OPTIMIZATION OF IN VITRO SELECTIVE MEDIA FOR SELECTING ANTHRACNOSIS-RESISTANT FLAX CELLS

Цель исследований – оптимизация селективных сред для проведения отбора in vitro каллусных клеток льна, устойчивых к культуральному фильтрату штаммов возбудителя антракноза и создание in vitro новых генотипов, устойчивых к болезни. В результате исследований уточнен состав культурального фильтрата штаммов антракноза. Выявлено, что токсичность культуральных фильтратов не зависела от вирулентности используемых штаммов – более токсичными оказались культуральные фильтраты штаммов 784 (сильновирулентного) и 780 (средневирулентного) (загнивание и отмирание первичных корешков на 5 сутки наблюдали у 67 – 88% проросших семян), менее токсичны – штаммы 793 (сильновирулентный) и 788 (слабовирулентный) (на 5 сутки загнивание и отмирание первичных корешков отмечено у 9 – 15% проросших семян). Установлено, что морфогенные очаги формировались активнее у генотипов, морфогенный каллус которых переносили на среду с аналогичной или более высокой концентрацией культурального фильтрата. Показано, что на 14 сутки во втором пассаже с большей частотой формировались морфогенные каллусы, почки и побеги при использовании в первом и втором пассажах селективной среды, содержащей культуральный фильтрат в концентрации 40 мл/л, или в первом пассаже – 40 мл/л, а во втором – 44 мл/л. Выделены генотипы, сохраняющие устойчивость к антракнозу в течение трёх поколений на уровне 50 – 60%: НО-78 х Ленок, HJI-103-2 х Ленок, НЛ-40-1 х Ленок, HЭ-38 х Росинка, НЭ-36 х Ленок, НЭ-17 х Ленок, HЭ-16-2 х Росинка. Research objective – optimization of selective media for in vitro selection of flax callus cells resistant to culture filtrate of anthracnose pathogen strains and in vitro creation of new disease-resistant genotypes. As a result of the research, the composition of the culture filtrate of anthracnose strains was clarified. It was revealed that the toxicity of cultural filtrates did not depend on the virulence of the strains used - cultural filtrates of strains 784 (highly virulent) and 780 (medium virulent) turned out to be more toxic (decay and death of primary roots on day 5 was observed in 67 - 88% of germinated seeds), less toxic - strains 793 (strongly virulent) and 788 (weakly virulent) (on the 5th day, decay and death of primary roots was noted in 9-15% of germinated seeds). It was found that morphogenic foci were formed more actively in genotypes, the morphogenic callus of which was transferred to a medium with a similar or higher concentration of the culture filtrate. It was shown that on the 14th day in the second passage, morphogenic callus, buds and shoots were formed with a greater frequency when using in the first and second passages a selective medium containing a culture filtrate at a concentration of 40 ml/l, or in the first passage - 40 ml/l, and in the second - 44 ml/l. Genotypes were identified that retain resistance to anthracnose for three generations at a level of 50 - 60%: NO-78 x Lenok, HJI-103-2 x Lenok, NL-40-1 x Lenok, NE-38 x Rosinka, NE-36 x Lenok, NE-17 x Lenok, NE-16-2 x Rosinka.

In Vitro Regeneration of Miscanthus x giganteus through Indirect Organogenesis: Effect of Explant Type and Growth Regulators

Miscanthus x giganteus is a spontaneous sterile hybrid therefore the creation of useful genetic diversity by conventional breeding methods is restricted. Plant regeneration through indirect organogenesis may be a useful approach to create genetic variability of this important agricultural crop. The present study aimed to evaluate the effect of the explant type and growth regulators on indirect organogenesis of Miscanthus x giganteus and to determine the ploidy level of plant regenerants by flow cytometry. On average, the highest percentage of morphogenic callus tested explants formed in the medium supplemented with 2.5 mg L–1 IBA + 0.1 mg L–1 BAP + 4.0 mg L–1 l-proline. The most intensive secondary differentiation of callus cells was observed in the medium supplemented with 4.0 mg L–1 ZEA + 1.0 mg L–1 NAA. The highest root formation frequency with the highest number of roots was determined in the MS nutrient medium supplemented with 0.4 mg L–1 IBA, where more than 95% of plant regenerants survived and were growing normally.

