Жасмонаты участвуют в регуляции защитных механизмов в растениях при неблагоприятных условиях среды, а также их роста и развития. В основном, знания о путях биосинтеза и передачи сигналов жасмонатов получены благодаря исследованию модельного двудольного растения Arabidopsis thaliana, однако их функции и молекулярные механизмы действия у однодольных растений, а именно у пшеницы, до сих пор остаются малоизученными. В данной работе мы исследовали устойчивость к стрессовым воздействиям мягкой пшеницы сорта Саратовская-60 и созданной на ее основе трансгенной линии (Tr-3) со сверхэкспрессией гена биосинтеза жасмонатов, 12-оксофитодиеноатредуктазы AtOPR3 (12-OXOPHYTODIENOATE REDUCTASE 3) из Arabidopsis thaliana. Несмотря на высокий уровень экспрессии перенесенного гена AtOPR3, содержание жасмоновой кислоты и ее конъюгата с изолейцином оказалось неизменным в листьях интактных трансгенных растений. После механического повреждения листьев, содержание жасмоновой кислоты в изучаемой трансгенной линии Tr-3 оказалось даже ниже в сравнении с поврежденными нетрансгенными растениями пшеницы Саратовская-60. Различий в содержании 12-оксофитодиеновой кислоты и жасмоноил-изолейцина не было. При этом стресс-индуцированный уровень экспрессии собственных генов алленоксидсинтазы, регулируемых жасмонатами, в трансгенных растениях Tr-3 стал выше, в сравнении с нетрансгенным контролем. Трансгенные растения пшеницы проявили повышенную устойчивость к заражению некротрофным грибом Botrytis cinerea, а также к осмотическому стрессу, вызванному полиэтиленгликолем при прорастании семян. В листьях трансгенной линии Tr-3, зараженных B. сinerea, наблюдалась более высокая активность каталазы, фермента антиоксидантной системы, в сравнении с зараженными листьями нетрансгенных растений, что свидетельствует о ее возможной роли в повышении устойчивости Tr-3 к фитопатогену. Таким образом, немногочисленные данные об особенностях функционирования жасмонатной системы в пшенице дополнены новыми данными о роли экспрессии одного из ключевых генов биосинтеза жасмонатов, а именно 12-OXOPHYTODIENOATE REDUCTASE, в регуляции защитных ответов при пониженном стресс-индуцированном уровне жасмоновой кислоты.

Алленоксидсинтазная (AOS) и гидропероксидлиазная (HPL) ветви пути биосинтеза оксилипинов ведут к образованию разных продуктов из одного субстрата – 13-гидроперокси-(9,11,15)-октадекатриеновой кислоты (13-ГПОТ). До сих пор остается неизвестным, как у растений регулируется распределение 13-ГПОТ субстрата между этими ветвями, как метаболиты каждой ветви влияют на активность параллельной ветви. В данной работе была исследована активность HPL ветви в листьях мягкой пшеницы (Triticum aestivum L.) сорта Саратовская-60, а также полученных на ее основе трансгенных растений со сверхэкспрессией гена биосинтеза жасмонатов из Arabidopsis thaliana AtOPR3 (12-OXOPHYTODIENOATE REDUCTASE 3). В ходе исследований выявлено высокое содержание метаболитов HPL ветви в листьях, что свидетельствует о высокой активности HPL пути биосинтеза оксилипинов у пшеницы. Показано, что сверхэкспрессия гена AtOPR3 приводит к повышению содержания метаболитов HPL ветви в листьях, в первую очередь цис-3-гексеналя, а также к изменению профиля летучих соединений HPL ветви, выделяемых поврежденными растениями. После механического повреждения листья трансгенной пшеницы со сверхэкспрессией AtOPR3 выделяют больше цис-3-гексенола и цис-3-гексенил ацетата, но меньше транс-2-гексеналя в сравнении с нетрансгенными растениями. Изменения содержания метаболитов HPL ветви в листовых тканях и в смеси выделяемых летучих соединений проявляются ярче в трансгенных линиях с более высоким уровнем экспрессии AtOPR3. Таким образом, впервые показано, что генетическая модификация AOS ветви биосинтеза оксилипинов приводит к изменению активности HPL ветви в растениях пшеницы.

