Physiological, biochemical and molecular basis of drought and osmotic stress tolerance in the early stages of vegetative development of selected Pisum genotypes

Suša i osmotski stres, pored efekata na rastenje i razviće biljaka, dovode i do oksidativnog stresa i nastajanja velike količine reaktivnih kiseoničnih vrsta (ROS). Kako bi neutralisale štetne efekte ROS, biljke su razvile antioksidativne sisteme zaštite. Cilj istraživanja ove disertacije je bio da se detaljno prouče fiziološki i biohemijski procesi koji su u osnovi reakcija biljaka stočnog graška na sušu i osmotski stres. Cilj je bio i da se u ova ispitivanja uključe i molekularna istraživanja, koja bi, uz analizu procesa rastenja i drugih fizioloških procesa (aktivnost antioksidativnih enzima, akumulacije i distribucije stres hormona abscisinske kiseline), značajno doprinela razumevanju efekata, ne samo osmotskog i stresa suše, već i oksidativnog stresa na biljke stočnog graška. Istraživanja su vršena na 7 odabranih genotipova roda Pisum iz kolekcije Instituta za ratarstvo i povrtarstvo iz Novog Sada. Tretmani su obuhvatili dva eksperimentalna sistema: osmotski stres i sušu. Za indukciju osmotskog stresa su korišćene različite koncentracije rastvora osmotikuma PEG 6000 osmotskog potencijala (od -0,1MPa i -0,2MPa) primenjene u kraćem (10 dana) i dužem vremenskom intervalu (15 dana) kod klijanaca i ponika biljaka. Kao kontrolna varijanta je korišćena voda bez dodatka PEG 6000. Eksperiment suše je obavljen kod biljaka gajenih u kontrolisanim uslovima (u fitotronskoj komori) i to tako da su biljke zalivane optimalno do pojave trećeg para listova, a potom je suša u zemlištu izazvana prekidanjem zalivanja biljaka do 18% (umerena suša) i 9% sadržaja vode u spustratu (jaka suša). Biljke u kontroli su zalivane do optimalnog vodnog režima zamljišta (36% sadržaja vode u supstratu). U ogledu osmotskog stresa u fazi klijanja su obavljena fiziološka ispitivanja uticaja osmotskog stresa na % klijavosti i srednje vreme klijanja ili Mean Germination Time, dok su u fazi ponika obavljena ispitivanja dužine i biomase nadzemnog dela i korena, kao i odnosa suve mase nadzemnog dela i korena, a izračunat je i indeks tolerantnosti za nadzemni deo i koren...Drought and osmotic stress, in addition to the effects on plant growth and development, lead to oxidative stress and the production of a large quantity of reactive oxygen species (ROS). Plants have developed antioxidant defense mechanisms in order to neutralise the damaging effects of ROS. The aim of this dissertation was to investigate the physiological and biochemical processes which are in the base of field pea reaction to drought and osmotic stress. The aim was also to include molecular research in the study, along with the analysis of growth and other physiological processes (activity of antioxidant enzymes, accumulation, and distribution of plant stress hormons abscisic acid), which would significantly improve understanding of not only osmotic and drought stresses, but also about the effects of oxidative stress on field pea plants. Studies were conducted on seven selected genotypes of the genus Pisum from the collection of the Institute of Field and Vegetable Crops, Novi Sad. Treatments consisted of two experimental systems: osmotic and drought stresses and drought in the environmental test chamber. For induction of osmotic stress different concentrations of osmotic solutions PEG 6000 (-0.1MPa and -0.2MPa) were used in a shorter (10 days) and longer time interval (15 days). Water without PEG 6000 was used as control. The drought experiment was conducted in control conditions (phytotron chamber) in a such waz that the plants were irrigated at the optimal soil water content until the third pair of leaves began emergence, after which soil drought was induced by termination of irrigation up to 18% (moderate drought), and 9% substrate water content (severe drought). Control plants were irrigated at the optimal level of soil water regime (36% substrate water content). In osmotic stress experiment in the germination stage physiological tests included measurements of germination percentage and Mean Germination Time, while in the seedling stage was performed testing of the length and biomass of plant shoot and root, as well as shoot dry weight and root dry weight ratio (shoot/root ratio) and calculation of the tolerance indexes for the shoots and roots..

