Vol 21 (2023): Спецвыпуск

Today, thanks to use the modern methods of biotechnology, the molecular mechanism underlying different aspects of plant development are started to open up. Along with the traditional methods of genetic analysis, plant developmental genetics actively uses the technics of genetic engineering and “omics” technologies. One of the problems of plant developmental genetics is the study of tumor growth in plants as a model for revealing the mechanisms of systemic control of cell division. Tumor is a pathological structure emerging as a result of uncontrolled proliferation of a group of cells leaving the systemic control of growth rate, cell differentiation and proliferation. Therefore, the elucidation of the mechanisms of tumor formation may help to identify the key regulators of systemic mechanisms controlling cell proliferation and differentiation. Tumor-like structures are found in almost all multicellular organisms, including higher plants. Pathogen-induced tumors, which make up the majority of neoplasms in higher plants, develop under the influence of infectious agents (bacteria, viruses, fungi, nematodes, insects, etc.) which create a niche for their own habitation in the host plant’s organism mostly by shifting the phytohormonal balance and sometimes activation of the meristematic competence of plant cells or modulation of plant cell cycle. At the same time, much rarer spontaneous tumors of higher plants are formed in plants with certain genotypes (mutants, interspecific hybrids, inbred lines) in the absence of any pathogen, which makes them closer to animal tumors. In particular, in the genetic collection of radish (Raphanus sativus L.), the inbred lines that form spontaneous tumors on the taproot during the flowering period were obtained many years ago. The connection between the spontaneous tumor formation in these lines and the altered balance of the main phytohormones, as well as ectopic expression of meristem-specific genes, was previously demonstrated. We have analyzed the differential gene expression in the spontaneous tumors of radish versus the lateral roots using the RNA-seq method. Data were obtained indicating the increased expression of genes associated with cell division and growth (especially genes that regulate G2-M transition and cytokinesis) in the spontaneous tumor. Among genes downregulated in tumor tissue, genes participating in the response to stress and wounding, as well as in the biosynthesis of glucosinolates, were enriched. Subsequently, we also performed whole genome sequencing of two closely related radish lines, contrasting in their ability to spontaneous tumor formation. In the coding genes of tumorous line, we have identified numerous SNPs and InDel which lead to frameshift and are probably associated with the tumor formation trait. Thus, spontaneous tumor formation in inbred lines of radish is probably under complex polygenic control. Testing of the relationship of these polymorphisms with tumor formation has begun. Our data will help to elucidate the mechanisms of spontaneous tumor development in higher plants.

The conference was held with support of the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 date 22.04.2022 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center “Agrotechnologies for the Future”.

Somatic embryogenesis is the formation of embryos from plant’s somatic cells. It is widely used in biotechnology for reproduction of plants, studying of regeneration process and it also represents a convenient way to obtain transgenic plants. Currently, a solid medium is usually used for the formation of transgenic somatic embryos, which has a number of disadvantages.

We are developing a system for cultivating explants in a liquid medium for the transformation and formation of somatic embryos for Medicago truncatula. Unlike a solid medium, it should allow using petioles as explants, simplify the renewal of the medium, replace disposable cultivation containers with reusable ones, and also reduce the time required for the formation of somatic embryos.

Currently, the optimal concentration of hygromycin as a selective agent in such a system was found. Interestingly, it appeared to be lower than the selective hygromycin concentration in a solid medium.

The addition of cefotaxime to the medium reduces the number of somatic embryos formed, but does not completely suppress their formation. Thus, cefotaxime can be used to eliminate agrobacteria during transformation using this cultivation system.

Embryos with GUS overexpression transformed with this method were successfully obtained.

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form embryos in a process called somatic embryogenesis (SE). SE can occur naturally in some plant species and it is widely used for the plant’s genetic transformation and regeneration.

This process is regulated by hormone treatment and many proteins, among which WUSCHEL-related homeobox (WOX) transcription factors are believed to play crucial roles. Our previous studies have shown that MtWOX9-1 stimulates SE in Medicago truncatula. The aim of the present research was to search for new MtWOX genes regulating SE. In this study, using transcriptomic data and literature data, we had selected several genes with an increased expression level during SE or in the generative organs and examined the overexpression effect of these genes on the SE ability. It was found that explants of the M. truncatula embryogenic line, transformed with the construct for MtWOX6-like overexpression, develop more somatic embryos compared to the control.

