Morphogenic Callus Research Articles

Morphogenesis <i>in vitro</i> in calluses of lavender <i>Lavandula angustifolia</i> Mill.: histological aspects

Histological events occurring in the calluses of Lavandula angustifolia Mill. at the initial stages of in vitro culture (1 passage) were described for the first time. It was found that the non-morphogenic callus is mainly represented by parenchymal tissue with few morphogenetic foci, mostly degenerated. In morphogenic calluses the morphogenesis pathways such as de novo organogenesis and indirect in vitro somatic embryogenesis have been identified and multiple developing morphogenetic foci have been noted also. The question of the realization of the pluri- and totipotency properties of callus cells in vitro is discussed. The histological data can be used in choosing the direction of application of regenerants obtained from calluses of this valuable essential oil and medicinal plant in various cell technologies.

H3K4me3 changes occur in cell wall genes during the development of Fagopyrum tataricum morphogenic and non-morphogenic calli.

Epigenetic changes accompany the dynamic changes in the cell wall composition during the development of callus cells. H3K4me3 is responsible for active gene expression and reaction to environmental cues. Chromatin immunoprecipitation (ChIP) is a powerful technique for studying the interplay between epigenetic modifications and the DNA regions of interest. In combination with sequencing, it can provide the genome-wide enrichment of the specific epigenetic mark, providing vital information on its involvement in the plethora of cellular processes. Here, we describe the genome-wide distribution of H3K4me3 in morphogenic and non-morphogenic callus of Fagopyrum tataricum. Levels of H3K4me3 were higher around the transcription start site, in agreement with the role of this mark in transcriptional activation. The global levels of methylation were higher in the non-morphogenic callus, which indicated increased gene activation compared to the morphogenic callus. We also employed ChIP to analyse the changes in the enrichment of this epigenetic mark on the cell wall-related genes in both calli types during the course of the passage. Enrichment of H3K4me3 on cell wall genes was specific for callus type, suggesting that the role of this mark in cell-wall remodelling is complex and involved in many processes related to dedifferentiation and redifferentiation. This intricacy of the cell wall composition was supported by the immunohistochemical analysis of the cell wall epitopes' distribution of pectins and extensins. Together, these data give a novel insight into the involvement of H3K4me3 in the regeneration processes in F. tataricum in vitro callus tissue culture.

Protoplast Isolation, Culture, and Regeneration in Common and Tartary Buckwheat.

Techniques based on the use of plant protoplasts are a convenient model for better understanding and observing developmental changes in the cells. The establishment of research tools based on protoplasts consists of many steps needed for optimization. Here, we describe the culture of morphogenic callus (MC)- and hypocotyl-derived protoplasts of common (Fagopyrum esculentum Moench) and Tartary (F. tataricum (L.) Gaertn.) buckwheat. Protoplasts embedding in agarose matrix and application of plant hormones, including phytosulfokine (PSK), enable the development of protoplast cultures and plant regeneration.

Влияние липополисахаридов грамотрицательных бактерий различных таксономических групп на морфогенетическую активность каллусов мягкой пшеницы

In vitro tissue culture is actively used in modern plant breeding for genetic transformation, effective selection, rapid production and propagation of genetically homogeneous lines. For many crops, the low yield of regenerated plants is limiting for the use of calluses in the breeding process. Some bacterial lipopolysaccharides are able to increase the morphogenetic activity of plant calluses, increasing the efficiency of in vitro technology. In this work, we investigated the effect of lipopolysaccharides (LPS) from the rhizobacterial strains Ensifer adhaerens T1Ks14, Ochrobactrum quorumnocens T1Kr02, and Pseudomonas chlororaphis K3 on the processes of callusogenesis, the formation of morphogenic calluses and regenerant plants of common wheat (Triticum aestivum L.) of a low-morphogenic line with the Rht-B1a dwarfing gene, obtained on the basis of the cultivar Saratovskaya 29. It was found that LPS preparations at a concentration of 10 μg/ml had different effects on wheat tissue cultures. The yield of calluses from explants obtained from immature embryos significantly increased by 2.5% when exposed to LPS of the O. quorumnocens strain T1Kr02 and decreased by 4% in the variant with LPS from P. chlororaphis K3. The yield of morphogenic calluses increased under the influence of LPS from E. adhaerens T1Ks14 (+57%) and P. chlororaphis K3 (+41%). LPS strain O. quorumnocens T1Kr02 had a negative effect on the yield of regenerated plants, while the other two LPS preparations did not differ from the control in this parameter. As a result, the effectiveness of using the LPS preparations from explants to regenerants was significantly higher only in the variant of the E. adhaerens strain T1Ks14 (+64%). The data obtained indicate significant differences in the response of plant tissue cultures to LPS of various rhizosphere bacteria.

