Differing In Vitro Rooting and Flowering Responses of the Persian Violet to Low and High UV-C Irradiation

Persian violet flowers are considered esthetically attractive, leading to the high economic value of this plant. Plant breeding is fundamental to crop improvement, and the induction of mutation by tissue culture technology in combination with irradiation has been beneficially applied to generate plants with novel desirable characteristics. In this research, single or double rounds of UV-C irradiations were carried out on plant tissue cultures to initiate the in vitro rooting and mutation of Persian violets. It was found that single low-intensity UV-C exposure, when applied to Persian violet microshoots for 4 h, could induce the maximum number of roots and the highest root length without the use of a plant growth regulator. Overall, the single and double UV-C irradiation of Persian violet microshoots led to 44 different types of Persian violet flower mutations. Under single high-intensity UV-C irradiation for 6 h, up to nine petals were initiated, whereas single low-intensity UV-C irradiation did not influence the morphological variation of Persian violet flowers. Thus, Persian violet microshoots respond differently in terms of in vitro rooting and flowering depending on the UV-C intensity and exposure duration. These outcomes may be applied to micropropagation and in vitro plant breeding.

Stimulation of Nicotiana tabacum L. In Vitro Shoot Growth by Endophytic Bacillus cereus Group Bacteria

In vitro plant tissue cultures face various unfavorable conditions, such as mechanical damage, osmotic shock, and phytohormone imbalance, which can be detrimental to culture viability, growth efficiency, and genetic stability. Recent studies have revealed a presence of diverse endophytic bacteria, suggesting that engineering of the endophytic microbiome of in vitro plant tissues has the potential to improve their acclimatization and growth. Therefore, the aim of this study was to identify cultivated tobacco (Nicotiana tabacum L.) endophytic bacteria isolates that are capable of promoting the biomass accumulation of in vitro tobacco shoots. Forty-five endophytic bacteria isolates were obtained from greenhouse-grown tobacco plant leaves and were assigned to seven Bacillus spp. and one Pseudomonas sp. based on 16S rRNA or genome sequence data. To evaluate the bacterial effect on in vitro plant growth, tobacco shoots were inoculated with 22 isolates selected from distinct taxonomic groups. Four isolates of Bacillus cereus group species B. toyonensis, B. wiedmannii and B. mycoides promoted shoot growth by 11–21%. Furthermore, a contrasting effect on shoot growth was found among several isolates of the same species, suggesting the presence of strain-specific interaction with the plant host. Comparative analysis of genome assemblies was performed on the two closely related B. toyonensis isolates with contrasting plant growth-modulating properties. This revealed distinct structures of the genomic regions, including a putative enzyme cluster involved in the biosynthesis of linear azol(in)e-containing peptides and polysaccharides. However, the function of these clusters and their significance in plant-promoting activity remains elusive, and the observed contrasting effects on shoot growth are more likely to result from genomic sequence variations leading to differences in metabolic or gene expression activity. The Bacillus spp. isolates with shoot-growth-promoting properties have a potential application in improving the growth of plant tissue cultures in vitro.

CG Demethylation Leads to Sequence Mutations in an Anther Culture of Barley Due to the Presence of Cu, Ag Ions in the Medium and Culture Time

During plant tissue cultures the changes affecting regenerants have a broad range of genetic and epigenetic implications. These changes can be seen at the DNA methylation and sequence variation levels. In light of the latest studies, DNA methylation change plays an essential role in determining doubled haploid (DH) regenerants. The present study focuses on exploring the relationship between DNA methylation in CG and CHG contexts, and sequence variation, mediated by microelements (CuSO4 and AgNO3) supplemented during barley anther incubation on induction medium. To estimate such a relationship, a mediation analysis was used based on the results previously obtained through metAFLP method. Here, an interaction was observed between DNA demethylation in the context of CG and the time of culture. It was also noted that the reduction in DNA methylation was associated with a total decrease in the amount of Cu and Ag ions in the induction medium. Moreover, the total increase in Cu and Ag ions increased sequence variation. The importance of the time of tissue culture in the light of the observed changes resulted from the grouping of regenerants obtained after incubation on the induction medium for 28 days. The present study demonstrated that under a relatively short time of tissue culture (28 days), the multiplication of the Cu2+ and Ag+ ion concentrations (‘Cu*Ag’) acts as a mediator of demethylation in CG context. Change (increase) in the demethylation in CG sequence results in the decrease of ‘Cu*Ag’, and that change induces sequence variation equal to the value of the indirect effect. Thus, Cu and Ag ions mediate sequence variation. It seems that the observed changes at the level of methylation and DNA sequence may accompany the transition from direct to indirect embryogenesis