Uncertainty analysis of trypsin inhibitor activity determination using microtiter plate method

Measurement of proteinase inhibitor activity of legume seeds is widely used since some of these exert toxic and antinutritional effects. Standard method assay for trypsin inhibitor activity (TIA) determination is based on the hydrolysis of benzoyl-DL-arginine-p-nitroanilide (BAPA) by trypsin and includes spectrophotometric measurement of the reaction products. Assaying of trypsin inhibitor activity is usualy performed using reaction mixture volumes of 4-10 ml, however, it was shown that microtitration method can also be used for TIA determination. Although the results obtained by microtitration method were consistent with those obtained by French Association for Normalization reference method (AFNOR), it was noted that any experimental error in absorbance values was increasingly magnified as the sample size was diminshed. The aim of this study was to analyse measurement uncertanty of TIA determination using microtiter plate method. Assaying procedure was conducted at 37 °C on sample (soybean variety Vojvoñanka), positive and negative control, which were set in four replicats in each row of the microtiter plate, with preincubation time of 10 minutes and incubation time of 30 minutes. The trypsin inhibitor activity was expresed in number of trypsin units inhibited (TUI) per miligram of sample, taking into account the fact that one trypsin unit is defined as an increase of 0.01 absorbance units at 405 nm. Large number of experiments was conducted and uncertainty analysis was obtained using uncertainty budget. Values of calculated trypsin inhibitor activity were approximately 70 TUI/mg and estimated expanded measurement uncertainty (with coverage factor k=2) was aproxitemately 15%

Determination of Kunitz trypsin inhibitor types in soybean (Glycine max) and wild soybean (Glycine soja)

Among the antinutritional factors present in the soybean seed, the main ones ate the protease inhibitors. Soybean seeds contain two major protease inhibitor classes - the Kunitz (KTI) trypsin inhibitor and the Bowman-Birk (BB) trypsin-chymotrypsin inhibitor. Approximately 80% of the trypsin inhibition is caused by KTI. Soybean KTI has several polymorphic types, which are controlled by codominant multiple alleles at a single locus. Of these types, Tia and Tib are predominant types. In total 10 soybean varieties (7 cultivated and 3 wild) was analyzed. In order to determine the type of KITT present, PCR-RFLP using restriction enzyme Mse I (Tru1 I) was performed. Tib type was found in cultivated as well as in wild soybean varieties. Using a pair of primers for KTI3 gene fragment of about 700 bp was amplified in all analyzed varieties, including the Kunitz variety, lacking active trypsin inhibitor. DNA extracted from the Kunitz variety and other varieties mixed equally and used for SNP analysis

Yield and quality of rapeseed seeds depending on genotype and cultivation conditions

U radu je dat prikaz rezultata pokusa s različitim sortimentom i agrotehničkim mjerama u cilju proučavanja postizanja visokih prinosa i dobivanja kvalitetnog sjemena uljane repice, kao i ocjena mogućnosti monitoringa genetički modificiranih biljaka, koji se kontinuirano provode u Institutu za ratarstvo i povrtarstvo u Novom Sadu. Osim poznavanja genotipova i uvjeta vanjske sredine, neophodni su uvjeti za postizanje tih ciljeva prilagođavanje načina pripreme zemljišta, vremena sjetve, žetve i gnojenja zemljišnim i klimatskim uvjetima svake regije. Također, vrlo važan dio u postupku proizvodnje sjemena dobre kvalitete jest i stalno praćenje prisustva genetske modiikacije u sjemenu i prehrambenim proizvodima od uljane repice.The paper presents the results of the experiment with different assortment and agrotechnical measures in the goal of studying the achievement of high yields and obtaining rapeseed seed of high quality, as well as evaluating the possibility of monitoring genetically modified crops, which are conducted continuously in the Institute for field crops and vegetable growing in Novi Sad. Except for having the knowledge of the genotypes and conditions of external environment, adjusting the manner of preparing the soil, time of sowing, harvesting and fertilizing to soil and weather conditions of each area are necessary for achieving these goals. Also, a very important part in the procedure of producing the seed of good quality is also a constant monitoring of the presence of genetic modification in the seed and food products of rapeseed

