Способы получения стерильных клонов тополя и осины (Populus ssp.) с использованием генной инженерии последовательность генов идентичности цветковой меристемы у тополя -гена LEAFY (LFY) и двух ортологов AGAMOUS (AG) [42].Для модельного вида генетики древесных P. trichocarpa было установлено удвоение последовательности всего генома, которое, вероятно, произошло примерно 60-65 млн лет назад.В результате почти 8 тысяч генов P. trichocarpa представлены в геноме двумя копиями (паралогами) [39].Таким образом, ген AGAMOUS (AG) также представлен в геноме двумя паралогичными последовательностями, в то время как LEAFY -уникален.Elorriaga с соавт.[14] сконструировали четыре направляющих (или гидовых) РНК (single guide RNA, sgRNA), чтобы оценить возможности системы CRISPR/Cas9 вносить целевые изменения (мутации) в последовательности генов LFY и AG.Авторы сконструировали шесть плазмид для доставки конструкций в клетки объекта.В результате экспериментов им удалось получить сотни трансгенных событий, в результате которых изменились последовательности генов-мишеней.Таким образом, была установлена высокая эффективность использования CRISPR/ Cas9 для нейтрализации генов цветения у тополей и получения стерильных клонов.Эта публикация содержит ценные методические рекомендации, однако в данном исследовании доставка C R I S P R / C a s о с у щ е с т в л я л а с ь п у т е м Agrobacterium-опосредованной генетической трансформации, что переводит полученные отредактированные растения в категорию ГМО.Размер генома тополя составляет всего около 550 Mb (млн пар оснований) [39], полногеномная сборка доступна для нескольких видов: P. trichocarpa [39], P. euphratica [23], P. tremula [22] и P. deltoides [5].Геномы разных видов Populus весьма сходны и коллинеарны [9].Поэтому для выявления последовательностей ДНК, подходящих в качестве мишени для редактирования с целью получения стерильных форм, в качестве старта можно использовать последовательность гена PLFY у P. trichocarpa (GenBank accession number U93196, Potri.015G106900),гомологичного гену LEAFY в геноме Arabidopsis, депонированную в БД Phytozome [17].Метод конструирования гидовых РНК для генов-мишеней при использовании системы CRISPR/Cas9 Дезоксирибонуклеаза Cas9 (CRISPR associated protein 9) -компонент иммунитета бактерий, который обеспечивает их защиту от чужеродной ДНК вирусов и плазмид.Уникальная особенность этого фермента заключается в том, что его специфичность можно программировать с помощью особой crРНК (CRISPR РНК).При этом для каталитической активности нуклеазы Cas9 нужна еще одна РНК -tracrРНК (trans-activating CRISPR РНК), которая частично комплементарна crРНК.Рибонуклеопротеиновый комплекс Cas9/ crРНК/tracrРНК способен распознавать участки ДНК, идентичные 20 нуклеотидам на 5'-конце crРНК.Эти нуклеотиды и составляют целевую последовательность редактируемого гена (или спейсер).При этом важно, чтобы к 5'-концу этой 20-нуклеотидной последовательности (спейсера) непосредственно примыкал так называемый PAM-мотив, распознаваемый Cas нуклеазой (например, CCN, в случае нуклеазы Cas9 бактерии Streptococcus pyogenes).Распознав PAM-мотив, белок Cas9 связывается с ним, а затем расщепляет обе цепи ДНК между 3 и 4 нуклеотидом (если считать в направлении 3'-5' от PAM-мотива).Направляющая РНК состоит из двух компонентов.Первый -консервативный компонентвключает последовательность crРНК (CRISPR РНК), подобную повторяющимся палиндромам в бактериальной клетке, переходящую в последовательность тракрРНК (tracrRNA) -транс-активирующую РНК, необходимую для присоединения Cas9.В современных конструкциях последовательности crРНК и тракрРНК объединены в единую последовательность -sgRNA скаффолд (single guide RNA scaffold).Вторая «часть» направляющей РНК -вариабельная, называемая спейсером, синтезируется комплементарно выбранному участку редактируемого гена и состоит из 17-20 нуклеотидов.Для дизайна спейсеров существует не