Phenolic Determination in Proembryogenic Cell Complexes of Buckwheat Morphogenic Cell Culture with Osmium Tetroxide, Toluidine Blue O Dye, and Iron Chloride.

The study of the localization of secondary metabolites in both plants and the cell cultures on the intravital sections is hampered by the difficulty of obtaining thin, correctly oriented sections. Techniques for fixing tissues in resins allow these difficulties to be overcome. Properly selected tissue fixation techniques allow using different dyes to identify the compound of interest. In addition, some components of tissue fixation can act as fixatives and as a dye for identifying secondary metabolites. For example, osmiumtetroxide, which fixes lipids in tissues, stains phenolic compounds black. This paper describes methods for the detection of phenolic compounds in morphogenic callus culture of buckwheat using osmium tetroxide, ToluidineBlue O dye, and ferric chloride as dyes in epoxy resin-embedded cell culture with double fixation of the material and when material fixed in Karnovsky's fixative.

Advancing gene editing: multiplex mutagenesis in hexaploid triticale

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.

Global epigenetic analysis revealed dynamic fluctuations in levels of DNA methylation and histone modifications in the calli of Fagopyrum with different capacity for morphogenesis

Buckwheat characterises with high susceptibility to in vitro tissue culture conditions, which have been researched extensively to study a plethora of processes. F. tataricum morphogenic callus (MC) is characterised by its capacity for morphogenesis for up to ten years of culture, displaying an extraordinary level of genome stability, and comprises of proembryogenic cell complexes (PECC),which are the structures resembling somatic embryos arrested on the pre-globular stage. The non-morphogenic callus (NC) that appears on the surface of MC after approximately two years of culture due to endoreduplication cycles, is characterised by aneuploidy, rapid growth rate and high level of oxidative stress. F. esculentum embryogenic callus (EC) has different morphological and histological features, remains stable for up to three years of culture, has a dense, globular structure, and is capable of forming embryoids from the masses of embryogenic cells, but does not produce a non-embryogenic clone. In this work, immunocytochemical analyses revealed dynamic epigenetic changes in Fagopyrum calli. We demonstrated that; decreased level of H3K4me2 seems to be associated with pluripotency acquisition in F. esculentum EC and F. tataricum MC; DNA hypomethylation appears to be connected with the acquisition of the embryogenic potential and PECC reinitiation in F. tataricum MC. Moreover, we observed that H4K16ac and H4K5ac exhibited the highest variability during the course of passage in NC. Elevated levels of these modifications on day zero and day six for H4K16ac and H4K5ac, respectively, seem to be connected with endoreplication peaks, the processes which are characteristic of this callus.