WHEAT ANDROCLINIC CALLI: DATA OF SCANNING ELECTRONIC MICROSCOPY

The processes of formation different types of calli, as well as the morphogenesis pathways in morphogenic calli, were studied by scanning electron microscopy (SEM) during anther culture in vitro in hybrid line Fotos of spring soft wheat. The microspore haploid origin of calli has been proven. The morphological status of the obtained calli was determined. It was shown that morphogenic callus consists of small densely packed meristematic cells covered with extracellular substance. This type of calli was obtained using a variant of the Potato II induction culture medium, added by 1.0 mg/l synthetic auxin 2,4-D. Nonmorphogenic callus consists of large, elongated, loosely located cells with a smooth surface. This type of calli was obtained using a variant of the Potato II culture medium, added by 2.0 mg/l 2,4-D. It was found that the introduction of various IAA concentrations into the Blaydes nutrient medium for regeneration in morphogenic calli implements the following pathways of morphogenesis in vitro: embryoidogenesis (without IAA addition), gemmorhizogenesis (0.5 mg/l), and rhizogenesis (1.5 mg/l). Revealed degenerative changes in cells of nonmorphogenic calli. The fundamental possibility of regulating of the morphogenesis pathways of in vitro of morphogenic calli in the direction necessary for research in biotechnological research has been confirmed.

Influence of plant growth regulators on morphogenic response, biomass and camptothecin production in the callus cultures of Chonemorpha fragrans (moon) Alston

In vitro morphogenic response of mature seed embryo-derived callus cultures of Chonemorpha fragrans was studied using solid and liquid Murashige and Skoog medium amended with cytokinins or their combinations with naphthalene acetic acid at 0.5 mg L-1. The plant growth regulators (PGRs) combination and concentrations tested could not stimulate organogenesis after three subcultivations of the callus cultures on the same PGRs-amended solid medium and when cultivated in the liquid but, formation of morphogenic callus was observed. Evaluation of biomass and camptothecin production showed that the PGRs influenced biomass and CPT yield of the callus cultures. The alkaloid yield of various explants of 3-4 weeks old axenic seedlings was higher in roots (0.019% CPT) followed by mature seed embryos (0.0053%), cotyledons (0.0039%), hypocotyls (0.0024%) and leaves (0.0017%), and no significant difference was observed in yield of CPT from callus induced from the various explants. Camptothecin yield of morphogenic callus cultures cultivated in liquid medium was lower than that of solid due to extracellular leaching effect of the alkaloid. Amount of synthesized CPT in the callus cultures also varied with PGR type and concentration amended in the cultivation medium, and was association with biomass production. Results of the present study suggest that callus cultures offer alternative tissue source for in vitro CPT yield enhancement through biotechnological approaches, with application in the large-scale production of the alkaloid to conserve the ever-decimated natural population of the medicinal woody climber for CPT.

Aprecierea potențialului morfogenetic și regenerativ al genotipurilor de triticale în cultura in vitro

Triticale is an important cereal crop grown throughout the world. Research showed that the regen-eration of young plants from mature embryos triticale depended on genotypic characteristics. The fre-quency of callusogenesis varied depending on the genotype and was: 188 TR5027 - 80.19%, Ingen 93 standard - 92.02% and Ingen 35 - 98.45%, and the frequency of rhizogenesis compared to embryogenesis proved to be high and constituted on average 57.35%. Only in 34.53% of the morphogenic callus, the de-velopment was of the embryoid type. The average frequency of regeneration was 35.07%. The dispersive analysis of the obtained results shows a significant influence of the genotype in establishing a positive callusogenetic response (P <0.001), the influence power being 76.04%.

Induction of Morphogenic Callus and In Vitro Regeneration of Sugarcane (Saccharum Officinarum L.)

