Artificial Hybrids Research Articles

Interspecific hybridization and cell engineering of lettuce (Lactuca L.)

Lactuca sativa L. is a leafy vegetable crop of the Asteraceae family, widely cultivated throughout the world. The main breeding trends for lettuce include higher yields, better taste quality, earliness, and resistance to abiotic and biotic stressors. Some wild Lactuca spp. have actively been employed by lettuce breeders as donors of resistance to various diseases. Conventional and biotechnological breeding methods are both currently used to develop new lettuce cultivars. This is an overview of the main advances in the production of interspecific Lactuca hybrids, including the use of cell and tissue culture techniques, and genetic engineering. Studying artificial hybridization and natural populations makes it possible to identify evolutionary relationships among various Lactuca spp. Somatic hybridization is an overlooked but promising technology in Lactuca breeding: it allows a breeder to obtain a wider range of variations, and is beyond the strict control by GMO laws. This technique faces problems associated with complicated protoplast regeneration and the loss of reproductive ability in hybrids. Genome-editing methods are more effective and better controllable, but society is still wary of any interference with the plant genome and legally regulates the sale of GM products as food. Thus, researchers are challenged with the task to improve these techniques.

Effectiveness of the use of chickpea collection samples in breeding

Aim. To determine the breeding value the large number of collection chickpea (Cicer arietinum L.) varieties obtained from the International Crops Research Institute for the Semi-Arid Tropics (India, Patancheru, ICRISAT) and the National Center for Plant Genetic Resources of Ukraine (Kharkiv) was evaluated in 1995-2022 under the conditions of the steppe zone of Ukraine. Methods. Standard field and laboratory methods (phenological observations, hybridization, structural analysis, biochemical analysis of protein content in seeds according to the Kjeldahl method), statistical analysis. Results. Donors and sources of such economically valuable features as early maturity, increased productivity, protein content in seeds, large-seeded, resistance against pathogens on an artificially created infectious background were identified. The set of 12 varieties of chickpea were developed by artificial hybridization, which are recommended for cultivation in all zones of Ukraine. Conclusions.The characteristics of the created varieties are given, the most valuable collection genotypes for use in crossing programs are recommended.

Checklist of Ornamental Trees, Shrubs, and Succulents of Apulia (Southern Italy).

In this study we focused on the need to fill a knowledge gap among Italian botanical studies namely that of ornamental species census. In particular, we addressed one of the regions in southern Italy with less knowledge in the field of such studies and with an obvious presence of non-native species. A widespread census of the Apulian territory was carried out between 2021 and 2024 in both urban and suburban areas including street trees, parks, and private and historic gardens. The inventory of ornamental trees, shrubs, and succulents of Apulia (southern Italy) was carried out in six provinces, i.e., Bari, Barletta-Andria-Trani, Brindisi, Foggia, Lecce, and Taranto. The checklist comprises 287 taxa (including 265 species, 6 varieties, 5 subspecies, and 11 forms) included in 179 genera belonging to 78 families. We evaluated the number of taxa per families and genera, the presence of each taxa in the provinces of Apulia, the number of taxa per occurrence status, growth forms, geographical origin, and the number of native and alien taxa and also the artificial hybrids. Remarks of the most significant taxa and evaluation of geographical distribution in Italy were also taken into consideration. A large number of surveyed taxa (51.74%) are comprised in the list reported in the recent study on allochthonous vascular flora in Italy with a marked prevalence of Neophyte Casual Alien and Neophyte Naturalized Alien species.

Phylotranscriptomic analyses reveal the evolutionary complexity of Paris L. (Melanthiaceae), a morphologically distinctive genus with significant pharmaceutical importance.

