Cytokinin homeostasis and expression of the genes involved in cell cycle control during kohlrabi (Brassica oleracea L. var. gongylodes) regeneration in vitro

Regeneracija in vitro gajenog biljnog tkiva kontrolisana je regulatorima rastenja, naročito citokininima i auksinima. Egzogeno primenjeni biljni regulatori rastenja mogu interagovati sa endogenim biljnim hormonima i uticati na njihovu biosintezu ili distribuciju, odnosno menjati hormonalnu homeostazu koja utiče na razviće in vitro. Proces regeneracije biljaka in vitro podrazumeva intenzivne ćelijske deobe, a centralnu ulogu u kontroli ćelijskog ciklusa i razvića organa imaju kompleksi ciklina i kinaza zavisnih od ciklina na čiju sintezu, odnosno aktivnost, utiču pomenuti biljni hormoni. U tom smislu, ciljevi ovog rada bili su uspostavljanje efikasnih protokola za regeneraciju biljaka kelerabe (Brassica oleracea L. var. gongylodes) in vitro, sa posebnim osvrtom na identifikaciju i kvantifikaciju različitih grupa citokinina i indol-3-sirćetne kiseline (IAA) u eksplantatskim tkivima sa regenerisanim izdancima, kao i analiziranje obrazaca i intenziteta ekspresije gena koji kodiraju cikline i kinaze zavisne od ciklina tokom de novo organogeneze izdanaka. Efikasan sistem za repetitivnu somatsku embriogenezu (SE) kod kelerabe, sorta Bečka Plava (BP), je uspostavljen iz nezrelih zigotskih embriona. Najveća frekvencija SE detektovana je kod nezrelih zigotskih embriona u ranom kotiledonarnom stadijumu razvića gajenih na hranljivoj podlozi bez regulatora rastenja. Uspešna indukcija de novo organogeneze izdanaka kelerabe, sorte BP i Bečka Bela (BB), postignuta je kod odsečaka hipokotila i intaktnih klijanaca, na podlogama sa pojedinačnim citokininima (CK): 6-benzilaminopurin (BAP), tidiazuron (TDZ), transzeatin (transZ) ili cis-zeatin (cisZ). Frekvencija regeneracije kao i vijabilnost regenerisanih izdanaka obe sorte je bila veća kod intaktnih klijanaca, dok su među primenjenim citokininima BAP i TDZ bili najefikasniji u indukciji de novo organogeneze. Kvantifikacija endogenog sadržaja citokinina i IAA u odsečcima hipokotila sa regenerisanim izdancima (HRI) kao i u intaktnim klijancima sa regenerisanim izdancima (KRI) je ukazala na to da uočene razlike u organogenom odgovoru ova dva tipa početnog biljnog materijala mogu biti povezane sa njihovim hormonskim statusom...vitro plant regeneration is under control of plant growth regulators (PGRs), especially cytokinins and auxins. Exogenously applied PGRs can affect the homeostasis of endogenous plant hormones by influencing their biosynthesis and distribution, subsequently altering in vitro development. Intensive cell divisions represent the basis of in vitro plant regeneration, and they are controlled by complexes of cyclins and cyclin-dependent kinases. The activity of these complexes during cell cycle and organ development is affected by plant hormones. Respecting this, the aims of this study were developing efficient protocols for in vitro regeneration of kohlrabi (Brassica oleracea L. var. gongylodes) with an emphasis on identification and quantification of different groups of endogenous cytokinins and indole-3-acetic acid (IAA) in explant tissue with respective regenerated shoots, as well as analyzing expression of genes encoding cyclins and and cyclin-dependent kinases during de novo shoot organogenesis. Efficient system for repetitive somatic embryogenesis (SE) was developed for kohlrabi cv. Vienna Purple (VP) using immature zygotic embryos. The highest frequency of SE was obtained from immature zygotic embryos at the early cotyledonary stage grown on PGR-free medium. Successful induction of de novo shoot organogenesis in kohlrabi cv VP and cv. Vienna White (VW) was achived using hypocotyl explants and intact seedlings, cultivated on media supplemented with single cytokinin (CK): 6-benzylaminopurine (BAP), thidiazuron (TDZ), trans-zeatin (transZ) or cis-zeatin (cisZ). Higher frequency of shoot regeneration as well as viability of regenerated shoots was detected for intact seedlings, with BAP and TDZ being the most efficient in de novo organogenesis induction. Quantification of endogenous cytokinin and IAA contents in both hypocotyl explants and seedlings with regenerated shoots (HRSs and SRSs, respectively) suggested that the observed differences in organogenic response between these two types of starting plant material were related to their cytokinin and IAA contents. The level of total CKs was higher in HRSs, while SRSs displayed a higher IAA/bioactive CKs ratio..