ЭМБРИКУЛЬТУРА IN VITRO КАК БИОТЕХНОЛОГИЧЕСКИЙ МЕТОД: ВОЗМОЖНОСТИ И ОГРАНИЧЕНИЯ (НА ПРИМЕРЕ ХЛЕБНЫХ ЗЛАКОВ)

The review article considers the use of the embryo culture in vitro method, which consists in the culture in vitro of zygotic embryos of different ages, in modern biotechnological studies of cereals, using the example of the literature and own data. Special attention is paid to the analysis of immature embryos culture in vitro as more effective explants in comparison with mature embryos, with a possible explanation for this effectiveness. The analysis of the cereal embryogenesis periodization and the identification of the critical stage of the relative autonomy of immature embryos on its basis is given. The method for obtaining regenerants from immature embryos under in vitro and ex vitro conditions through the stage of morphogenic callus formation, as well as the method for directly obtaining regenerants by passing the stage of callus formation, are discussed. Examples of successful application of the method in the elaboration of techniques to increase the adaptation of immature embryos/morphogenic callus/regenerants to the stress factor of drought are given. Both the possibilities and limitations of embryo culture in vitro as a biotechnological method are evaluated. The principle of universality of plant morphogenesis processes in natural and experimental conditions, put forward by T. B. Batygina (2014 and earlier), is confirmed.

The reaction of fiber flax (<i>Linum usitatissimum</i> L.) to the content of aluminum ions <i>in vitro</i> culture

Relevance. The influence of unfavorable environmental factors is one of the reasons for the low realization of the biological capabilities of modern flax varieties in production conditions. Cultivation of flax on soils with strongly acidic (pHKCl 4.5 and below) and neutral (pHKCl over 6.0) reactions leads to a significant decrease in the productivity of fiber and seeds. Biotechnological methods are effective in creating new forms with altered features and properties.Methods. The studies were conducted in vitro. The source material is seeds and hypocotyl segments of flax varieties-dolguntsa Impulse, Phoenix, Leader, S-108, Soyuz. The selective agent in the studies was aluminum salts in the form of AlCl3 at concentrations of 0 (control — H2O dist. / nutrient medium (without selective agent), 44 mg/l, 64 mg/l, 84 mg/l.Results. The toxic effect of AlCl3 on the germination of fiber flax seeds, the formation of primary roots and seedlings — hypocotyls of the varieties used was revealed. The indicator of the value indicating the length of primary roots in germinated fiber flax seeds decreased on the seventh day with an increase in the concentration of the AlCl3 solution. The highest sensitivity to the content of aluminum ions was shown by varieties S-108, Leader and Soyuz. This was expressed in a decrease in the length of the hypocotyl with an increase in the concentration of aluminum ions in the solution. In genotypes in all variants of studies on a selective medium containing AlCl3, a morphogenic callus was formed. The frequency of formation of morphogenic calli was different depending on the concentration of AlCl3 in the cultivation medium. A trend towards a decrease in the number of morphogenic cells with an increase in the concentration of aluminum ions was observed in all varieties. In less sensitive varieties (Impulse and Phoenix), the amount of morphogenic callus formed was higher than in more sensitive ones (C-108, Soyuz, Leader). Under selective conditions, shoots of flax of the studied varieties resistant to AlCl3 were obtained.

Promotive effect of phytosulfokine - peptide growth factor - on protoplast cultures development in Fagopyrum tataricum (L.) Gaertn

BackgroundFagopyrum tataricum (Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of Fagopyrum. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat.ResultsIn this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 106 protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts.ConclusionsThis study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of Fagopyrum tataricum used as source material. These findings suggest that Tartary buckwheat’s protoplast cultures have potential implications for the species’ somatic hybridization and genetic improvement.

FEATURES OF GROWTH AND INULIN CONTENT IN CALLUS CULTURES Cichorium intybus L. in vitro

