Albendazole (ABZ), a widely used anthelmintic drug, enters the environment mainly via livestock excrements. To evaluate the environmental impact of ABZ, the knowledge of its uptake, effects and metabolism in all non-target organisms, including plants, is essential. The present study was designed to identify the metabolic pathway of ABZ and to test potential ABZ phytotoxicity in fodder plant alfalfa, with seeds and in vitro regenerants used for these purposes. Alfalfa was chosen, as it may meet manure from ABZ-treated animals in pastures and fields. Alfalfa is often used as a feed of livestock, which might already be infected with helminths. The obtained results showed that ABZ did not inhibit alfalfa seed germination and germ growth, but evoked stress and a toxic effect in alfalfa regenerants. Alfalfa regenerants were able to uptake ABZ and transform it into 21 metabolites. UHPLC-MS/MS analysis revealed three new ABZ metabolites that have not been described yet. The discovery of the parent compound ABZ together with the anthelmintically active and instable metabolites in alfalfa leaves shows that the contact of fodder plants with ABZ-containing manure might represent not only a danger for herbivorous invertebrates, but also may cause the development of ABZ resistance in helminths.

• Klíčová slova
• UHPLC-MS/MS, anthelmintics, drug metabolism, drug phytotoxicity, drugs in the environment,
• MeSH
• albendazol farmakologie   MeSH
• anthelmintika farmakologie   MeSH
• klíčení MeSH
• krmivo pro zvířata MeSH
• Medicago sativa účinky léků   růst a vývoj   metabolismus   MeSH
• metabolom * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• albendazol MeSH
• anthelmintika MeSH

Current research needs to be more focused on agronomical plants to effectively utilize the knowledge obtained from model plant species. Efforts to improve legumes have long employed common breeding tools. Recently, biotechnological approaches facilitated the development of improved legumes with new traits, allowing them to withstand climatic changes and biotic stress. Owing to its multiple uses and profits, alfalfa (Medicago sativa L.) has become a prominent forage crop worldwide. This review provides a comprehensive research summary of tissue culture-based genetic transformation methods, which could be exploited for the development of transgenic alfalfa with agronomically desirable traits. Moreover, advanced bio-imaging approaches, including cutting-edge microscopy and phenotyping, are outlined here. Finally, characterization and the employment of beneficial microbes should help to produce biotechnologically improved and sustainable alfalfa cultivars.

• Klíčová slova
• Advanced microscopy, bio-imaging, phenotyping, symbiotic interaction, tissue culture, transformation techniques, transgenic alfalfa,
• MeSH
• biotechnologie metody   MeSH
• elektroporace MeSH
• fixace dusíku MeSH
• geneticky modifikované rostliny genetika   MeSH
• Medicago sativa genetika   MeSH
• mikrobiota MeSH
• mikroskopie metody   MeSH
• symbolismus MeSH
• techniky tkáňových kultur metody   MeSH
• transformace genetická * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Nitrogen-fixing rhizobia and legumes have developed complex mutualistic mechanism that allows to convert atmospheric nitrogen into ammonia. Signalling by mitogen-activated protein kinases (MAPKs) seems to be involved in this symbiotic interaction. Previously, we reported that stress-induced MAPK (SIMK) shows predominantly nuclear localization in alfalfa root epidermal cells. Nevertheless, SIMK is activated and relocalized to the tips of growing root hairs during their development. SIMK kinase (SIMKK) is a well-known upstream activator of SIMK. Here, we characterized production parameters of transgenic alfalfa plants with genetically manipulated SIMK after infection with Sinorhizobium meliloti. SIMKK RNAi lines, causing strong downregulation of both SIMKK and SIMK, showed reduced root hair growth and lower capacity to form infection threads and nodules. In contrast, constitutive overexpression of GFP-tagged SIMK promoted root hair growth as well as infection thread and nodule clustering. Moreover, SIMKK and SIMK downregulation led to decrease, while overexpression of GFP-tagged SIMK led to increase of biomass in above-ground part of plants. These data suggest that genetic manipulations causing downregulation or overexpression of SIMK affect root hair, nodule and shoot formation patterns in alfalfa, and point to the new biotechnological potential of this MAPK.