The quality of digested residue after anaerobic digestion of chicken manure

Cilj ovog rada je utvrditi kakvoću digestiranog ostatka nakon anaerobne digestije pilećeg gnoja. Istraživanja su provedena na postrojenju za proizvodnju bioplina u Republici Austriji anaerobnom digestijom pri 35°C. Istraživanja su obuhvatila kemijske analize (pH, elektroprovodljivost, suha tvar, žareni ostatak i gubitak žarenjem, organska tvar, C, N, P, K, Ca, Mg, Na, Mn, Zn, Cu, Fe, Pb, Cd) i bakteriološke pretrage uzoraka. Utvrđeno je da digestirani ostatak u svim uzorcima, koji su blago alkalni, sadrži niski udio suhe tvari od koje je oko 75% organska tvar. Biogeni elementi su u svim istraživanim uzorcima u umjerenoj koncentraciji, vrijednost teških metala nalazi se u dopuštenim granicama. Analizom N:P:K, vrijednosti biogenih elemenata i teških metala može se zaključiti da se digestirani ostatak nakon anaerobne digestije pilećeg gnoja može primijeniti u poljoprivrednoj proizvodnji, posebice u ratarstvu i travnjaštvu. U digestiranom ostatku su pronađeni mezofilni i termofilni mikroorganizmi, dok kriofilnih nije bilo. Ni u jednom uzorku nisu pronađene patogene bakterije. Sve izolirane bakterije potpuno su uništene nakon sterilizacije.The aim of this paper is to determine the quality of the digested residue after the process of anaerobic digestion by using chicken manure as a raw material. The research was conducted in the Republic of Austria in a plant for biogas production by means of anaerobic digestion at 35°C. The research comprised chemical analysis (pH, electrical conductivity, dry matter, burning residue, burning loss, organic matter, C, N, P, K, Ca, Mg, Na, Mn, Zn, Cu, Fe, Pb, Cd) and bacteriological tests of samples. .It was found that the digested residue in all of the samples, all of which were mildly alkaline, contained a low level of dry matter, 75% of which was organic matter. Biogenic elements in all investigated samples were present in moderate concentration; the values of heavy metals were within approved limits. The analysis of the N:P:K ratio, values of the biogenic elements and heavy metals, leads to the conclusion that the digested residues of chicken manure can be used in agricultural production, especially in plant production and grassland cultivation. Mesophilic and thermophilic microorganisms were found in the digested residue samples, but there were no criophilic microorganisms. Not one sample showed proof of pathogenic bacteria. All isolated bacteria were destroyed in the process of sterilization