In vitro propagation is a method to produce massive healthy new planting materials quickly. An experiment was carried out for morphogenic callus induction and regeneration of a domestic sugarcane variety. The Explants used was an inert folded leaf and incubated on modified MS medium augmented with 1 mg/l, 2.5 mg/l, and 5 mg/l of 2,4-D for callus induction. The leaf calluses were subcultured on MS medium enriched with different growth regulators for shoot initiation and multiplication. The highest percentage of callus formation was achieved in the medium containing 2.5 mg/l of 2,4-D, while the fastest callus initiation was noticed in MS medium supplemented with 5 mg/l 2,4-D, and maximum proliferation and the morphogenic response of callus were obtained in 3rd subculture. Two types of callus observed on the induction medium were dry nodular friable and smooth compact. This highly morphogenic callus was white to white creamy in color and easy to separate. The highest shoot proliferation rate was found on the medium containing 2 mg/l Kinetin + 1 mg/l IAA and no growth was noticed on the medium containing Kinetin alone. Therefore, the study suggests that the growth hormone of cytokinin in combination with auxin is necessary for in vitro regeneration of sugarcane callus culture.

USAGE OF CULTURAL FILTRATES IN IN VITRO SELECTION FOR ANTHRACNOSE RESISTANCE

The research was carried out on the basis of the laboratory of selection technologies of the Federal State Budgetary Scientific Institution “Federal Scientific Center of Fiber Crops” (Tver region) in 2018–2020. The aim of the research is in vitro development of new flax genotypes resistant to anthracnose, one of the most harmful fungal diseases. As a result of the research, the composition of the cultural filtrate of the anthracnose causative agent was clarified. It was revealed that toxicity of the cultural filtrates did not depend on the virulence of the strains used in the present studies, the cultural filtrates of strains 784 (highly virulent) and 780 (medium virulent) turned out to be more toxic (decay and death of radicle was observed on the 5th day in 67 - 88% of germinated seeds), less toxic are strains 793 (highly virulent) and 788 (weakly virulent) (decay and death of radicle was observed on the 5th day in 9-15% of germinated seeds). It was found that morphogenic foci were formed more actively in genotypes the morphogenic callus of which was transferred to a medium with a higher concentration of the cultural filtrate; it was shown that in the second passage, when transferring morphogenic calli from a selective medium, which contains 40 ml / L of cultural filtrate on a selective medium also containing 40 ml / L of cultural filtrate, as well as on a selective medium containing 44 ml / L of cultural filtrate, the number of formed morphogenic calli and green buds on the 14th day is significantly higher than in case of transferring on a selective medium containing 36 ml / L of cultural filtrate. Viable regenerant plants were obtained and genotypes were isolated, which retained resistance to anthracnose for three generations at a level of 50 - 60%: NO-78 x Lenok, NL-103-2 x Lenok, NL-40-1 x Lenok, NE-38 x Rosinka, NE-36 x Lenok, NE-17 x Lenok, NE-16-2 x Rosinka.

Creation of clary sage cultivar using cell engineering methods. 1. Obtaining of plant-regenerants in callus culture in vitro

To increase the efficiency in agricultural plant breeding, including clary sage – one of the main essential oil crops grown in Russia, it is necessary to use biotechnological methods. One of these techniques is based on the induction of somaclonal variability in the callus tissue culture. To develop it, it is necessary to optimize the conditions for obtaining plant-regenerants in vitro and their analysis. The aim of this work was to study the features of morphogenesis and regeneration of plants from callus cultures to develop cell technologies for creating an initial breeding material based on somaclonal variability in Salvia sclarea L. In the course of the research, we found that the optimal explants for obtaining morphogenic callus, from which shoots were regenerated, were segments of buds and stems with a node (isolated from seedlings in vitro). Cytological analysis of callus cultures revealed two types of morphogenesis – organogenesis (gemmogenesis) and somatic embryogenesis. The features of the morphogenic callus formation of six sage cultivars and samples during the long-term cultivation were studied. The maximum frequency of morphogenesis was noted in the 2nd passage (from 32.4 to 85.2 %, depending on the genotype). Then, to the 8–10th passage, this indicator decreased to 0.0–3.9 %.‘S-785’ and ‘Taigan’ cultivars showed the highest morphogenesis frequency (81.5–85.2 %) and duration of callus regeneration potential (up to the 10th passage). The analysis of callus cultures of six donor plants of ‘S-785’ cultivar helped us to reveal their heterogeneity in morphogenesis induction ability. The maximum frequency of morphogenic callus formation (76.3–91.5 % in the 2nd passage) and the duration of the morphogenic potential preservation (up to the 12th passage) were observed in plants No. 3 and 9, whereas in No. 2, regeneration with a frequency of 3.6–9.7 % was observed only during three passages. Analysis of plants obtained from calli showed their variability in morphology – up to 12.5 % of the samples had deviations compared to the initial cultivar ‘S-785’ in leaf shape, inflorescence structure, flower color, etc. Somaclonal changes in morphological and economically useful traits revealed in regenerants indicate that they are promising for use in sage breeding.