Our findings could be a helpful point for searching and studying new morphogenic regulators controlling SE and could have a positive impact on plant biotechnology in improving the transformation and regeneration capacity for other legumes.

This work was supported by the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 date 22.04.2022 on providing a grant in the form of subsidies from the Federal budget of Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center “Agrotechnologies for the Future”.

Komagataella phaffii (Pichia pastoris) is known to be an excellent producer of recombinant proteins for industrial and research purposes. Protein synthesis improvement in this yeast includes selection of optimal cultivation parameters [1, 2]. Therefore, much attention is paid to the influence of media components on physiology of this yeast [3–5].

One of the essential media components is biotin. In yeast cells it plays a crucial role as a cofactor of enzymes, providing carboxylation reactions in lipo-, gluconeogenesis, and nitrogen metabolism. K. phaffii is biotin auxotrophic organism unable to synthesize this vitamin de novo. Thus, it necessarily requires adding of biotin in the media.

In this study, we analyzed the effect of biotin starvation on gene expression in K. phaffii cells during its growth on methanol- and glycerin-containing media. These carbon sources are the most commonly used in standard protocols for recombinant protein biosynthesis in K. phaffii.

It was shown, that biotin starvation cell response significantly depends on carbon source. In glycerol-containing media biotin deficiency enhanced transcription of genes involved in biotin and thiamine metabolism, glyoxylate cycle, synthesis of acetyl-CoA in cytoplasm and its carnitine-mediated transport into mitochondria. Genes involved in biosynthesis of lipids and glucose were repressed in media with glycerol. In methanol-containing media the biotin deficiency effect was more pronounced and led to repression of numerous genes involved in protein and amino acids synthesis and activation of cell response to oxidative stress.

The obtained results are thought to be important for optimizing the culture conditions in the K. phaffii expression systems.

CRISPR/Cas9-based genome editing systems are widely used for genetic modification of different organisms. One of the drawbacks of CRISPR/Cas9 methods is the non-specific activity of Cas9, which can lead to accumulation of unwanted mutations in the edited genome [1]. Therefore, the development of in vivo models for high-throughput analysis of factors influencing the frequency of mutagenesis associated with the use of CRISPR/Cas9 technologies is a relevant task. Yeast Saccharomyces cerevisiae is a convenient object for developing such models [2].

Here we represent a yeast in vivo model that allows us to evaluate the effects of nucleotide sequence of the protospacer adjacent motif (PAM) and the guide RNA (gRNA) on the efficiency of binding between the gRNA/Cas9 complex and the target sequence in the genome. Since the Cas9 activity is lethal in cells lacking a donor sequence for homologous repair of double-strand breaks caused by this endonuclease, in the proposed test-system, the reduced efficiency of transformation by a plasmid encoding Cas9 and various gRNA variants reflects the efficiency of recognition of the target gene by the gRNA/Cas9 complex.

To study the influence of different PAM variants, with a consensus of NGG, on CRISPR/Cas9 activity, we obtained four isogenic strains that differ in their PAM sequence (AGG, TGG, CGG, GGG) in the codon 202 of the chromosomal copy of the reporter gene URA3. To evaluate the effect of incomplete matching between gRNA and the target site sequences, we propose using a series of plasmids based on the pML107 vector, encoding Cas9 and one of the 20 possible gRNA variants with single nucleotide substitutions at each of the 20 positions. The results obtained so far indicate the potential of the proposed approach.

CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease from Lachnospiraceae bacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNA in vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice.

In conclusion, new PAM TTAA greatly increases specificity of DNA cleavage allowing to use this system as an instrument for rapid detection if microdeletions in mice.

