Suspension culture of stem cells established of Calendula officinalis L.

Plant stem cell cultures have so far been established in only a few plant species using cambial meristematic cells. The presence of stem cells or stem cell-like cells in other organs and tissues of the plant body, as well as the possibility of de novo generation of meristematic cells from differentiated cells, allow to consider the establishment of stem cell cultures in a broader range of species. This study aimed to establish a stem cell culture of the medicinal plant Calendula officinalis L. Callus tissues were induced from leaf and root explants, and already at this stage, stem and dedifferentiated cells could be identified. The cell suspension cultures established both from the root- and leaf-derived calli contained a high proportion of stem cells (92-93% and 72-73%, respectively). The most effective combination of growth regulators for the development of stem cells in calli as well as cell cultures was 1.0 mg/L 2,4-D and 0.5 mg/L BAP. The highest amount of stem cells (5.60-5.72 × 105) was in cell suspension derived from the roots. An effective protocol for the establishment of marigold stem cell suspension culture was developed. The ratio of root-derived stem cells against dedifferentiated cells exceeded 90%.

UPTAKE AND DISTRIBUTION OF RADIOSTRONTIUM IN TOMATO TREATED WITH ARBUSCULAR MYCORRHIZAL FUNGI.

Arbuscular mycorrhizal fungi (AMF) were inoculated into the root system of edible tomato plants to investigate their effect on the uptake and distribution of strontium radionuclide in the tissues of plants grown under laboratory conditions. The experiments were carried out in a sterile mixture of topsoil and sand, where in one series of experiments contaminated soil with 85Sr was used. Seeds were inoculated with a mixture of AMF at sowing, the second, control series was without application of AMF. Determination of radioactivity in soil and in different parts of tomato plants was carried out by gamma-spectrometric measurement. The obtained results and statistical analysis indicated a significant association between AMF inoculation of plants and the change in the distribution of 85Sr. The presence of AMF reduced the translocation of 85Sr from the root system to the aerial parts of tomato plants.

Establishment of Stem Cell-like Cells of Sida hermaphrodita (L.) Rusby from Explants Containing Cambial Meristems.

The in vitro cultures of plant stem cells and stem cell-like cells can be established from tissues containing meristematic cells. Chemical compounds-as well as their production potential-is among the emerging topics of plant biotechnology. We induced the callus cell biomass growth and characterized the parameters indicating the presence of stem cells or stem cell-like cells. Four types of explants (stem, petiole, leaf, root) from Sida hermaphrodita (L.) Rusby and various combinations of auxins and cytokinins were tested for initiation of callus, growth of sub-cultivated callus biomass, and establishment of stem cells or stem cell-like cells. Induction of callus and its growth parameters were significantly affected both by the explant type and the combination of used plant growth hormones and regulators. The responsibility for callus initiation and growth was the highest in stem-derived explants containing cambial meristematic cells. Growth parameters of callus biomass and specific characteristics of vacuoles confirmed the presence of stem cells or stem cell-like cells in sub-cultivated callus cell biomass. Establishment of in vitro stem cell or stem cell-like cell cultures in S. hermaphrodita can lead to the development of various applications of in vitro cultivation systems as well as alternative applications of this crop.

The impact of sewage sludge on the fungal communities in the rhizosphere and roots of barley and on barley yield.

Current problems with sewage sludge (SS) disposal could be solved by application to agricultural land considering its fertilizer properties and ability to improve soil condition. However, SS may contain heavy metals as well as pathogenic microorganisms. In this study, molecular analysis of partial 18S rRNA gene was used to study the impact of SS application into the soil on the genetic diversity of fungal communities, especially arbuscular mycorrhizal fungi in the rhizosphere and roots of barley. These samples were collected on three dates from the control soil without SS and from the soil with the addition of SS at the concentrations of 5 and 15 t ha-1. Fungal alpha diversity in the rhizosphere of barley was affected by SS differently than in barley roots. In addition, principal component analysis and cluster analysis revealed that fungal communities were strongly influenced by the SS addition into the soil, sample type, and the sampling date. This approach was complemented by an evaluation of the basic parameters of barley production and the response of these parameters to the presence of SS in the soil. The plant height increased with increasing SS concentration and the thousand seed weight significantly increased at the concentration of 5 t ha-1 SS but significantly decreased in 15 t ha-1.

Superabsorbent Polymer Seed Coating Reduces Leaching of Fungicide but Does Not Alter Their Effectiveness in Suppressing Pathogen Infestation.