Structural features of explant cells in vivo and morphogenic callus formation in vitro (Review).

Morphogenic callus is an integrated system that is formed in vitro from an explant both exogenously (as a result of proliferation of surface cells of various tissues) and endogenously (in the depth of these tissues), initially consisting of homogeneous meristematic cells, which are gradually transformed into groups of heterogeneous morphogenetically competent cells. Under optimal conditions of further in vitro cultivation the potencies of such cells are realized by various pathways of morphogenesis, including the formation of full-fledged regenerated plants, which is the goal of a number of biotechnologies. The advantage of using morphogenic calli in biotechnological research, in addition to a number of undoubted methodological usability, is the similarity of morphogenetic processes in plants in vivo and in cultured calli in vitro, which should be regarded as the manifestation of the universality of morphogenesis in various plant reproduction systems. The formation of morphogenic calli from explants in in vitro conditions is determined by a complex of interrelated endogenous and exogenous factors. In general, endogenous factors are regarded as the presence in explants in vivo of target cells capable of perceiving the inducer (so-called initial callus cells), while exogenous (usually stressful) factors – as an inducer of the process of callus formation in vitro. The review article uses the example of representatives of various plant families to analyze the literature and own data on the identification and characterization of structural features of initial morphogenic callus cells in explants in vivo. The available literature provides answers to the fundamental questions: what are the initial cells of callus (having the properties of meristematicity, pluri- and totipopotency and, possibly, stemness) and how do they structurally differ from other explant cells; whether the initial cells have the competence to callus formation under in vivo conditions or whether the conditions of preliminary stress conditions in situ and/or the initial stages of in vitro culture induce the acquisition of these cells the ability to reprogramming. The positive role of the positional arrangement of initial callus cells in the explant cell and tissue system is suggested.

USE OF SUCROSE AND MANNITOL FOR DIFFERENTIATION OF FLAX GENOTYPES BY RESISTANCE TO OSMOTIC STRESS

The studies were carried out with the aim of studying the effect of various concentrations of sucrose and mannitol on seeds, immature embryos, and callus cultures of flax to develop a method for obtaining genotypes resistant to osmotic stress. The work was carried out in the Tver region in the laboratory of breeding technologies in 2017–2019. Flax varieties Barbara, Belinka, LM-98, Aurore, Tverskoy, Svetoch, Diplomat, Symfonia were used as objects of research. The seeds were obtained from the National Flax Collection of the Federal Scientific Center for Bast Crops. The effect of sucrose solution on the length of the primary root was detected at concentrations - 0; 8.7; 14.9%. To assess the germination energy of seeds under osmotic stress, the concentration of sucrose was reduced and the range 0 (control) ... 9% was considered. Immature embryos removed from the capsules on the 10th day after pollination were cultivated on MS medium with sucrose, as a selective agent, at a concentration of 5.0 ... 7.0%. Callus tissues were cultured using mannitol as an osmotic at concentrations of 0; 30.0; 36.4; 37.0; 37.4; 38.0 mg/l. Concentrations of 5.0, 6.0 and 7.0% of sucrose can be used as an osmotic differentiator for seeds (10 ... 80% of seeds germinated in the Belinka variety, 80 ... 100% in the Varbara variety, 80 ... 90% in the variety LM-98). Sucrose, as a selective agent, in a culture of immature flax embryos in vitro at a concentration of 5.0 ... 7.0% can be selective only for certain genotypes, for example, the Aurore variety. The selection of resistant callus cells, followed by the formation of adventive buds and shoots in the meristematic foci, can be carried out on media containing 30.0 or 36.4 mg / L of osmosis, which allows obtaining morphogenic callus, buds, shoots in all studied genotypes, as well as in the Aurore variety 1.1 ... 1.2 byp./callus, in the Tverskoy variety - 0.6 ... 0.8, in the Barbara variety - 1.0 ...1.1