Magdalena Dyduch-Siemińska

The aim of this study was to molecularly identify and assess the genetic diversity of 12 Mentha genotypes using, for the first time, two types of DNA markers simultaneously – ISSR (inter simple sequence repeats) and SCoT (start codon targeted). Selected genotypes representing various Mentha species and varieties were analyzed to determine the level of genetic similarity and phylogenetic relationships between them. The level of polymorphism obtained for ISSR markers was 71%, while for SCoT it was 88.7%. The obtained data were analyzed, allowing for the assessment of the level of genetic similarity and the construction of dendrograms illustrating the genetic structure of the studied population. Studies indicate that the use of SCoT markers enables the identification of the following genotypes: Plectranthus amboinicus (Lour.) Spreng, Mentha pulegium L., Mentha spicata L. cv. Moroccan, Mentha suaveolens Ehrh. var. variegata (pineapple mint), Mentha spicata L., and Mentha longifolia L. ISSR markers, due to the generation of only monomorphic and polymorphic bands, do not allow for the direct identification of any of the studied genotypes. Data from both marker systems indicate significant genetic diversity among the analyzed genotypes, which may be important for breeding programs and the conservation of genetic resources of the Mentha genus.

Introduction: Stevia rebaudiana Bertoni has recently gained significant attention due to the presence of intensely sweet yet low-calorie steviol glycosides (SGs) in its leaves, making it a promising natural sugar alternative with applications in the food, pharmaceutical, and cosmetics industries. The primary goal of this study was to determine whether generating somaclonal variation from plant material obtained by indirect regeneration results in further genetic changes identifiable using the SCoT marker (Start Codon Targeted). Methods: In the first stage, callus tissue was initiated from first-generation somaclones on MS medium supplemented with 4.0 mg/L 6-benzylaminopurine (BAP), 2.0 mg/L 1-naphthaleneacetic acid (NAA), and 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Their morphogenetic potential was analyzed on four media with different BAP and Kinetin concentrations. Donor plants, first and second generation somaclones, were also analyzed for genetic diversity using SCoT markers. Results: All first-generation somaclones demonstrated a very high callus initiation capacity, ranging from 95 to 100%. It was found that for most of the studied somaclones, the greatest number of shoots were developed by explants grown in a medium supplemented with 0.5 mg/L BAP and 0.25 mg/L Kin. The studied group of somaclones exhibits a high degree of polymorphism (55.2%). The analysis of genetic similarity of somaclones presented in the form of individual dendrograms indicates that in most cases, greater genetic diversity was revealed as a result of indirect regeneration in the first generation of somaclones compared to the second. Indirect organogenesis allows for the production of subsequent generations of genetically unstable somaclones, creating the potential for obtaining new phenotypic variants useful in plant breeding.

The use of in vitro cultures in plant breeding allows for obtaining cultivars with improved properties. In the case of Stevia rebaudiana Bert., genotypes with an appropriate rebaudioside A/stevioside ratio are desirable. The use of indirect organogenesis allows for the induction of somaclonal variation, which, consequently, results in obtaining variability within the regenerants. The Murashige and Skoog medium containing 4.0 mg × dm⁻³ 6-benzylaminopurine (BAP), 2.0 mg × dm⁻³ 1-naphthaleneacetic acid (NAA), and 2.0 mg × dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in obtaining plants that were biochemically and genetically diverse. The obtained regenerants were characterized by an increased content of rebaudioside A and a better rebaudioside A/stevioside ratio. Genetic analysis using SCoT (start-codon-targeted) markers showed their diversity at the molecular level. Moreover, this study showed that genotype multiplication through six subsequent re-cultures does not cause variability at the genotype level and does not affect the steviol glycoside profile. This study is the first report on obtaining genotypes with higher rebaudioside A content and a more attractive rebaudioside A to stevioside ratio through the use of in vitro cultures. The improved regenerants can be used as parents in hybridization programs or directly as valuable new genotypes.

