Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat.

This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2 mg/L and kinetin 2 mg/L; for shoot initiation 6-benzylaminopurine 2 mg/L, kinetin 0.2 mg/L, and indole-3-acetic acid 0.2 mg/L; for shoot multiplication 6-benzylaminopurine 3 mg/L and indole-3-acetic acid 0.2 mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1 mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.

Arabidopsis as a model for translational research.

Arabidopsis thaliana is currently the most-studied plant species on earth, with an unprecedented number of genetic, genomic, and molecular resources having been generated in this plant model. In the era of translating foundational discoveries to crops and beyond, we aimed to highlight the utility and challenges of using Arabidopsis as a reference for applied plant biology research, agricultural innovation, biotechnology, and medicine. We hope that this review will inspire the next generation of plant biologists to continue leveraging Arabidopsis as a robust and convenient experimental system to address fundamental and applied questions in biology. We aim to encourage lab and field scientists alike to take advantage of the vast Arabidopsis datasets, annotations, germplasm, constructs, methods, molecular and computational tools in our pursuit to advance understanding of plant biology and help feed the world's growing population. We envision that the power of Arabidopsis-inspired biotechnologies and foundational discoveries will continue to fuel the development of resilient, high-yielding, nutritious plants for the betterment of plant and animal health and greater environmental sustainability.

A rapid and scalable approach to build synthetic repetitive hormone-responsive promoters.

Advancement of DNA-synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high-complexity DNA sequences containing tandems of short repeats are still notoriously difficult to produce synthetically, with commercial DNA synthesis companies usually rejecting orders that exceed specific sequence complexity thresholds. To overcome this limitation, we developed a simple, single-tube reaction method that enables the generation of DNA sequences containing multiple repetitive elements. Our strategy involves commercial synthesis and PCR amplification of padded sequences that contain the repeats of interest, along with random intervening sequence stuffers that include type IIS restriction enzyme sites. GoldenBraid molecular cloning technology is then employed to remove the stuffers, rejoin the repeats together in a predefined order, and subclone the tandem(s) in a vector using a single-tube digestion-ligation reaction. In our hands, this new approach is much simpler, more versatile and efficient than previously developed solutions to this problem. As a proof of concept, two different phytohormone-responsive, synthetic, repetitive proximal promoters were generated and tested in planta in the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene-responsive promoter 10x2EBS-S10 fused to the GUS reporter gene uncovered several developmentally regulated ethylene response maxima, indicating the utility of this reporter for monitoring the involvement of ethylene in a variety of physiologically relevant processes. These encouraging results suggest that this reporter system can be leveraged to investigate the ethylene response to biotic and abiotic factors with high spatial and temporal resolution.

Evaluation of Seed Germination of Six Rare Stipa Species following Low Temperature Stress (Cryopreservation).

Cryopreservation is one way to preserve rare, endangered species. However, during the cryopreservation process, plant cells undergo considerable stress, which may lead to cell death. In our work, orthodox Stipa seeds of six rare species were cryopreserved: S. sareptana, S. ucrainica, S. tirsa, S. dasyphylla, S. adoxa, and S. pulcherríma. Short-term cryopreservation (14 days) stimulated germination of all Stipa species studied. Prolonged cryopreservation (70 days and more) decreased the germination of all Stipa seeds except S. sareptana. The decrease in germination progressed over time as a result of the cumulative stress of cryopreservation rather than the initial stress. To stimulate germination, seeds were stratified and treated with GA3, KNO3, NaOH, and H2O2. After four years of seed cryopreservation, it was possible to obtain seedlings of all the Stipa species studied with 30 days of stratification and 180 days of germination. After five years of cryopreservation and seed treatment with 30% NaOH for one hour, the best germination was obtained in S. adoxa and S. pulcherrima. After treatment with 5% H2O2 for 20 min, the best germination was obtained in S. sareptana, S. ucrainica, and S. dasyphylla. S. sareptana seeds germinated in all the aforementioned experiments. S. sareptana has a non-deep physiological dormancy and is the most widespread and drought-tolerant Stipa species studied. The best habitat adaptation and stress tolerance correlated with this species'cryotolerance. S. sareptana was recommended for further cryopreservation, while storage protocols for the other Stipa species studied need further improvements.