В статье представлен обзор литературных данных по вопросу молекулярно-генетических механизмов устойчивости березы и дуба к дефициту воды и азота. Анализ транскриптомной активности генов, реагирующих на абиотические стрессы дает возможность осуществления целенаправленной селекции генетически улучшенных форм для нужд эффективного лесовосстановления, а также создания лесных плантаций. Молекулярно-генетический ответ на засуху более изучен для древесных растений, чем дефицит азота. По сравнению с Betula sp., ранний ответ Quercus sp. на засуху менее интенсивный и выражается в подавлении метаболической активности и активации систем поддержания роста после стресса. При длительном воздействии засухи наблюдается интенсивное восстановление клеточной стенки, связанное с биосинтезом целлюлозы и лигнина. Наблюдаются активные процессы нейтрализации активных форм кислорода и биосинтез антиоксидантов. Во время раннего ответа Betula sp. на засуху наблюдается высокий уровень экспрессии генов, связанных со снижением потерь воды. Защита от засухи также включает контроль закрытия устьиц и активацию генов, ассоциированных с ответом на осмотический стресс. Среди генов-кандидатов ответа на засуху у видов дуба были идентифицированы транскрипционные факторы, белки теплового шока, белки-транспортеры и др. По результатам аналитического обзора литературы составлен список дифференциально экспрессируемых генов (ДЭГ), идентифицированных у березы и дуба в ответ на засуху. Если молекулярно-генетические механизмы засухоустойчивости древесных растений изучены в достаточной степени, определено множество генов-кандидатов, участвующих в реакции на стресс, и могут быть в дальнейшем рекомендованы для целенаправленной селекции, то вопрос адаптации древесных растений к дефициту азота слабо изучен и представляется весьма актуальным для дальнейших исследований. The article presents a review of the literature about molecular and genetic mechanisms of birch and oak resistance to water and nitrogen deficiency. Analysis of the transcriptome activity of genes that respond to abiotic stresses makes it possible to carry out targeted selection for the purpose of effective reforestation, as well as the creation of forest plantations. The molecular genetic response to drought has been more studied in woody plants than nitrogen deficiency. Compared to Betula sp., the early response of Quercus sp. to drought is less intense and is expressed in the suppression of metabolic activity and activation of growth maintenance systems after stress. With prolonged exposure to drought, intensive сell wall remodeling is observed, associated with the biosynthesis of cellulose and lignin. Active processes of neutralization of reactive oxygen species and biosynthesis of antioxidants are observed. During the early response of Betula sp. drought, a high level of expression of genes associated with a decrease in water loss is observed. Drought protection also includes control of stomatal closure and activation of genes associated with the response to osmotic stress. Among candidate genes for response to drought in oak species, transcription factors, heat shock proteins, transporter proteins, etc. were identified. Based on the results of an analytical review of the literature, a list of differentially expressed genes (DEGs) identified in birch and oak in response to drought was compiled. If the molecular genetic mechanisms of drought resistance in woody plants have been studied to a sufficient extent, many candidate genes involved in the stress response have been identified and can be further recommended for targeted breeding, then the issue of adaptation of woody plants to nitrogen deficiency is poorly studied and seems to be very relevant for further research.

Lignin is the second most common terrestrial biopolymer. It provides mechanical strength to plants, confers waterproof properties to the vascular system, and plays an important role in protection against biotic and abiotic stresses. The chemical resistance of lignin impedes the conversion of plant biomass into cellulose and biofuels; this circumstance led to intense research on lignin biosynthesis. For a long time, it was believed that lignin consists almost exclusively of three monolignols. However, about thirty more minor monomers of diverse chemical nature have been discovered to date. Using genetic engineering methods, a number of transgenic trees with altered expression of lignin biosynthesis genes and the transcription factor genes that regulate this process have been obtained. Changes in the content and/or composition of lignin allowed researchers to significantly raise the efficiency of delignification and enzymatic hydrolysis of woody biomass, but these changes often led to retarded growth and distorted plant development. In search of a balance between the industrial needs and plant viability, new strategies have been proposed that are based on the inclusion of new monolignols in lignin as well as on the use of lignin-deficient natural tree forms. New physicochemical properties of lignin are expected to increase its extractability. At the same time, growth, development, and stress resistance of such transgenic plants should be studied under field conditions. The review presents the current state of research on properties and modification of lignin in woody plants. In addition, the relations between these modifications and plant viability, as well as the prospects for their commercial use, are discussed.

The plant phenotyping is an integral part of both fundamental and applied research.In recent years, automated image analysis has been actively used, but mainly for herbaceous plants.Since 2010 we are studying the phenotype of transgenic aspen, birch and pear plants using imaging technology.The effects of the genotype and environmental conditions on size and shape of leaves and fruits of transgenic trees were investigated.

