Prospects for Knockout of MYB60, a Transcriptional Repressor of Anthocyanin Biosynthesis, in Brassicaceae Plants by Genome Editing

The Brassicaceae plant family contains many economically important crops such as Brassica napus L., Brassica rapa L., Brassica oleracea L., Brassica juncea L., Eruca sativa Mill., Camelina sativa L. and Raphanus sativus L. Insufficient data on the genetic regulation of agronomic traits in these species complicates the editing of their genomes. In recent years, the attention of the academic community has been drawn to anthocyanin hyperaccumulation. This trait is not only beneficial for human health, but can also increase plant resistance to stress. MYB transcription factors are the main regulators of flavonoid biosynthesis in plants. Some of them are well studied in Arabidopsis thaliana. The AtMYB60 gene is a transcriptional repressor of anthocyanin biosynthesis, and it also negatively impacts plant responses to drought stress. Myb60 is one of the least studied transcription factors with similar functions in Brassicaceae. There is a high degree of homology between predicted MYB60 genes of A. thaliana and related plant species. However, functions of these homologous genes have never been studied. Gene knockout by CRISPR/Cas technology remains the easiest way to perform genome editing in order to discover the role of individual plant genes. Disruption of genes acting as negative regulators of anthocyanin biosynthesis could result in color staining of plant tissues and an increase in stress tolerance. In the present study, we investigated the AtMYB60 gene and its homologs in Brassicaceae plants and suggested universal gRNAs to knockout these genes. Keywords: CRISPR, Brassicaceae, MYB60, knockout, anthocyani

Quantitative Determination of Inulin Content in Hairy Roots of Common Chicory Cichorium intybus L.

Cichorium intybus L. – многолетнее травянистое растение, представляющее интерес в качестве источника инулина. Инулин – полисахарид, состоящий из остатков фруктозы в форме фуранозы, являющийся перспективным компонентом функционального питания. Производство инулина традиционным способом сопряжено с рядом трудностей, таких как необходимость тщательной очистки корней и зависимость от климатических условий, но их можно преодолеть с помощью биотехнологии. Поскольку в России на данный момент промышленное биотехнологическое производство инулина отсутствует, представляет интерес получение культур волосовидных корней C. intybus, способных продуцировать инулин, что в перспективе может привести к созданию в России биотехнологического производства. Целью данного исследования было получение волосовидных корней C. intybus и анализ количественного содержания в них инулина. Двенадцать линий волосовидных корней диких растений C. intybus и сорта Кофеек были получены путем агробактериальной трансформации штаммом A4 Agrobacterium rhizogenes с бинарным вектором pCambia 1301 листовых эксплантов растений, культивированных в условиях in vitro. Содержание инулина определяли спектрофотометрическим методом. Содержание инулина в волосовидных корнях от диких растений C. intybus и сорта Кофеек в среднем составило 1,46±0,46 % и 1,34±0,34 % от сырой массы соответственно, в то время как в нативных корнях диких растений C. intybus содержалось 13,13±1,9 % инулина, а сорта Кофеек – 11,55±2,32 %. Полученные результаты свидетельствуют о том, что содержание инулина в волосовидных корнях существенно ниже, чем в нативных. Получение инулина из волосовидных корней в условиях промышленного культивирования в биореакторах может иметь ряд преимуществ, таких как независимость от природных условий, отсутствие необходимости предварительной очистки корней, а также возможность круглогодичного культивирования, что в перспективе может частично компенсировать низкое содержание инулина. Однако требуется проведение дальнейших исследований, направленных на повышение содержания инулина в культуре волосовидных корней цикорияChicory (Cichorium intybus L.) is a perennial herbaceous plant, a promising source of inulin in functional nutrition. Inulin is a polysaccharide consisting of fructofuranose residues. Its traditional production is associated with a number of difficulties, such as the need for thorough cleaning of the roots and dependence on climatic conditions, but these can be overcome by means of biotechnology. As biotechnological production of inulin is currently non-existent in Russia, obtaining cultures of chicory hairy roots capable of producing inulin is of paramount value. It can provide an opportunity for launching biotechnological production of inulin in Russia in the future. In this study, twelve lines of hairy root culture of C. intybus ‘Kofeek’ and wild type plants were obtained and the content of inulin in them was quantified. For this purpose, leaf explants obtained from in vitro plants were subjected to transformation with the A4 strain Agrobacterium rhizogenes containing the binary vector pCambia 1301. The content of inulin was determined by the spectrophotometric method. The inulin content in hairy root culture of wild type chicory plants and chicory ‘Kofeek’ averaged 1.46±0.46 % and 1.34±0.34 %, respectively, while the native roots of wild chicory contained 13.13±1.9 % inulin, and ‘Kofeek’ 11.55±2.32 %. The results indicate that the content of inulin in hairy roots is significantly lower than in native ones. Obtaining inulin from hairy roots under conditions of industrial cultivation in bioreactors may have a number of advantages, such as independence of weather conditions, no need for preliminary cleaning of the roots, and the possibility of year-round cultivation, which may partially compensate for the low content of inulin. However, further research aimed at increasing inulin content in hairy root culture is of immense importanc