In vitro callus cultures of common chicory (Cichorium intybus L.) were obtained and their growth and biochemical characteristics depending on the hormonal composition of the MS medium and the spectral composition of light were studied. The study of the effect of light culture on callus tissue formation and inulin accumulation in it was carried out in opaque grow tents with radiation aligned with the flux density of pho- tosynthetic photons and different ratios of its levels in the region of 660 nm (R, red) and 730 nm (FR, far red). The control variant was placed under white linear fluorescent lamps. The resulting cultures were character- ized by high proliferative activity and the capability for morphogenesis. It has been established that the inter- action of two factors—the presence of auxins in the nutrient medium (IAA or NAA at a concentration of 7.5 mg/L in combination with BAP 0.5 mg/L) and cultivation under light culture conditions (FR R, FR = R, FR R)—had a significant impact on the biosynthetic potential of cell cultures. In the obtained cultures, a study of the quantitative content of inulin was carried out. It has been shown that the high content of inulin (7.55–7.95%) in callus cultures was on the MS medium in combination with IAA at FR R illumination. This is probably due to the fact that well proliferating and highly morphogenic callus tissue was formed under these conditions. The obtained results confirm the hypothesis about the specificity of cultured cells to in vitro synthesize and accumulate secondary metabolites in dedifferentiated cells and the dependence of this process on factors of chemical and physical nature.

Indirect regeneration in Ficus lyrata Warb. and metabolite profiles influenced by nitric oxide and Plant growth regulators

BackgroundTo establish an indirect regeneration protocol in Ficus lyrata, a three-phase experiment (callus induction, morphogenic callus induction, and plant regeneration) based on auxin, cytokinin, and nitric oxide interactions was designed and implemented using leaf explants. The metabolite profiles (amino acid profile, total phenolic content, total soluble sugars, and total antioxidant activity) alteration patterns were also investigated to determine the metabolites contributing to the progress of each phase.ResultsResults demonstrated that 11 out of 48 implemented treatments resulted in morphogenic callus induction (morphogenic treatments), and nitric oxide played a key role in increasing efficiency from 13 to 100%. More importantly, nitric oxide cross-talk with cytokinins was necessary for shoot regeneration from morphogenic calli. Only 4 out of all 48 implemented treatments were capable of shoot regeneration (regenerative treatments), and among them, PR42 treatment led to the highest shoot regeneration rate (86%) and maximum mean number of shoot/explant (10.46). Metabolite analyses revealed that the morphogenic and regenerative treatments followed similar metabolite alterations, which were associated with increased biosynthesis of arginine, lysine, methionine, asparagine, glutamine, histidine, threonine, leucine, glycine, serine amino acids, total soluble sugars content, and total antioxidant activity. On the contrary, non-morphogenic and non-regenerative treatments caused the accumulation of a significantly greater total phenolic content and malondialdehyde in the explant cells, which reflexed the stressful condition of the explants.ConclusionsIt could be concluded that the proper interactions of auxin, cytokinins, and nitric oxide could result in metabolite biosynthesis alterations, leading to triggering cell proliferation, morphogenic center formation, and shoot regeneration.

Histological Approach to the Study of Morphogenesis in Callus Cultures In Vitro: A Review

The use of in vitro callus cultures as experimental model systems allows us to get closer to understanding the patterns and features of morphogenesis in intact plants. In this regard, the problem of realizing the morphogenetic potential of callus cells due to their pluri- and totipotency properties is of great interest. To solve this problem, it is important to use the histological approach, which involves studying the structures of developing tissues, organs and organisms in their interactions and relationships. This review article analyzes data devoted to the study of the histological features of formed primary morphogenic calli (formation of morphogenetic centers and superficial meristematic zones), as well as the in vitro morphogenesis pathways in calli that lead to the formation of regenerants (de novo organogenesis and in vitro somatic embryogenesis). The terminology used is considered. Some questions for discussion are raised. The opinion is expressed that histological (structural) studies should be considered as a methodologic basis for further investigation of various morphogenetic scenarios in in vitro callus cultures, especially in economically valuable plants and for biotechnological purposes.