TiO2 Phase Ratio's Contribution to the Photocatalytic Activity.

Photocatalysis is one of the approaches for solving environmental issues derived from extremely harmful pollution caused by industrial dyes, medicine, and heavy metals. Titanium dioxide is among the most promising photocatalytic semiconductors; thus, in this work, TiO2 powders were prepared by a hydrothermal synthesis using titanium tetrachloride TiCl4 as a Ti source. The effect of the hydrochloric acid (HCl) concentration on TiO2 formation was analyzed, in which a thorough morpho-structural analysis was performed employing different analysis methods like XRD, Raman spectroscopy, SEM/TEM, and N2 physisorption. EPR spectroscopy was employed to characterize the paramagnetic defect centers and the photogeneration of reactive oxygen species. Photocatalytic properties were tested by photocatalytic degradation of the rhodamine B (RhB) dye under UV light irradiation and using a solar simulator. The pH value directly influenced the formation of the TiO2 phases; for less acidic conditions, the anatase phase of TiO2 crystallized, with a crystallite size of ≈9 nm. Promising results were observed for TiO2, which contained 76% rutile, showing a 96% degradation of RhB under the solar simulator and 91% under UV light after 90 min irradiation, and the best result showed that the sample with 67% of the anatase phase after 60 min irradiation under the solar simulator had a 99% degradation efficiency.

Major Contribution of c.[1622T>C;3113C>T] Complex Allele and c.5882G>A Variant in ABCA4-Related Retinal Dystrophy in an Eastern European Population.

Inherited retinal diseases (IRDs) constitute a prevalent group of inherited ocular disorders characterized by marked genetic diversity alongside moderate clinical variability. Among these, ABCA4-related eye pathology stands as a prominent form affecting the retina. In this study, we conducted an in-depth analysis of 96 patients harboring ABCA4 variants in the European part of Russia. Notably, the complex allele c.[1622T>C;3113C>T] (p.Leu541Pro;Ala1038Val, or L541P;A1038V) and the variant c.5882G>A (p.Gly1961Glu or G1961E) emerged as primary contributors to this ocular pathology within this population. Additionally, we elucidated distinct disease progression characteristics associated with the G1961E variant. Furthermore, our investigation revealed that patients with loss-of-function variants in ABCA4 were more inclined to develop phenotypes distinct from Stargardt disease. These findings provide crucial insights into the genetic and clinical landscape of ABCA4-related retinal dystrophies in this specific population.

Pathogenic Variants and Genotypes of the CFTR Gene in Russian Men with Cystic Fibrosis and CBAVD Syndrome.

Pathogenic CFTR variants cause cystic fibrosis (CF), and CF-related disorders (CF-RD), including bilateral aplasia of the vas deferens (CBAVD). The spectrum of clinical manifestations depends on the CFTR genotype. The frequency and spectrum of the CFTR variants vary between populations and clinical groups. CFTR variants and genotypes were analyzed in Russian men with CF (n = 546) and CBAVD syndrome (n = 125). Pathogenic variants were detected in 93.95% and 39.2% of the CF and CBAVD alleles, respectively. The most frequent c.1521_1523del (F508del; p.Phe508del) variant was found in 541 (49.5%) CF alleles. A total of 162 CFTR genotypes were revealed in CF patients, including 152 homozygous and 394 compound-heterozygous. The most common CF-genotype was F508del/F508del (24.9%). Other frequent CF-genotypes were F508del/3849+10kbC>T, F508del/CFTRdele2,3, and F508del/E92K. CF-causing variants and/or 5T allele were found in 88% of CBAVD patients: 5T/CFTRmut (48.0%), CFTRmut/N (17.6%), CFTRmut/CFTRmut (6.4%), 5T/5T (10.4%), 5T/N (5.6%) and N/N (12.0%), with the most common CBAVD-genotype being F508del/5T (29.6%). The allele frequencies of F508del, CFTRdele2,3 394delTT, and 3849+10kbC>T were significantly higher in CF patients. L138ins/L138ins, 2184insA/E92K, and L138ins/N genotypes were found in CBAVD, but not in CF patients. The results indicate certain differences in the frequency of some CFTR variants and genotypes in Russian CF and CBAVD patients.