Superabsorbent polymers (SAPs) applied to soil have been recognized as water reservoirs that allow plants to cope with periods of drought. Their application as a seed coat makes water available directly to the seeds during their germination and early growth phase, but on the other hand, it can affect the efficiency of plant protection substances used in seed dressing. In our experiments, we evaluated the effect of seed coating with SAP on fungicide leaching and changes in their effectiveness in suppressing Fusarium culmorum infestation. Leaching of fungicide from wheat seeds coated with SAP after fungicide dressing, as measured by the inhibition test of mycelium growth under in vitro conditions, was reduced by 14.2-15.8% compared to seeds without SAP coating. Germination of maize seeds and growth of juvenile plants in artificially infected soil did not differ significantly between seeds dressed with fungicide alone and seeds treated with SAP and fungicide. In addition, plants from the seeds coated with SAP alone grew significantly better compared to untreated seeds. Real-time PCR also confirmed this trend by measuring the amount of pathogen DNA in plant tissue. Winter wheat was less tolerant to F. culmorum infection and without fungicide dressing, the seeds were unable to germinate under strong pathogen attack. In the case of milder infection, similar results were observed as in the case of maize seeds.

Responses of Rhizosphere Fungal Communities to the Sewage Sludge Application into the Soil.

Due to the increasing sewage sludge production in the world and problems with its disposal, an application of sludge to the soil appears to be a suitable solution considering its fertilizer properties and ability to improve the soil physical conditions. On the other hand, the sludge may also contain undesirable and toxic substances. Since soil microorganisms are sensitive to environmental changes, they can be used as indicators of soil quality. In this study, we used sewage sludge (SS) from two municipal wastewater treatment plants (SS-A and SS-B) in the dose of 5 t/ha and 15 t/ha in order to determine possible changes in the fungal community diversity, especially arbuscular mycorrhizal fungi (AMF), in the rhizosphere of Arundo donax L. Rhizosphere samples were collected in summer and autumn for two consecutive years and the fungal diversity was examined using terminal restriction fragment length polymorphism and 18S rDNA sequencing. Fungal alpha diversity was more affected by SS-A than SS-B probably due to the higher heavy metal content. However, based on principal component analysis and ANOSIM, significant changes in overall fungal diversity were not observed. Simultaneously, 18S rDNA sequencing showed that more various fungal taxa were detected in the sample with sewage sludge than in the control. Glomus sp. as a representative of AMF was the most represented. Moreover, Funneliformis in both samples and Rhizophagus in control with Septoglomus in the sludge sample were other representatives of AMF. Our results indicate that the short-term sewage sludge application into the soil does not cause a shift in the fungal community composition.

Progress in the genetic engineering of cereals to produce essential polyunsaturated fatty acids.

Essential polyunsaturated fatty acids with more than two double bonds and length of carbon chain 18-22 must be taken in the diet to prevent diseases and imbalances caused by their deficiency. Terrestrial sources of polyunsaturated fatty acids are limited to only a few plant species whose large-scale cultivation is not possible and the production of their seeds and oil is ineffective. The complete biosynthetic pathway of fatty acids is known in organisms, including plants. After the first gene encoding the enzyme catalysing the initial steps of PUFA biosynthesis (ω-3 desaturase, Δ6-desaturase) were isolated, isolation of other genes encoding relevant enzymes of the PUFA pathway from different donor organisms followed. Genetic transformations of model plants by the desaturase- and elongase-encoding genes opened the way for the genetic engineering of oilseed crop species. Some of the developed transgenic plants produced PUFAs, including eicosapentaenoic and docosahexaenoic acids. Seed oils extracted from them were similar to fish oil. Tools of the synthetic biology can be applied in modifications of the PUFA pathway and also in overcoming of limitations when the gene and its expression product are absent in the pathway. Such progress in cereals (barley, wheat, maize) has been made only recently, when the first successful modifications of the ω-3 and ω-6 PUFA pathways succeeded. This review focuses on genetic modifications of the PUFA biosynthetic pathway in cereals in relation to the status reached in model plants and oilseed crops.

Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Biosynthesis of Essential Polyunsaturated Fatty Acids in Wheat Triggered by Expression of Artificial Gene.

The artificial gene D6D encoding the enzyme ∆6desaturase was designed and synthesized using the sequence of the same gene from the fungus Thamnidium elegans. The original start codon was replaced by the signal sequence derived from the wheat gene for high-molecular-weight glutenin subunit and the codon usage was completely changed for optimal expression in wheat. Synthesized artificial D6D gene was delivered into plants of the spring wheat line CY-45 and the gene itself, as well as transcribed D6D mRNA were confirmed in plants of T0 and T1 generations. The desired product of the wheat genetic modification by artificial D6D gene was the γ-linolenic acid. Its presence was confirmed in mature grains of transgenic wheat plants in the amount 0.04%-0.32% (v/v) of the total amount of fatty acids. Both newly synthesized γ-linolenic acid and stearidonic acid have been detected also in leaves, stems, roots, awns, paleas, rachillas, and immature grains of the T1 generation as well as in immature and mature grains of the T2 generation. Contents of γ-linolenic acid and stearidonic acid varied in range 0%-1.40% (v/v) and 0%-1.53% (v/v) from the total amount of fatty acids, respectively. This approach has opened the pathway of desaturation of fatty acids and production of essential polyunsaturated fatty acids in wheat.