genotypes on callus formation, mean number of shoot per anther, mean number of shoot per callus and percentage of complete plant regeneration in anther culture of marigold were studied. The results of this experiment showed that buds with the length of 5-10 mm have anthers which their microspores are at the proper growth and development stage for callogenesis and shoot production. The T3 and T4 genotypes, (both of them belonging to French species, Tagetes patula), produced the highest percentage of plant regeneration among the various cultivars. In the second experiment, we explored the impact of mannitol treatment on androgenic traits in marigold anther culture. Specifically, we examined two concentrations: 0.1 M and 0.2 M mannitol, both applied in the form of solid culture medium. Additionally, we investigated two concentrations, 0.0 M and 0.2 M mannitol, when applied as a pre-treatment in a liquid medium containing mannitol. These treatments yielded the highest percentage of callus formation. While the pre-treatment of anthers with a liquid culture medium containing 0.5 M mannitol led to the highest mean number of shoot per anther and the mean number of shoots per callus. Also, the pre-treatment with liquid medium containing 0.2 M mannitol showed the highest percentage of complete plant regeneration. Conclusion Results showed that in marigold, buds with the size of 5-10 mm contained microspores with mid-uninucleate stage to early bi-nucleate stage showed the highest response to the induction of androgenesis. Also, T3 and T4 genotypes belong to the French species showed the highest response to the regeneration. In another experiment, the pre-treatment of anthers with 0.2 and 0.5 M mannitol by using mannitol in a liquid culture medium for 24 hours, respectively showed the highest percentage of complete plant regeneration and the highest mean number of shoot per callus and anther. Chromosome counting results showed that 3 out of 5 examined plants were dihaploid and had 24 chromosomes in their root tip cells, while examined mother plants were tetraploid and showed 48 chromosomes in their root tip cells.

The search for new, effective methods for broadening the genetic polymorphism of the original breeding material remains one of important problems of hybrid maize breeding. Globally, breeding of commercial maize varieties and hybrids is carried out using diploid genotypes, whereas tetraploid sources of initial material and wild relatives of maize are poorly involved in the breeding process. The direct heteroploid crosses between diploid and tetraploid genotypes lead to the formation of weakly fertile or completely sterile triploid hybrids, which are cytologically unstable in subsequent generations. Tetraploid maize (2n=40), as well as some wild relatives with tetraploid genome, such as Zea perennis Hitchk. (2n=40) and Tripsacum dactiloides (L.) L. (2n=72), are attractive to breeders as sources for improving economically valuable traits. The attractiveness of resynthesized diploid lines is explained by the fact that unequal crossing over between homologous chromosomes forming polyvalent associations of chromosomes, more chromosomal rearrangements occur in tetraploids than in diploid genotypes, the chromosomes of which form bivalents. Synthetic tetraploid populations of maize and its tetraploid wild relatives have great potential of variability for improving diploid maize. The authors proposed a direct method for the resynthesis of doubled haploid lines using haploid induction and an indirect method for obtaining diploid lines by heteroploid crossing and subsequent segregation of a triploid hybrid in the progeny. The method of resynthesizing the tetraploid maize genome to diploid serves as an ideal model for studying the processes of crossing over in meiosis between multivalent associations of homologous chromosomes; it is promising for obtaining diploid lines with an increased frequency of recombination between the homologous chromosomes of different genomes, combined into a common one, and can serve as a source for obtaining aneuploid series.

The efficient doubled haploid (DH) plant production methods play a key role in accelerating the breeding of new varieties and hybrids in cultivated plants. Consequently, DH plant production methods are continuously improving for barley (Hordeum vulgare L.) breeding and research programs. Two plant regeneration (FHGR and K4NB) and three rooting media (MSr, N6I and ½N6I + Ca) were compared with four F1 barley cross-combinations to clarify the effect of medium on the regeneration of green and albino plantlets and acclimatization. The plant regeneration efficiency was higher using K4NB medium (74.53 green plantlets/100 anthers and 30.85 albino/100 anthers) compared to FHGR (55.77 green plantlets/100anthers and 21.32 albino/100 anthers). The percentage of acclimatization was highest when the K4NB regeneration medium was combined with the MSr rooting medium. Altogether, 61.83% of the anther culture-derived plantlets of 8 cross-combinations acclimatized to the greenhouse conditions, and 1403 acclimatized plantlets were produced from the F1 cross-combinations. Haploid (22.52%), diploid (69.37%) and tetraploid (8.11%) plantlets were identified among the 111 tested green plantlets by flow cytometric analyses. The tetraploid lines can be explored to offer new scopes for future barley research and breeding directions. Nearly one thousand DH plants have been integrated into our barley breeding program.