A Molecular Genetic Analysis of RPE65-Associated Forms of Inherited Retinal Degenerations in the Russian Federation.

Pathogenic variants in the RPE65 gene cause the only known form of inherited retinal degenerations (IRDs) that are prone to gene therapy. The current study is aimed at the evaluation of the prevalence of RPE65-associated retinopathy in the Russian Federation, the characterization of known variants in the RPE65 gene, and the establishment of the specificities of the mutation spectrum in Russian patients. METHODS: The analysis was carried out on blood samples obtained from 1053 non-related IRDs patients. The analysis, which consisted of 211 genes, was carried out based on the method of massive parallel sequencing (MPS) for all probands. Variant validation, as well as biallelic status verification, were carried out using direct automated Sanger sequencing. The number of copies of RPE65 exons 1-14 was analyzed with quantitative MLPA using an MRC-Holland SALSA MLPA probemix. RESULTS: Out of 1053 non-related patients, a molecular genetic diagnosis of IRDs has been confirmed in 474 cases, including 25 (5.3%) patients with RPE65-associated retinopathy. We detected 26 variants in the RPE65 gene, nine of which have not been previously described in the literature. The most common mutations in the Russian population were c.304G>T/p.(Glu102*), c.370C>T/p.(Arg124*), and c.272G>A/p.(Arg91Gln), which comprised 41.8% of all affected chromosomes. CONCLUSIONS: The current study shows that pathogenic variants in the RPE65 gene contribute significantly to the pathogenesis of IRDs and comprise 5.3% of all patients with a confirmed molecular genetic diagnosis. This study allowed for the formation of a cohort for target therapy of the disorder; such therapy has already been carried out for some patients.

L138ins Variant of the CFTR Gene in Russian Infertile Men.

(1) Introduction: Pathogenic variants in the CFTR (Cystic Fibrosis Transmembrane conductance Regulator, OMIM: 602421) gene cause Cystic Fibrosis (CF, OMIM: 219700) and CF-related disorders (CF-RD), often accompanied by obstructive azoospermia due to congenital bilateral aplasia of vas deferens (CBAVD, OMIM: 277180) in male patients. The L138ins (c.413_415dup; p. (Leu138dup)) is a mild variant in the CFTR gene that is relatively common among CF-patients in Slavic populations. The frequency of this variant in Russian infertile men has not been sufficiently studied; (2) Materials and Methods: The sample consisted of 6033 Russian infertile men. The patients were tested for 22 common in Russian populations pathogenic variants of the CFTR gene and the IVS9Tn-polymorphic locus of the intron 9. Molecular-genetic studies were performed using amplified fragment length polymorphism (AFLP-PCR), multiplex ligation-dependent probe amplification (MLPA), and nested PCR (for analysis of the IVS9Tn-polymorphic locus); (3) Results: Pathogenic variants in the CFTR were detected in 3.9% of patients. The most frequent variants were F508del and CFTRdele2.3(21kb), accounted for 61.0% and 7.1% of detected variants, respectively. The L138ins variant was detected in 17 (0.28%) individuals: one of them was homozygous, 10 patients were heterozygous, and 6 patients were compound-heterozygous (F508del/L138ins, n = 4; L138ins/N1303K, n = 1; L138ins/5T, n = 1). Two pathogenic CF-causing variants in the CFTR gene were detected in 8 patients, including 7 compound heterozygous (F508del/L138ins, n = 4; F508del/N1303K, n = 1; 2184insA/E92K, n = 1; 3849+10kbC>T/E92K, n = 1) and one homozygous (L138ins/L138ins). The L138ins variant was found in 7 out of 16 (43.75%) chromosomes in six of these patients. The most common pathogenic variant, F508del, was identified in five out of them, in 5 of 16 (31.25%) chromosomes. The allele frequency (AF) of the L138ins variant in the sample has been found to be 0.0014.; (4) Conclusions: The L138ins variant of the CFTR gene is the third most common variant after F508del and CFTRdele2.3(kb) among Russian infertile men.

