Optimization of Barley Mature Embryo Regeneration and Comparison with Immature Embryos of Local Cultivars
Regeneration ability of plant cells or tissues in explant culture is one of the key factors affecting success of genetic transformation. In experiments, the effect of explant type (whole embryo, scutellum embryonic axis, meristematic/central zone of embryonic axis) and plant growth regulators (BAP or TDZ) on mature embryo regeneration was determined. Explant type significantly affected regeneration efficiency. While no regenerants were observed using mature scutella, whole embryos or embryonic axes produced the highest number of regenerants. Using embryonic axes with discarded apical and basal parts, regeneration efficiency dramatically decreased. No statistical differences in regeneration were observed between BAP and TDZ added to the regeneration medium in concentration 0.1 or 1 mg l-1. At last, regeneration ability of mature embryos of nine Slovak spring barley cultivars (Donaris, Ezer, Levan, Ludan, Nitran, Pribina Sladar, Orbit, Pax) and Golden Promise as a model cultivar was examined and compared with regeneration ability of immature embryos which have been usually used for genetic transformation of barley. Although the regeneration from mature embryos was very weak, the same cultivars Golden Promise, Pribina and Levan showed the best regeneration ability by using both, immature and mature embryos. On the other hand cultivars Ezer and Pax belonged to the weakest ones in both experiments.
Marcela Gubišová, Jozef Gubiš and Alžbeta Žofajová
The effect of plant propagation method on growth parameters and the yield of above-ground biomass in two species of gigantic grasses were measured during three growing seasons. Plants were multiplied in explant culture and through traditional methods – by rhizome segments (Miscanthus × giganteus) or by stem cuttings (Arundo donax). In the case of M. × giganteus, in vitro-multiplied plants produced more shoots with significantly lower diameter, but the differences in the number of shoots, plant height and the yield of dry biomass were not statistically significant. Different results were observed for A. donax, where in vitro-multiplied plants showed significantly weaker results in all parameters, with the exception of the number of shoots in the first measured season. In both the species, there was observed the strong effect of the year. While in M. × giganteus the yield of dry biomass gradually decreased during the measured years, it increased in the case of giant reed.
Katarína Ondreičková, Mária Babulicová, Daniel Mihálik, Marcela Gubišová and Jozef Gubiš
Terminal restriction fragment length polymorphism was used to determine the bacterial community diversity in crop rotations with different proportion of cereals (40%, 60% and 80%) and various fertilisation treatments (F1 - mineral fertiliser amendment + the application of organic fertiliser Veget® and F2 - mineral fertilisation) sampled on two dates (July and October 2012). No statistically significant differences in the number of terminal restriction fragments were detected by Fisher´s least significant difference between two types of fertilisation or among samples with different proportion of cereals. In contrast, statistically significant differences were detected between samples collected in July and October, when in October there was a 43.5% reduction in the number of bacterial species in comparison with July. Principal component analysis as well as cluster analysis showed that a higher similarity in composition of bacterial communities was present among all soil samples collected in July and two samples collected in October. Other samples collected in October were separated from each other. The results of this study demonstrated that bacteria abundance and community composition were not affected by the proportion of cereals and fertiliser used.
Katarína Ondreičková, Alžbeta Žofajová, Michaela Piliarová, Jozef Gubiš and Martina Hudcovicová
In this study, bacterial genetic diversity from the rhizosphere of barley and wheat were studied. The plants were sown in pots with aliquot amount of 15 t/ha concentration of soil additive derived from sewage sludge and agricultural by-products represented by wastes from grain mill industry and crushed corn cobs. The plants sown in pots without the addition of soil additive represented control samples. The rhizosphere samples were collected on two dates (plant flowering and maturity) and the composition of bacterial communities were detected using two molecular fingerprinting methods – automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (T-RFLP). Microbial biomass expressed as the amount of metagenomics DNA was higher in soils with addition of soil additive, except during maturity stage in barley rhizosphere. Nevertheless, statistically significant differences between control and sludge samples were not detected in any case. Similarly, no changes were detected in the composition of bacterial community between control and sludge samples in barley and wheat rhizosphere by using cluster analysis. Only minor temporal changes in the composition of bacterial community between flowering and maturity periods were observed. These changes were related to the samples collected in the plant maturity stage. In this stage, plants were completely mature and their impact on the rhizosphere bacterial communities in the form of root exudates was limited. Statistically significant differences between ARISA and T-RFLP methods were detected in all measured values of diversity indices. Despite these differences, both methods gave results leading to similar conclusions.