• Klíčová slova
• Medicago sativa, SIMK, SIMKK, infection thread, nodule, root hair,
• MeSH
• biomasa MeSH
• Medicago sativa * genetika   MeSH
• mitogenem aktivované proteinkinasy kinas MeSH
• rostlinné proteiny * genetika   MeSH
• shluková analýza MeSH
• symbióza genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitogenem aktivované proteinkinasy kinas MeSH
• rostlinné proteiny * MeSH

Leguminous plants have established mutualistic endosymbiotic interactions with nitrogen-fixing rhizobia to secure nitrogen sources in root nodules. Before nodule formation, the development of early symbiotic structures is essential for rhizobia docking, internalization, targeted delivery, and intracellular accommodation. We recently reported that overexpression of stress-induced mitogen-activated protein kinase (SIMK) in alfalfa affects root hair, nodule, and shoot formation, raising the question of how SIMK modulates these processes. In particular, detailed subcellular spatial distribution, activation, and developmental relocation of SIMK during early stages of alfalfa nodulation remain unclear. Here, we characterized SIMK distribution in Ensifer meliloti-infected root hairs using live-cell imaging and immunolocalization, employing alfalfa stable transgenic lines with genetically manipulated SIMK abundance and kinase activity. In the SIMKK-RNAi line, showing down-regulation of SIMKK and SIMK, we found considerably decreased accumulation of phosphorylated SIMK around infection pockets and infection threads. However, this was strongly increased in the GFP-SIMK line, constitutively overexpressing green fluorescent protein (GFP)-tagged SIMK. Thus, genetically manipulated SIMK modulates root hair capacity to form infection pockets and infection threads. Advanced light-sheet fluorescence microscopy on intact plants allowed non-invasive imaging of spatiotemporal interactions between root hairs and symbiotic E. meliloti, while immunofluorescence detection confirmed that SIMK was activated in these locations. Our results shed new light on SIMK spatiotemporal participation in early interactions between alfalfa and E. meliloti, and its internalization into root hairs, showing that local accumulation of active SIMK modulates early nodulation in alfalfa.

• Klíčová slova
• Ensifer meliloti, Alfalfa, MAPKs, SIMK, immunolocalization, infection pocket, infection thread, light-sheet fluorescence microscopy, root hairs, subcellular localization,
• MeSH
• Medicago sativa genetika   metabolismus   MeSH
• mikroskopie MeSH
• mitogenem aktivované proteinkinasy * metabolismus   MeSH
• rostliny metabolismus   MeSH
• Sinorhizobium meliloti * metabolismus   MeSH
• symbióza fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitogenem aktivované proteinkinasy * MeSH

Global efforts are in rapid progress to tackle the emerging conundrum of climate change-induced heat stress in grassland ecosystems. Zinc oxide nanoparticles (n-ZnO) are known to play a crucial role in plants' abiotic stress regulation, but its response in alfalfa against heat stress has not been explored. This study aimed at assessing the effects of n-ZnO on alfalfa under heat stress by various morpho-physiological and cellular approaches. Five-week-old alfalfa seedlings were subjected to foliar application of n-ZnO as a pretreatment before the onset of heat stress (BHS) to evaluate its effect on heat tolerance, and as a post-treatment after heat stress (AHS) to evaluate recovery efficiency. In vitro studies on Zn release from n-ZnO by Inductively coupled plasma mass spectroscopy (ICPMS) disclosed that the particle uptake and Zn release were concentration dependent. The uptake and translocation of n-ZnO examined by transmission electron microscope (TEM) reveling showed that n-ZnO was primarily localized in the vacuoles and chloroplasts. TEM images showed that ultrastructural modifications to chloroplast, mitochondria, and cell wall were reversible by highest dose of n-ZnO applied before heat stress, and damages to these organelles were not recoverable when n-ZnO was applied after heat stress. The results further enlightened that 90 mg L-1 n-ZnO better prevented the heat stress-mediated membrane damage, lipid peroxidation and oxidative stress by stimulating antioxidant systems and enhancing osmolyte contents in both BHS and AHS. Although, application of 90 mg L-1 n-ZnO in BHS was more effective in averting heat-induced damages and maintaining better plant growth and morpho-physiological attributes compared to AHS. Conclusively, foliar application of n-ZnO can be encouraged as an effective strategy to protect alfalfa from heat stress damages while minimizing the risk of nanoparticle transmission to environmental compartments, which could happen with soil application.

