REGULATION OF CEREAL EMBRYONIC ORGANOGENESIS IN VITRO CONDITIONS

The article provides the brief review of the literature and own works devoted to the peculiarities of the cereal embryonic organogenesis at the early stages of ontogenesis in the conditions of in vitro culture (the so-called somatic embryogenesis, or embryoidogenesis in vitro). Particular attention is paid to the issues of hormonal regulation of the development of somatic cereal embryos from initial cells to mature structures in vitro. A comparison of somatic embryogenesis in vitro with similar events in zygotic embryogenesis in vivo confirms the validity of the principle of universality of morphogenesis processes in vivo and in vitro (Batygina, 2014). The prospects of using somatic embryogenesis in vitro as a model for studying the most complex biological phenomenon – zygotic plant embryogenesis in vivo – are discussed.

Embriogénesis y organogénesis de aguacate criollo mexicano

La variedad Drymifolia de aguacate (Persea americana Mill.) es usada generalmente como portainjerto por lo que es importante implementar las técnicas de embriogénesis somática y la organogénesis in vitro para su mejora genética, conservación y propagación clonal. En este trabajo se evaluaron embriogénesis somática directa, indirecta y organogénesis de la variedad Drymifolia. La germinación de los embriones somáticos maduros se indujo con 0.5 mg L-1 de 6-N-bencil amino purina (BAP) y 1 mg L-1 de ácido giberélico (GA3). De las seis accesiones evaluadas, Celaya 79, Comonfort 53, San Miguel, BG24, BG181 y Zutano, solo San Miguel respondió al proceso de embriogénesis. En la embriogénesis directa la eficiencia más alta de regeneración se obtuvo con 0.2 mg L-1 de picloram (46%) y 10 mg L-1 de ANA (40%). En la embriogénesis indirecta la accesión San Miguel formó callos con 0.2 mg L-1 de picloram y tuvo una eficiencia de regeneración de 45% conservando su potencial de regeneración hasta por seis meses. Con respecto a la organogénesis, se cultivaron embriones cigóticos inmaduros decapitados en medio con reguladores de crecimiento o sin ellos y las seis accesiones respondieron positivamente a ambas condiciones. La accesión Comonfort 53 tuvo mayor eficiencia de regeneración (54%) con reguladores de crecimiento. Este estudio realizado del 2016 a 2018, proporciona un enfoque nuevo y prometedor para la regeneración y multiplicación de plantas de P. americana var. Drymifolia a través de embriones cigóticos.

The application of microbial originating bioregulators to obtain in vitro lines of potato with increased resistance to parasitic nematodes

Aim. Obtaining in vitro new lines of potato (Solanum tuberosum L.) cultivar Vernissage with genetically mediated resistance to the parasitic nematode H. schachtii on nutrient MS media with microbial bioregulators. Methods. In vitro conditions the process of organogenesis of potato plants on nutrient MS media containing microbial bioregulators used at concentrations 25-100 μl/l in combination with phytohormones 2 mg/l BAP and 0,1 mg/l NAA was investigated. Using dot blotting method the degree of hybridization between cytoplasmic si/miRNA isolated from cells of potato plants-regenerants, grown on the artificial invasive background, and nematode mRNA was studied. In the wheat seedlings cell-free system in vitro the silencing activity of si/miRNA isolated from cells of potato plants-regenerants on the template of nematode mRNA was investigated. Results. The experiments conducted in vitro conditions showed that the addition of microbial bioregulators at concentrations 25-100 μl/l in combination with phytohormones 2 mg/l BAP and 0,1 mg/l NAA into MS media increased the efficiency of regeneration of potato shoots to 43–47 % as compared with similar indices obtained on control MS media. The increase of the degree of hybridization to 19-38 % between cytoplasmic mRNA isolated from cells of nematode H. schachtii and si/miRNA isolated from cells of experimental potato plants-regenerants grown in vitro conditions on nutrient media containing bioregulators on the artificial invasive background was shown. Conclusions. Using microbial bioregulators in vitro conditions as components of nutrient MS medium increases potato shoot regeneration efficiency and enhances RNAi-mediated resistance of plants-regenerants to parasitic nematode H. schachtii. Keywords: microbial bioregulators, potato organogenesis in vitro, potato resistance to nematode Heterodera schachtii, RNA interference.