Stevia (Stevia rebaudiana Bertoni) is an essential herbal plant used as a sweetener. The demand for stevia is growing due to its low caloric and medicinal value, hence the need for a more thorough investigation of its nutritional and biological properties. Nanoparticles of metal oxides have been found to have broad applications in agriculture for the stimulation of plant growth and development. The study aimed to assess the effect of various zinc oxide nanoparticles (ZnONPs) concentrations on stevia plants’ quantitative and qualitative traits obtained in in vitro cultures. Micropropagation of two stevia varieties, Candy and Morita, was carried out using explants of shoot tips placed on MS medium supplemented with 1.0 mg dm–3 BA and 0.1 mg dm–3 IBA and with ZnONPs at concentrations of 0 (control), 10, 20, 30 and 40 mg dm–3. The obtained results indicated that high concentrations of ZnONPs stimulated the propagation of shoots. On the other hand, they negatively influenced shoot length, root number and length, and the fresh weight of the plantlets. The presence of zinc oxide nanoparticles in the medium increased the potassium, calcium, magnesium, and zinc content while decreasing the sodium and iron content in the regenerated stevia plantlets. The total phenolic content in the Candy variety was higher in the treatments with ZnONPs than in the control plants, while it was varied in the Morita variety. In both varieties, total antioxidant content measured by the ABTS method showed significantly higher in the treatments with 20–30 mg dm–3 ZnONPs than in the control. The content of chlorophyll a, chlorophyll b and chlorophyll a + b in the Morita variety was higher in the treatments with 10 and 20 mg dm–3 ZnONPs than in the control. On the other hand, high concentrations of ZnONPs negatively affected the content of carotenoids in both varieties. The study showed that stevia plants obtained in in vitro cultures on control media and media containing ZnONPs had a high content of valuable minerals, phytocompounds with antioxidant properties, and photosynthetic pigments.

The efficiency and method of regeneration in in vitro culture conditions depend primarily on the plant growth regulators (PGRs) used. Even growth regulators belonging to one group may have different effects, stimulating the process of direct or indirect organogenesis, thus possibly disturbing the genetic stability among regenerants. The main aim of this study was to identify the genetic stability of Scutellaria baicalensis regenerates obtained by in vitro culture method using start codon targeted (ScoT) markers. S. baicalensis nodal explants were regenerated on MS medium supplemented with kinetin (KIN) at concentrations of 0.25, 0.5, 1.0, and 2.0 mg × dm⁻³ or benzylaminopurine (BAP)—0.25, 0.5, 1.0, and 2.0 mg × dm⁻³. The effects of the number of propagated shoots, length, number of nodes, and fresh mass of regenerants were assessed. Moreover, the genetic stability of the regenerants was analyzed using start codon targeted (SCoT) markers. Direct shoot organogenesis was observed on an MS medium containing kinetin, while indirect shoot induction occurred on an MS medium supplemented with BAP. The highest average number of shoots (3.6) was achieved for the MS + KIN medium at a concentration of 0.25 and 5.8 for the MS + BAP 1.0 medium. The average length and average number of nodes were the highest on the MS + BAP 0.25 medium (50.0 and 6.0, respectively), while the lowest values of these features were observed on the MS + KIN 2.0 medium (40.3 and 4.9, respectively). A total of 111 amplified bands were exhibited by SCoT primers. Three of the analyzed primers revealed four unique genotype-specific markers. The average percentage of polymorphism obtained was 36.7%. The analysis of genetic similarity revealed a high level of genetic similarity between the donor plant and regenerants obtained on MS “0” (medium without the addition of phytohormones). A slightly lower value of genetic similarity was observed for regenerants obtained by direct organogenesis (MS + KIN medium at all concentrations). Indirect shoot organogenesis observed on the MS + BAP medium (all concentrations) resulted in the highest differentiation, both in relation to the donor plant and MS “0” regenerants. The results of our work indicate that, in the case of S. baicalensis, the maintenance of genetic stability depends primarily on the presence of the cytokinin type in the medium.