Members of DOMAIN OF UNKNOWN FUNCTION 679 membrane proteins (DMPs) have the DUF679 domain, which plays an important role in the process of plant fertilization. In this study, bioinformatics methods were used to identify and analyze the DMP gene family in pepper. The location of the expression of the DMP gene family was explored according to the transient expression of Nicotiana benthamiana, and its expression patterns in different tissues and abiotic stress treatments were analyzed by qRT-PCR. A total of 17 CaDMP genes were identified from the three capsicum varieties, and sub-cellular localization prediction showed that CaDMPs were located on the cell membrane. Phylogenetic analysis showed that CaDMP5 in subgroup Ⅳ was highly homologous with haploid induction genes in Arabidopsis and maize, and its expression level in reproductive organs was significantly higher than that in other tissues, suggesting that CaDMP5 could be a candidate gene for haploid induction in pepper. The expression of CaDMPs increased to varying degrees after different stress treatments, indicating that the DMP gene plays an important role in plant growth and development. The CaDMP gene family was systematically analyzed in this study, which provided preliminary insights for the further research of Capsicum haploid breeding.

Cytopenia due to the abnormal regulation of GATA1 could manifest as varying degrees of thrombocytopenia and/or anemia and more severely in male children than in female children. Here, we describe the case of pancytopenic and transfusion-dependent twin brothers at our center whose bone marrow puncture revealed low bone marrow hyperplasia. Whole-exome sequencing revealed that the twins had a new germline GATA1 mutation (nm_002049: exon 3:c.515 T >C:p.F172S), which confirmed the diagnosis of GATA1 mutation–related pancytopenia. The mutation was inherited from their mother, who was heterozygous for the mutation. Sanger sequencing verified the pathogenicity of the mutation. Further family morbidity survey confirmed that GATA1 mutation–related pancytopenia is an X-linked recessive genetic disorder. We developed haploid hematopoietic stem cell transplantation programs for twins, with the father as the only donor, and finally, the hematopoietic reconstruction was successful. Although they experienced acute graft-versus-host disease, hemorrhagic cystitis, and a viral infection in the early stage, no abnormal manifestations or transplant-related complications were observed 3 months after transplantation. Through hematopoietic stem cell transplantation technology for one donor and two receptors, we eventually cured the twins. The p.F172S variant in the new germline GATA1 mutation may play an essential role in the pathogenesis of GATA1 mutation–related cytopenia. [ABSTRACT FROM AUTHOR]

Chrysanthemum (Chrysanthemum × morifolium (Ramat.) Hemsl.) holds a prominent position in the market of ornamental plants. To further advance chrysanthemum breeding efforts, the development of haploids may be useful. Therefore, the effect of various chemical and thermal treatments on regeneration efficiency and ploidy level in chrysanthemum was studied. Ovaries and ovules of three chrysanthemum cultivars, i.e., 'Brasil,' 'Capitola,' and 'Jewel Time Yellow,' were cultured either on a medium with 1 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and different concentrations (0.5–1.5 mg·L−1) of thidiazuron (TDZ) or subjected to thermal shock (pretreatment temperature of 4 °C or 32 °C) and cultured on a medium with 1 mg·L−1 2,4-D and 1 mg·L−1 6-benzylaminopurine (BAP). It was found that ovaries had a greater organogenic potential (both in terms of callogenesis and shoot formation) than ovules. Microscopic analyses revealed that shoots mainly developed via indirect somatic embryogenesis from a callus developed from the ovary wall. The highest number of shoots was produced in cooled (at 4 °C) ovaries of chrysanthemum 'Brasil' and in 'Jewel Time Yellow' ovaries cultured on a medium with 1.0–1.5 mg·L−1 TDZ. The latter cultivar also had the highest potential to produce plants with an altered ploidy level (doubled and halved the number of chromosomes). This study demonstrates that manipulating factors such as temperature and thidiazuron concentration can enhance regeneration efficiency and induce altered ploidy levels in selected cultivars, offering valuable insights for chrysanthemum breeding programs. [ABSTRACT FROM AUTHOR]