Precipitirajući čimbenici i simptomi u bolesnika s dijabetičkom ketoacidozom

The aim of the study was to determine the most common precipitating factors and symptoms of diabetic ketoacidosis and the possible difference according to age, gender and severity of diabetic ketoacidosis. Medical records from January 1, 2017 until December 31, 2019 were reviewed and patients diagnosed with diabetic ketoacidosis were selected. The study included 52 patients, median age 34 (interquartile range 21-56) years. There was no statistically significant difference between male and female gender. The severity of diabetic ketoacidosis was moderate in the majority of cases (65.4%; p=0.005). The most common precipitating factor was infection (61.7%). In patients with moderate diabetic ketoacidosis, respiratory infections were more common, while gastrointestinal infections were more common in severe diabetic ketoacidosis (33% and 25%, respectively; p=0.03). Nausea (median age 32 years; p=0.004) and vomiting (median age 31 years; p=0.01) were more common in younger age groups, while altered mental status was more common in the older age group (median age 61 years; p=0.001). Infection was the most common precipitating factor. The most common symptoms in younger age groups were nausea and vomiting, and altered mental status in the older age group.Cilj istraživanja je bio utvrditi najčešće precipitirajuće čimbenike i simptome dijabetičke ketoacidoze te postoji li razlika s obzirom na dob, spol i stupanj dijabetičke ketoacidoze. Pregledana je medicinska dokumentacija od 1. siječnja 2017. do 31. prosinca 2019. godine te su izdvojeni bolesnici s dijagnozom dijabetičke ketoacidoze. U istraživanje je uključeno 52 bolesnika. Medijan dobi bio je 34 godine (interkvartilni raspon 21-56 godina). Nije postojala statistički značajna razlika muškog i ženskog spola. U najvećem broju slučajeva radilo se o umjerenom stupnju dijabetičke ketoacidoze (65,4%; p=0,005). Najčešći precipitirajući čimbenik bila je infekcija (61,7%). Kod umjerenog stupnja najčešće se radilo o respiracijskim infekcijama, a u teškom stupnju o gastrointestinalnim infekcijama (33% i 25%; p=0,03). U mlađim dobnim skupinama češće su se pojavljivale mučnina (medijan dobi 32 godine; p=0,004) i povraćanje (medijan dobi 31 godina; p=0,01), a u starijoj dobnoj skupini poremećaj svijesti (medijan dobi 61 godina; p=0,001). Infekcije su bile najčešći precipitirajući čimbenik dijabetičke ketoacidoze. Najčešći simptomi u mlađim dobnim skupinama bili su mučnina i povraćanje, a u starijoj dobnoj skupini poremećaj svijesti

Serum glucose, sodium and potassium concentrations in patients with diabetic ketoacidosis

Aim To investigate possible differences in serum glucose and sodium and potassium concentrations with respect to age, gender and severity of diabetic ketoacidosis. Methods Medical records from 1 January 2017 to 31 December 2019 were reviewed and patients with the diagnosis of diabetic ketoacidosis were selected. Results The study included 52 patients. Glucose concentration was significantly higher in the age group of 25-44 and >65 years compared to the group of 18-24 years (p=0.02). Sodium concentration was significantly higher in the age group 18-24 and >65 years compared to groups 25-44 and 45-65 years (p=0.002). Females had significantly higher sodium concentration than males (p=0.002). Potassium concentration was significantly higher in the age group 25-44 years compared to other groups (p=0.01). Males had significantly higher potassium concentration (p =0.01). Conclusion This study showed that significant differences exist in electrolyte concentration between specific age groups, male and female gender as well as DKA severity. Knowing these differences could help clinicians to promptly recognize and treat electrolyte derangements, leading to better outcome of patients with DKA

The effects of β-lactam antibiotics and hygromycin B on de novo shoot organogenesis in apple cv. Golden Delicious

Since the genetic transformation of the apple is strongly genotype-dependent and generally inefficient, the evaluation of factors affecting shoot regeneration are crucial for the establishment of a successful transformation process. In this report, we evaluated the effects of the β-lactam antibiotics meropenem and timentin on in vitro regeneration via de novo shoot organogenesis from leaf explants of apple cv. Golden Delicious, as well as on the growth of the Agrobacterium tumefaciens strain EHA 105, and compared them with the commonly used β-lactam cefotaxime. Also, we report for the first time the effect of hygromycin B as a selective agent in the domesticated apple, as regards shoot regeneration and shoot multiplication efficiency. We observed that cefotaxime and timentin at concentrations higher than 100 mg L-1 were sufficient to prevent Agrobacterium growth during a two-week period, while meropenem exhibited an inhibitory effect on bacterial growth at all tested concentrations (25-150 mg L-1). Cefotaxime at a concentration of 300 mg L-1 increased the number of regenerated shoots per explant (9.39) in comparison with the control (7.67). In contrast to cefotaxime, meropenem and timentin caused a decrease in shoot regeneration efficiency, but larger and more developed shoots were obtained on meropenem (25-125 mg L-1) after the same period of cultivation. Hygromycin B at a concentration of 5 mg L-1 or higher completely inhibited shoot regeneration and induced explant tissue necrosis. Therefore, the selection procedure with a final concentration of 4 mg L-1 throughout organogenesis and 10 mg L-1 for further shoot growth and multiplication is recommended for an efficient transformation process in apple cv. Golden Delicious