Previous phylogenetic studies on the pharmaceutically significant genus Paris (Melanthiaceae) have consistently revealed substantial cytonuclear discordance, yet the underlying mechanism responsible for this phenomenon remains elusive. This study aims to reconstruct a robust nuclear backbone phylogeny and elucidate the potential evolutionarily complex events contributing to previously observed cytonuclear discordance within Paris. Based on a comprehensive set of nuclear low-copy orthologous genes obtained from transcriptomic data, the intrageneric phylogeny of Paris, along with its phylogenetic relationships to allied genera were inferred, using coalescent and concatenated approaches. The analysis of gene tree discordance and reticulate evolution, in conjunction with an incomplete lineage sorting (ILS) simulation, was conducted to explore potential hybridization and ILS events in the evolutionary history of Paris and assess their contribution to the discordance of gene trees. The nuclear phylogeny unequivocally confirmed the monophyly of Paris and its sister relationship with Trillium, while widespread incongruences in gene trees were observed at the majority of internal nodes within Paris. The reticulate evolution analysis identified five instances of hybridization events in Paris, indicating that hybridization events might have recurrently occurred throughout the evolutionary history of Paris. In contrast, the ILS simulations revealed that only two internal nodes within sect. Euthyra experienced ILS events. Our data suggest that the previously observed cytonuclear discordance in the phylogeny of Paris can primarily be attributed to recurrent hybridization events, with secondary contributions from infrequent ILS events. The recurrent hybridization events in the evolutionary history of Paris not only drove lineage diversification and speciation but also facilitated morphological innovation, and enhanced ecological adaptability. Therefore, artificial hybridization has great potential for breeding medicinal Paris species. These findings significantly contribute to our comprehensive understanding of the evolutionary complexity of this pharmaceutically significant plant lineage, thereby facilitating effective exploration and conservation efforts.

ANN based optimization of nano-beam oscillations with intermolecular forces and geometric nonlinearity

In this study, we investigate the effect of Van der Waals and Casimir forces on the mathematical model of nano-electromechanical systems (NEMS) such as nano-beam actuators that contain cantilever and double cantilever beams. The singular nonlinear boundary value problem governing the beam-type actuators, including geometric nonlinearity is solved by using an intelligent strength of feedforward artificial neural networks (ANNs) and hybridization of optimization algorithms such as arithmetic optimization algorithm (AOA) and active set algorithm (ASA). The proposed ANN-AOA-AS algorithm is employed to quantify the effect of changes in applied voltage, dispersion forces, geometric nonlinearity parameters, and initial axial strain on the deflection of the beam. Furthermore, to validate the results obtained by the proposed algorithm, statistical analyses are conducted to compare the approximate solutions with state-of-the-art methodologies available in the latest literature. In addition, performance indicators are defined such as mean square error (MSE), Nash–Sutcliffe efficiency (NSE), mean absolute deviations (MAD), root mean square error (RMSE), and Error in Nash–Sutcliffe efficiency (ENSE) to study the accuracy and efficiency of the solutions. The results show that these indicators’ mean percentage values lie around 10−4 to 10−6 which reflects the perfect modeling of the approximate solutions.

Unravelling the novel sex determination genotype with 'ZY' and a distinctive 2.15-2.95 Mb inversion among poplar species through haplotype-resolved genome assembly and comparative genomics analysis.

Populus tomentosa, an indigenous tree species, is widely distributed and cultivated over 1,000,000 km2 in China, contributing significantly to forest production, ecological conservation and urban-rural greening. Although a reference genome is available for P. tomentosa, the intricate interspecific hybrid origins, chromosome structural variations (SVs) and sex determination mechanisms remain confusion and unclear due to its broad and even overlapping geographical distribution, extensive morphological variations and cross infiltration among white poplar species. We conducted a haplotype-resolved de novo assembly of P. tomentosa elite individual GM107, which comprises subgenomes a and b with a total genome size of 714.9 Mb. We then analysed the formation of hybrid species and the phylogenetic evolution and sex differentiation across the entire genus. Phylogenomic analyses suggested that GM107 likely originated from a hybridisation event between P. alba (♀) and P. davidiana (♂) which diverged at approximately 3.8 Mya. A total of 1551 chromosome SVs were identified between the two subgenomes. More noteworthily, a distinctive inversion structure spanning 2.15-2.95 Mb was unveiled among Populus, Tacamahaca, Turaga, Aigeiros poplar species and Salix, highlighting a unique evolutionary feature. Intriguingly, a novel sex genotype of the ZY type, which represents a crossover between XY and ZW systems, was identified and confirmed through both natural and artificial hybrids populations. These novel insights offer significant theoretical value for the study of the species' evolutionary origins and serve as a valuable resource for ecological genetics and forest biotechnology.