KEY MESSAGE:The first-time generation of hexaploid triticale plants harbouring variable panels of novel mutations in gene families involved in starch biosynthesis has been achieved by the subgenome-independent multiplexed CRISPR/Cas9-mediated editing.

Jasmonates are involved in the regulation of protective mechanisms of plants against unfavorable environments as well as in control of their growth and development. Main data on jasmonate biosynthesis and signals transduction pathways were obtained on model dicotyledonous plant Arabidopsis thaliana. Meanwhile, functions and molecular mode of action of these compounds are still poorly investigated in monocotyledons, including wheat. In the present study, the stress tolerance of transgenic line (Tr-3) and nontransgenic plants of bread wheat cv. Saratovskaya-60 was studied. Transgenic line Tr-3 overexpresses AtOPR3 (12-OXOPHYTODIENOATE REDUCTASE 3) gene from A. thaliana encoding the jasmonate biosynthesis enzyme 12-oxophytodienoate reductase. In spite of the high expression level of AtOPR3, the content of jasmonic acid and its conjugate with isoleucine in intact leaves of the transgenic plants remained unchanged. Furthermore, in the mechanically wounded leaves, the content of jasmonic acid in the transgenic line Tr-3 was even lower than in nontransgenic Saratovskaya-60, while the levels of 12-oxophytodienoic acid and jasmonoil-isoleucine did not differ. In the transgenic plants, the stress-induced expression of the endogenous jasmotate-regulated allene oxide synthase gene was, however, higher than in the nontransgenic control. The transgenic wheat plants were more tolerant to infection by necrotrophic fungus Botrytis cinerea and to osmotic stress caused by polyethylene glycol applied to germinating seeds. The Botrytis-inoculated leaves of the transgenic line Tr-3 manifested higher activity of antioxidant enzyme catalase than inoculated leaves of nontransgenic plants. This fact points to the possible role of catalase in the transgene-associated tolerance to the pathogen. Therefore, the available scarce information concerning the peculiarities of functions of the jasmonate system in wheat plants has been amended with the new data on the role of expression of one of the key genes of jasmonate biosynthesis, namely, 12-OXOPHYTODIENOATE REDUCTASE, in control of defense responses even though the stress-induced level of jasmonic acid is decreased.

The allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches of the oxylipin biosynthesis pathway lead to the formation of different products from one substrate-13-hydroperoxy-(9,11,15)-octadecatrienoic acid (13-HPOT). It remains unknown how the distribution of 13-HPOT substrate between these branches is regulated in plants, and how the metabolites of each branch influence the activity of the parallel branch. In this work, the activity of HPL branch in the leaves of bread wheat (Triticum aestivum L.) of the Saratovskaya-60 variety, as well as transgenic plants overexpressing the gene for the biosynthesis of jasmonates from Arabidopsis thalianaAtOPR3 (12-OXOPHYTODIENOATE REDUCTASE 3), was investigated. The studies revealed a high content of HPL metabolites in the leaves, indicating a high activity of the HPL pathway of oxylipin biosynthesis in wheat. Overexpression of the AtOPR3 gene has been shown to lead to an increased content of HPL metabolites in the leaves, primarily cis-3-hexenal, as well as to changes in the profile of volatile compounds of the HPL branch released by damaged plants. After mechanical damage, leaves of transgenic wheat overexpressing AtOPR3 produced more cis-3-hexenol and cis-3-hexenyl acetate, but less trans-2-hexenal compared to nontransgenic plants. Changes in the content of HPL branch metabolites in leaf tissues and the mixture of released volatile compounds were more pronounced in transgenic lines with higher expression of AtOPR3. Thus, it was shown for the first time that genetic modification of the AOS branch of oxylipin biosynthesis leads to a change in the activity of the HPL branch in wheat plants.