Sulforaphane affects morphological changes and cell viability of colon cancer cells: p53 mutation-dependent effect

Colorectal carcinoma (CRC) is an increasing cause of morbidity and mortality worldwide. Among biologically active compounds which have been shown to exert cytotoxic effects on human cancer cells, including CRC, is sulforaphane (SFN), an isothiocyanate compound extracted from cruciferous vegetables, especially broccoli. This in vitro study aimed to investigate the effect of SFN on the growth of human CRC cells and its dependency on the expression of p53. Two human CRC cell lines: HT-29 (a p53 mutated line) and HT-116 (a p53 wild-type line) were treated with SFN at concentrations of 0, 4, 8, 16, and 32 μmol/L for 72 h to test its anticancer effect. Results indicated that SFN induced cell morphological changes and decreased the total number of viable cells. Treatment of CRC cells with SFN for 72 h resulted in moderate dose-dependent cytotoxicity. HCT-116 cells, with a p53-wt, were more sensitive (IC50 = 8.41 μM) than p53-mutated HT-29 cells (IC50 = 24.83 μM). These results indicate that SFN exhibits the anticancer effect against CRC cells in p53 mutation- dependent manner (Serbia-China project: 451-03-1203/2021-09)

Toxicity testing of D, L-sulforaphane in a zebrafish model

Sulforaphane (SFN) belongs to the group of isothiocyanates and is present in many cruciferous plants, especially broccoli. The positive properties of this bioactive molecule in the form of extracts have been shown in numerous studies and include antibacterial, cardioprotective, and neuroprotective effects, antioxidant and immunomodulatory effects, as well as positive effects in various cancers. However, the potential for harmful effects of SFN has not been sufficiently investigated, particularly for chemically obtained D,L-sulforaphane. This study aimed to investigate the toxicity of D,L-sulforaphane on zebrafish (Danio rerio) model. Wild (AB) strain embryos and zebrafish transgenic lines with fluorescently labeled liver cells (Tg(fabp:EGFP)) and endothelial cells of blood vessels (Tg(fli1:EGFP)) were used, treated with different concentrations of SFN (1 to 20 μg/mL). The survival of embryos, developmental toxicity, hepatotoxicity and cardiotoxicity were monitored for five days. A concentration of 20 μg/mL of D,L-sulforaphane caused the death of all embryos, while the median lethal concentration (LC50) was found to be 14.2 μg/mL. D,L-sulforaphane exhibited toxic effects at concentrations higher than 3 μg/mL, primarily on the development of the swim bladder (4 μg/mL), and growth and development of embryos (4.58 μg/mL), while harmful effects on the liver (liver size and yolk resorption) were observed at a concentration of 10 μg/mL. Effects on the cardiovascular system were not observed at concentrations from 1 to 10 μg/mL. The investigation of D,L-sulforaphane on zebrafish embryos showed that harmful effects occur at very low concentrations, indicating the need for further investigation of toxicological potential of this molecule

Induction of potato (Solanum tuberosum L.) defense genes by French marigold (Tagetes patula L.) essential oil

When attacked, plants activate defense mechanisms including volatile emission. Besides havinga pest repellent effect, volatiles can induce defense response in surrounding unharmed plants.French marigold (Tagetes patula L.) essential oil (EO) was isolated from the above-ground partsof plants collected from nature by hydrodistillation using Clevenger apparatus. GC/MS analysisrevealed the presence of 42 compounds, of which 97% (32 compounds) have been identified.The monoterpene fraction was dominant (76.9%) with terpinolene (32.4%) and limonene (14.7%)as the most abundant compounds. In order to investigate the potential of marigold EO to inducethe defense of potato, we analyzed 3 genes related to the induced resistance (IR): pathogenesisrelated PR-2 and PR-5 genes, and proteinase inhibitor Pin2. The potato plants were exposed to EOvolatiles during 4, 8 and 12 h. Two leaves of EO-treated or untreated control plants were collected,and plants were then additionally injured by mechanically wounding leaves across the leaf surface.After 24 h of wounding, another two leaves from each plant were collected and expressionof the IR related genes was analyzed by qPCR method. EO induced altered expression of PR genes,while Pin2 wasn’t affected by the treatment. Mechanical wounding increased the expression ofall genes after 4 and 8 h of EO treatment, with up to 4-fold change in PR-2. However, in plantsexposed to EO for 12 h before wounding, the level of transcription in PR-2 and Pin2 was similar tothat detected in untreated controls. These findings support the efforts for environmentally-safepest control in crops by using natural compounds.Uzelac B, editor. Book of abstracts. 3rd International Conference on Plant Biology (22nd SPPS Meeting); 2018 Jun 9-12; Belgrade, Serbia. Belgrade: Serbian Plant Physiology Society; Institute for Biological Research "Siniša Stanković"; Faculty of Biology; 2018. p. 58

