Taxonomic structure of tissue endophytic bacterial microbiome in clonal apple rootstock grown on sod-podzolic soils with variant properties

The taxonomic structure of tissue endophytic bacterial microbiome was comparatively studied in microplants (undifferentiated explant callus tissues, passage 25) and 5-year clonal apple rootstocks 57-490 and 54-118 cultured from corresponding tissues (passage 1) on sod-podzolic soils with variant granulometry, chemical, physical and physicochemical properties. Proteobacteria (91.6 %) predominated in vitro tissues among other endophytic bacterial phyla in rootstock 57-490, while Proteobacteria (52.5 %) and Firmicutes (47.4 %) — in rootstock 54-118. The endophytic Firmicutes ratio vs. in vitro tissues decreases (0.7-2.0 %) in roots and more severely (0-0.2 %) in leaves. Endophytic Actinobacteriota are revealed in 11.7 % in roots of the study rootstock in heavy loam soil, whilst in medium loam their ratio drops to 2.74.1 % in roots and 0.1-0.2 % in leaves. The phylogenetic diversity indices estimation for main endophytic bacterial phyla in apple rootstock tissue recovers their essentially lower diversity and evenness in culture endosphere (Shannon index 0.42-1.00) vs. open soil roots (1.34-2.08). The leaves Shannon index is typically low (0.06-0.13) indicating poor diversity and evenness of the main endophytic bacterial phyla.

Transformation of Teosinte (Zea mays ssp. parviglumis) via Biolistic Bombardment of Seedling-Derived Callus Tissues

Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of Zea mays identified as teosinte, Zea mays ssp. parviglumis is the most closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the maize domestication process and facilitate breeding for future crop improvement. However, a protocol of genetic transformation, which is essential for gene functional analyses, is not available in teosinte. In this study, we report the establishment of a robust callus induction and regeneration protocol using whorl segments of seedlings germinated from mature seeds of Zea parviglumis. We also report, for the first time, the production of fertile, transgenic teosinte plants using the particle bombardment. Using herbicide resistance genes such as mutant acetolactate synthase (Als) or bialaphos resistance (bar) as selectable markers, we achieved an average transformation frequency of 4.17% (percentage of independent transgenic events in total bombarded explants that produced callus). Expression of visual marker genes of red fluorescent protein tdTomato and β-glucuronidase (gus) could be detected in bombarded callus culture and in T1 and T2 progeny plants. The protocol established in this work provides a major enabling technology for research toward the understanding of this important plant in crop domestication.

The Effect of Phenyl Acetic Acid (PPA) On Micropropagation of Date Palm Followed By Genetic Stability Assessment

Date palm is propagated by the offshoots, the number of which is limited. Therefore, adult date palms produce shoot tips and axillary buds meristems through the use of tissue culture. The micropropagation of the date palm also still faces many obstacles. Here, we established an efficient plant-regeneration system for (Phoenix dactylifera L.) cv. Ashgar by tissue culture. Murashige and Skoog medium containing 6-benzyladenine (BA) (2.0 and 5.0 mg L− 1) and a-naphthaleneacetic acid (NAA) or phenylacetic acid (PAA) (0.05–2.00 mg L− 1) was used to initiate shoot formation from callus tissues. The maximum number of shoots per jar was produced on a medium containing 5.0 mgL− 1 BA and 0.5 mg L− 1 PAA. The medium supplemented with 2.0 mgL− 1 BA in combination with 0.05 m L− 1 PAA gave the highest callus induction (+++). A decrease in browning percentage was observed in the tissue cultured in the media supplied with BA in combination with NAA or PAA compared to the media provided with BA alone. In comparison with other treatments, the total amount of phenolic compounds was significantly reduced to 0.45 mg g− 1 in buds cultured in the media supplemented with 5.0 mg L− 1 BA and 0.5 mg L− 1 PAA. The RAPD DNA-based fingerprinting technique confirmed the genomic stability of this protocol. RAPD binding patterns showed no difference between the tissue culture-derived plants tested. The in vitro micropropagation protocol reported here can be presented to produce genetically stable date palm plants.

Reaction of flax seeds and explants to osmotic stress

Purpose. Obtaining experimental data for the development of methods for obtaining fiber flax forms resistant to stress factors (drought) by cell selection methods. Methods. The work was carried out in the Tver region in the laboratory of breeding technologies in 2017–2019. The flax varieties Varbara, Belinka, LM-98, Svetoch, Diplomat, Symfonia were used as objects of research. The seeds were obtained from the National Flax Collection Federal state budgetary scientific institution “Federal scientific center of lubya kultur”. The effect of sucrose solution on the length of the primary root was detected at concentrations of 0, 8.7, 14.9 %. To assess the energy of seed germination under osmotic stress, the concentration of sucrose was reduced and the range from 0 (control) to 9 % was considered. Results. Sucrose as a selective agent in the 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 Svetoch variety. When culturing callus tissues, using mannitol as an osmotic at concentrations of 0; 30.0; 36.4; 37.0; 37.4; 38.0 mg/l, selection of resistant callus cells with subsequent formation in meristematic foci of adventive buds and shoots can be carried out on media containing 30.0 or 36.4 mg/l osmotic. The scientific novelty of the research lies in the fact that the method for obtaining in vitro resistant explants to osmotic stress for flax is being developed for the first time.