The transformation protocol based on the dual selection approach (fluorescent protein and herbicide resistance) has been applied here to produce transgenic plants of two cereal species, emmer wheat and bread wheat, with the goal of activating the synthesis of the stress hormone jasmonates by overexpressing ALLENE OXIDE SYNTHASE from Arabidopsis thaliana (AtAOS) and bread wheat (TaAOS) and OXOPHYTODIENOATE REDUCTASE 3 from A. thaliana (AtOPR3) under the strong constitutive promoter (ZmUbi1), either individually or both genes simultaneously. The delivery of the expression cassette encoding AOS was found to affect morphogenesis in both wheat species negatively. The effect of transgene expression on the accumulation of individual jasmonates in hexaploid and tetraploid wheat was observed. Among the introduced genes, overexpression of TaAOS was the most successful in increasing stress-inducible phytohormone levels in transgenic plants, resulting in higher accumulations of JA and JA-Ile in emmer wheat and 12-OPDA in bread wheat. In general, overexpression of AOS, alone or together with AtOPR3, negatively affected leaf lamina length and grain numbers per spike in both wheat species. Double (AtAOS + AtOPR3) transgenic wheat plants were characterized by significantly reduced plant height and seed numbers, especially in emmer wheat, where several primary plants failed to produce seeds.

The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for the initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. The development of strategies enabling the generation of marker-free transgenic plants presents an alternative to address public concerns regarding the safety of biotech crops. This study examined the capabilities of highly regenerative potato cultivars to develop transgenic plants without the presence of selective substances in their media. Internodal segments of in vitro potato plants were inoculated with the Agrobacterium strain AGL0 carrying plasmids, which contained the GFP or RFP gene driven by the CaMV 35S promoter to monitor the transformation process by observing in vivo green or red fluorescence. Despite the absence of selective pressure, inoculated explants demonstrated comparable or even higher transient expression compared to experiments based on antibiotic assistant selection. Consequently, under non-selective conditions, non-transgenic, chimeric, and fully fluorescent potato plantlets were concurrently developed. Among the five tested cultivars, the regeneration efficiency of non-chimeric transgenic plants varied from 0.9 (‘Chicago’) to 2.7 (#12-36-42) plants per 100 detached plantlets. Depending on the regenerative characteristics of potato varieties (early, intermediate, or late), a specific time interval can be determined when a blind collection of transgenic plantlets is more successful, streamlining the transformation procedure. The results indicate that the outlined procedure is simple and reproducible, consistently achieving the transformation efficiency of 7.3–12.0% (per 100 inoculated explants) in potato cultivars without selective pressure. The described transformation procedure holds the potential for obtaining cisgenic or intragenic potato plants with new valuable traits that do not carry marker genes.

Среди разновидностей твердой пшеницы, полба (Triticum dicoccum) занимает особое место как ценнейшая крупяная культура, потребность в которой растет в России с каждым годом. Приоритетный интерес наших исследований - применение современных биотехнологических методов, а именно геномного редактирования, для повышения качества зерна пшеницы.

12-Oxophytodienoate reductase is the enzyme involved in the biosynthesis of phytohormone jasmonates, which are considered to be the major regulators of plant tolerance to biotic challenges, especially necrotrophic pathogens. However, we observe compromised tolerance to the necrotrophic fungal pathogen Botrytis cinerea in transgenic hexaploid bread wheat and tetraploid emmer wheat plants overexpressing 12-OXOPHYTODIENOATE REDUCTASE-3 gene from Arabidopsis thaliana, while in Arabidopsis plants themselves, endogenously produced and exogenously applied jasmonates exert a strong protective effect against B. cinerea. Exogenous application of methyl jasmonate on hexaploid and tetraploid wheat leaves suppresses tolerance to B. cinerea and induces the formation of chlorotic damages. Exogenous treatment with methyl jasmonate in concentrations of 100 µM and higher causes leaf yellowing even in the absence of the pathogen, in agreement with findings on the role of jasmonates in the regulation of leaf senescence. Thereby, the present study demonstrates the negative role of the jasmonate system in hexaploid and tetraploid wheat tolerance to B. cinerea and reveals previously unknown jasmonate-mediated responses.