МОРФОГИСТОЛОГИЧЕСКИЕ ОСОБЕННОСТИ РИЗОГЕНЕЗА IN VITRO В ЗАРОДЫШЕВЫХ КАЛЛУСАХ ПШЕНИЦЫ

In modern biotechnological studies of economically valuable plants, numerous developments aimed at obtaining calluses are presented, in which various pathways and types of morphogenesis are induced in vitro. At the same time, such the type of morphogenesis as rhizogenesis in vitro, consisting in the formation and development of roots, but without further regeneration of plants, has not been studied sufficiently. Nevertheless, it is important to identify the tissue mechanisms of formation and development in calluses the roots as rhizogenesis structures. Using the example of calluses obtained from immature wheat embryos, it was revealed that the formation of morphogenetic foci is the basis of rhizogenesis in vitro. Anatomical characteristics of the emerging roots have been revealed, which largely coincide with similar characteristics of the cereal roots under in planta conditions. It is proposed to use morphogenic calluses in vitro as model systems for studying the rhizogenesis of intact plants.

ФОРМИРОВАНИЕ МОРФОГЕНЕТИЧЕСКИХ ОЧАГОВ КАК ОСНОВА ГЕММОРИЗОГЕНЕЗА IN VITRO В ЗАРОДЫШЕВЫХ КАЛЛУСАХ ПШЕНИЦЫ

Biotechnological elaborations for breeding purposes aimed at creating highly productive wheat varieties require a significant number of morphogenic calli, in which the manifestation of various pathways of morphogenesis in vitro are demonstrated. Of great practical interest is the gemmorhizogenesis in vitro, associated with multiple regeneration of full–fledged plants. It is methodically important to identify the dependence of the induction of this morphogenesis pathway in wheat calli in vitro on the duration of their cultivation as well cell/tissue mechanisms of gemmorhizogenic structures formation. On the example of morphogenic calli obtained from immature wheat embryos the process of gemmorhizogenic structures forming is significantly stretched over time, and the formation of morphogenetic zones lays at the basis of in vitro gemmorhizogenesis.

INFLUENCE OF MINERAL COMPOSITION OF SELECTIVE MEDIUM ON FLAX MORPHOGENESIS IN IN VITRO CULTURE

The studies were carried out on the basis of the laboratory of breeding technologies of Federal State Budgetary Scientific Institution "Federal Scientific Center of Bast Crops" (Tver region) in 2016, 2018, 2021. The purpose of the research is to determine appropriate mineral composition of the selective medium for effective in vitro selection for resistance to anthracnose. Various mineral composition media were studied. It was found that it is possible to use nutrient media Gamborg, MS, SH-2, which do not contain vitamins, chelate complex and phytohormones, in order to obtain cultural filtrates of fungus strains - the causative agent of flax anthracnose. The culture filtrates obtained on the basis of these media were highly toxic and it was possible to use them for in vitro selection. It was noted that the callus formed on the basis of hypocotyl segments had a higher ability for morphogenesis compared to the callus formed on the basis of immature embryos. As for selective medium consisting of mineral salts of Gamborg medium and cultural filtrate, the proportion of formed morphogenic callus was the smallest in all genotypes taken in the study and amounted to 0.1–6.8%. Mineral composition of the selective medium and the morphogenetic potential of the genotype influenced the formation of morphogenic callus based on primary callus. Depending on the genotype, the proportion of morphogenic calli formed on Gamborg medium + CF was 0.1 - 2.2%, on MS medium + CF - 3.3 - 8.0%, on Sh-2 medium + CF - 3.4 – 8.1%, respectively. Mineral composition of the selective medium had no significant effect on formation of morphogenic cells based on transplanted callus. The proportion of morphogenic callus was 3.0–5.2% for Lenok variety; 3.0 - 5.4% - Rosinka; 0.1 - 4.3% - Zaryanka; 3.1 - 5.5% - for L 2053-5-11 line; 6.1 - 7.4% - L 957-8-7. The breeding lines L 2053-5-11 and L 957-8-7 had a high morphogenetic potential.

Lipopolysaccharide and flagellin of Azospirillum brasilense Sp7 influence callus morphogenesis and plant regeneration in wheat.