X-Linked Myotubular Myopathy in a Female Patient with a Pathogenic Variant in the MTM1 Gene.

X-linked centronuclear myopathy is caused by pathogenic variants in the MTM1 gene, which encodes myotubularin, a phosphatidylinositol 3-phosphate (PI3P) phosphatase. This form of congenital myopathy predominantly affects males. This study presents a case of X-linked myotubular myopathy in a female carrier of a pathogenic c.1261-10A>G variant in the MTM1 gene.

Cleavable crystals, crystal structure, and magnetic properties of the NbFe1+xTe3 layered van der Waals telluride.

Transition metal-based two-dimensional nanomaterials with competing magnetic states are at the cutting edge of spintronic and low-power memory devices. In this paper, we present a Fe-rich NbFe1+xTe3 layered telluride (x ≈ 0.5), which shows an interplay of spin-glass and antiferromagnetic states below the Néel temperature of 179 K. The compound has a layered crystal structure, where the NbFeTe3 layers are terminated by the Te atoms and van der Waals gaps. Bulk single crystals grown by chemical vapor transport reactions possess the (1̄01) cleavage plane suitable for the exfoliation of two-dimensional nanomaterials. Combination of high-resolution transmission electron microscopy and powder X-ray diffraction reveals the zigzag ladders of Fe atoms inside the structural layers, as well as complementary zigzag chains of the partially occupied Fe positions in the interstitial region. Fe atoms carry large effective magnetic moment of 4.85(3)µB per atom in the paramagnetic state yielding intriguing magnetic properties of NbFe1+xTe3. They include frozen spin-glass state at low temperatures and spin-flop transition in high magnetic fields indicating promising flexibility of the magnetic system and its potential control by magnetic field or gate tuning in the spintronic devices and heterostructures.

Spectrum of Genes for Non-GJB2-Related Non-Syndromic Hearing Loss in the Russian Population Revealed by a Targeted Deafness Gene Panel.

Hearing loss is one of the most genetically heterogeneous disorders known. Over 120 genes are reportedly associated with non-syndromic hearing loss (NSHL). To date, in Russia, there have been relatively few studies that apply massive parallel sequencing (MPS) methods to elucidate the genetic factors underlying non-GJB2-related hearing loss cases. The current study is intended to provide an understanding of the mutation spectrum in non-GJB2-related hearing loss in a cohort of Russian sensorineural NSHL patients and establish the best diagnostic algorithm. Genetic testing using an MPS panel, which included 33 NSHL and syndromic hearing loss (SHL) genes that might be misdiagnosed as NSHL genes, was completed on 226 sequentially accrued and unrelated patients. As a result, the molecular basis of deafness was found in 21% of the non-GJB2 NSHL cases. The total contribution pathogenic, and likely pathogenic, variants in the genes studied among all hereditary NSHL Russian patients was 12%. STRC pathogenic and likely pathogenic, variants accounted for 30% of diagnoses in GJB2-negative patients, providing the most common diagnosis. The majority of causative mutations in STRC involved large copy number variants (CNVs) (80%). Among the point mutations, the most common were c.11864G>A (p.Trp3955*) in the USH2A gene, c.2171_2174delTTTG (p.Val724Glyfs*6) in the STRC gene, and c.107A>C (p.His36Pro) and c.1001G>T (p.Gly334Val) in the SLC26A4 gene. Pathogenic variants in genes involved in SHL accounted for almost half of the cases with an established molecular genetic diagnosis, which were 10% of the total cohort of patients with non-GJB2-related hearing loss.