In light of recent climate change, with its rising temperatures and precipitation changes, we are facing the need to increase the valuable crop's tolerance against unfavorable environmental conditions. Emmer wheat is a cereal crop with high nutritional value. We investigated the possibility of improving the stress tolerance of emmer wheat by activating the synthesis of the stress hormone jasmonate by overexpressing two genes of the jasmonate biosynthetic pathway from Arabidopsis thaliana, ALLENE OXIDE SYNTHASE (AtAOS) and OXOPHYTODIENOATE REDUCTASE 3 (AtOPR3). Analyses of jasmonates in intact and mechanically wounded leaves of non-transgenic and transgenic plants showed that the overexpression of each of the two genes resulted in increased wounding-induced levels of jasmonic acid and jasmonate-isoleucine. Against all expectations, the overexpression of AtAOS, encoding a chloroplast-localized enzyme, does not lead to an increased level of the chloroplast-formed 12-oxo-phytodienoic acid (OPDA), suggesting an effective conversion of OPDA to downstream products in wounded emmer wheat leaves. Transgenic plants overexpressing AtAOS or AtOPR3 with increased jasmonate levels show a similar phenotype, manifested by shortening of the first and second leaves and elongation of the fourth leaf, as well as increased tolerance to osmotic stress induced by the presence of the polyethylene glycol (PEG) 6000.

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.

The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. Development of strategies enabled the generation of marker-free transgenic plants is a possible alternative to deal with public concerns over the safety of biotech crops. Leveraging the high in vitro regeneration and transformation capabilities of potato, this study examined the factors associated with the development of transgenic plants without presence of selective substances in medium. To compare the antibiotic-assisted with the antibiotic-free transformation, nodal explants from five highly regenerative potato cultivars were inoculated with the Agrobacterium strain AGL0 carrying the pBIN-mGFP5-ER (35SGFP/nosNPTII) plasmid. The efficiency of transient expression and the formation of chimeric and completely transgenic shoots were assessed through vital GFP observations. Under non selective conditions, the regeneration efficiency of stable non-chimeric transgenic shoots varied from 0.9 (‘Chicago’) to 2.7% (#12-36-42). In order to streamline transformation protocol, a specific timeline for collecting transgenic plantlets was established for each analyzed cultivar. This timeline strategy was implemented to generate transgenic potato plants, which demonstrated the suppression of homologous SteIF4Es family genes as a result of the successful introduction of the marker-free hpRNAi construct.

In modern horticulture Plum pox virus (PPV) imposes serious threats to commercial plantations of a wide range of fruit species belonging to genera Prunus. Given the lack of natural genetic resources, which display reliable resistance to PPV infection, there has been considerable interest in using genetic engineering methods for targeted genome modification of stone fruit trees to control Sharka disease caused by PPV. Among the many virus defense mechanisms, RNA interference is shown to be the most promising transgenic disease-control strategy in plant biotechnology. The present study describes the production of transgenic PPV resistant European plum `Startovaya` (P. domestica L.) through the Agrobacterium-mediated transformation of in vitro leaf explants. Due to organogenesis from leaves, the established protocol allows the genetic engineering of the plum genome without losing clonal fidelity of original cultivar. Seven independent transgenic plum lines containing the self-complementary fragments of PPV-CP gene sequence separated by a PDK intron were generated using hpt as a selective gene and uidA as a reporter gene. The transformation was verified through the histochemical staining for β-glucuronidase activity, PCR amplification of appropriate vector products from isolated genomic DNA and Southern blot analysis of hairpin PPV-CP gene fragments. To clarify the virus resistance, plum buds infected by PPV-M strain were grafted onto 1-year-old transgenic plants, which further were grown into mature trees in the greenhouse. As evaluated by RT-PCR, DAS-ELISA, Western blot, Immuno Strip test, and visual observations, GM plum trees remained uninfected over 9 years. Infected branches that developed from grafted buds displayed obvious symptoms of Sharka disease over the years and maintained the high level of virus accumulation, whereby host transgenic trees had been constantly challenged with the pathogen. Since the virus was unable to spread to transgenic tissues, the stable expression of PPV-derived gene