Meiosis in Plasmodium is post-zygotic, unlike that in most model eukaryotes. Key components involved in meiosis are either highly divergent or absent in Plasmodium. There is DNA replication and recombination of homologous chromosomes without karyokinesis during meiosis in Plasmodium. Meiosis is essential for malaria parasite transmission. Meiosis is sexual cell division, a process in eukaryotes whereby haploid gametes are produced. Compared to canonical model eukaryotes, meiosis in apicomplexan parasites appears to diverge from the process with respect to the molecular mechanisms involved; the biology of Plasmodium meiosis, and its regulation by means of post-translational modification, are largely unexplored. Here, we discuss the impact of technological advances in cell biology, evolutionary bioinformatics, and genome-wide functional studies on our understanding of meiosis in the Apicomplexa. These parasites, including Plasmodium falciparum, Toxoplasma gondii , and Eimeria spp. , have significant socioeconomic impact on human and animal health. Understanding this key stage during the parasite's life cycle may well reveal attractive targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Background: Okra [Abelmoschus esculentus (L.) Moench.] is a nutrient-rich vegetable crop widely grown in the tropics and subtropics mainly for its edible pods. The haploid technique has been used in plant breeding for the improvements of plants and to develop new varieties in relatively a short time. Hence, we have optimized several factors such as plant growth regulators (PGR), sucrose concentration, cold treatment, type of media and culture conditions for callus induction from the anther and ovary of okra (557 F1 hybrid). Methods: The flower buds of different sizes were collected to determine various stages of development and then subjected to cold pretreatments. The explants were then cultured on various combinations of PGRs i.e., naphthyloxy acetic (NOA), Indole acetic acid (IAA), 2, 4-Dichlorophenoxy acetic acid (2, 4-D), Benzyl amino purine (BAP), isopentenyl adenine 2iP, Kinetin (KIN) and Thidiazuron (TDZ). Result: The optimum developmental stage of microspore for callus initiation was achieved from flower buds of okra and its size was about 11 mm long. Flower buds that emerged one week after the flowering showed significantly higher percentage of callus induction. The optimum stage for ovary and ovule culture were one or two days prior to anthesis and the flower buds stage was 35±1mm. In conclusion, our study investigated the effect of several factors that affects callus induction in okra and optimized cultured conditions for future haploid development for okra. [ABSTRACT FROM AUTHOR]

The efficiency of the anther culture method in vitro largely depends on the responsiveness of varieties and hybrids to the induction of androgenesis. The purpose of the current study was to estimate the efficiency of haploproduction of regenerated lines (R0) of winter bread wheat developed by anther culture in vitro and grown in a greenhouse. To achieve this goal, there was set a task to conduct a morphometric estimation of the developed heads (R0) to identify their fertility level. The study was carried out in the laboratory of cell breeding of the FSBSI Agricultural Research Center “Donskoy” in 2021–2022. The objects of the study were 7 hybrid combinations of winter wheat, which were responsive to androgenesis in vitro in the culture of isolated anthers. As a result of induced androgenesis on an artificial nutrient medium, there were identified 333 neoplasms, represented by calluses, embryoids and embryogenic complexes. Green regenerated plants were vernalized in test tubes, cloned, diloidized, then planted into soil. There were obtained 13 haploid lines, after diploidization only 34 plants survived. The highest haploproductive capacity was established in the hybrid combination F3 ‘Rostovchanka 7xBezostaya 100’. In this combination, there were formed 8 green regenerants, 21 plants of which were developed together with clones, 18 of which survived. The cytometric estimation has shown, that 25 dihaploid (2n = 42) and 16 haploid (n = 21) wheat plants have been identified by hybrid combinations, including 13 dihaploids from the combination ‘Rostovchanka 7xBezostaya 100’. As a result of the structural analysis and morphometric estimation of the head there has been found a different fertility degree of androgenic regenerated plants. There have been established prospects of using the dihaploid line ‘79.2.1.2’ in further breeding work based on the hybrid combination F3 ‘Rostovchanka 7xBezostaya 100’ with the highest values of such yield elements as ‘number of heads’, ‘number and weight of grains per head’.