Limitation of Cytokinin Export to the Shoots by Nucleoside Transporter ENT3 and Its Linkage with Root Elongation in Arabidopsis

The trans-membrane carrier AtENT3 is known to transport externally supplied cytokinin ribosides and thus promote uptake by cells. However, its role in distributing either exogenous or endogenous cytokinins within the intact plant has not hitherto been reported. To test this, we used ent3-1 mutant Arabidopsis seedlings in which the gene is not expressed due to a T-DNA insertion, and examined the effect on the concentration and distribution of either endogenous cytokinins or exogenous trans-zeatin riboside applied to the roots. In the mutant, accumulation of endogenous cytokinins in the roots was reduced and capacity to deliver externally supplied trans-zeatin riboside to the shoots was increased suggesting involvement of equilibrative nucleoside (ENT) transporter in the control of cytokinin distribution in the plants. Roots of ent3-1 were longer in the mutant in association with their lower cytokinin concentration. We concluded that the ENT3 transporter participates in partitioning endogenous cytokinins between the apoplast and the symplast by facilitating their uptake by root cells thereby limiting cytokinin export to the shoots through the xylem. Dilution of the mineral nutrient solution lowered endogenous cytokinin concentration in the roots of both wild type (WT) and ent3-1 plants accompanied by promotion of root elongation. Nevertheless, cytokinin content was lower, while roots were longer in the ent3-1 mutant than in the WT under either normal or deficient mineral nutrition suggesting a significant role of ENT3 transporter in the control of cytokinin level in the roots and the rate of their elongation

Evaluation of genetic polymorphism and drought and salinity tolerance of Amaranthus cruentus L. mutants

Background. Drought and salinization are serious environmental problems in agriculture. Therefore, it is important to grow cultivated plants resistant to abiotic environmental factors in the fields. Due to its high nutritional value and adaptation to diverse environmental conditions, amaranth is considered a promising crop for cultivation, including in arid zones. In connection with modern climate change, the urgency of breeding more stressresistant amaranth varieties is increasing.The purpose of the study is to assess the genetic diversity and stress resistance of mutant forms of red amaranth Amaranthus cruentus L. obtained using sodium azide. Materials and methods. Using microsatellite markers at three SSR loci, the molecular genetic polymorphism of seven mutant forms of amaranth generation M3 was evaluated. According to SSR markers GB-AM-132 and GB-AM-137, mutant plants did not differ from each other and from the wild type. Using the SSR marker GBAM-099, three alleles with three combinations of them were identified among the analyzed mutant plants. Results. According to the results of morphophysiological analysis under conditions of abiotic stress, one of the mutant lines during drought had the best indicator of the relative water content, was characterized by the highest results in terms of stem height and biomass, and in these mutants an increase in the activity of ascorbate peroxidases and glutathione-S-transferases was recorded, compared with wild type. The results obtained indicate a high drought resistance of this mutant line. Another mutant line showed high salt tolerance. Thus, under saline conditions, these mutants were characterized by an increased stem height, an increase in the total antioxidant capacity, as well as an increase in the activities of ascorbate peroxidases and glutathione-S-transferases compared to the wild type. Conclusions. Thus, as a result of our study, mutant forms of amaranth were identified that are resistant to lack of water and excessive salt content in the soil. These forms of amaranth can be used as a material for breeding of stress-resistant varieties