• Klíčová slova
• Alfalfa tolerance and recovery, Antioxidant system, Chloroplast, Heat stress, Medicago sativa L., Oxidative stress,
• MeSH
• antioxidancia metabolismus   MeSH
• ekosystém MeSH
• Medicago sativa MeSH
• nanočástice * MeSH
• oxid zinečnatý * chemie   toxicita   MeSH
• semenáček MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antioxidancia MeSH
• oxid zinečnatý * MeSH

In the production of fermented feed, each crop can be contaminated with a variety of microorganisms that may produce natural pollutants. Biogenic amines, mycotoxins, and undesirable organic acids can decrease health feed safety. The aim of this study was to compare the counts of microorganisms, levels of biogenic amines, and the mycotoxins in forage legumes, and also to compare the occurrence of microorganisms and levels of mycotoxins in green fodder and subsequently produced silage and the influence of additives on the content of natural harmful substances in silage. The experimental plot was located in Troubsko and Vatín, in the Czech Republic. Two varieties of Medicago sativa and one variety of Trifolium pratense were compared. Green fodder and subsequently produced silage reaching up to 23% of dry matter were evaluated and prepared using a bio-enzymatic additive and a chemical additive. Green fodder of Medicago sativa was more contaminated by Enterococci than Trifolium pratense fodder. The obvious difference was determined by the quality of silage leachate. The silage prepared from Medicago sativa fodder was more contaminated with butyric acid. Fungi were present in higher counts in the anaerobic environment of green fodder and contaminated it with zearalenone and deoxynivalenol. Lower counts of fungi were found in silage, although the zearalenone content did not change. Lower content of deoxynivalenol was detected in silage, compared with green fodder. Silages treated with a chemical additive were found not to contain butyric acid. Lower ethanol content was determined, and the tendency to reduce the risk of biogenic amines occurrence was evident. The additives proved to have no influence on the content of mycotoxins.

• Klíčová slova
• biological additives, butyric acid, cadaverine, chemical additives, deoxynivalenol, enterococci, fungi, green matter, putrescine, silage, spermine, tyramine, zearalenone,
• MeSH
• biogenní aminy chemie   MeSH
• fermentace MeSH
• kontaminace potravin prevence a kontrola   MeSH
• krmivo pro zvířata mikrobiologie   MeSH
• Medicago sativa chemie   mikrobiologie   MeSH
• mykotoxiny chemie   MeSH
• potravinářské přísady farmakologie   MeSH
• siláž analýza   mikrobiologie   MeSH
• Trifolium chemie   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• biogenní aminy MeSH
• mykotoxiny MeSH
• potravinářské přísady MeSH

Fourteen genetically modified lines of alfalfa (Medicago sativa) containing the gene Ov from Japanese quail, coding for a methionine-rich protein ovalbumin, were evaluated for nodulation ability and concentration of aerobic bacteria in the rhizosphere. The transgenic lines were derived from a highly regenerable genotype Rg9/I-14-22, selected from cv. Lucia. On selective media, a higher concentration of ammonifying bacteria, bacterial spores, denitrifying and nitrifying bacteria were observed in the rhizosphere of transgenic clonesand, on the other hand, lower concentration of cellulolytic bacteria and Azotobacter spp. compared with the rhizosphere of non-transgenic clone SE/22-GT2. A statistically significant difference in the concentration of all the bacterial types was found between samples taken from two types of substrates (i.e. sterile vs. nonsterile). Higher bacterial concentration (measured as colony forming units per g soil dry mass) were observed for all tested groups of culturable bacteria in the non-sterile substrate. The presence of Azotobacter spp. was found only in the rhizosphere of plants grown in non-sterile soil in which the highest number of fertile soil particles (97 %) was observed in transgenic clones SE/22-9-1-12 and SE/22-11-1-1S.1. Concentration of bacteria involved in the N cycle in the soil was increased in the rhizosphere of transgenic clones and decreased in the rhizosphere of non-transgenic plants compared with the average value. In spite of some differences in colony numbers in samples isolated from the root rhizosphere of transgenic and nontransgenic alfalfa plants, we could not detect any statistically significant difference between individual lines.

• MeSH
• aerobní bakterie klasifikace   růst a vývoj   izolace a purifikace   metabolismus   MeSH
• Azotobacter klasifikace   izolace a purifikace   MeSH
• celulosa metabolismus   MeSH
• geneticky modifikované rostliny mikrobiologie   MeSH
• kořeny rostlin genetika   mikrobiologie   MeSH
• kvartérní amoniové sloučeniny metabolismus   MeSH
• Medicago sativa genetika   mikrobiologie   MeSH
• ovalbumin genetika   MeSH
• půdní mikrobiologie * MeSH
• spory bakteriální izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• celulosa MeSH
• kvartérní amoniové sloučeniny MeSH
• ovalbumin MeSH