BackgroundDespite marked recent improvements in long-read sequencing technology, the assembly of diploid genomes remains a difficult task. A major obstacle is distinguishing between alternative contigs that represent highly heterozygous regions. If primary and secondary contigs are not properly identified, the primary assembly will overrepresent both the size and complexity of the genome, which complicates downstream analysis such as scaffolding.ResultsHere we illustrate a new method, which we call HapSolo, that identifies secondary contigs and defines a primary assembly based on multiple pairwise contig alignment metrics. HapSolo evaluates candidate primary assemblies using BUSCO scores and then distinguishes among candidate assemblies using a cost function. The cost function can be defined by the user but by default considers the number of missing, duplicated and single BUSCO genes within the assembly. HapSolo performs hill climbing to minimize cost over thousands of candidate assemblies. We illustrate the performance of HapSolo on genome data from three species: the Chardonnay grape (Vitis vinifera), with a genome of 490 Mb, a mosquito (Anopheles funestus; 200 Mb) and the Thorny Skate (Amblyraja radiata; 2650 Mb).ConclusionsHapSolo rapidly identified candidate assemblies that yield improvements in assembly metrics, including decreased genome size and improved N50 scores. Contig N50 scores improved by 35%, 9% and 9% for Chardonnay, mosquito and the thorny skate, respectively, relative to unreduced primary assemblies. The benefits of HapSolo were amplified by down-stream analyses, which we illustrated by scaffolding with Hi-C data. We found, for example, that prior to the application of HapSolo, only 52% of the Chardonnay genome was captured in the largest 19 scaffolds, corresponding to the number of chromosomes. After the application of HapSolo, this value increased to ~ 84%. The improvements for the mosquito's largest three scaffolds, representing the number of chromosomes, were from 61 to 86%, and the improvement was even more pronounced for thorny skate. We compared the scaffolding results to assemblies that were based on PurgeDups for identifying secondary contigs, with generally superior results for HapSolo.

BackgroundDespite marked recent improvements in long-read sequencing technology, the assembly of diploid genomes remains a difficult task. A major obstacle is distinguishing between alternative contigs that represent highly heterozygous regions. If primary and secondary contigs are not properly identified, the primary assembly will overrepresent both the size and complexity of the genome, which complicates downstream analysis such as scaffolding.ResultsHere we illustrate a new method, which we call HapSolo, that identifies secondary contigs and defines a primary assembly based on multiple pairwise contig alignment metrics. HapSolo evaluates candidate primary assemblies using BUSCO scores and then distinguishes among candidate assemblies using a cost function. The cost function can be defined by the user but by default considers the number of missing, duplicated and single BUSCO genes within the assembly. HapSolo performs hill climbing to minimize cost over thousands of candidate assemblies. We illustrate the performance of HapSolo on genome data from three species: the Chardonnay grape (Vitis vinifera), with a genome of 490 Mb, a mosquito (Anopheles funestus; 200 Mb) and the Thorny Skate (Amblyraja radiata; 2650 Mb).ConclusionsHapSolo rapidly identified candidate assemblies that yield improvements in assembly metrics, including decreased genome size and improved N50 scores. Contig N50 scores improved by 35%, 9% and 9% for Chardonnay, mosquito and the thorny skate, respectively, relative to unreduced primary assemblies. The benefits of HapSolo were amplified by down-stream analyses, which we illustrated by scaffolding with Hi-C data. We found, for example, that prior to the application of HapSolo, only 52% of the Chardonnay genome was captured in the largest 19 scaffolds, corresponding to the number of chromosomes. After the application of HapSolo, this value increased to ~ 84%. The improvements for the mosquito's largest three scaffolds, representing the number of chromosomes, were from 61 to 86%, and the improvement was even more pronounced for thorny skate. We compared the scaffolding results to assemblies that were based on PurgeDups for identifying secondary contigs, with generally superior results for HapSolo.

