ACTIVITIES
2014
Identification and study of areas affected by salinity
In-situ Field Evaluation - Field screening is the best way to identify the most suitable genotypes because salt tolerance is very complex phenomenon but spatial variability in the field sometimes give the false positive results due to escape. Field trips will be made in saline areas of the five counties to collect soil samples by all project partners,and analysis of the soil will be made where the land is affected by salt excess, to determine the concentration and type of salts. The electrical conductivity of soil solution and the nature and concentration of ions in the soil will be determined.We have prevoiusly identified this type of soils by analyzing their composition in Cl-, Na+, Ca++, Mg++, K+, SO4--.amd precise mapping. (Ardelean et al., 2003; 2004).
Collection of genotypes tolerant to osmotic stress
There will be collected local landraces of bean, tomato, onion, garlic, circa 100 local landraces from the following counties: Timis, Arad, Bihor, Botosani, Iasi.
For plant material collection several trips will be organized to specific location know for thei saline soils. Time alocated for each location will be 4-5 days. In order to identify and collect the biological material (around 20 samples for each target species: tomato, bean, onion and garlic) we will take samples directly form the filed but also form the previuos production. The collection will be done according to the following steps: a paper sheet will be formulated with questions relevant to the project aim; the plant material will be deposited in special paper or plastic bags to ensure air flow; each bag will be labelled; after collection the samples will be stored in a special room for drying. Each evaluation sheet will contain the collection no., genus; species; description of the collection/origin site; latitude and logitude of collection site; date; sample status at the time of collection and the source.
Characterization of collected biological material
It will be characterized based on the information from the provider and by visual appreciations (color, shape, aspect, etc.) and biometrical measurement (weight, diameter, length, etc.). The collected seed will be photographed for the construction of the data base.
Publication of the scientific results
It will be realized during international conferences and symposiums in the specialized field of science ( Croatia, Bulgaria, Hungary, Germany).
Deliverables (brief description and month of delivery)
Publication of 5 papers in international scientific field, containing information about the results of studies regarding sampled soils and identified, collected and characterized local landraces – month 10; identification, collection and characterization of local populations of tomatoes, beans, onions and garlic (100/species)
Testing salt stress tolerance by determining the main physiological parameters:
Harvested leaf blades will be rinsed with deionised water, dried at 70 ◦C for 3 days, weighed and extracted in 500 mM HCl at 80 ◦C for 1 h and analysed for Na+ and K+ by an atomic absorption spectrophotometer. Phosphoruswas wil bel analysed on dried (70 ◦C for 3 days), finely ground and pelleted leaf material using an X-ray fluorescence spectrometer according to the method described by Norrish and Hutton (1977).
- germination test of seeds on saline medium; assessment by biometrical measurements of growing rate (assessment of osmotic stress intensity on dry matter content, assessment of leaf area dynamics); determination of transpiration intensity dynamics; determination of photosynthetic intensity.-In addition to various protective mechanisms (Niyogi 1999, Munekage et al. 2002), the PSII repair cycle functions to replace the damaged reaction centre protein D1 with a de novo synthesized copy (Aro et al. 1993, Takahashi et al. 2008). By studing the efficiency of electron transoport chain function the effect of various stresses on photosynthesis is estimated and can be correlated with yield. One of the most used techniques to investigate photosynthetic function is the study of ChlA fluorescence, and we aim to use a variant of this methode in order to characterise in detail the effect of osmotic stress upon various genetic types of legumes. ; Evaluation of tolerance to NaCl will be realized by the culture of sterilezed seeds on different salt concentrations with concentration between: 0-300 mM. There will be purchased the following ecquipments necessary for experiments: porometer, germination chamber, chlorophyllmeter, portable photosynthesis analyzer, (3) climatic chambers.