Drought tolerance of Vicia sp. at germination stage

Drought is considered the most important abiotic stress which limits growth and productivity of plants and degrades agricultural soils. Therefore, testing drought tolerance of different species, particularly in the early stages of development, is of great importance. The aim of the experiment was to examine the tolerance of vetch to water stress during the germination stage. Three species of vetch, Vicia sativa L., Vicia villosa Roth and Vicia pannonica Crantz, produced at the Institute of Field and Vegetable Crops in Novi Sad, were tested. The substrates for germination were moistened with the PEG solution of 0, 0.3, 0.6 and 0.9 MPa concentrations. As indicators of drought tolerance, germination and growth parameters (shoot and root length, fresh and dry weight of seedlings) were determined. With an increasing concentration of PEG, all the tested parameters were reduced, but the results showed the interaction between the species and drought level

Evaluation of bean and snap bean populations collected on western part of Fruska gora Mt.

Seed of old varieties is an important source of genetic variability and plants adaptability. Broad genetic diversity is the base for successful breeding processes and adaptations to various environmental conditions and pathogens. In last five to six decades disappearance of old cultivars has been accelerated, mainly due to introduction of new cultivars based on principles of modern genetics into agricultural production. There was a cessation of cultivated plants previously used, and agricultural production and human diet is based on four main crops: wheat, corn, rice and potatoes. Large arable areas have been planted with single genotype of newly created cultivars resulting in great genetic impoverishment, especially, of the most cultivated plants. In that way, certain traits, particularly those connected with high yield, have been favored. Thus, the maintenance and evaluation of old cultivar’s seeds have been recognized as extremely important for all of humanity and its future. The research presented in this paper has been conducted on the territory of southwestern Fruska gora Mt. as a part of the project „Za Vojvodjanskog paora” (2011-2012) financed by Provincial Secretariat for Science and Technological Development, Republic Serbia. Considering the extreme importance of Fruska gora Mt. for Vojvodina Province and the ongoing Master Plan for the mountain’s Sustainable Development, idea of the project was to collect seeds and planting material of crops, vegetables and wild plants and to examine the state of the mountain’s genetic resources. Of all the collected samples during the project, 14 samples of snap beans and 21 samples of beans have been analyzed in this paper. For all the accessions, 1000-seed mass, seed color, seed shape and phaseolin type was determined. Seed color was determined visually. Based on the ratios of: (i) seed length and seed width, and (ii) seed thickness and seed width, collected seed samples were classified into five basic groups of seed shape: round, ellipsoid, cylindrical, semi-flat and kidney-shaped. Seeds of collected bean samples have three, while snap bean seeds have four different shapes, and none of them was round. Seed coats of bean accessions were of five different colors, while in the snap bean group seed coats were of three different colors. Seeds of collected bean and snap bean accessions were predominantly white and cylindrical in shape. Mass of 1000 seeds ranged between 104.9 and 634.96 g. Averaged 1000-seed mass of bean accessions was 379.44g. Snap beans had smaller seed, with 247.98 g of 1000-seed mass in average. T phaseolin type dominated, while S type of phaseolin was present in six bean samples, and in snap bean accessions numbered NK2/12 and NK40/12. T phaseolin type was mostly present in seeds of snap beans that were white, while no significant correlation between seed color and phasolin type was determined in the beans

Physiological, biochemical and molecular basis of drought and osmotic stress tolerance in the early stages of vegetative development of selected Pisum genotypes