Coccolithophores play a significant role in marine calcium carbonate production and carbon cycles, attributing to their unique feature of producing calcareous plates, coccoliths. Coccolithophores also possess a haplo-diplontic life cycle, presenting distinct morphology types and calcification states. However, differences in nutrient acquisition strategies and mixotrophic behaviors of the two life phases remain unclear. In this study, we conducted a series of phagocytosis experiments of calcified diploid and non-calcified haploid strains of coccolithophore Gephyrocapsa huxleyi under light and dark conditions. The phagocytosis capability of each strain was examined based on characteristic fluorescent signals from ingested beads using flow cytometry and fluorescence microscopy. The results show a significantly higher phagocytosis percentage on fluorescent beads in the bacterial prey surrogates of the non-calcified haploid Gephyrocapsa huxleyi strain, than the calcified diploid strain with or without light. In addition, the non-calcified diploid cells seemingly to presented a much higher phagocytosis percentage in darkness than under light. The differential phagocytosis capacities between the calcified diploid and non-calcified haploid Gephyrocapsa huxleyi strains indicate potential distinct nutritional strategies at different coccolithophore life and calcifying stages, which may further shed light on the potential strategies that coccolithophore possesses in unfavorable environments such as twilight zones and the expanding coccolithophore niches in the natural marine environment under the climate change scenario.

Maize is one of the most successful crops with regard to the utilization of heterosis. The haploid induction technique is one of the fastest methods to obtain pure maize material at the present stage. However, the molecular mechanism of haploid doubling is rarely reported. In this study, we treated B73 and ZNC442 haploid young shoots with colchicine for 0 h, 6.2 h, and 10 h, and analyzed the differentially expressed genes (DEGs). We found that colchicine treatment for 6.2 h and 10 h compared to 0 h resulted in a total of 4868 co-DEGs. GO enrichment analysis and KEGG metabolic pathway analysis found significantly enriched 282 GO terms and 31 significantly pathways, respectively. Additionally, The GO term and KEGG pathway genes of spindle, cytoskeleton, microtubules and nuclear division were selected for analysis, and three candidate genes were screened by taking intersections. Zm00001d033112, Zm00001d010525, and Zm00001d043386 were annotated as kinesin-associated protein 13, kinesin-like protein KIN-10C, and kinesin light-chain LC6, respectively. The real-time fluorescence quantification (RT-PCR) results revealed that Zm00001d033112, Zm00001d010525, and Zm00001d043386 had the same trends as RNA-seq. Interestingly, Zm00001d033112 is homologous gene AT3G20150 in Arabidopsis, which was involved in the regulation of chromosome movement and mitotic spindle assembly. Our study suggests that kinesin genes may play an important role in doubling chromosomes, thus providing valuable information for future studies on the molecular mechanisms of chromosome doubling in maize.

Double haploid production methods have great potential for accelerating breeding programs for cultivar development. Parthenogenesis, an embryo arising from unfertilized egg cells, is the most efficient technique for Cucumis sativus L. breeding programs. This study focused on the creation of haploid plants in cucumbers through the fertilization of female flowers with gamma radiation–treated pollen. The anthers were treated with different doses of gamma rays (200.0, 300.0, and 500.0 Gray). Pollen viability and vigor were investigated using the TTC (2,3,5-triphenyl tetrazolium chloride) and germination tests, respectively. Isolated female flowers were pollinated with irradiation-affected pollen. According to the results, pollination is most effective in producing haploid offspring when pollen has been exposed to radiation at a dose of 300.0 Gy. The ploidy levels of the regenerated plantlets were determined and confirmed using flow cytometry. In most plants, 78.39% were found to have only half the normal number of chromosomes (haploid), and 21.6% had a mix of normal and half chromosomes (mixoploid). The number of chromosomes in haploid plants was doubled by in vitro treatment of nodal explants with different colchicine concentrations. The use of 500.0 mg L−1 colchicine for 24 h led to the most successful regeneration rate of doubled haploid cells at 85.4%. The optimized double haploid production protocol would be applicable for producing 100% homozygous lines for utilization in breeding programs. [ABSTRACT FROM AUTHOR]

The great economic importance of sugar beet determines the ongoing biotechnological studies conducted worldwide to improve the technology of obtaining doubled haploids (DHs) using the method of unpollinated ovule culture in vitro. To improve the induction of gynogenesis, we tested the effect of thidiazuron (TDZ), temperature bud pretreatment, different concentrations of sucrose, and culturing on liquid or solid medium. Three genotypes were tested in this study. The use of TDZ at a concentration of 0.4 mg/L in solid IMB (induction medium for Beta vulgaris) induction nutrient medium with 3 g/L phytagel, 50 g/L sucrose, 200 mg/L ampicillin and cultivation at 28◦C in the dark produced up to 16.7% induced ovules. The liquid nutrient medium of the same composition induced up to 8% ovules. Increasing TDZ concentration to 0.8 mg/L resulted in reduction or total inhibition of gynogenesis, depending on the genotype. Reducing the sucrose concentration to 20 g/L or increasing it to 80 g/L was not effective. In all three genotypes, the absence of temperature pretreatment of buds (5–6 °C) showed the best results. The plant regeneration with MS nutrient medium of 20 g/L sucrose, 3 g/L phytagel, 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L gibberellic acid (GA3) resulted in up to seven shoots from one induced ovule in the most responsive genotype. We showed by flow cytometry, chromosome counting and chloroplast number assessment that all regenerant plants were haploid (2n = x = 9). [ABSTRACT FROM AUTHOR]