In Vitro Callus Culture of Dianthus Chinensis L. for Assessment of Flavonoid Related Gene Expression Profile

Dianthus chinensis L. is an edible, ornamental herb used to prepare the Dianthi Herba, a Chinese traditional rejuvenating medicine. Owing to the rapid proliferation of callus tissues, in vitro production of flavonoids has their own specific importance. Callus cultures raised followed by auxin directed biosynthesis of flavonoid through related transcript profile were carried out. Murashige and Skoog (MS) medium fortified with 2,4- Dichlorophenoxy acetic acid (2,4- D) or picloram induced formation of friable callus from internode derived cultures of D. chinensis. Culture medium containing 2,4- D (10 µM) produced the highest flavonoid content, 4.44 mg quercetin equivalent per gram (QE g− 1) under incubation in continuous dark condition, while maximum dry weight yield (0.38 g/ culture) was obtained from 10 µM 2,4- D under 16 h light / 8 h dark condition (50 µmol m− 2 s− 1 irradiance) at 60 days of incubation. The callus raised in light condition in 10 µM 2,4- D selected to analyze flavonoid related gene expression profile viz., chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and flavonol synthase (FLS) at specific time intervals. The transcript abundance of CHS, F3H, or FLS gene was higher at 60 days old callus cultures and reaching 11.59, 48.31, and 114.63-fold relative expression than that of initial callus tissues respectively. These understandings are critical for the regulation of targeted phytochemicals as well as their wide exploitation in the field of biological research.

Tree-ring reconstruction of snow avalanches in Şureanu Mountains (Southern Carpathians, Romania)

<p>Snow avalanches (SAs) are a widespread natural hazard in the Carpathians, damaging forests and threatening properties, tourism infrastructures and people. In Şureanu Mountains (Southern Carpathians), SA activity is not documented in the historical archives and consequently information regarding the SA frequency and their spatial extent is lacking. Along the forested avalanche paths, disturbed trees record selectively in their annual rings evidence of past events. Tree rings represent therefore a natural archive which can provide valuable information about the past SA activity. The aim of the present study is to reconstruct the occurrence and spatial extent of past SA activity with tree rings in Şureanu Mts. For this purpose, two avalanche paths adjacent to a ski area located in the central part of Şureanu Mts., have been investigated. Samples (cores and discs) collected from 121 and 141 Norway spruce (Picea abies (L.) Karst.) trees damaged by SAs along both paths have been analyzed. Tree-growth anomalies (e.g. scars, callus tissues, onset sequences of tangential rows of traumatic resin ducts, compression wood and growth suppression sequences) associated with the mechanical impact produced by SAs on trees were identified and used to reconstruct the SA history. Within the investigated paths, the reconstructed SA chronology spans the period of the last century. The minimum SA frequency and maximum extent reconstructed served to define the return periods within the two paths investigated. Tree-ring derived records provided the most consistent SA chronology in the study area, and can further be integrated in the avalanche hazard zoning assessment.</p>

Introduction of hyssopus officinalis l . into in vitro culture to optimize the conditions for obtaining callus tissues and microclonal propagation as a promising metod of innovative agrobiotechnologies

The sterilization process of plant explants of H. officinalis was optimized when introduced into an in vitro culture, the most effective sterilization modes, optimal sterilizing agents, their exposure time and concentration were selected. Callus tissues and mini-plants of H. officinalis were obtained in vitro and the most optimal nutrient media were determined both for microclonal propagation and for the induction of callus tissue H. officinalis, which can be further used for mass cultivation of cell and culture and obtaining safe bio-additives with active substances for livestock and crop production as a part of the development of modern agrobiotechnologies.

ВМІСТ ФЛАВОНОЇДІВ І КCАНТОНІВ У КАЛЮСНИХ КУЛЬТУРАХ РОСЛИН ВИДІВ РОДУ GENTIANA L. ЗА ВИРОЩУВАННЯ У РІДКОМУ ЖИВИЛЬНОМУ СЕРЕДОВИЩІ НА ПОРОЛОНОВИХ ПІДКЛАДКАХ

The content of flavonoids and xanthones in callus cultures derived from the roots of plants of six species of Gentiana L. genus was studied during the cultivation of these cultures in liquid growth media on foam substrates. The research findings indicate that for most callus cultures, which were grown on both agar and foam substrates, the content of biologically active substances (BAS) was higher or close to that in the roots of wild plants, but lower compared to their shoots. The content of flavonoids and xanthones in tissue cultures grown in liquid nutrient media exceeded (G. punctata, Mt. Breskul, G. asclepiadea, Mt. Pozhyzhevska, G. cruciata, Krenychi village and G. lutea, Mt. valley Rohnechska), was close (G acaulis, Mt. Turkul) or lower (G. cruciata, «Medobory» Nature Reserve, G. lutea, Mt. Troyaska) compared to those in the corresponding calluses on agar substrates. In the callus of G. pneumonanthe (Vyhoda village) during cultivation on agar medium and in liquid medium on foam substrates, flavonoids and xanthones were not detected. For most callus cultures of gentians: G. punctata (Mt. Breskul), G. asclepiadea (Mt. Pozhyzhevska), G. cruciata (Krenychi village), G. lutea (Mt. valley Rohnechska), cultivation in a liquid growth medium on foam substrates can increase the growth of callus biomass (1.3–1.7 times) and the content of flavonoids (1.2–1.6 times) and xanthones (1.2–2.3 times) in comparison with the same cultures on agar media. For the callus of G. cruciata («Medobory» Nature Reserve) and G. lutea (population of Mt. Troyaska) on liquid media with foam substrates, both the growth index by fresh weight and the content of secondary metabolites are lower compared to cultures from agar medium. The growth of callus G. acaulis (Mt. Turkul) on the nutrient medium with foam substrates is more intense than on agar, but with lowered BAS. Thus, the developed method of cultivating callus tissues of gentians in liquid nutrient media on foam substrates can reduce costs by replacing agar with foam substrates, as well as increase both the yield of biomass of most callus cultures and their ability to synthesize and accumulate flavonoids and xanthones. Key words: Gentiana L. species, in vitro cultures, foam substrates, flavonoids, xanthones.

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