Assessment of osmotic stress tolerance with determination of enzimatic activity:
The determination of enzymatic activity in plants subjected to salt stress is a valuable biochemical method for characterizing their tolerance level. The effects of increasing NaCl concentrations on biomass, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and phenylalanine ammonia-lyase (PAL) in tomatoes, beans, onoin and garlic seedlings will be investigated. In plant cell, antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) have been considered as a defensive team, whose combined purpose is to protect cells from oxidative damage. Increased SOD, POD and CAT activities are closely related to salt tolerance of many plants as reported in various researches (Rahnama and Ebrahimzadeh 2005, Azevedo Netoet al. 2006, Koca et al. 2007). These findings suggest that the induction of ROS-scavenging enzymes, such as SOD, POD and CAT, is the most common mechanism of salt tolerance for detoxifying ROS synthesized. Enzyme extraction - For SOD, POD and CAT extraction, leaf samples (0.5g) were homogenized in ice cold 0.1 M phosphate buffer (pH=7.5) containing 0.5 mM EDTA with pre-chilled pestle and mortar. Each homogenate was transferred to centrifuge tubes and was centrifuged at 4°C inBeckman refrigerated centrifuge for 15 min at 15000×g. The supernatant was used for enzyme activity assay (Esfandiari et al., 2007). Enzyme activity analysis - SOD activity will be estimated by recording the decrease in absorbance of superoxidenitroblue tetrazolium complex by the enzyme (Sen Gupta et al., 1993). CAT activity will be measured according to Aebi (1984) and POD activity will be performed according to the Sakarov and Aridilla (1999) method. Free proline concentration in plant tissues will be determined according to Bates (1973).
Publication of scientific results
It will be realized during international conferences and simposions from the specialized field (Austria, Croatia, Hungary, Germany).
Deliverables (brief description and month of delivery)
Publication of 8 articles in international scientific field, containing informations about the results of physiological and biochemical studies on assessed local landraces
Identification and selection of populations that show high tolerance to salinity (15 / species) as well as those sensitive (5/species) to study them at molecular level and value their potential
The value of investitions in equipments: 171,2 thousands
Extraction and molecular analysis of DNA from selected genotypes
Molecular markers should not be considered as normal genes, as they usually do not have any biological effect, and instead can be thought of as constant landmarks in the genome. They are identifiable DNA sequences, found at specific locations of the genome, and transmitted by the standard laws of inheritance from one generation to the next.
Will be made DNA extraction for the selected species, 4 x 80 genotypes = 320 DNA extraction samples. Genotyping using different types of molecular markers: RAPD (Random Amplified Polymorphic DNA), and ISSR (Intersimple Sequence Repeats);
Total RNA will be isolated from 500 mg of three weeks old seedlings, using the extraction method originally described by Chomczynski and Sacchi, (1987) tha uses Trireagent and then redissolved in RNase-free distilled water. RNA concentration will be determined on the basis of absorbance at 260 nm. Traces of contaminating genomic DNA will be removed by treatment with RNase-free DNase For Reverse transcriptase – PCR (RT-PCR) analysis transcript levels will be monitored by semi-quantitative RT-PCR analysis. cDNA templates were generated from DNase-treated total RNA (5 μg) samples by reverse transcription using SuperScriptTM II Rnase H- reverse transcriptase kit, according to the instructions of the supplier. PCR reactions were performed in 50 μl volume, using 2 μl cDNA template and Dupla-TaqTM polymerase Gene specific primers (two pairs) will be used for detection of P5CS1 and P5CS2 transcripts, respectively. Primers will be used for amplification of UBIQUITIN 10 cDNA (Bancos et al., 2002), which serves as constitutive reference.
Assessment of genetic relatedness between the selected local landraces and preparation of dendrograms
The analysis of genetic diversity and relatedness between the different landraces collected in this project is a central task of this phase.