This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 94030690) and the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663)

The most common application of CRISPR-Cas9 genome editing system is a gene knockout via indel mutations introducing. It is obvious, because this approach has minimum critical conditions in guide RNA design: available PAM sequence and conservative 19–25 nucleotides within all alleles of a target gene. Precise nucleotide changing with base editing systems has more limitations: target nucleotide should locate in editing window of adenine- or cytidine-deaminase, besides this, all undesired adenines or cytosines in editing window will be likely changed. However, there is a more fundamental issue — it is very difficult to find a single aminoacid substitution, which changes protein features in a desirable side. One of the good examples of base editing target will be considered in this work.

Nicotiana tabacum L. is a plant from Solanaceae family, the same as potato, tomato and pepper. All these plants are strongly affected by potato virus Y (PVY). It is known, that PVY recruits host translation initiation factor eIF4E by the viral protein VPg in order to start synthesis its proteins. If eIF4E can’t interact with VPg, plant will be resistant.

In our work, we established an aminoacid substitution in tobacco eIF4E factor, which disrupted interaction with PVY VPg in yeast two-hybrid conditions, but didn’t influence the factor functionality. Then we designed two genetic constructions with different sgRNAs for introducing this mutation in tobacco plants using cytidine-deaminase system. These constructions were used to plant transformation and development of edited tobacco plants.

This work is supported by State Task No. 0431-2022-0004.

The presence of several sets of chromosomes in polyploid crops is a serious problem for the application of gene and genome editing systems. Efficient CRISPR/Cas-based mutagenesis of series of genes involved in the grain starch biosynthesis of hexaploid triticale has been developed. Triticale (×Triticosecale), is a hybrid of rye (Secale) and wheat (Triticum) and consists of three subgenomes. Four genes were targeted and to ensure efficient editing of all subgenomes, a trio of guide RNAs for each target genes were designed. To enable simultaneous editing of 36 genetic loci at once (three sgRNAs × four genes × three subgenomes), an expression cassette was constructed, assembled as an array of twelve sgRNAs. The polysitron vector was delivered to morphogenic calli using a gene gun [1] together with a vector encoding Cas9 nuclease [2] to induce mutations. A number of transgenic plants of spring and winter triticale carrying both Cas9 and sgRNAs inserts have been generated. The efficiency of native gene editing varied depending on the target gene and sgRNA activity. Using a trio of sgRNAs for each target gene, we successfully mutated all three subgenome copies, thereby modifying seed starch synthesis. It can be expected that the described approach will make an important contribution to the future breeding of polyploid crops to produce various combinations of new genetic alleles encoding desired traits.

The study is supported by Kurchatov Genomics Center of All-Russia Research Institute of Agricultural Biotechnology, agreement No. 075-15-2019-1667.

CRISPR/Cas systems are presently the most attractive genome editing technology, that is widely used for genetic engineering of various crops and industrial microorganisms. Currently, application of the CRISPR/Cas based genome editing promises advances in microalgae biotechnology aimed at boosting the output of biofuels and valuable bioactive compounds. However, algae remain relatively complex objects for genetic manipulation [1]. The main problems are associated with the need of a species-oriented approach when creating a transformation toolbox due to the peculiarities in the structure of membranes and the cell wall of a particular taxon. The proper selection and design of a CRISPR construct is also required due to the possible presence of a powerful silencing system against introduced genetic constructs in the cell. These difficulties explain the low efficiency of microalgae transformation and the meager list of successfully edited species [1, 2].

The first instance of genome editing in microalgae using CRISPR/Cas was reported in Chlamydomonas reinhardtii P.A. Dang [3]. To date, four transformation methods (Agrobacterium-mediated, particle bombardment, glass beads agitation, electroporation) have been successfully used for editing (knock-in and knock-out) the C. reinhardtii genome with two types of CRISPR constructs (plasmid and ribonucleoprotein). The developed protocols make it possible to achieve high efficiency of genomic editing — for example, in our study it varied from 10.6% to 68.8% [4]. These benefits along with completely sequenced genome, well-studied genetics, accessibility and haplontic life cycle makes C. reinhardtii an outstanding model organism for CRISPR/Cas application in microalgae research [5].

Eggplants, known scientifically as Solanum melongena L., are renowned for their health benefits, largely attributed to phenolic acids. Chlorogenic acid stands out as one of the most prevalent phenolic acids in eggplants. The enzyme hydroxycinnamoyl CoA-quinate transferase (SmHQT) plays a pivotal role in the production and concentration of this acid in the fruit. However, until this study, the exact function and influence of SmHQT on the eggplant’s composition remained elusive [1–3].