Changing expression of a single gene by introducing mutations or transformation often affects expression of other genes, which results in the modification of the plant phenotype. We have obtained aspen plants with reduced expression levels of 4CL gene (4-coumarate-CoA ligase). Change in qualitative composition and 11-23% reduced lignin content were observed in the wood of the greenhouse plants. In the study of rhizogenesis in vitro it was found that the micro shoots of transgenic plants formed a greater number of adventive roots than in the control, 30%. Studying the transgenic aspen plants grown in a greenhouse (3 months) and passed the semi-natural conditions trial (4 months) has revealed 5-10% reduction in the lignin content, alteration of the phenotype – decrease in biometrical values (height and diameter of stem). Analysis of RNA from the plants grown under semi-natural conditions indicated changed expression levels of monolignol biosynthesis genes, 4CL, CCR1 (cinnamoyl-CoA reductase), CAD6 (cinnamyl-alcohol dehydrogenase), and CCoAOMT (caffeoyl-CoA O-methyltransferase). For the lines PtXVI4CL9a and PtXIII4CL2c grown under semi-natural conditions, weight of the root system was 1.5-2 fold reduced in the transgenic plants as compared to the control. The conductive: feeding root ratio was increased. Morphology of the root system was changed.

The effects of transformation of downy birch (Betula pubescens Ehrh.) with the GS1 gene encoding the cytosolic form of glutamine synthetase on the rooting of plants in vitro was studied. The transgenic plants had an elevated content of glutamine as well as glutamic and aspartic acids and rooted more rapidly than the control plants. Rooting on a medium containing the glutamine synthetase inhibitor phosphinothricin prevented the accumulation of auxin in birch plants carrying the GS1 gene, indicating the involvement of this enzyme in raising the level of auxins in the transgenic plants. The correlation between the increase in the auxin levels in the transgenic plants carrying the glutamine synthetase gene and the increase in the rooting rate is shown for the first time.

Aspen Populus tremula L. (Salicaceae) is the fast-growing tree species of environmental and economic value. Aspen is capable of reproduction by both seeds and vegetative means, forming root sprouts. In an adult stand, identification of ramets of one clone among the trees of seed origin based on their morphology is difficult. A panel of 14 microsatellite loci developed for individual identification of aspen was applied for the clonal structure analysis in four natural aspen stands of the European part of Russia: Moscow and Voronezh oblasts, the Mari-El Republic, and the Republic of Tatarstan. In 52 trees from the Moscow sample, 41 multilocus genotypes were identified; in the Voronezh sample, among 30 individuals, 25 different genotypes were detected; and in the sample from Mari-El, 32 trees were represented by 13 genotypes. In the stand from Sabinsky Forestry, Tatarstan, all of the examined 29 trees were represented by a single genotype. The ancestral tree carrier of this genotype which was the most heterozygous (0.929) among all studied aspen individuals (sample mean, 0.598) obviously has spread over a large territory during several cutting and reproduction cycles, currently occupying the area of 2.2 ha. For aspen, usually suffering from Aspen trunk rot, such high viability is evidence of resistance to the main pathogens. The revealed superclone deserves further study with karyological methods and flow cytometry to determine ploidy level and analysis of the growth rate and the quality of wood for possible use in plantation forest production.

Obtaining herbicide resistant plants is an important task in the genetic engineering of forest trees. Transgenic European aspen plants (Populus tremula L.) expressing the bar gene for phosphinothricin resistance have been produced using Agrobacterium tumefaciens-mediated transformation. Successful genetic transformation was confirmed by PCR analysis for thirteen lines derived from two elite genotypes. In 2014-2015, six lines were evaluated for resistance to herbicide treatment under semi-natural conditions. All selected transgenic lines were resistant to the herbicide Basta at doses equivalent to 10 l/ha (twofold normal field dosage) whereas the control plants died at 2.5 l/ha. Foliar NH4-N concentrations in transgenic plants did not change after treatment. Extremely low temperatures in the third ten-day period of October 2014 revealed differences in freeze tolerance between the lines obtained from Pt of f2 aspen genotypes. Stable expression of the bar gene after overwintering outdoors was confirmed by RT-PCR. On the basis of the tests, four transgenic aspen lines were selected. The bar gene could be used for retransformation of transgenic forest trees expressing valuable traits, such as increased productivity.