Summary: Production of in planta haploid embryos that inherit chromosomes from only one parent can greatly increase breeding efficiency via quickly generating homozygous plants, called doubled haploid. One of the main players of in planta haploid induction is a pollen‐specific phospholipase A, which is able, when mutated, to induce in vivo haploid induction in numerous monocots. However, no functional orthologous gene has been identified in dicots plants.Here, we show that loss‐of‐function of gynoecium‐expressed phospholipase AII (pPLAIIγ) triggers maternal haploid plants in Arabidopsis, at an average rate of 1.07%. Reciprocal crosses demonstrate that haploid plants are triggered from the female side and not from the pollen, and the haploid plants carry the maternal genome.Promoter activity of pPLAIIγ shows enriched expression in the funiculus of flower development stages 13 and 18, and pPLAIIγ fused to yellow fluorescent protein reveals a plasma‐membrane localizationInterestingly, the polar localized PIN1 at the basal plasma membrane of the funiculus was all internalized in pplaIIγ mutants, suggesting that altered PIN1 localization in female organ could play a role in maternal haploid induction. [ABSTRACT FROM AUTHOR]

The discovery of the ability of some mutations to stimulate haploidy during hybridization made it possible to create one of the most promising and sought-after trends in the field of reproductive biology. Haploid inducers created on their basis are capable of increasing the frequency of haploidy up to 15 %. The improvement of the existing haploid inducer lines and the search for new genes that contribute to a high frequency of haploidy are underway. Along with these studies, the field of application of haploid inducers in genetics and plant breeding is expanding. Haploid inducers carrying R1-nj genes for anthocyanin pigmentation of the seed and embryo are able not only to mark the hybrid embryo and identify haploid genotypes, but also to detect genes that suppress the anthocyanin color of the grain, like C1-I, C2-Idf, and In1-D. Depending on their quantity, the phenotypic manifestation of the gene in the seed varies. Haploidy is widely used for accelerating hybrid breeding and obtaining both new maize lines with improved traits and their sterile counterparts. By introducing certain genes into the genome of the improved line, breeders can use the doubled haploid (DH) breeding technology to accelerate the creation of pure lines carrying the desired gene. Haploid inducer maize lines and their tetraploid analogs are used in the selection of rediploid maize lines by their resynthesis from tetraploid genotypes. In 2019, Syngenta Company synthesized a haploid inducer maize line carrying a CRISPR/cas construct capable of simultaneously stimulating haploidy and editing the genome at a specified DNA site. Thanks to this technology, it became possible to improve haploid inducers by introducing various CRISPR/cas constructs into the haploid inducer genome for editing any DNA site. Maize haploid inducers are widely used in doubled haploid wheat breeding. The first experiments showed that the most effective haploid inducer for stimulating haploidy in wheat is maize pollen. Researchers are intensively searching for other ways of using maize haploid inducers in plant breeding.

Relevance. To create an effective technology for obtaining doubled haploids (DH-technology) of zucchini in unpollinatedseedpod culture in vitro it is necessary to select the optimal values of many factors, the degree of influence of each of which on gynogenesis can vary significantly. The aim of the study was to optimize the individual stages of the technology.Methods. Liquid and agarized (7 g/L) IMC medium with different sucrose concentrations (20 to 80 g/L) and different plant growth regulators (2 mg/L 2,4 D; 0.2 mg/L TDZ ; 0.8 mg/L 2,4 D and 1.2 mg/L NUC) were used for induction of embryogenesis.Results. Optimal for the studied zucchini genotypes was pre-isolated from the evening, plucked in the morning opened bud. Sterilization of zucchini ovaries by short-term burning after treatment with 96% alcohol, allows significant reduction of the time required for this step without loss of embryogenic potential. IMC nutrient medium with sucrose (20 to 40 g/l) can be used for induction of gynogenesis in the unpollinatedzucchini ovary culture. The use of nutrient media with 2 mg/l 2,4 D for most genotypes was more effective and resulted in higher number of embryoids compared to nutrient media containing 0.2 mg/l TDC and media with 0.8 mg/l 2,4 D and 1.2 mg/l NAA. Embryoid formation was observed after 5 weeks of cultivation.Conclusion. We were able to complete the full cycle of technology for obtaining doubled haploids in unpollinatedseedpod culture in vitro for 30 zucchini genotypes and obtain DHplants, which are valuable source material for both breeders and genetic research. Optimization of the individual steps of the technology made it possible to achieve the maximum result for individual genotypes – 55 embryoids per 100 cultivated ovules.