For several decades, researchers are working to develop improved major crops with better adaptability and tolerance to environmental stresses. Forage legumes have been widely spread in the world due to their great ecological and economic values. Abiotic and biotic stresses are main factors limiting legume production, however, alfalfa (Medicago sativa L.) shows relatively high level of tolerance to drought and salt stress. Efforts focused on alfalfa improvements have led to the release of cultivars with new traits of agronomic importance such as high yield, better stress tolerance or forage quality. Alfalfa has very high nutritional value due to its efficient symbiotic association with nitrogen-fixing bacteria, while deep root system can help to prevent soil water loss in dry lands. The use of modern biotechnology tools is challenging in alfalfa since full genome, unlike to its close relative barrel medic (Medicago truncatula Gaertn.), was not released yet. Identification, isolation, and improvement of genes involved in abiotic or biotic stress response significantly contributed to the progress of our understanding how crop plants cope with these environmental challenges. In this review, we provide an overview of the progress that has been made in high-throughput sequencing, characterization of genes for abiotic or biotic stress tolerance, gene editing, as well as proteomic and metabolomics techniques bearing biotechnological potential for alfalfa improvement.

• Klíčová slova
• Medicago sativa, alfalfa, genomics, metabolomics, proteomics, stress resistance genes,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Twenty multiparous Friesian cows, 60 to 100 days postpartum, were allotted to two groups of 10 cows according to calving date, lactation number, and daily milk production and were assigned randomly to one of two diets in a crossover design experiment. The control diet (maize diet) was 55% maize silage (dry basis), 20% soybean meal, 22% ground maize, and 3% mineral and vitamin mix. The treatment diet (alfalfa diet) was 55% alfalfa silage, 10% soybean meal, 32% ground maize, and 3% mineral and vitamin mix. The two diets contained similar quantities of crude protein and metabolizable energy. The diets were offered individually as total mixed rations in two equal portions at 09:00 and 20:00 h in amounts to achieve ad libitum intake. The two groups of cows were allowed exercise in an open lot without shade from 11:00 to 16:30 h and 22:00 to 05:30 h. The cows were milked daily at 06:00 and 17:00 h. The dry matter, metabolizable energy and crude protein intakes, milk production, and milk fat, protein, lactose, total solids and solids not fat contents, as well as milk fat and protein yields and body condition score, were not significantly affected when maize silage was given to lactating cows, compared with that of cows given alfalfa silage. No differences were observed in blood serum concentrations of glucose, total protein, albumin, urea, triglycerides, cholesterol, Na, K, Ca, P and Mg between maize and alfalfa silages.

• MeSH
• krmivo pro zvířata * MeSH
• kukuřice setá MeSH
• laktace * MeSH
• Medicago sativa MeSH
• mléko chemie   MeSH
• skot krev   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• skot krev   fyziologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell suspension cultures were studied over their growth cycles. The total content of PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with high level of free putrescine (Put) (in exponential growth phase it represented about 65-73% of the intracellular Put pool). In contrast, the high content of soluble Put conjugates was found in tobacco cells (in exponential phase about 70% of the intracellular Put). Marked differences occurred in the amount of PAs excreted into the cultivation medium: alfalfa cells excreted at the first day after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight (FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively, while at the same time tobacco cells excreted only small amount of Put and Spd (12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad, respectively. In the course of lag-phase and the beginning of exponential phase the relative contents of extracellular PAs continually decreased (with the exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs in tobacco cells represented from day 3 only 0.1% from their total pools. The possible role of PA excretion into the cultivation medium in maintenance of intracellular PA contents in the cells of the two cell culture systems, differing markedly in growth rate and PA metabolism is discussed.

• MeSH
• buněčné kultury MeSH
• histaminasa metabolismus   MeSH
• kadaverin metabolismus   MeSH
• karboxylyasy metabolismus   MeSH
• Medicago sativa cytologie   metabolismus   MeSH
• ornithindekarboxylasa metabolismus   MeSH
• polyaminy metabolismus   MeSH
• putrescin metabolismus   MeSH
• spermidin metabolismus   MeSH
• spermin metabolismus   MeSH
• tabák cytologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arginine decarboxylase MeSH Prohlížeč
• histaminasa MeSH
• kadaverin MeSH
• karboxylyasy MeSH
• ornithindekarboxylasa MeSH
• polyaminy MeSH
• putrescin MeSH
• spermidin MeSH
• spermin MeSH