Suša i osmotski stres, pored efekata na rastenje i razviće biljaka, dovode i do oksidativnog stresa i nastajanja velike količine reaktivnih kiseoničnih vrsta (ROS). Kako bi neutralisale štetne efekte ROS, biljke su razvile antioksidativne sisteme zaštite. Cilj istraživanja ove disertacije je bio da se detaljno prouče fiziološki i biohemijski procesi koji su u osnovi reakcija biljaka stočnog graška na sušu i osmotski stres. Cilj je bio i da se u ova ispitivanja uključe i molekularna istraživanja, koja bi, uz analizu procesa rastenja i drugih fizioloških procesa (aktivnost antioksidativnih enzima, akumulacije i distribucije stres hormona abscisinske kiseline), značajno doprinela razumevanju efekata, ne samo osmotskog i stresa suše, već i oksidativnog stresa na biljke stočnog graška. Istraživanja su vršena na 7 odabranih genotipova roda Pisum iz kolekcije Instituta za ratarstvo i povrtarstvo iz Novog Sada. Tretmani su obuhvatili dva eksperimentalna sistema: osmotski stres i sušu. Za indukciju osmotskog stresa su korišćene različite koncentracije rastvora osmotikuma PEG 6000 osmotskog potencijala (od -0,1MPa i -0,2MPa) primenjene u kraćem (10 dana) i dužem vremenskom intervalu (15 dana) kod klijanaca i ponika biljaka. Kao kontrolna varijanta je korišćena voda bez dodatka PEG 6000. Eksperiment suše je obavljen kod biljaka gajenih u kontrolisanim uslovima (u fitotronskoj komori) i to tako da su biljke zalivane optimalno do pojave trećeg para listova, a potom je suša u zemlištu izazvana prekidanjem zalivanja biljaka do 18% (umerena suša) i 9% sadržaja vode u spustratu (jaka suša). Biljke u kontroli su zalivane do optimalnog vodnog režima zamljišta (36% sadržaja vode u supstratu). U ogledu osmotskog stresa u fazi klijanja su obavljena fiziološka ispitivanja uticaja osmotskog stresa na % klijavosti i srednje vreme klijanja ili Mean Germination Time, dok su u fazi ponika obavljena ispitivanja dužine i biomase nadzemnog dela i korena, kao i odnosa suve mase nadzemnog dela i korena, a izračunat je i indeks tolerantnosti za nadzemni deo i koren...Drought and osmotic stress, in addition to the effects on plant growth and development, lead to oxidative stress and the production of a large quantity of reactive oxygen species (ROS). Plants have developed antioxidant defense mechanisms in order to neutralise the damaging effects of ROS. The aim of this dissertation was to investigate the physiological and biochemical processes which are in the base of field pea reaction to drought and osmotic stress. The aim was also to include molecular research in the study, along with the analysis of growth and other physiological processes (activity of antioxidant enzymes, accumulation, and distribution of plant stress hormons abscisic acid), which would significantly improve understanding of not only osmotic and drought stresses, but also about the effects of oxidative stress on field pea plants. Studies were conducted on seven selected genotypes of the genus Pisum from the collection of the Institute of Field and Vegetable Crops, Novi Sad. Treatments consisted of two experimental systems: osmotic and drought stresses and drought in the environmental test chamber. For induction of osmotic stress different concentrations of osmotic solutions PEG 6000 (-0.1MPa and -0.2MPa) were used in a shorter (10 days) and longer time interval (15 days). Water without PEG 6000 was used as control. The drought experiment was conducted in control conditions (phytotron chamber) in a such waz that the plants were irrigated at the optimal soil water content until the third pair of leaves began emergence, after which soil drought was induced by termination of irrigation up to 18% (moderate drought), and 9% substrate water content (severe drought). Control plants were irrigated at the optimal level of soil water regime (36% substrate water content). In osmotic stress experiment in the germination stage physiological tests included measurements of germination percentage and Mean Germination Time, while in the seedling stage was performed testing of the length and biomass of plant shoot and root, as well as shoot dry weight and root dry weight ratio (shoot/root ratio) and calculation of the tolerance indexes for the shoots and roots..