Cytogenetic analysis of meiotic behaviour and stability in a trigeneric hybrid (triticale x trigopiro).

Trigeneric hybrids in Triticeae may help to establish evolutionary relationships among different genomes present in the same cellular genetic background and to transfer different alien characters into cultivated wheat. In the present study, a trigeneric hybrid involving species of Triticum, Secale, and Thinopyrum was synthesized by crossing hexaploid triticale with hexaploid trigopiro. The meiotic behaviour of chromosomes belonging to different genomes was analyzed, using routine and in situ hybridization techniques in F1, F2, and F3 generations of the trigeneric hybrid. The purpose of this study was to determine the chromosome number and genomic constitution and to discuss the mechanisms involved in the stabilization of the artificial tricepiro hybrids. The chromosome number of the trigeneric F1 hybrid was 2n = 42. Between 12 and 16 bivalents were observed in the central zone of the equatorial meiotic plate and between 9 and 18 univalents were found in the periphery of the MI equatorial plate. Seven of these univalents showed hybridization signals with rye DNA. Lagging rye and non-rye chromosomes and separation of sister chromatids were found in anaphase I. Tetrads with a maximum of six micronuclei, with and without hybridization signals of rye DNA, were observed. After three generations, meiotic cells revealed the presence of 42 chromosomes and 21 bivalents in diakinesis cells. The presence of 14 rye (Secale cereale) chromosomes and the complete pairing of chromosomes in F3 hybrids suggest that rye chromosomes would be preferentially transmitted to the progeny and that an elimination mechanism would act on chromosomes of Thinopyrum and wheat D genome.

A robust system of hybridogenesis that increases genetic variability and promotes evolutionary succession in greenlings (Teleostei: Hexagrammidae, genus Hexagrammos): Regeneration of a new hemiclonal lineage.

Unisexual hybrids that reproduce either clonally or hemiclonally are considered to be evolutionarily short-lived as they lack the ability to reduce deleterious mutations and increase genetic diversity. In the greenling (Teleostei: Hexagrammidae, genus Hexagrammos), unisexual hybrids that produce haploid eggs containing only the H. octogrammus (maternal species) genome generate hemiclonal offspring by fertilization with haploid sperm of H. agrammus (paternal species). When hemiclonal hybrids are backcrossed to a male of the maternal species, the offspring (BC-Hoc) are phenotypically similar to the maternal species and produce recombinant gametes through conventional meiosis. BC-Hoc (recombinant generation) individuals referred to as carriers harbor the genetic factor for hybridogenesis, thereby facilitating the production of new hemiclonal lineages through hybridization. Previous studies based on field research have suggested that the carriers produced by two-way backcrossing (mating pattern in which hemiclonal hybrids are backcrossed with both parental species) may overcome the evolutionary dead end imposed by the lack of recombination. The present study verified this hypothesis by regenerating a newly hemiclonal lineage through artificial hybridization. To clarify the genetic mode of hybrids produced by crosses between BC-Hoc and Hag, mature eggs were obtained from 16 individuals and fertilized with either Hag or Hoc sperm. Hybridogenesis was confirmed in one of the 16 individuals. Based on the low occurrence rate, these findings suggest that hemiclonal lineages can be regenerated, and that the hemiclonal factors are likely distributed across multiple genes on different chromosomes. The findings provide important evidence for the retention of a robust system for increasing genetic variability and maintaining evolutionary succession in unisexual hybrids that reproduce hemiclonally.

Genome Variability in Artificial Allopolyploid Hybrids of Avena sativa L. and Avena macrostachya Balansa ex Coss. et Durieu Based on Marker Sequences of Satellite DNA and the ITS1-5.8S rDNA Region.