Svest o korišćenju veterinarskih lekova u regionu Sjenice kao potencijalnih zagađivača životne sredine

The aim of this study was to evaluate the awareness of livestock breeders on the used veterinary drugs in order to assess potential environmental contamination in the region of Sjenica. Veterinary drugs can enter the environment mostly due to widespread usage, but also as metabolites eliminated from the organisms. In order to evaluate the awareness of livestock breeders, a survey questionnaire was used. Questions were related to the most frequently used veterinary drugs without the supervision of a veterinary medical doctor. The obtained data show that the awareness of domestic animal breeders on the use of veterinary drugs is very low in the region of Sjenica. The most commonly used veterinary medicines were antibiotics, streptomycin, and benzylpenicillin-procaine, antihelmintic, albendazole. Unexpectedly, the animal breeders identified formaldehyde as veterinary medicine and used it as a disinfectant in poultry raising. The results also show that breeders frequently treat animals with drugs on their own, without control by veterinary medical doctors. Because of the wide use of veterinary medicines in the region of Sjenica, it monitoring of the environmental concentration of streptomycin, benzylpenicillin-procaine, and albendazole, as well as metabolites in soil and water should be recommended. Moreover, human exposure to these veterinary medicines could occur because of the abuse of veterinary medicines, no consultations with veterinary medical doctors, and non-proper handling of waste from animals.Veterinarski lekovi dospevaju u životnu sredinu najčešće usled neadekvatnog odlaganja otpada, ali i kao metaboliti iz organizma životinja. Cilj studije je bio procena nivoa svesti uzgajivača stoke o upotrebi veterinarskih lekova u regionu Sjenice sa ciljem procene potencijalne kontaminacije životne sredine. Za procenu nivoa svesti uzgajivača stoke korišćen je anketni upitnik. Pitanja iz upitnika su se odnosila na najčešće korišćene veterinarske lekove i korišćenje bez nadzora veterinara. Dobijeni podaci pokazuju da je nivo svestu uzgajivača stoke u regionu Sjenice nizak jer koriste veterinarske lekove bez nadzora veterinara. Najčešće koriste antibiotike, streptomicin i benzoil-penicilin, antihelmintik – abendazol i, iznenađujuće, koriste formaldehid za dezinfekciju prostorija za smeštaj životinja, pri čemu ga smatraju veterinarskim lekom. Zbog rasprostranjene upoutrebe veterinarskih lekova u regionu Sjenice od strane uzgajivača stoke, a bez kontrole veterinara preporučuje se monitoring streptomicina, benzoilpenicilina i abendazola kao i njihovih metabolita u životnoj sredini, zemljištu i vodi. Potrebno je preduzeti dodatne mere opreza da zbog niskog nivoa svesti ne bi došlo do izloženosti ljudi ovim veterinarskim lekovima i njihovim metabolitima naročito zbog neadekvatnog tretiranja stajnjaka i drugog otpada.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Supplementary data for the article: Stupar, S.; Dragićević, M.; Tešević, V.; Stanković-Jeremić, J.; Maksimović, V.; Ćosić, T.; Devrnja, N.; Tubić, L.; Cingel, A.; Vinterhalter, B.; Ninković, S.; Savić, J. Transcriptome Profiling of the Potato Exposed to French Marigold Essential Oil with a Special Emphasis on Leaf Starch Metabolism and Defense against Colorado Potato Beetle. Plants 2021, 10 (1), 172. https://doi.org/10.3390/plants10010172.

Supplementary material for: [https://doi.org/10.3390/plants10010172]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4499