Physiological, biochemical and molecular basis of drought and osmotic stress tolerance in the early stages of vegetative development of selected Pisum genotypes

Suša i osmotski stres, pored efekata na rastenje i razviće biljaka, dovode i do oksidativnog stresa i nastajanja velike količine reaktivnih kiseoničnih vrsta (ROS). Kako bi neutralisale štetne efekte ROS, biljke su razvile antioksidativne sisteme zaštite. Cilj istraživanja ove disertacije je bio da se detaljno prouče fiziološki i biohemijski procesi koji su u osnovi reakcija biljaka stočnog graška na sušu i osmotski stres. Cilj je bio i da se u ova ispitivanja uključe i molekularna istraživanja, koja bi, uz analizu procesa rastenja i drugih fizioloških procesa (aktivnost antioksidativnih enzima, akumulacije i distribucije stres hormona abscisinske kiseline), značajno doprinela razumevanju efekata, ne samo osmotskog i stresa suše, već i oksidativnog stresa na biljke stočnog graška. Istraživanja su vršena na 7 odabranih genotipova roda Pisum iz kolekcije Instituta za ratarstvo i povrtarstvo iz Novog Sada. Tretmani su obuhvatili dva eksperimentalna sistema: osmotski stres i sušu. Za indukciju osmotskog stresa su korišćene različite koncentracije rastvora osmotikuma PEG 6000 osmotskog potencijala (od -0,1MPa i -0,2MPa) primenjene u kraćem (10 dana) i dužem vremenskom intervalu (15 dana) kod klijanaca i ponika biljaka. Kao kontrolna varijanta je korišćena voda bez dodatka PEG 6000. Eksperiment suše je obavljen kod biljaka gajenih u kontrolisanim uslovima (u fitotronskoj komori) i to tako da su biljke zalivane optimalno do pojave trećeg para listova, a potom je suša u zemlištu izazvana prekidanjem zalivanja biljaka do 18% (umerena suša) i 9% sadržaja vode u spustratu (jaka suša). Biljke u kontroli su zalivane do optimalnog vodnog režima zamljišta (36% sadržaja vode u supstratu). U ogledu osmotskog stresa u fazi klijanja su obavljena fiziološka ispitivanja uticaja osmotskog stresa na % klijavosti i srednje vreme klijanja ili Mean Germination Time, dok su u fazi ponika obavljena ispitivanja dužine i biomase nadzemnog dela i korena, kao i odnosa suve mase nadzemnog dela i korena, a izračunat je i indeks tolerantnosti za nadzemni deo i koren...Drought and osmotic stress, in addition to the effects on plant growth and development, lead to oxidative stress and the production of a large quantity of reactive oxygen species (ROS). Plants have developed antioxidant defense mechanisms in order to neutralise the damaging effects of ROS. The aim of this dissertation was to investigate the physiological and biochemical processes which are in the base of field pea reaction to drought and osmotic stress. The aim was also to include molecular research in the study, along with the analysis of growth and other physiological processes (activity of antioxidant enzymes, accumulation, and distribution of plant stress hormons abscisic acid), which would significantly improve understanding of not only osmotic and drought stresses, but also about the effects of oxidative stress on field pea plants. Studies were conducted on seven selected genotypes of the genus Pisum from the collection of the Institute of Field and Vegetable Crops, Novi Sad. Treatments consisted of two experimental systems: osmotic and drought stresses and drought in the environmental test chamber. For induction of osmotic stress different concentrations of osmotic solutions PEG 6000 (-0.1MPa and -0.2MPa) were used in a shorter (10 days) and longer time interval (15 days). Water without PEG 6000 was used as control. The drought experiment was conducted in control conditions (phytotron chamber) in a such waz that the plants were irrigated at the optimal soil water content until the third pair of leaves began emergence, after which soil drought was induced by termination of irrigation up to 18% (moderate drought), and 9% substrate water content (severe drought). Control plants were irrigated at the optimal level of soil water regime (36% substrate water content). In osmotic stress experiment in the germination stage physiological tests included measurements of germination percentage and Mean Germination Time, while in the seedling stage was performed testing of the length and biomass of plant shoot and root, as well as shoot dry weight and root dry weight ratio (shoot/root ratio) and calculation of the tolerance indexes for the shoots and roots..