Artificial hybrids between cultivated Avena species and wild Avena macrostachya that possess genes for resistance to biotic and abiotic stresses can be important for oat breeding. For the first time, a comprehensive study of genomes of artificial fertile hybrids Avena sativa × Avena macrostachya and their parental species was carried out based on the chromosome FISH mapping of satellite DNA sequences (satDNAs) and also analysis of intragenomic polymorphism in the 18S-ITS1-5.8S rDNA region, using NGS data. Chromosome distribution patterns of marker satDNAs allowed us to identify all chromosomes in the studied karyotypes, determine their subgenomic affiliation, and detect several chromosome rearrangements. Based on the obtained cytogenomic data, we revealed differences between two A. macrostachya subgenomes and demonstrated that only one of them was inherited in the studied octoploid hybrids. Ribotype analyses showed that the second major ribotype of A. macrostachya was species-specific and was not represented in rDNA pools of the octoploids, which could be related to the allopolyploid origin of this species. Our results indicate that the use of marker satDNAs in cytogenomic studies can provide important data on genomic relationships within Avena allopolyploid species and hybrids, and also expand the potential for interspecific crosses for breeding.

Breeding value of potato hybrid clones from the VIR collection revealed in the environments of Kamchatka Territory

Background. Potato breeding involves planned artificial hybridization between previously selected parental lines. A way to make potato breeding more efficient is to use the diversity of the germplasm and broaden the gene pool of parental lines. Interspecific potato hybrids originated on the elite potato germplasm display a set of valuable traits. The current challenge is to assess the performance of potato hybrids under specific soil and climate conditions to clarify their breeding value.The objective of the study was to evaluate potato hybrids in the environments of Kamchatka Territory according to a set of traits, identify the best parental lines, develop new hybrid combinations, and select promising breeding material.Materials and methods. Eleven potato hybrids from VIR were studied over three years (2020–2022) in the fields of Kamchatka Research Institute of Agriculture. The hybrids were benchmarked against two reference potato cultivars, ‘Fresco’ and ‘Vulkan’, belonging to the early and mid-early maturity groups, respectively. Crosses between the hybrids and 28 potato cultivars were carried out in the greenhouse. The new hybrid material was tested in a preliminary trial nursery for yield and earliness.Results. Hybrid 117-2 was better in yield (899 g/plant) than cvs. ‘Vulkan’ and ‘Fresco’ (700–739 g/plant). Hybrids 94-5, 99-10- 1, 8-1-2004, 8-3-2004, 8-5-2004, 135-3-2005, 135-5-2005, 99-6-6, and 134-2-2006 in most traits matched the potato cultivars, manifesting good berry formation and seed setting when crossed. Out of 140 crosses between 11 hybrids and 28 cultivars, 35 crosses between 10 hybrids and 13 cultivars were successful. Most hybrids were effectively used in crossings as maternal forms.Conclusion. VIR’s hybrids 94-5, 8-1-2004, and 8-5-2004 proved the most promising as source material for the development of new early potatoes under the conditions of Kamchatka Territory.

Study on the Short-Term Preservation of Gametes, Cross-Stimulation of Oocytes by Distant Sperm, and the Impact of Cold-Stimulated Fertilized Eggs on Eyes in the Celestial Goldfish

This study layed the groundwork for the creation of haploid and triploid celestial goldfish, presenting essential data derived from preliminary investigations. The research delved into three main areas: the short-term preservation of celestial goldfish gametes, the interaction between oocytes and foreign sperm, and the effects of temperature on fertilized eggs concerning hatching rates and late-stage ocular development. Initially, the study explored the optimal semen dilution ratio for celestial goldfish under microscopic examination. And the hybridization of the largemouth bass and celestial goldfish was investigated: largemouth bass sperm was crossbred with celestial goldfish eggs, and it was found that their sperm could not stimulate the development of celestial eye eggs. At last, celestial goldfish fertilized eggs were stimulated at 4 °C and −20 °C, respectively, to observe their impact on the hatching rate and later celestial eye rate. The results revealed no significant differences in hatching rate and celestial eye rate between the cold stimulus groups and the control group, but numerically, the 4 °C cold stimulation reduced the celestial eye rate of celestial goldfish fertilized eggs. The research provided fundamental data for artificial breeding and hybridization experiments in celestial goldfish.