We developed an efficient system for agro-bacterial transformation of plum ( Prunus domestica L.) leaf explants using the PMI/mannose and GFP selection system. Th e variety `Startovaya` was transformed using Agrobacterium tumefaciens strain CBE21 carrying the vector pNOV35SGFP. Leaf explants were placed onto a nutrient medium containing various concentrations and combinations of mannose and sucrose to develop an efficient selection system. Nine independent transgenic lines of plum plants were obtained on a regeneration medium containing 20 g/L sucrose and 15 g/L mannose. The highest transformation frequency (1.40 %) was produced using a delayed selection strategy. Starting from the 1st days after transformation and ending by regeneration of shoots from the transgenic callus, selection of transgenic cells was monitored by GFP fluorescence that allowed avoid ing formation of escapes. Integration of the manA and gfp transgenes was confi rmed by PCR and Southern blotting. On the whole, no direct correlation between the fluorescence level and the copy numbers of the transgenes was found in the present study, though the most intensive fluorescence was observed in line #9 with a single-copy insert. The difference of GFP expression level may have been caused by the integration site or by other factors such as DNA methylation and varying copy number. The described transformation protocol using a positive PMI/mannose system is an alternative selection system for production of transgenic plum plants without genes of antibiotic and herbicide resistance, and the use of leaf explants enables retention of variety traits of plum plants.

In cereals, the vernalization-related gene network plays an important role in regulating the transition from the vegetative to the reproductive phase to ensure optimal reproduction in a temperate climate. In hexaploid bread wheat (Triticum aestivum L.), the spring growth habit is associated with the presence of at least one dominant locus of VERNALIZATION 1 gene (VRN-1), which usually differs from recessive alleles due to mutations in the regulatory sequences of the promoter or/and the first intron. VRN-1 gene is a key regulator of floral initiation; various combinations of dominant and recessive alleles, especially VRN-A1 homeologs, determine the differences in the timing of wheat heading/flowering. In the present study, we attempt to expand the types of VRN-A1 alleles using CRISPR/Cas9 targeted modification of the promoter sequence. Several mono- and biallelic changes were achieved within the 125-117 bp upstream sequence of the start codon of the recessive vrn-A1 gene in plants of semi-winter cv. ‘Chinese Spring’. New mutations stably inherited in subsequent progenies and transgene-free homozygous plants carrying novel VRN-A1 variants were generated. Minor changes in the promoter sequence, such as 1–4 nucleotide insertions/deletions, had no effect on the heading time of plants, whereas the CRISPR/Cas9-mediated 8 bp deletion between −125 and −117 bp of the vrn-A1 promoter shortened the time of head emergence by up to 2-3 days. Such a growth habit was consistently observed in homozygous mutant plants under nonvernalized cultivation using different long day regimes (16, 18, or 22 h), whereas the cold treatment (from two weeks and more) completely leveled the effect of the 8 bp deletion. Importantly, comparison with wild-type plants showed that the implemented alteration has no negative effects on main yield characteristics. Our results demonstrate the potential to manipulate the heading time of wheat through targeted editing of the VRN-A1 gene promoter sequence on an otherwise unchanged genetic background.

Triticum urartu Thum. ex Gandil. is a wild diploid wheat species (2n = 2x = 14) that is an Au genome donor of modern polyploid cultivars of durum and bread wheat. In the last decade, this relict species has attracted breeders as donors of various agronomically important characteristics to broaden the genetic diversity of cultivated wheat. In addition, T. urartu can be considered as a model species for studying the evolution, biology and genomics of wheat without the cross-influence of homologous sub-genomes. Various genetic engineering technologies, including transgenesis and genome editing, may be applied to facilitate the functional characterization of genes located in A chromosomes. Such biotechnological techniques are still required for the efficient tissue culture systems to allow easy plant regeneration. The objective of our study was to assess the abilities of in vitro plant regeneration from zygotic immature embryo-derived tissues of spring and winter types of T. urartu. Three synthetic auxins, 2,4-D, Dicamba and Picloram, at four concentrations were studied to stimulate morphogenic responses in spring T. urartu. The induction medium supplemented with 4 mg·L−1 Dicamba stimulated the highest frequency of regenerable callus production (65.8%), promoting the generation of 5.7 plants. Although the presence of 2 mg·L−1 2,4-D was less effective in stimulating regenerable callus formation (53.2%) than Dicamba, it allowed the regeneration of more plants from one regenerable callus (9.3 plants). These two treatments also successfully initiated morphogenesis in winter assertions; however, their regenerative capacity was generally lower. The frequency of regenerable callus production was accession-dependent and fluctuated within 31.3 to 49.2%, with a formation of an average 2.2–5.8 plants per callus. The relatively simple and fast regeneration system described in this study could be further used as the basis for regenerating transgenic plants of T. urartu.