This research aimed to explore SmHQT’s role by overexpressing it in the eggplant’s flesh using agroinfiltration, a technique that transiently introduces genes into plants. This method offers insights into potential changes in the plant’s chemical makeup. Advanced techniques like quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography (HPLC) revealed that the chlorogenic acid content in the genetically altered eggplants was over twice that of the unaltered ones.

The study also investigated the cascading effects of this overexpression. The qRT-PCR results showed variations in the expression of genes linked to the chlorogenic acid pathway, hinting at SmHQT’s wider role in phenolic acid biosynthesis in eggplants. Comprehensive analyses of protein interactions and cis-regulating elements were undertaken to grasp SmHQT’s full impact.

Phenolic acids, like chlorogenic acid, offer therapeutic benefits against conditions such as diabetes, cancer, and arthritis in humans. In plants, they enhance natural defenses against pests and diseases. While there have been attempts to boost the phenolic acid content in eggplants using genes from wild variants, this study’s approach proved more effective.

Another notable achievement of this research was the introduction of an improved agroinfiltration protocol. This method is promising for future studies focused on transient gene expression in fruits, facilitating swift genetic modification prototyping. In essence, this research underscores the immense potential of bioengineering in augmenting the nutritional profiles of crops by enhancing their inherent phytochemicals.

Microalgae contain a wide range of useful substances: antioxidants, lipids, proteins, carbohydrates and secondary metabolites which could be used in nutraceuticals and dietary supplements. Green microalgae Chlorella containing highest amount of chlorophylls of any known plant, 60% protein, 18 amino acids, 20 vitamins and minerals [1]. Microalgae are exceptionally rich source of pharmacologically active metabolites with antineoplastic, antitumor, antibacterial, antifungal and antiviral properties and, also capable of wastewater treatment, and biomass production.

The genetic information can improve the scenario of metabolic engineering in microalgae. Green algae C. reinhardtii, a reference organism for understanding the basic algal genetics and metabolism is usually used to work out various genetic strategies, including omics resources and mutant libraries, for the enhancement of beneficial properties of microalgae. The synergy of microalgal multi-omics datasets (genomic, transcriptomic and proteomic) offer a rapid and predictable strategic path for the strain improvement [2]. The algal nuclear or chloroplast engineering (transformation and CRISPR/CAS editing) has been carried out using synthetic biology approach for the production of recombinant proteins having therapeutic properties. More than 100 recombinant proteins have been expressed in microalgae, mainly in C. reinhardtii, including: the vaccines, antibodies, immunotoxins and therapeutic proteins (human erythropoietin, fibronectin, interferon B1, proinsulin, endothelial growth factor and others [3]. Thus, the wide taxonomic and biochemical diversity among the microalgae when using modern biotechnologies, makes them suitable resource of abundant biomolecules with industrial and biomedical importance.

The paper covers the questions of secondary metabolite modulation in medicinal plants by means of gene engineering. It is demonstrated that cutting-edge tools of contemporary biotechnology tools made it possible to manage the biosynthesis of important bioactive substances, modify the secondary metabolism, enabling plants to synthesize and produce new compounds, as well as eliminate metabolic pathways of synthesizing harmful substances.

Currently, large-scale production of bioactive substances (BAS) requires highly productive plants to produce them. Applying methods of gene engineering to medicinal plants is a promising way to reduce the resource consumption and increase their productivity, quality and the product’s marketability [1]. Traditional growing and collecting techniques are challenged by resource shortage, environmental damage, etc. [2]. Gene engineering helps to increase pest, disease and herbicide resistance, gain greater yields and higher BAS content [3].