Artificial interspecific hybridization of the two distant anemonefish species and phenotypic characterization of the F1 hybrid

Artificial interspecific hybridization of the two distant anemonefish species and phenotypic characterization of the F1 hybrid

Optimized Protocol for in vitro Pollen Germination in Brinjal (Solanum melongena L.)

Background: Understanding floral and pollen biology is essential for efficient artificial hybridization in hybrid seed production. This knowledge helps ensure proper fertilization of the ovary and optimal fruit and seed set. Selecting suitable pollen parents and preserving pollen for future use allows for better control and optimization of hybridization practices in brinjal breeding programs. To achieve this, a robust protocol for pollen collection and viability testing is necessary to monitor pollen quality in the field and during storage. This information is crucial for maintaining desired characteristics and genetic traits in the resulting hybrid seeds. Methods: This study; aimed at optimizing the pollen germination assessment protocol for brinjal. The step wise modification standard medium composition was done, the optimal growth condition was tested and in vivo predictions were made. So, the study aimed to standardization of media for in vitro pollen germination evaluate and viability in brinjal in ambient as well as cold storage conditions. The pollen germination was tested in vitro as well in vivo, by incubating the pollen grains for 3 hours at 25°C in different concentrations of sucrose, boron and agar media and distilled water at interval of 12 hours. Result: The most favorable germination of fresh pollen was achieved with a treatment consisting of 1% Agar, 12% Sucrose and 300 ppm Boron. Comparing the two storage conditions, pollen stored in the refrigerator exhibited higher in vitro germination percentages compared to ambient storage conditions. The highest in vitro pollen germination and viability percentages were observed in fresh pollen collected within 12 hours (95.00%). As the duration of pollen storage increased in both storage conditions, the in vitro pollen germination decreased. This decline in in vitro pollen germination was reflected in the in vivo fertilization fruit set percentage. The highest fruit set percentage was observed when pollen was stored in the refrigerator (64.52%) and when female flowers were pollinated with pollen stored for less than 12 hours (84.66%).

The use of genetic resources in rose breeding and creation of new rose cultivars through hybridization and selection

Despite the existence of more than 37,000 different varieties of roses worldwide, there remains a significant interest and high relevance in rose breeding. The rose is the most beloved flower in the world, commonly named the “queen of flowers”. In addition to its remarkable ornamental significance, the rose possesses various ecological, environmental, landscaping, food, pharmaceutical and medicinal, cosmetic, and industrial attributes, among others, thereby contributing to its overall socio-economic value. The current study investigated the results of rose breeding in a horticulture research center in the northwest of Romania with over 70 years of experience. The main method used to generate variability for the selection of new genotypes was artificial hybridization. After 1970, an average of 15-20 crossings per year were carried out, in different parental formulas or types of crosses, with approximately 1000-2000 flowers per pollinated per parental combination. Thousands of F1 hybrids were obtained annually (approximately 10,000-12,000 hybrids per year per hybrid combination), and dozens of elites were selected, grafted, and promoted as clonal selections in comparative trials. The success rate in obtaining new varieties through hybridization was 5.1% from total hybrid combinations, 0.72% from total hybrids, and 1.6% from total clonal selections. Of the total new varieties, hybrid teas represent 11.9%, floribundas 64.2%, shrubs 16.6%, and others 7.1%. 38 new cultivars suitable for open field cultivation were obtained, showing different habits, decorative value, flowering behavior, flower color, fragrance, resistance to frost or major diseases, and intended use. The exploration of new research directions aimed at harnessing the potential of roses, the region, and the socio-economic requirements was undertaken to enhance and beauty degraded, or landslide-prone lands, as well as to introduce damascene roses for the oil perfume and cosmetics industry.

