Development of haploids in the winter bread wheat anthers

In the modern world, the use of isolated anther cultivation technology is currently an integral part of the wheat breeding process. The development of haploids in the winter bread wheat anthers will allow obtaining new forms of wheat in the shortest possible time and without large areas. The purpose of the current study was to estimate the F3 winter bread wheat hybrids according to the anthers’ sensitivity to androgenesis and plant regeneration in vitro and to identify the factors affecting the yield of haploid production. There has been studied the ability to androgenesis in vitro in the anthers of four winter bread wheat hybrids of intensive and semi-intensive type of the FSBSI “ARC “Donskoy”. There has been assessed the role of the mineral composition of three induction nutrient media N6, W14 and NPB-99. There has been established a correlation between the main stages of development of haploids and a genotype. The highest regeneration rate of green plants was obtained in the sample F3 623 of intensive type (3.3%). The most suitable medium for androgenesis of the winter bread wheat anthers in vitro is NPB-99. Since the genotype F3 623 of intensive type demonstrated high values of haploid production capacity, it could be successfully used in breeding programs for the rapid production of homozygous wheat anther lines in vitro. Using two-way analysis of variance, there has been identified a correlation between the effects of a genotype, nutrient medium and their interaction with the main parameters of haploid formation in winter wheat. The formation of embryogenic structures is mainly associated with the effect of a genotype (46.52%). The proportion of the nutrient composition of the medium was low (1.82%), and the correlation factor was 2.1%. The genotype had the greatest effect on the indicator of the regenerants’ number. The nutrient medium had little effect. Regarding the regeneration of green plants, which is the main indicator of the haploid production, the share of a genotype effect was the largest (47.32%). The contribution of the medium and the correlation of factors were less important, but statistically significant.

The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

Molecular Evolution of Calcium Signaling and Transport in Plant Adaptation to Abiotic Stress

Adaptation to unfavorable abiotic stresses is one of the key processes in the evolution of plants. Calcium (Ca2+) signaling is characterized by the spatiotemporal pattern of Ca2+ distribution and the activities of multi-domain proteins in integrating environmental stimuli and cellular responses, which are crucial early events in abiotic stress responses in plants. However, a comprehensive summary and explanation for evolutionary and functional synergies in Ca2+ signaling remains elusive in green plants. We review mechanisms of Ca2+ membrane transporters and intracellular Ca2+ sensors with evolutionary imprinting and structural clues. These may provide molecular and bioinformatics insights for the functional analysis of some non-model species in the evolutionarily important green plant lineages. We summarize the chronological order, spatial location, and characteristics of Ca2+ functional proteins. Furthermore, we highlight the integral functions of calcium-signaling components in various nodes of the Ca2+ signaling pathway through conserved or variant evolutionary processes. These ultimately bridge the Ca2+ cascade reactions into regulatory networks, particularly in the hormonal signaling pathways. In summary, this review provides new perspectives towards a better understanding of the evolution, interaction and integration of Ca2+ signaling components in green plants, which is likely to benefit future research in agriculture, evolutionary biology, ecology and the environment.

EFFECT OF COMPLEXONS, DERIVATIVES OF SUCCINIC ACID, ON THE FORMATION OF CAROTENOIDS IN GREEN PLANTS

На основании обобщения результатов многолетних лабораторных и полевых опытов обнаружена зависимость уровня содержания жёлтых фотосинтетических пигментов в растениях от внекорневой обработки растворами комплексонов. Комплексоны, производные янтарной кислоты (иминодиянтарная кислота, этилендиаминдиянтарная кислота, диаминоциклогександиянтарная кислота) способствуют накоплению каротиноидов в зелёных растениях, а этилендиаминтетрауксусная кислота существенно не влияет на их содержание. Based on the generalization of the results of many years of laboratory and field experiments, the dependence of the content of yellow photosynthetic pigments in plants on foliar treatment with solutions of complexones was found. Complexones, derivatives of succinic acid (iminodisuccinic acid, ethylenediaminedisuccinic acid, diaminocyclohexanedisuccinic acid) contribute to the accumulation of carotenoids in green plants, and ethylenediaminetetraacetic acid does not significantly affect their content.

Structural Equation Modeling (SEM) Analysis of Sequence Variation and Green Plant Regeneration via Anther Culture in Barley

The process of anther culture involves numerous abiotic stresses required for cellular reprogramming, microspore developmental switch, and plant regeneration. These stresses affect DNA methylation patterns, sequence variation, and the number of green plants regenerated. Recently, in barley (Hordeum vulgare L.), mediation analysis linked DNA methylation changes, copper (Cu2+) and silver (Ag+) ion concentrations, sequence variation, β-glucans, green plants, and duration of anther culture (Time). Although several models were used to explain particular aspects of the relationships between these factors, a generalized complex model employing all these types of data was not established. In this study, we combined the previously described partial models into a single complex model using the structural equation modeling approach. Based on the evaluated model, we demonstrated that stress conditions (such as starvation and darkness) influence β-glucans employed by cells for glycolysis and the tricarboxylic acid cycle. Additionally, Cu2+ and Ag+ ions affect DNA methylation and induce sequence variation. Moreover, these ions link DNA methylation with green plants. The structural equation model also showed the role of time in relationships between parameters included in the model and influencing plant regeneration via anther culture. Utilization of structural equation modeling may have both scientific and practical implications, as it demonstrates links between biological phenomena (e.g., culture-induced variation, green plant regeneration and biochemical pathways), and provides opportunities for regulating these phenomena for particular biotechnological purposes.

Efficient Multi-Sites Genome Editing and Plant Regeneration via Somatic Embryogenesis in Picea glauca

Conifers are the world's major source of timber and pulpwood and have great economic and ecological value. Currently, little research on the application of CRISPR/Cas9, the commonly used genome-editing tool in angiosperms, has been reported in coniferous species. An efficient CRISPR/Cas9 system based on somatic embryogenesis (SEis) suitable for conifers could benefit both fundamental and applied research in these species. In this study, the SpCas9 gene was optimized based on codon bias in white spruce, and a spruce U6 promoter was cloned and function-validated for use in a conifer specific CRISPR/Cas9 toolbox, i.e., PgCas9/PaU6. With this toolbox, a genome-editing vector was constructed to target the DXS1 gene of white spruce. By Agrobacterium-mediated transformation, the genome-editing vector was then transferred into embryogenic tissue of white spruce. Three resistant embryogenic tissues were obtained and used for regenerating plants via SEis. Albino somatic embryo (SE) plants with mutations in DXS1 were obtained in all of the three events, and the ratios of the homozygous and biallelic mutants in the 18 albino mutants detected were 22.2% in both cases. Green plants with mutations in DXS1 were also produced, and the ratios of the DXS1 mutants to the total green plants were 7.9, 28, and 13.5%, respectively, among the three events. Since 22.7% of the total 44 mutants were edited at both of the target sites 1 and 2, the CRISPR/Cas9 toolbox in this research could be used for multi-sites genome editing. More than 2,000 SE plants were regenerated in vitro after genome editing, and part of them showed differences in plant development. Both chimerism and mosaicism were found in the SE plants of white spruce after genome editing with the CRISPR/Cas9 toolbox. The conifer-specific CRISPR/Cas9 system developed in this research could be valuable in gene function research and trait improvement.