Using in vitro androgenesis serves as a unique opportunity to produce doubled haploid (DH) plants in many species. More benefits of this biological phenomenon have kept these methods in the focus of fundamental research and crop breeding for decades. In common wheat (Triticum aestivum L.), in vitro anther culture is one of the most frequently applied DH plant production methods. The efficiency of in vitro wheat anther culture is influenced by many factors, such as the genotype, growing conditions, collection time, pre-treatments, and compositions of media and culture conditions. According to some critical review, the genotype dependency, low efficiency and albinism are mentioned as limitations of application of the anther culture method. However, some research groups have made significant efforts to diminish the effects of these bottlenecks. Due to the improvements, a well-established in vitro anther culture method can be an efficient tool in modern wheat breeding programs.
RNA extraction involves several main stages, regardless of the method of extraction: homogenization, effective denaturation of proteins from RNA, inactivation of ribonuclease and removal of any DNA, protein, and some residual contamination. Isolation of undamaged intact RNA is challenging when the related tissue contains high levels of polysaccharides and phenols. Several efforts have been made towards the comparison and optimization of extraction and purification methods for RNA from plant tissues. This is dictated by the necessity of obtaining RNA of a good quality and in a sufficient quantity for further molecular analyzes. Plant storage organs (such as bulbs or seeds) rich in polysaccharide and polyphenolic compounds present distinct challenges for total RNA isolation. Such components, considered in this case as contamination, may bind and co-precipitate with nucleic acids and negatively affect later assays. Since standard routine protocols yield unacceptable results in bulbs, we have designed a new method for RNA extraction. We used two modified procedures (based on CTAB and sarkosyl reagents) of RNA extraction from so called “difficult plant material” and compared them to a popular RNA isolation base on the column isolation kit and TriPure reagent. Our modified protocols dealt with problems of both RNA degradation and low yield caused by co-purification with polysaccharides present in plant bulbs. In this study we have shown that improvement of the CTAB and sarkosyl method with a lyophilization step of plant tissues leads to isolation of high quality RNA from difficult material like storage organs of bulbous plants. The main changes in the procedure compared to the previously described methods concerned the different order of lithium chloride and sodium acetate addition, lithium chloride concentration increase and modification of centrifugation conditions. Gel electrophoresis and spectrophotometer analysis confirmed the high quality and integrity of the obtained RNA. The modified procedures allowed for obtaining a satisfying amount of RNA concentration in the range from 280 to 950 ng/μl depending on the plant species. Thus, the demonstrated RNA isolation methods are efficient and can be used for plant material rich in polysaccharides, such as bulbs.
Hydro-ethanolic extracts of flowers from four Verbascum species were evaluated for the phenolic content, composition, and antioxidant activity using Folin-Ciocalteu assay, HPLC-DAD analysis, and DPPH assay, respectively. The highest flavonoid content was detected in V. sinuatum extract from Khoramabad (19.91 mg RE/g DW). The extract of V. pseudo-digitalis from Maymand yielded the highest amount of total phenols, together with the highest apigenin and luteolin levels (55.62 mg GAE/g DW, 12.18 and 88.13 μg/mg DW, respectively), while that of V. songaricum from Ardekan showed the highest naringin content (12.44 μg/mg DW). The extract of V. songaricum from Shirmard exhibited the highest quercetin and rutin levels (1.0 and 24.24 μg/mg DW, respectively), whereas that of V. sinuatum from Ardekan had the highest caffeic acid content (7.78 μg/mg DW). The antioxidant activity of Verbascum samples showed IC50 values from 45.12 to 226.62 μg/mL.
Chromosome numbers for 23 taxa of Hieracium L. from Bulgaria, Greece, Poland and Slovakia are given and their metaphase plates are illustrated. The ploidy level of 8 taxa was also confirmed by flow cytometry. Chromosome numbers are published for the first time for Hieracium bracteolatum subsp. koracis (Boiss.) Zahn (4x), H. marmoreum Pančić & Vis. (3x), H. ossaeum Zahn (3x), H. sartorianum Boiss. & Heldr. (3x), H. sericophyllum Nejčeff & Zahn (3x) as well as for five other undescribed species.
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