Collection of Hairy Roots as a Basis for Fundamental and Applied Research.

Due to population growth, instability of climatic conditions, and reduction of the areas of natural ecosystems, it becomes necessary to involve modern biotechnological approaches to obtain highly productive plant material. This statement applies both to the creation of plant varieties and the production of new pharmaceutical raw materials. Genetic transformation of valuable medicinal plants using Agrobacterium rhizogenes ensures the production of stable and rapidly growing hairy roots cultures that have a number of advantages compared with cell culture and, above all, can synthesize root-specific substances at the level of the roots of the intact plant. In this regard, special attention should be paid to the collection of hairy roots of the Institute of Plant Physiology RAS, Russian Academy of Sciences, the founder of which was Dr. Kuzovkina I.N. Currently, the collection contains 38 hairy roots lines of valuable medicinal and forage plants. The review discusses the prospects of creating a hairy roots collection as a basis for fundamental research and commercial purposes.

Mummified Wood of Juniperus (Cupressaceae) from the Late Miocene of Taman Peninsula, South Russia.

Juniperus L. is the second-largest genus of conifers, having the widest distribution of all conifer genera. Its phytogeographic history is, however, obscure due to its very poor fossil record. We described a wood of Juniperus sp. from the lower Maeotian sediments of the Popov Kamen section, Taman Peninsula, South Russia, in order to clarify its taxonomic position shedding light on the phytogeographic history of the genus. This fossil wood was well-preserved by mummification, which allowed for it to be studied by the same methods as used for the anatomical examination of modern woods. The wood from the Popov Kamen section shows the greatest similarity to the extant Mediterranean species J. excelsa, belonging to the section Sabina. This is the first reliable macrofossil evidence of the sect. Sabina from Eurasia convincingly dated to the Miocene. The age of the mummified wood from the Popov Kamen section is consistent with molecular dating of diversification of the lineage comprising juniper species of the sect. Sabina from Europe, Asia and eastern Africa. The wood of Juniperus sp. has not been buried in situ, as it was found in the relatively deep-water marine sediments. The available coeval pollen series and macrofossils of Cupressaceae from the surrounding regions suggest that this wood was likely transferred by sea current from the northwestern side of the Black Sea, which was a part of the Eastern Paratethys.

Fe-Rich Ferromagnetic Cleavable Van der Waals Telluride Fe5AsTe2.

Transition metal-based layered compounds with van der Waals gaps between the structural layers are a rich source of magnetic materials for spintronic applications. Bulk crystals can be cleaved, providing high-quality two-dimensional nanomaterials, which are promising for the manipulation of spins in spintronic devices and low power quantum logic interfaces. The layered van der Waals telluride Fe5AsTe2 can be synthesized by the high-temperature reaction of elements. In the crystal structure, Fe-rich structural layers with the composition of Fe4.58(4)AsTe2 are separated by the van der Waals gaps with no atoms in the interstitial region. Crystal growth employing chemical vapor transport reactions yields bulk cleavable crystals, which exhibit weak inherent ferromagnetism below the Curie temperature of TC = 48 K. In the ordered state, the magnetization shows a dual-slope behavior in low magnetic fields, indicating the compensated or canted nature of magnetism. Magnetic susceptibility and magnetization measurements reveal perpendicular magnetic anisotropy. The large Rhodes-Wohlfarth ratio of 4.6 indicates the itinerant nature of ferromagnetism in Fe5AsTe2.

Deciphering the molecular basis of tissue-specific gene expression in plants: Can synthetic biology help?

Gene expression differences between distinct cell types are orchestrated by specific sets of transcription factors and epigenetic regulators acting upon the genome. In plants, the mechanisms underlying tissue-specific gene activity remain largely unexplored. Although transcriptional and epigenetic profiling of individual organs, tissues, and more recently, of single cells can easily detect the molecular signatures of different biological samples, how these unique cell identities are established at the mechanistic level is only beginning to be decoded. Computational methods, including machine learning, used in combination with experimental approaches, enable the identification and validation of candidate cis-regulatory elements driving cell-specific expression. Synthetic biology shows great promise not only as a means of testing candidate DNA motifs but also for establishing the general rules of nature driving promoter architecture and for the rational design of genetic circuits in research and agriculture to confer tissue-specific expression to genes or molecular pathways of interest.