Evaluation of the evolutionary process within Populus caspica species from Hyrcanian forests by karyotype analysis

Caspian poplar (Populus caspica Bornm.) is distributed exclusively in the Hyrcanian forests. Hyrcanian forests are the final remnants of Tertiary temperate deciduous forests in Western Eurasia and worldwide. This species plays a significant ecological role in the protection of the natural environment in Hyrcanian forests. In this research, chromosome number and karyotype details of 11 populations of the species were investigated for the first time, using fresh root cuttings collected from mature trees in different parts of the forest, located in the northern parts of Iran. Pre-treatment, fixation, hydrolyzing, and staining were conducted by α-bromonaphthalene, carnoy’s solution, 1N HCl, and hematoxylin agent, respectively. Chromosomal data were analyzed according to a nested model based on a completely randomized design. Chromosome numbers of all of the populations were the same as 2n = 38, which mostly were medium region and sub-metacentric types. Significant differences were observed between the provinces and populations, based on chromosome length grand means, arm ratios and centromere indices. The results demonstrated that structural rearrangement has occurred within the studied populations and indicated an active evolutionary process within and between populations of the species due to natural hybridization. Also, these results showed that artificial inter-specific hybridization between the P. caspica and its relative species can be employed to broaden the ecological zone of the species.

Phylogenomics reveal Populusgonggaensis as a hybrid between P.lasiocarpa and P.cathayana (Salicaceae).

High levels of intra-specific polymorphism and frequent hybridisation make it difficult to define species and correctly apply their scientific names. Populus L. is a challenging genus with plentiful natural and artificial hybrids. This study is a part of the project 'Flora of Pan-Himalaya' and aims to determine the taxonomic identity of P.gonggaensis N. Chao & J.R. He and to find out whether it is of hybrid origin. Whole-genome sequencing data were obtained from 57 samples. The SNP matrix was developed for phylogenetic reconstruction, ABBA-BABA statistics, PCA and ADMIXTURE analysis. The results indicate that P.gonggaensis is a spontaneous hybrid between P.lasiocarpa and P.cathayana. This study points out the importance of SNP data and comprehensive analyses for discovering the potential interspecific hybridisation and clarifies the usage of the name. In addition, the lectotype of P.gonggaensis was designated.

Strong plastic responses in aerenchyma formation in F1 hybrids of Imperata cylindrica under different soil moisture conditions.

Hybrids can express traits plastically, enabling them to occupy environments that differ from parental environments. However, there is insufficient evidence demonstrating how phenotypic plasticity in specific traits mediates hybrid performance. Two parental ecotypes of Imperata cylindrica produce F1 hybrids. The E-type in wet habitats has larger internal aerenchyma than the C-type in dry habitats. This study evaluated relationships between habitat utilisation, aerenchyma plasticity, and growth of I. cylindrica accessions. We hypothesize that plasticity in expressing parental traits explains hybrid establishment in habitats with various soil moisture conditions. Aerenchyma formation was examined in the leaf midribs, rhizomes and roots of two parental ecotypes and their F1 hybrids in their natural habitats. In common garden experiments, we examined plastic aerenchyma formation in leaf midribs, rhizomes and roots of natural and artificial F1 hybrids and parental ecotypes and quantified vegetative growth performance. In the natural habitats where soil moisture content varied widely, the F1 hybrids showed larger variation in aerenchyma formation in rhizomes than their parental ecotypes. In the common garden experiments, F1 hybrids showed high plasticity of aerenchyma formation in rhizomes, and their growth was similar to that of C-type and E-type under drained and flooded conditions, respectively. The results demonstrate that F1 hybrids of I. cylindrica exhibit plasticity in aerenchyma development in response to varying local soil moisture content. This characteristic allows the hybrids to thrive in diverse soil moisture conditions.