Using transgenic medicinal plants (TMP) as BAS producers in the pharmaceutical industry is crucial for metabolic engineering. Current research of the secondary metabolism modulation in TMP enables to modify the key BAS biosynthesis and the secondary metabolism, so that plants can produce new substances, or, on the contrary, silence the metabolic pathways for harmful ones. This way, greater TMP biomass with higher BAS content can be obtained in bioreactors. This would require rather modest investments — an advantage for biopharmacy. Nowadays, TMP are grown in vitro as calluses or suspension cell cultures. Biotechnology can modify the secondary metabolism in TMP to produce surplus amounts of necessary BAS, reduce the content of toxic compounds or even synthesize new substances. The versatility of transcription and translation mechanisms in medicinal plants enables them to accumulate homologous substances and synthesize heterologous ones. It is known that in TMP, MYB transcription factors are involved in gene regulation in secondary metabolic pathways, regulation of genes engaged in developmental processes, etc. [4]. In conclusion, we should emphasize the relative biosafety of BAS obtained from TMP, for human use, as they are chemically pure and are not connected with biological hazards.

Amyloids are protein aggregates characterized by their insolubility in detergents and ability to form fibrils. They are often associated with various diseases, including neurodegenerative disorders, type 2 diabetes and certain forms of cancer. Amyloids also play important roles in bacteria and different physiological processes in both lower and higher eukaryotes.

Together with the laboratory of Prof. Y.O. Chernoff we have developed a comprehensive approach for screening new potentially amyloidogenic proteins. This involves using bioinformatics algorithms to predict protein amyloidogenicity and further verifying using a yeast model. We have created a yeast test system specifically designed to study changes in phenotype in genetically modified Saccharomyces cerevisiae strains [1]. This system involves the production of recombinant amyloidogenic proteins fused with reporter proteins Sup35N or YFP. Using yeast assay, we have investigated 22 human proteins that were predicted to be amyloidogenic by ArchCandy algorithm [2]. Currently, additional in vitro biochemical tests are underway with proteins that have shown the potential to form amyloids in yeast models. There are also plans to evaluate the amyloid-forming ability of specific human proteins in mammalian cell cultures. These various approaches appear to be enhancing our comprehension of the impact of amyloid formation in health and disease.

Pathogenic viruses cause severe tomato losses around the world despite the development of both classical breeding and biotechnological methods. Since replication of phytoviruses involves the interaction between viral components and host plant factors, therefore loss-of-function mutations in the latters can confer viral resistance in plants. There are evidences that eukaryotic translation elongation factor 1 (eEF1) proteins are involved in the replication of some plant viruses. However, the involvement of individual subunits of the eEF1B in the viral cycle is still poorly understood.

This work is devoted to the study of the role of the eEF1B factor in the development of tomato virus infection. The contribution of each of the α, β and γ subunits of the eEF1B factor to tomato viral resistance will be determined by CRISPR-Cas9-induced targeted mutagenesis of corresponding gene sequences. As an applied aspect, we expect to find ways to create tomato plants with increased resistance to certain viral diseases. A series of binary vectors contained sequences encoded different RNAs targeting the eEF1B subunit genes was constructed. As a result of Agrobacterium-mediated transformation of tomato, more than 300 independent transgenic lines were obtained. The presence of expression cassettes with functional genes (Cas9 and sgRNAs) was confirmed by PCR. The presence of mutations in target sequences was detected using T7E1 analysis and sequencing. It turned out that the majority of transgenic lines carrying mutations have a chimeric genotype, and mutations of the target genes in the homozygous state were not detected. The propagation of self-pollinated transgenic plants under greenhouse condition and following analyses of target genes to segregate the insertion of foreign DNA and obtain homozygous mutations in eEF1B subunit sequences are in progress.

CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION-related) peptides are known as systemic regulators of legume-rhizobium symbiosis that negatively control the number of nitrogen-fixing nodules. These regulatory peptides are produced in the root in response to inoculation with rhizobia, and are transported through the xylem to the shoot, where they are recognized by their receptor, CLV1-like (CLAVATA1-like) kinase, active in leaf phloem cells. After that, a shoot-derived signaling pathway is activated that inhibits subsequent nodule development in the root. Previously, we found that in Medicago truncatula, the expression of the MtCLE35 gene is activated in response to rhizobia and nitrate treatment, and its overexpression systemically inhibits nodulation. However, little is known about the downstream target genes regulated by a MtCLE35 signaling pathway in the root. Moreover, it is not completely clear which stage of symbiosis development is affected by MtCLE35-activated pathway. In order to identify genes regulated by the MtCLE35-induced signaling pathway, we performed a transcriptomic analysis of the roots overexpressing the MtCLE35 gene. Totally, 1122 genes were found to be differentially expressed between MtCLE35-overexpressing and control roots after rhizobial inoculation, among them 185 genes were upregulated and 937 genes were downregulated. Among downregulated genes, many known regulators of legume-rhizobia symbiosis were found. In addition to this, we analyze early steps of interaction between M. truncatula overexpressing the MtCLE35 gene and Sinorhizobium meliloti labeled with fluorescent reporter. We did not observe penetration of S. meliloti into host plant roots with MtCLE35 overexpression. Our data suggest that overexpression of the MtCLE35 gene inhibits nodulation at the very early stages of symbiosis development.

Legume plants are important for ecosystems due to their ability to form root nodules in symbiosis with rhizobia, where nitrogen fixation takes place. The number of symbiotic nodules is regulated by the CLE peptides inhibiting excessive nodule formation. Previously, we have identified four genes encoding CLE peptides, activated in response to rhizobia inoculation in pea. Three of them, PsCLE13, PsCLE12 and PsNIC-like, were also activated by nitrate, and, therefore, they could mediate nitrate-dependent inhibition of nodulation [1]. Overexpression of PsCLE13 and PsCLE12 inhibited nodulation on transgenic roots: however, the role of PsNIC-like and PsCLE12-like have not been investigated.

In this study, we constructed vectors for overexpression of the PsCLE12-like and PsNIC-like genes to study their possible role in nodulation, and also analyzed the expression levels of nodulation-related genes in transgenic roots overexpressing four PsCLEs genes. Moreover, vectors for CRISPR-Cas9-mediated gene editing of the PsCLE12 and PsCLE13 genes were constructed to further explore the role of these genes in nodulation. Overexpression of PsCLE12-like, PsCLE13 and PsCLE12 resulted in increased expression levels of TOO MUCH LOVE (PsTMLs) genes known as root-acting regulators of nodule number. In addition, in the roots overexpressing four PsCLEs genes, down regulation of the PsSYM37 gene (encoding the receptor for Nod-factors) was observed, suggesting that the CLE peptides might inhibit the development of symbiotic nodules at the earliest stages of symbiosis development upon Nod-factor perception.

Naturally transgenic plants are plants that have been subjected to “Agrobacterium” mediated transformation in natural conditions without any human impact. They contain T-DNA-like sequences, called cellular T-DNA (cT-DNA) in their genomes and transfer them from generation to generation [1].

At the moment, several dozen species of natural GMOs are known, and this list is constantly updated. Based on the available data on the diversity of natural GMOs, it can be concluded that in each case, plants have their own set of functionally active transgenes. Accordingly, each cT-DNA performs its own functions. This set of active transgenes will define promising areas for nGMO research, such as:

• description of the structures and functions of opine synthesis genes and the biological activity of their products in the regulation of plant-microbial interactions [2];
• description of the effect of oncogenes on plant morphogenesis, their primary and secondary metabolism [1].

In addition, sequences, newly acquired by plants, can be successfully used in phylogenetic studies [3].

These topics will be the subject of a report at the conference.

The work was supported by a grant from the Russian Science Foundation using the equipment of the resource centers of St. Petersburg State University “Biobank” “Chemical Analysis and Materials” and “Development of molecular and cellular technologies”.

Keywords: natural GMO; cellular T-DNA; phylogenetic studies; opines; T-DNA oncogenes.

Cultivated peanut is an allotetraploid species that received the A and B genomes from Arachis duranensis and A. ipaensis. Homologs of the agrobacterial cucumopine synthase gene were previously found in both genomes as a result of horizontal transfer [1]. These sequences are found both in ancestral species and in cultivated peanuts. In addition to them, natural GMOs are A. monticola and A. stenosperma. How widespread natural GMOs are within the genus Arachis is currently unknown. The aim of our study was to search for natural GMOs within the genus Arachis and to analyze the polymorphism of natural transgenes out the studied species.