Katarína Ondreičková, Marcela Gubišová, Jozef Gubiš, Lenka Klčová and Miroslav Horník
Application of sewage sludge to soil is a potentially inexpensive source of nutrition for plants, but may contain undesirable and toxic substances, e.g. heavy metals. Alterations in microbial communities can serve as an environmental indicator of possible soil contamination. We used two molecular fingerprinting methods (Automated Ribosomal Intergenic Spacer Analysis, ARISA and Terminal Restriction Fragment Length Polymorphism, T-RFLP) to study changes in the genetic diversity of bacterial communities in the rhizosphere of Arundo donax L. cultivated in the soil fertilised with additive based on sewage sludge from wastewater treatment plant and agricultural by-products represented by crushed corn hobs and wastes from grain mill industry. The metagenomic DNA extracted from rhizosphere samples were collected in August and November 2014. The amount of mgDNA was statistically higher in samples with additive than in control samples without it in both dates. The Venn diagrams showed that operational taxonomic units which were common to all samples were represented in 32.8% in ARISA and 43.4% in T-RFLP. However, based on Principal component analysis and subsequent PERMANOVA statistical tests did not confirm significant differences in the rhizosphere of control plants and plants grown in the soil supplemented with sewage sludge in dose 5 and 15 t/ha present in the additive.
Zuzana Dürešová, Anna Šuňovská, Miroslav Horník, Martin Pipíška, Marcela Gubišová, Jozef Gubiš and Juraj Lesný
The aim of the present work was to compare the accumulation and translocation of Cd and Zn in plants of tobacco (Nicotiana tabacum L.), celery (Apium graveolens L.), maize (Zea mays L.), giant reed (Arundo donax L.), and alpine pennycress (Noccaea caerulescens L.) under conditions of short-term hydroponic experiments using nutrient solutions spiked with radionuclides 109Cd or 65Zn, and direct gamma-spectrometry. It was found that the time-course of metals accumulation in studied plants was not different in terms of target metal, but it was significantly different on the level of plant species. The highest values of Cd accumulation showed plants of giant reed, whereby the accumulation decreased in the order: giant reed > tobacco > alpine pennycress >> maize and celery. On the basis of concentration ratios (CR) [Me]shoot / [Me]root calculation for both metals, it was found that Cd and Zn were in prevailing part accumulated in the root tissues and only partially accumulated in the shoots, where the amount of accumulated Cd and Zn increased from the oldest developed leaves to the youngest developed leaves. The CR values corresponding to these facts were calculated in the range 0.06 – 0.27 for Cd and for Zn 0.06 – 0.48. In terms of plant species, the CR values obtained for Cd decreased in the order: maize > celery > tobacco and giant reed > alpine pennycress. The similarity between studied objects – individual plant species on the basis of the obtained variables defining Cd or Zn accumulation at different conditions of the experiments as well as the relationships between obtained variables and conditions of the experiments were subjected to multivariate analysis method – cluster analysis (CA). According to the findings and this analysis, it can be expected that plants of tobacco and giant reed will dispose with similar characteristics as plants of alpine pennycress, which are classified as Zn/Cd hyperaccumulators, in terms of Cd or Zn accumulation and other positive parameters for their utilization in phytoremediation processes and techniques.
Denisa Partelová, Klára Kuglerová, Yevheniia Konotop, Miroslav Horník, Juraj Lesný, Marcela Gubišová, Jozef Gubiš, Peter Kováč and Ildikó Matušíková
The current findings show that positron emission tomography (PET), primarily developed for medical diagnostic imaging, can be applied in plant studies to analyze the transport and allocation of wide range of compounds labelled with positronemitting radioisotopes. This work is focused on PET analysis of the uptake and transport of 2-deoxy-2-fluoro[18F]-D-glucose (2-[18F]FDG), as a model of photoassimilates, in tissues of giant reed (Arundo donax L. var. versicolor) as a potential energy crop. The absorption of 2-[18F]FDG and its subsequent transport in plant tissues were evaluated in both acropetal and basipetal direction as well. Visualization and quantification of the uptake and transport of 2-[18F]FDG in plants immersed with the root system into a 2-[18F]FDG solution revealed a significant accumulation of 18F radioactivity in the roots. The transport rate in plants was increased in the order of plant exposure through: stem > mechanically damaged root system > intact root system. PET analysis in basipetal direction, when the plant was immersed into the 2-[18F]FDG solution with the cut area of the leaf of whole plant, showed minimal translocation of 2-[18F]FDG into the other plant parts. The PET results were verified by measuring the accumulated radioactivity of 18F by direct gamma-spectrometry.