In response to the challenges posed by modern plant micropropagation techniques, a promising technology for growing shoots temporary immersed in nutrient solution (temporary immersion system, TIS) using SETIS™ bioreactors has been developed. In this experiment, the suitability of this technology for the propagation of Chlorophytum comosum (Thunb.) Jacques was assessed. In vitro culture was carried out using a conventional technique on solid media and liquid media using the SETIS™ bioreactor. In addition, two culture media differing in macro- and micronutrient content (Murashige & Skoog and Rugini OM), while having the same set of phytohormones were evaluated in both systems. Explants obtained from the flower stalk of the plants were used to establish the culture. The effectiveness of the cultures after the first and second subculture was assessed. The study has demonstrated that the efficiency of liquid culture carried out using the SETIS™ bioreactor is higher compared to the conventional culture. The highest multiplication coefficient, fresh weight of regenerants and RGR index value in bioreactor cultures was recorded on Rugini OM medium. No statistically significant differences were found between MS medium and Rugini OM medium in terms of shoot length and vigour with this method of culture. When using the conventional method, better results can be achieved with MS medium. This research can be considered as a first step towards the production of Chlorophytum comosum (Thunb.) Jacques on a larger scale. In response to the challenges posed by modern plant micropropagation techniques, a promising technology for growing shoots temporary immersed in nutrient solution (temporary immersion system, TIS) using SETIS™ bioreactors has been developed. In this experiment, the suitability of this technology for the propagation of Chlorophytum comosum (Thunb.) Jacques was assessed. In vitro culture was carried out using a conventional technique on solid media and liquid media using the SETIS™ bioreactor. In addition, two culture media differing in macro- and micronutrient content (Murashige & Skoog and Rugini OM), while having the same set of phytohormones were evaluated in both systems. Explants obtained from the flower stalk of the plants were used to establish the culture. The effectiveness of the cultures after the first and second subculture was assessed. The study has demonstrated that the efficiency of liquid culture carried out using the SETIS™ bioreactor is higher compared to the conventional culture. The highest multiplication coefficient, fresh weight of regenerants and RGR index value in bioreactor cultures was recorded on Rugini OM medium. No statistically significant differences were found between MS medium and Rugini OM medium in terms of shoot length and vigour with this method of culture. When using the conventional method, better results can be achieved with MS medium. This research can be considered as a first step towards the production of Chlorophytum comosum (Thunb.) Jacques on a larger scale.

The in vitro culture technique can be used for micropropagation of medicinal plants as well as for creating genotypes with an improved profile of phytochemical compounds. For this purpose, somaclonal variability may be used for the induction of genetic diversity among regenerants. The paper presents a protocol for obtaining Scutellaria baicalensis regenerants by indirect organogenesis and the assessment of their genetic variability with the use of start codon-targeted markers. The most intense process of indirect shoot organogenesis was observed on Murashige and Skoog medium supplemented with kinetin and 6-Benzylaminopurine (0.5 mg × dm⁻³ each)—7.4 shoot per explant on average. The callogenesis process occurred on the medium supplemented with TDZ, while the medium supplemented with GA₃ allowed for direct shoot organogenesis and was used for the micropropagation of regenerants. In the analysis of plantlets obtained by indirect organogenesis, 11 ScoT markers generated a total of 130 amplicons, 45 of which were polymorphic. This analysis showed genetic diversity of regenerants in relation to the donor plant as well as within them, with mean similarity among the analyzed genotypes at the level of 0.90. This study confirms that the use of in vitro cultures allows for the possibility to generate genetic variability in Scutellaria baicalensis, which can be effectively revealed with the use of the SCoT marker.

Functional properties and biological activities of plant-derived polyphenolic compounds have gained great interest due to their epidemiologically proven health benefits and diverse industrial applications in the food and pharmaceutical industry. Moreover, the food processing conditions and certain chemical reactions such as pigmentation, acylation, hydroxylation, and glycosylation can also cause alteration in the stability, antioxidant activity, and structural characteristics of the polyphenolic compounds. Since the (poly)phenols are highly reactive, to overcome these problems, the formulation of a complex of polyphenolic compounds with natural biopolymers is an effective approach. Besides, to increase the bioavailability and bioaccessibility of polyphenolic compounds, milk proteins such as whey protein concentrate, sodium caseinate, and milk protein concentrate act as natural vehicles, due to their specific structural and functional properties with high nutritional value. Therefore, milk proteins are suitable for the delivery of polyphenols to parts of the gastrointestinal tract. Therefore, this review reports on types of (poly)phenols, methods for the analysis of binding interactions between (poly)phenols–milk proteins, and structural changes that occur during the interaction.

The strawberry fruit market increasingly demands new cultivars producing berries with enhanced bioactive attributes. In this research the suitability of twelve strawberry cultivars for effective breeding aimed at the enhancement of bioactive fruit attributes was studied. The group of genotypes with different pedigree was examined in respect of variation, breeding parameters, correlation between the content of bioactive compounds and the antioxidant capacity of fruit. Pre-breeding research showed the highly significant differences between genotypes regarding all traits analysed. Relationship between the bioactive phytochemicals content and antioxidant capacity of fruit were mostly positive and significant. Only correlation between vitamin C and flavonoids was negative (−0.482). Path analysis exhibited the highest positive direct effect of total phenolic content on antioxidant capacity (0.609). Heritability of traits was very high, reaching values above 0.90. The highest genetic advance was observed for flavonoids. Results suggested that the genotype plays the main role in shaping of fruit antioxidant potential. This study indicated that the efficient strawberry breeding focused on obtaining the forms with enhanced bioactive berry properties could be highly possible.