When developing a variety, along with the classical breeding methods, there are increasingly being used bio-technological and molecular genetic methods. These methods include a cultivation technology of isolated anthers, based on the phenomenon of androgenesis. The current paper has presented the results of developing androgenetic rice lines from 7 hybrids of the first generation. The purpose of the work was to study the ability of rice hybrids to form callus on artificial nutrient media and develop regenerated plants followed by a visual estimation of ploidy. Developing dihaploid homozygous lines will speed up the breeding process. The selection of rice panicles was carried out in the field in the shooting phase at the stage of mononuclear pollen. There were cultivated 7218 anthers, of which there were obtained 259 calli. The combinations ‘Akustik x Nerica 1’, ‘Nerica 1 x Akustik c’, ‘Kapitan x Akustik’ were the most responsive to callus formation. The regeneration frequency to the number of calli was 9.13 %. There were identified 794 new formations, from which there were formed 46 plants, including 26 albino and 20 green. The largest number of plants was formed in the hybrid ‘Kapitan x Akustik’ (22 pcs., 14 of which are green). Among the survived green regenerated plants, according to a complex of morphological traits, two plants turned out to be haploids with small sterile flowers and anthers, thin and narrow leaves; five plants were dihaploids with well-grained panicles and medium-sized leaves; two plants were tetraploids that had a wide leaf, a large panicle with very large flowers, but with low fertility. Plants and seeds were handed over to breeders for further work.

Anther culture is an efficient biotechnological tool in modern plant breeding programs to produce new varieties and parental lines in hybrid seed productions. However, some bottlenecks—low induction rate, genotype dependency, albinism—restrict the widespread utilization of in vitro anther culture in rice breeding, especially in Oryza sativa ssp. indica (indica) genotypes, while an improved efficient protocol can shorten the process of breeding. Three different induction media (N6NDK, N6NDZ, Ali-1) and four plant regeneration media (mMSNBK1, MSNBK3, MSNBKZ1, MSNBKZ2) were tested with five indica rice genotypes to increase the efficiency of in vitro androgenesis (number of calli and regenerated green plantlets). The production of calli was more efficient on the N6NDK medium with an average 88.26 calli/100 anthers and N6NDZ medium with an average of 103.88 calli/100 anthers as compared to Ali-1 with an average of 6.96 calli/100 anthers. The production of green plantlets was greater when calli was produced on N6NDK medium (2.15 green plantlets/100 anthers) compared when produced on to N6NDZ medium (1.18 green plantlets/100 anthers). Highest green plantlets production (4.7 green plantlets/100 anthers) was achieved when mMSNBK1 plant regeneration medium was used on calli produced utilizing N6NDK induction medium. In the best overall treatment (N6NDK induction medium and mMSNBK1 plant regeneration medium), four tested genotypes produced green plantlets. However, the genotype influenced the efficiency, and the green plantlets production ranged from 0.4 green plantlets/100 anthers to 8.4 green plantlets/100 anthers. The ploidy level of 106 acclimatized indica rice plantlets were characterized with flow cytometric analyses to calculate the percentage of spontaneous chromosome doubling. Altogether, 48 haploid-, 55 diploid-, 2 tetraploid- and 1 mixoploid plantlets were identified among the regenerant plantlets, and the spontaneous chromosome doubling percentage was 51.89%. Utilization of DH plants have been integrated as a routine method in the Hungarian rice breeding program. The tetraploid lines can be explored for their potential to offer new scopes for rice research and breeding directions in the future. [ABSTRACT FROM AUTHOR]

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