Case Report: Phenotype-Driven Diagnosis of Atypical Dravet-Like Syndrome Caused by a Novel Splicing Variant in the SCN2A Gene.

Febrile-associated epileptic encephalopathy is a large genetically heterogeneous group that is associated with pathogenic variants in SCN1A, PCDH19, SCN2A, SCN8A, and other genes. The disease onset ranges from neonatal or early-onset epileptic encephalopathy to late-onset epilepsy after 18 months. Some etiology-specific epileptic encephalopathies have target therapy which can serve as a clue for the correct genetic diagnosis. We present genetic, clinical, electroencephalographic, and behavioral features of a 4-year-old girl with epileptic encephalopathy related to a de novo intronic variant in the SCN2A gene. Initial NGS analysis revealed a frameshift variant in the KDM6A gene and a previously reported missense variant in SCN1A. Due to lack of typical clinical signs of Kabuki syndrome, we performed X-chromosome inactivation that revealed nearly complete skewed inactivation. Segregation analysis showed that the SCN1A variant was inherited from a healthy father. The proband had resistance to multiple antiseizure medications but responded well to sodium channel inhibitor Carbamazepine. Reanalysis of NGS data by a neurogeneticist revealed a previously uncharacterized heterozygous variant c.1035-7A>G in the SCN2A gene. Minigene assay showed that the c.1035-7A>G variant activates a cryptic intronic acceptor site which leads to 6-nucleotide extension of exon 9 (NP_066287.2:p.(Gly345_Gln346insTyrSer). SCN2A encephalopathy is a recognizable severe phenotype. Its electro-clinical and treatment response features can serve as a hallmark. In such a patient, reanalysis of genetic data is strongly recommended in case of negative or conflicting results of DNA analysis.

A Ribo-Seq Method to Study Genome-Wide Translational Regulation in Plants.

Protein production from mRNA is one of the fundamental molecular processes in a cell. Accurate genome-wide information on the levels of translation and ribosome distribution on mRNA can be gathered by carrying out ribosome footprinting, aka Ribo-seq. Herein, we present a detailed protocol describing the construction of parallel Ribo-seq and RNA-seq libraries from Arabidopsis seedlings treated with the plant hormone auxin. The improved protocol for ribosome footprint library generation can be easily adapted to analyzing the effects on translation of genetic perturbations and various abiotic and biotic factors to shed the much-needed light on translational regulation in plants.

Scaled in Cartesian Coordinates Ab Initio Molecular Force Fields of DNA Bases: Application to Canonical Pairs.

The model of Regularized Quantum Mechanical Force Field (RQMFF) was applied to the joint treatment of ab initio and experimental vibrational data of the four primary nucleobases using a new algorithm based on the scaling procedure in Cartesian coordinates. The matrix of scaling factors in Cartesian coordinates for the considered molecules includes diagonal elements for all atoms of the molecule and off-diagonal elements for bonded atoms and for some non-bonded atoms (1-3 and some 1-4 interactions). The choice of the model is based on the results of the second-order perturbation analysis of the Fock matrix for uncoupled interactions using the Natural Bond Orbital (NBO) analysis. The scaling factors obtained within this model as a result of solving the inverse problem (regularized Cartesian scale factors) of adenine, cytosine, guanine, and thymine molecules were used to correct the Hessians of the canonical base pairs: adenine-thymine and cytosine-guanine. The proposed procedure is based on the block structure of the scaling matrix for molecular entities with non-covalent interactions, as in the case of DNA base pairs. It allows avoiding introducing internal coordinates (or coordinates of symmetry, local symmetry, etc.) when scaling the force field of a compound of a complex structure with non-covalent H-bonds.