In vitro somatic callus culturing is used widely in plant biotechnology, but its effectiveness depends largely on the donor plant genotype. Bacteria or components of their cells are rarely used to activate morphogenesis. In this work, inoculation of explants from immature wheat (Triticum aestivum L.) embryos with a suspension of living cells of the bacterium Azospirillum brasilense Sp7 resulted in callus death after 7days of growth, in contrast to explant treatment with a suspension of heat-killed whole cells of Sp7. The experiments used two wheat lines, LRht-B1a and LRht-B1c, which differ in morphogenic activity. Growing calluses with the lipopolysaccharide of A. brasilense Sp7 increased the yield of regenerated plants 2- to 3.5-fold in both lines. This increase was through the activation of regenerant formation from morphogenic calluses. We have demonstrated for the first time the effects of bacterial flagellin on plant tissue culture. The polar-flagellum flagellin of A. brasilense Sp7 leveled the genotypic differences in the morphogenic ability of callus tissue. Specifically, it increased the yield of morphogenic calluses in the weakly morphogenic line LRht-B1a to the yield value in the highly morphogenic line LRht-B1c but lowered the yield of regenerants in the highly morphogenic line LRht-B1c to the yield value in the weakly morphogenic line LRht-B1a. Thus, bacterial lipopolysaccharides and flagellins can be used to regulate the formation of morphogenic calluses and regenerants in plant tissue culturing in vitro.

Особенности индуцированного морфогенеза и регенерации растений из соматических тканей люцерны сортов отечественной селекции в культуре in vitro

The authors have studied Morphogenesis and regeneration features of domestic breeding cultivars of alfalfa and conditions for obtaining regenerant plants from the morphogenic callus tissues. As a result of the study, the efficient sterilization protocol of alfalfa seeds was established with the use of sterilizing agents as 70% ethanol and 10% sodium hypochlorite which resulted in the production of 100% aseptically viable seedlings for the further in vitro studies. Callus initiation from cotyledons and hypocotyls was implemented on a Gamborg B5 nutrient medium containing 2,4-dichlorophenoacetic acid (2,4-D), kinetin in a concentration of 5,0 mg/l, and naphthaleneacetic acid (NAA) of 0,1 mg/l. Induction of morphogenic callus was achieved on a Gamborg B5 medium with 0,2 mg/l 6-benzylaminopurine (BAP). Further shoot initiation occurred on a hormone-free Gamborg B5 medium with the addition of Gamborg B5 vitamins, and on a half-strength medium with the addition of 0,2mg/l kinetin. It was found that established method of obtaining regenerated plants from the callus cultures of Medicago Varia proved itself effective.

ПРОДОЛЖИТЕЛЬНОСТЬ КУЛЬТИВИРОВАНИЯ IN VITRO ЗАРОДЫШЕВЫХ КАЛЛУСОВ ПШЕНИЦЫ ВЛИЯЕТ НА ПРОЯВЛЕНИЕ ИХ МОРФОГЕНЕТИЧЕСКОГО И РЕГЕНЕРАЦИОННОГО ПОТЕНЦИАЛА

Biotechnological developments for breeding purposes aimed at creating highly productive wheat varieties require a significant number of morphogenic callus, in which the manifestation of various pathways of morphogenesis in vitro associated with the regeneration of full-fledged plants is induced. It is methodically important to identify the dependence of the induction of a specific pathway of morphogenesis in wheat callus in vitro on the duration of their cultivation. On the example of morphogenic calli obtained from immature wheat embryos at the stage of early organogenesis on an induction medium containing 2.0 mg/l of synthetic auxin 2,4-D, depending on the duration of cultivation, the following sequence of induction of morphogenesis pathways in vitro was established: gemmogenesis, rhizogenesis, gemmorhizogenesis, somatic embryogenesis (or embryoidogenesis). These pathways of morphogenesis were identified morphologically by the appearance of the corresponding morphogenic structures: buds, roots, gemmorhizogenic structures, somatic embryos. It is shown that the process of laying and forming morphogenic structures is significantly stretched over time, and morphogenic structures may be present in the same callus at different stages of development.

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.