METHODS: Gene sequencing for various Arachis species was determined using the bwa [2], GATK [3] and samtools [4] packages based on NGS data aggregated in the SRA NCBI database.

RESULTS: We have found homologues of the cucumopine synthase gene in the genomes of A. appressipila, A. batizocoi, A. cardenasii, A. correntina, A. diogoi, A. duranensis, A. glandulifera, A. helodes, A. hoehnei, A. ipaensis, A. macedoi, A. magna, A. monticola, A. paraguariensis, A. pintoi, A. pusilla, A. rigonii, A. stenophylla, A. stenosperma, A. trinitensis, A. valida, A. villosa, and also characterized the intraspecific variability of the gene in cultivated peanuts. In 16 of the 22 species studied, the gene is full-length. The report will consider the possibility of using the cucumopine synthase gene in peanut phylogenetic studies.

CONCLUSION: The list of species of natural GMOs within the genus Arachis today includes 23 species.

The presence of foreign DNA is the main obstacle to the application of biotechnological plant varieties. However, transgene-free technologies in the field of genome editing make it possible to overcome this problem. In most countries that already have legislation in this area, plants without foreign DNA do not require field trials and safety tests.

The easiest way to avoid integration of transgenes is the delivery of RNP complexes directly into the cell without the use of plasmids. However subsequent selection of edited cells in the absence of a selective marker and plant regeneration are quite difficult. Therefore, traditional genetic constructs are used more often, despite that the elements of the CRISPR system are integrated into the genome. Backcrossing and cross-pollination are used to get rid of unwanted inserts. There are opportunities to accelerate the selection process, such as the Transgene Killer CRISPR system, which ensures the death of plants carrying transgenes in the early embryonic stages [1].

Constructs based on viral replicons integrated into T-DNA are an alternative option. They provide a high level of transient expression of CRISPR elements which are not integrated into the genome. Such vectors were created on the basis of geminiviruses, rhabdoviruses, potexviruses, potyviruses, bunyaviruses [2]. The ability of viruses to move between cells can be both preserved and lost due to the removal of the corresponding proteins.

One of the newest approaches is grafting of shoots onto roots expressing Cas and guide RNA [3]. The addition of tRNA-like motifs to the transcripts ensured their mobility and dispersal along the shoot. Heritable edits were observed in the progeny of grafted plants.

Thus, for transgene-free editing technologies of plant genomes are rapidly developing, which will accelerate the commercialization of new varieties with economically valuable traits.

Entomopathogenic fungi (EPF) of the genus Akanthomyces (formerly Lecanicillium) are one of the most common and important fungal entomopathogens, infecting sucking insects of the order Hemiptera mainly. The fungi can also parasitize on phytopathogenic fungi (rust, powdery mildew). The entomopathogens from these genera reported as endophytes in various plants under natural conditions [1–2], contributing to an increase in plant immunity to pathogens, as well as a decrease in plant colonization by pests. Endophytic colonization of plants by the fungus Akanthomyces lecanii can suppress the growth of the peach aphid [3]. Akanthomyces muscarius strains caused the death of moth when feeding on cabbage colonized by the fungus [4]. Endophytic properties were assessed using the A. muscarius (= Lecanicillium muscarium) strain Vl 72-GFP fluorescently labeled with GFP [5]. The transformation was done by electroporation of germinated conidia of the high-virulent “wild” strain Vl 72 by the pBARGPE1 vector harboring an eGFP gene, showed an expression of fluorescent protein without affecting fungal growth and virulence. The influence of the fungus on the growth rates of beans was revealed when leaves, sterile soil and seeds were treated with a suspension of conidia of 10⁸ spores/ml. On the 7^th day, stimulation of the growth of the stems and roots of the beans was observed when the seeds were soaked in a spore suspension of the fungus. When spraying the leaves, only the stem’s elongation was observed. The studied strain colonizes beans irregularly. When treating the seeds, the fungus was isolated in greater quantities from the roots (26%), when spraying the leaves — from the stem (36%), when watering the soil — also from the stem (43%). Infection of A. muscarius plants by spilling the soil was most effective. No effect of endophytization was found on the number of aphids after 14 days of aphid plant colonization. As a result of the introduction of the spores of Vl 72-GFP strain by shedding the soil under flower crops (lantana, gerbera, acanthus) in the greenhouse of Saint Petersburg Botanical Garden, this strain was isolated from the leaves of the Acanthus mollis L. after one month, which confirms the ability of this species to endophytic colonization of plants in greenhouse conditions. Analysis of hyphae Vl 72-GFP in the plant performed on an AxioImager M1 fluorescent microscope demonstrated the same level of fluorescence as in A. muscarius hyphae growing on the media.