The properties of transgenic aspen (Populus tremula) clones carrying the recombinant gene of xyloglucanase sp-Xeg from Penicillium canescens have been analyzed. Complex modifications were revealed both in the composition of the wood and in the plant phenotype. Biometric analysis showed that shoot dimensions increased by 24.8%, 25% and 26% in the PtXIV-Xeg1a, PtXVXeg1a and PtXVXeg1b lines, respectively. The number of internodes in some transgenic clones also increased. Modifications in rhizogenesis have been shown for the first time in the plants with the recombinant gene of xyloglucanase: in vitro rooting efficiency exceeded the control value in 13 out of 25 lines. Maximum rooting efficiency was observed in the PtXVXeg1a line (3.2-fold higher than in the control). A reliable increase in the root system mass (by 20% to 52%) under greenhouse conditions was observed for 8 out of 25 clones. A lower pentosan content in the wood was shown for all lines. The data on xyloglucanase activity and pentosan content generally correlated with phenotypic modifications.

Plant xyloglucans play an important role in the processes of cell wall extension, determine their mechanical properties, thus affecting growth and morphology of individual cells and whole organs. Being one of the main components of hemicellulose, xyloglucans play a particular physiological role in woody plants. To study xyloglucan physiological role, transgenic aspen (Populus tremula L.) plants with a recombinant sp-Xeg gene from the fungus Penicillium canescens were produced. Constitutive expression of this gene in the heterologous surrounding was confirmed by RT-PCR method. The analysis of protein extracts from the leaves of greenhouse-grown plants and microshoots grown in vitro showed activation of xylogluconase in transgenic lines. The strongest activation (1.6-fold) was observed in the leaf extracts (clone PtXVXeg1b) and in vitro microshoots (clone PtXVXeg1c). In transgenic plants, the relative content of pentosans in the wood was declined. In control plants (Pt genotype), it was equal to 148 mg/g dry wt, whereas in tested clones (PtXVXeg1a, PtXVXeg1b, and PtXVXeg1c), it varied from 100 to 140 mg/g dry wt. The strongest decrease (by 31%) in the content of pentosans was observed for the line PtXVXeg1c; the content was equal to 102.1 ± 1.5 mg/g dry wt. A comparative analysis of leaf morphology revealed an increase in the length of petiole and a decrease in the length of the main vein in transgenic lines. In control plants, the ratio of the petiole length to the length of the main vein was equal to 0.49, whereas in transgenic plants, it varied from 0.51 to 0.66. A significant increase of this index was observed in 12 from 14 transgenic lines.

Conditions of cultivation and micropropagation of selected biotypes of five willow species ( Salyx dasyclados Wimm., S. caspica Pall., S. triandra L., S. purpurea L., and S. viminalis L.) and two hybrids (× S. acuminata S. and × S. palustris Host.) were optimized. Data on in vitro propagation of S. caspica , S. triandra , S. purpurea together with hybrids S. acuminata and S. palustris were obtained for the first time. It has been demonstrated that the outcome of cultivation and propagation of willows strongly depends on genotypic peculiarities of initial plants. The optimal terms of isolation and sterilization of single-node segments for obtaining 50–75% of aseptic viable developing cultures were estimated. The nutritive media were selected providing induction of stem development (to 67%), their rooting (to 91%), elongation (to 3–6 cm), and multiplication (propagation coefficient of 4). The designed method (adopted to different genotypes) can be applied for obtaining aseptic in vitro cultures serving as initial plant material for genetic transformation and mass propagation of plants with new agriculturally valuable characteristics which are of interest for construction of bioenergetic plantations and for needs of the paper industry.

Transgenic aspen plants containing the glutamine synthetase gene GSI from pine have been produced. Among 37 transformed lines, 34 were found to possess GSI. The RT-PCR analysis of GSI transcripts confirmed the presence of specific transcripts in 32 lines. The phenotypic effect of a glutamine synthetase activity in transgenic plants was evaluated by in vitro cultivation of plants at the presence of a sublethal dose (0.5 mg/l) of phosphinothricin, which inhibits this enzyme. It was shown that the sublethal dose of this herbicide provides a predictable inhibiting effect on the nontransformed aspen plants, including the inhibition of their rhizogenesis, whereas transgenic plants demonstrated various responses. In most transgenic lines, we observed an unexpected stimulating effect of low herbicide doses on in vitro rhizogenesis; this effect was manifested through the increased radication frequency, increased average number of roots per plant, and increased total length of roots.