Phenotypic traits of reciprocal tetraploid hybrids derived from tetraploid Crassostrea gigas and tetraploid Crassostrea angulata

Allotetraploid has the potential to become an important germplasm resource for genetic breeding of organisms. Previously, autotetraploid oysters have played a great role in the worldwide triploid oyster industry by mating with diploids to produce triploid seeds. However, whether allotetraploid oysters can be produced and their performance is unknown. Here, we carried out an artificial interspecific cross between tetraploid Crassostrea gigas and teraploid C. angulata, forming 4 experimental groups, two autotetraploid groups (GGGG: tetraploid C. gigas♀ × C. gigas♂, AAAA: C. angulata♀ × C. anguata♂) and two allotetraploid groups (GGAA: C. gigas♀ × C. anguata♂, AAGG: C. angulata♀ × C. gigas♂). These two pure tetraploid species hybridized without barrier to gamete reproductive isolation, resulting in high fertilization rate (>93%) and acceptable hatching rate (>78%). Reciprocal tetraploid hybrid survival rates were higher than that of pure tetraploids. Some degree of growth heterosis was observed, especially for yearlings. Mid-parent heterosis of wet weight and shell height at day 360 was 31.58% and 16.11%, respectively, which were primarily influenced by egg origin and mating strategy. Overall, the order of performance of the four groups in the warm water area was as following: AAGG>GGAA>AAAA>GGGG. Our results demonstrate that artificial interspecific hybridization between two tetraploid oysters can produce heterosis and that teraploid hybrids have the potential to become new genetic resources for the improvement of polyploid oysters.

Selective miR-21 detection technology based on photocrosslinkable artificial nucleic acid-modified magnetic particles and hybridization chain reaction

Recently, microRNA (miRNA) detection in blood has attracted attention as a new early detection technology for cancer. The extraction of target miRNA is a necessary preliminary step for detection; however, currently, most extraction methods extract all RNA from the blood, which limits the detection selectivity. Therefore, a method for the selective extraction and detection of target miRNA from blood is very important. In this study, we utilized photocrosslinkable artificial nucleic acids and the hybridization chain reaction (HCR) in an attempt to improve upon the current standard method RT-qPCR, which is hampered by problems with primer design and enzymatic amplification. By introducing photocrosslinkable artificial nucleic acids to oligonucleotide probes modified with magnetic particles with a sequence complementary to that of the target miRNA and irradiating them with light, covalent bonds were formed between the target miRNA and the oligonucleotide probes. These tight covalent bonds enabled the capture of miRNA in blood, and intensive washing ensured that only the target miRNA were extracted. After extraction, two types of DNA (H1 and H2) modified with fluorescent dyes were added and the fluorescence signals were amplified by the HCR in the presence of the target miRNA bound to the photocrosslinkable artificial nucleic acids, allowing for isothermal and enzyme-free miRNA detection. The novel method is suitable for selective miRNA detection in real blood samples. Because the reaction proceeds isothermally and no specialized equipment is used for washing, this detection technology is simple and selective and suitable for application to point-of-care technology using microfluidic devices.

Stigma receptivity and flower development stage during emasculation in tomato affect artificial hybridization

ABSTRACT Determination of appropriate stages of flower emasculation and flower development when self-pollination occurs in tomato (Solanum lycopersicum L.) flowers is important to increase the degree of success of cross pollination. The experiment was conducted from July 2015 to June 2017. Seven tomato varieties (as female parents) and 1 genotype (as pollen source) were evaluated. Stages of flower emasculation (3 levels) were: 1) emasculation of immature flowers (light yellow petal) (Stage I); 2) emasculation of mature flowers (dark yellow petal, and/or petal not opened) (Stage II); and 3) emasculation of mature flowers (dark yellow petal, and/or petal partially opened) (Stage III). Complete success of cross fertilization was for the interaction of Stage II (dark yellow petal, and/or petal not opened) flower emasculation with pollination of emasculated flower after 24 h. The lowest (1.8%) percent of success of cross fertilization was at stage I (light yellow petal) flower emasculation with pollination of emasculated flower within 12 h. The low range, 1.8–5.3% of success of cross fertilization was at flower stage III (dark yellow petal, and/or petal partially opened) emasculation with no pollination of emasculated flower, indicating occurrence of an accidental self-fertilization. Stage of flower development stage I (light yellow petal) and pollination of emasculated flowers after 48 h could give the optimum percent of success of cross fertilization and avoid accidental self-pollination.