THE FEATURES OF THE ACCUMULATION OF FLAVONOIDS IN BIOTECHNOLOGICAL RAW MATERIAL OF IRIS SIBIRICA L. THE DEVELOPMENT OF METHODS OF QUANTIFICATION

Identification of scientific regularities of accumulation of physiologically active compounds is relevant to the issues of plant biology, as it can create conditions for the rapid development of biotechnological approaches and the solution of a number of environmental and economic problems associated with the use of plant raw materials. The aim of this work was to identify the features of the accumulation of flavonoids and the development of methods of differential spectrophotometry allows to assess the quality of biotechnological raw materials Iris sibirica L. (Siberian iris) on the content of flavonoids. The most important regulators of the synthesis of secondary compounds in plant tissue cultures include such components of nutrient media as hormones. As a result of our experiments it was noted for I. sibirica characteristic relationship between the accumulation of biomass and the content of quercetin and rutin. On the medium with 5.0 µm BAP, supplemented by auxins with an increase in the total height of the shoots, the quercetin content and rutin in the phytomass decreased dramaticly. To maintain a balance between biomass accumulation and flavonoid content for I. sibirica, we recommend using media with 2.5 µm BAP supplemented with auxins. The developed method allows to determine the content of the total of flavonoids in Iris sibirica raw materials in the presence of other compounds, is simple in execution and does not require expensive equipment. A validation evaluation of the technique indicates its suitability for quality control of biotechnological raw materials Iris sibirica.

Influence of Selected Antibiotics on the Tomato Regeneration in In Vitro Cultures

Generally, antimicrobial agents are frequently used in micropropagation techniques to obtain free elite clones or after genetic transformation to select putative transformants. Their successful application minimizes bacterial contamination however; they may be phytotoxic and may diversely affect the regeneration ability in plant tissue cultures. The objective of the current study was to estimate the effects of four antibiotics i.e. ampicillin, carbenicillin, cefotaxime and kanamycin on morphogenesis of three Polish tomato cultivars, cultured on MS medium with phytohormones. In this experiment the ability of tomato cotyledon explants to regenerate entire plants via indirect organogenesis was tested. Among four antibiotics tested, kanamycin was most harmful for the explants. This antibiotic, even at low doses (10-20 mg/L), inhibited tomato morphogenesis. On the other hand, the current study revealed significant influence of different concentrations of ampicillin, carbenicillin, cefotaxime on the frequency of bud formation. While the addition of cefotaxime at low concentration (100-200 mg/L) stimulated the bud formation, its increasing concentration adversely affected the organogenesis of tomato. The results clearly pointed out that carbenicillin and ampicillin at low concentrations (100-400 mg/L) were not only non-toxic, but they promoted bud regeneration. The obtained results show the crucial role not only of use of efficient antibiotics, but also of their proper doses in obtaining successful transformation and regeneration of tomato. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

Bacterial Contamination of Plant in vitro Cultures in Commercial Production Detected by High‑Throughput Amplicon Sequencing

The study overviews results of bacterial incidence in in vitro plant tissue cultures obtained from commercial laboratory dealing with plants micropropagation. For the exploration, the 454 pyrosequencing of the 16S rRNA gene was used. Three samples of plant in vitro cultures with visual bacterial contamination were subjected for identification of present bacteria. Eleven genera as Acinetobacter, Lactobacillus, Methylobacterium, Roseomonas, Microbacterium, Mycobacterium, Curtobacterium, Acidovorax, Magnetospirillum, Chryseobacterium and Ralstonia were detected. Obtained results confirmed the advantages of high‑throughput amplicon sequencing analysis for identification of bacterial communities in contaminated plant in vitro cultures what provides an important information for applying appropriate measures to eliminate bacterial contamination.