Induction in vitro of adventitious morphogenesis in isolated tissues of the genus Rubus plants using cytokinins from adenine and diphenylurea groups

The eff ect of cytokinins from the adenine and diphenylurea groups on adventitious organogenesis in vitro in isolated tissues of the genus Rubus plants was studied. Leaf explants and callus of red raspberry of the Volnitsa variety, blackberry of the Chester Thornless variety and the Rubus odoratus species were cultivated in the dark at a temperature of +25 ±2 °C on Murashige and Skoog medium. The medium contained 0.5 mg/l of gibberellic acid (HA), 0.5 mg/l of indolylacetic acid (IAA), and 6-benzylaminopurine (6-BAP) at concentrations of 0, 1, 2 and 4 mg/l or thidiazuron (TDZ) at concentrations of 0.1, 0.2 and 0.4 mg/l. The number of explants that formed adventitious shoots and the number of shoots per explant were taken into account. It has been established that for the induction of adventitious morphogenesis from leaf explants and calluses of red raspberry and blackberry the use of 6-BAP is more eff ective, thydiazuron is more eff ective for Rubus odoratus. The optimal concentration for 6-BAP is 2 mg/l and 0.2 mg/l for TDZ. Exceeding these concentrations of cytokinins can cause shoot vitrifi cation. For blackberry, it is possible to increase the content of 6-BAP in the medium for the induction of morphogenesis to 4 mg/l. The unequal ability to regenerate adventitious shoots in diff erent genotypes was revealed. In optimal variants of the experiment, the maximum frequency of their formation ranged from 13.3 % in Rubus odoratus to 40.0 % in blackberry. The use of the established optimal concentrations of growth regulators made it possible to obtain regenerated plants from callus that underwent long-term cultivation (for 10 months) on artifi cial nutrient media during tissue selection for tolerance to heavy metal salts and pesticides. Despite the reduced morphogenetic potential of tissues undergoing prolonged cultivation in vitro, 3 red raspberry regenerant plants and 1 Rubus odoratus plant were obtained from callus selected for tolerance to cobalt chloride. 9 blackberry plants and 7 red raspberry plants were regenerated from the tissues selected for pesticide tolerance.

In vitro callusogenesis and organogenesis of different Linum usitatissimum L. accessions

Goal. The study of different genetic origin collection samples of flax (Linum usitatissimum L.) for the frequency and intensity of callusogenesis and organogenesis in vitro conditions, the establishment of differences in convar. elongatum, intermedia and humile was the goal of our research. Results and Discussion. The L. usitatissimum L. species is significantly capable of forming callus and shoot regeneration in vitro under cultivation conditions with a photoperiod of 16 hours, relative humidity of 60 – 80 %, air temperature of 22 – 24°С and agarized Murashige and Skoog medium, supplemented 0.05 mg/L of 1‑naphthylacetic acid, 1.0 mg/L of 6‑benzylaminopurine and 30 g/L of sucrose. The frequency and intensity of callusogenesis and organogenesis depends on the genotype. Minimum and maximum values of signs were set for Глінум (UF0401603), Кром (UF0401494), Орион (UF0401867), Есмань (UF0402071), Белита (UF0402134), Nor Man (UF0401792), Mapun M.A. (UF0401819), Lisa (UF0401830), Taragvi (UF0401864), Visamo (1–356)/L. monnseo (UF0402178), Ручеек (UF0401897), Lirina (UF0401900), Опус (UF0402142), СКі‑1 (UF0402143), Ruta (UF0402228) samples. The frequency of callusogenesis was 15.0 – 100 %, the calus mass from one explant was 0.56 – 1.51 g, the frequency of organogenesis was 10.0 – 93.8 %, the number of shoots was 1.4 – 4.0 pieces and the height of the shoots was 0.78 – 2.37 cm. Conclusions. Collection samples of Глінум (Ukraine), Кром (Russia), Visamo (1‑356)/L. monnseo (Czech Republic), Ручеек (Russia) and СКі‑1 (USA) were the best of complex signs (callusogenesis frequency, organogenesis frequency and number of shoots). Elongata flax and humile flax are characterized by the highest frequency of callusogenesis and organogenesis on hypocotyl and epicotyl explants, intermedia flax forms the largest mass of callus from the explant, the number of regenerated shoots and their height, intermedia flax has the greatest variation of the studied signs.