In stone fruit trees, resistance to Plum pox virus (PPV) can be achieved through the specific degradation of viral RNA by the mechanism of RNA interference (RNAi). Transgenic virus-resistant plants, however, raise serious biosafety concerns due to the insertion and expression of hairpin constructs that usually contain various selective foreign genes. Since a mature stone tree represents a combination of scion and rootstock, grafting commercial varieties onto transgenic virus-tolerant rootstocks is a possible approach to mitigate biosafety problems. The present study was aimed at answering the following question: To what extent are molecular RNAi silencing signals transmitted across graft junctions in transgrafted plum trees and how much does it affect PPV resistance in genetically modified (GM)/non-transgenic (NT) counterparts? Two combinations, NT:GM and GM:NT (scion:rootstock), were studied, with an emphasis on the first transgrafting scenario. Viral inoculation was carried out on either the scion or the rootstock. The interspecific rootstock `Elita` [(Prunus pumila L..P. salicina Lindl.)x(P. cerasifera Ehrh.)] was combined with cv. Startovaya (Prunus domestica L.) as a scion. Transgenic plum lines of both cultivars were transformed with a PPV-coat protein (CP)-derived intron-separate hairpin-RNA construct and displayed substantial viral resistance. High-throughput sequence data of small RNA (sRNA) pools indicated that the accumulation of construct-specific small interfering RNA (siRNA) in transgenic plum rootstock reached over 2 %. The elevated siRNA level enabled the resistance to PPV and blocked the movement of the virus through the GM tissues into the NT partner when the transgenic tissues were inoculated. At the same time, the mobile siRNA signal was not moved from the GM rootstock to the target NT tissue to a level sufficient to trigger silencing of PPV transcripts and provide reliable viral resistance. Th e lack of mobility of transgenederived siRNA molecules was accompanied by the transfer of various endogenous rootstock-specific siRNAs into the NT scion, indicating the exceptional transitivity failure of the studied RNAi signal. The results presented here indicate that transgrafting in woody fruit trees remains an unpredictable practice and needs further in-depth examination to deliver molecular silencing signals.

ISHS VIII International Scientific and Practical Conference on Biotechnology as an Instrument for Plant Biodiversity Conservation (Physiological, Biochemical, Embryological, Genetic and Legal Aspects) Genetic modification of jasmonate biosynthesis pathway in wheat alters plant tolerance to necrotrophic fungi

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

A well-developed root system is an important characteristic of crop plants, which largely determines their productivity, especially under conditions of water and nutrients deficiency. Being Poaceous, wheat has more than one seminal root. The number of grown seminal roots varies in different wheat accessions and is regulated by environmental factors. Currently, the molecular mechanisms determining the number of germinated seminal roots remain poorly understood. The analysis of the root system development in germinating seeds of genetically modified hexaploid wheat plants with altered activity of jasmonate biosynthesis pathway and seeds exogenously treated with methyl jasmonate revealed the role of jasmonates in the regulation of sixth seminal root development. This regulatory effect strongly depends on the jasmonate concentration and the duration of the exposure to this hormone. The maximum stimulatory effect of exogenously applied methyl jasmonate on the formation of the sixth seminal root was achieved at 200 μM concentration after 48 h of treatment. Further increase in concentration and exposure time does not increase the stimulating effect. While 95% of non-transgenic plants under non-stress conditions possess five or fewer seminal roots, the number of plants with developed sixth seminal root reaches up to 100% when selected transgenic lines are treated with methyl jasmonate.