Horizontal gene transfer from agrobacteria to plants turned out to be a much more widespread phenomenon than previously thought. In the first it was established in 2019 due to the application of bioinformatic methods and databases, then about 30 species of naturally transgenic plants were discovered [1]. The deposition of new nucleotide sequences of various plant species makes it possible to periodically update the list of naturally GMOs. Analysis of genomic and transcriptomic databases in 2023 revealed more than 50 new naturally transgenic plants and, thus, more than 100 species of nGMOs are currently known. And the share of naturally transgenic plants in relation to deposited species of terrestrial dicotyledonous plants is about 7%. Interestingly, this indicator retains its value regardless of the change in the number of organisms in the studied databases [2].

Among the discovered new nGMOs there are species that have been cultivated by humans since ancient times and are important agricultural crops. Fruit crops include the following species of nGMOs: carambola (Averrhoa carambola L.), persimmon (Diospyros kaki Thunb.), wasabi (Eutrema japonicum (Miq.) Koidz.), raspberry (Rubus idaeus L.), Luffa acutangula (L.) Roxb. There are also many medicinal, ornamental and oilseed species among the naturally transgenic plants. Further study of these species would make it possible to establish what role horizontal gene transfer played in the appearance of traits in plants that were selected by humans.

The obtained data can be further used to study the molecular evolution and the role of transgenes in naturally transgenic plants.

The policy of the Republic of Tajikistan in the field of biosafety, regarding the issue of handling and use of living genetically modified organisms (LMOs or GMOs) is aimed at compliance with international legal acts, agreements and obligations to ratified Conventions. Tajikistan ratified the Convention on Biological Diversity in 1997 and the Cartagena Protocol on Biological Safety in 2004. After ratifying the protocol, the country has prepared three National Reports in accordance with the requirements of international agreements. Earlier in Tajikistan, the Law of the Republic of Tajikistan “On Biological Safety” (2005) was adopted. “The Law regulates the development, testing, production, import, export and release on the market and into the environment of GMOs, is aimed at reducing the risk of adverse effects of GMO on human health, biological diversity, ecological balance and the state of the environment”. Currently, this Law has been renamed into the Law “On Genetically Modified Organisms” and is under discussion, approval and adoption by the Parliament of the Republic of Tajikistan.

Among the urgent problems that the Republic of Tajikistan is currently facing, considering the prospects for the coming years, is the problem of food security, including issues related to ensuring food safety. Taking into account the importance of conducting research in the field of biological and food safety, scientifically based risk assessment of biological agents (including GMOs) and toxins, chemical contaminants in food products and crops by the Decree of the Presidium of the Academy of Sciences of the Republic of Tajikistan No. 108 dated 30.11.2015 the Laboratory of Biological Safety was established at the Institute of Botany, Plant Physiology and Genetics of Tajikistan National Academy of Science, the main tasks of which are the development and application of modern methods of analysis for the detection of biological agents and toxins, chemical contaminants in food products and crops, and analysis of GMO products.

It should be noted that at present there is no official information related to the production, use, distribution, sale, import and export of GMOs, as well as the registration of incoming GMO food products in Tajikistan. An analysis of the market for agricultural products in the capital city of Dushanbe showed that a number of GMO food products and genetically modified seed material are still imported from abroad in the form of technical and humanitarian assistance as well as international trade. In this regard, food safety activities should include risk assessment based on scientific evidence. Its emphasis should be on both process control and end product safety so that potentially unsafe foods can be identified early. GMO food can be considered safe if the risks associated with it are at an acceptable and acceptable level. It should be noted that an effective system for monitoring food products, including products containing GMOs, their compliance with quality standards is important for protecting the health and safety of the country’s population.