Genetically modified crops – A potential risk for sustainable agriculture

The concept of sustainability applied to agriculture has developed mainly as a result of growing awareness of negative impacts of intensive agriculture production systems on the environment and the quality of life. Intensive agriculture systems are based on genetically uniform crops which can be susceptible to various diseases and insect pests. Genetically modified (GM) crops have been developed in an attempt to expedite the process of crop improvement for food quality and solve some of the problems associated with commercial agriculture, including disease and weed management. The introduction of GM crops into agriculture has opened a great debate about their safety with respect to possible long-term adverse effects on the environment, human health and sustainability of this new agricultural technology. Environmental safety issues focus on the direct or indirect effects of GM crops on non-target organisms and the transfer of GM traits to populations of wild plants via gene flow. Food safety concerns relate to the potential allergenicity of food products. Herbicide tolerant Roundup Ready (RR) soybean is the dominant GM crop occupying 50% of the global biotech area. It is one of seven transgenic soybean lines authorized for the EU market, none of those for cultivation. The most serious problem for farmers who grow RR soybean is the outbreak of glyphosate-resistant weeds, or “superweeds” which can lead to overuse of selective broad-leaf herbicides or herbicide mixtures

Identifikacija prouzrokovača uvenuća i truleži plodova paradajza

Tomatoes are parasitized by a number of pathogens, including Fusarium oxysporum f. sp. lycopersici, causal agent of fusarium wilt. Fresh vegetable fruits can be contaminated with various fungi that produce mycotoxins, which is an important issue for human health. The objective of this paper was to isolate, determine, and identify causal organisms of tomato wilt and fruit rot, based on the pathogens morphological and molecular characteristics. Samples of diseased plants showing symptoms of tomato wilt were collected from different localities in the production region of Vojvodina. Fruits with symptoms of fusarium rot were collected from storage and warehouses. The isolation and morphological determination of the fungus were performed on PDA and Czapek’s nutrient media. Isolates from diseased plants growing in field, designated as TFW1-TFW12 and seven isolates from diseased tomato fruits (TFM1-TFM7) were chosen for further investigation. For identification of the fungal isolates, the polymerase chain reaction (PCR) was also used. The EF1/EF2 primer pair was used for molecular identification of Fusarium sp. Nine analyzed samples were found to contain DNA fragments 700 bp in size.Patogene gljive roda Fusarium, prouzrokovači uvenuća biljaka, pojavljuju se i kao paraziti povrća uzrokujući propadanje korena, prizemnog dela stabla, kao i uvenuće biljaka. Veći broj patogena parazitira paradajz, među kojima je i Fusarium oxysporum f. sp. Lycopersici, prouzrokovač fuzarioznog uvenuća. Fuzariozna trulež plodova paradajza redovno se javlja u skladištima i može naneti velike ekonomske gubitke. Cilj ovog rada bio je izolacija, utvrđivanje i identifikacija prouzrokovača uvenuća paradajza i truleži plodova, na osnovu morfoloških i molekularnih karakteristika patogena. Prikupljeni su uzorci obolelih biljaka sa simptomima uvenuća paradajza iz proizvodnih regiona Vojvodine. Sveži plodovi sa simptomima fuzariozne truleži prikupljeni su iz različitih magacina i skladišta. Za izolaciju i morfološku determinaciju gljive korišćene su PDA i Czapek hranljiva podloga. Za dalja proučavanja odabrano je 12 izolata (TFW1-TFW12) poreklom sa biljaka gajenih u polju i sedam izolata (TFM1-TFM7) dobijenih sa zaraženih plodova iz skladišta. Determinacija izolata gljiva obavljena je i pomoću metode, lančane reakcije polimeraze (PCR – Polymerase Chain Reaction). Za molekularnu identifikaciju izolata F. oxysporum korišćen je par prajmera EF1/EF2. U devet ispitivanih uzoraka potvrđeno je prisustvo DNK fragmenta veličine 700 bp