Traditional methods of cultivar identification frequently are based on the evaluation of sets of morphological characteristics. Although it is usually cost-effective, morphological assessments may have their limitations, including (1) insufficient variation among cultivars (especially if the cultivars to be compared share a closely related pedigree), (2) subjectivity in the analysis, (3) influence of the environment and management practice, and (4) expression of some characters only in certain developmental stages. These considerations triggered the exploration of alternative means of cultivar identification, including allozyme analyses, cytogenetics, analysis of secondary metabolites, and DNA profiling (Camlin et al., 2003). DNA from studied genotype (selected genotypes) will be analysed to establish the degree of relatedness between selected local populations. Phylogenetic relationships between them will be displayd in dendrograms.
Publication of scientific results
It will be realized during international conferences and symposiums in specialized field (Austria, Croatia, Hungary, Germany)
Deliverables (brief description and month of delivery).
Publication of 8 articles in international scientific field, containing information about the genetic fingerprint and phylogenetical relatedness for every species from the study. 80 genetic fingerprints of the local populations studied, 4 dendrograms (1/specie) regarding the phylogenetic relationships between them.
Creating an online database
The data base will be accessible online and will contain the following information:
Collecting organization
Names of collectors; Collecting number ; Collecting date; Collecting date; Origin of sample (name of farm); Origin of sample (name of farm); Type of material (seed in our case)
Sample identification
Genus, species, subspecies, botanical Genus, species, subspecies, botanical variety (taxon name); Photographs numbers and subjects ; Status of sample (landraces in our case)
Sampling information
Collecting source ; Size estimates of sample (sample area); Frequency of the target species in the area
Collecting site localization
Geographical coordinates (latitude, Geographical coordinates (latitude, longitude, altitude, map reference, site longitude, altitude, map reference, site number, farmer number, farmer’s name of collecting site s name of collecting site (village, region).
Use of the experimental results (obtained from physiological, molecular and genetic characterization of the collected populations) in order to obtain new genotypes (prebreeding material) with a high tolerance to saline stress.
Prebreeding includes the introduction, adaptation, evaluation, and improvement of germplasm resources for use in breeding programs. Prebreeding usually does not provide directly new cultivars for the growers. It rather develops germplasm resources that are either directly or indirectly used to develop new cultivars.
For every studied species, the landraces which present contrasting attributes in terms of yield and salinity tolerance traits will be introduced into a full diallel crossing system in order to obtain F1 hybrid populations. The study of the F1 hybrid populations allows to obtain useful information regarding the gene effects involved in the inheritance of the traits involved in salinity tolerance and establishing of the selective potential for these hybrid populations in terms of valuables recombinant lines for salinity tolerance.
Conservation of valuables genotypes for present and future use
In order to exploit the potential of valuables genotypes directly in production, breeding programs or research activities, it is necessary to retain their genetic variability and diversity. As such, the preservation of valuables genotypes of the studied species. As such, the preservation of valuables genotypes of the studied species will be made “ex situ” both as a seed in controlled environmental conditions, and "in vitro" under conditions of slow growing or cryopreservation in liquid nitrogen.
Utilization of germplasm in order to obtain the type approval
Legal procedures will be started for patenting (type approving) of 4 breeds tolerant to salinity.
Deliverables (brief description and month of delivery)
Four invention patents registered using the collected germplasm with salt tolerance; comprising a germplasm collection of 80 genotypes of which 60 salinity tolerant, a database on collected and characterized germplasm.
Management, training, dissemination and extension
Startup: Project team organization, selection of candidates for the available positions, brain-storming and workshops of the entire project team by videoconferences, assigning responsibilities for each member of the team, establishing agricultural land to be used in the project, establishing of the acquisition list and starting purchase of necessary materials.
Dissemination and extension: Results obtained in this project will be published in peer‐reviewed scientific journals and presented in conferences. The activities in this phase will include data publication and presentation, the set‐up of a project webpage and maintenance. The genotypes collected and developed from this project is considered to be available without restriction to others users for further research and breeding and can be use without any legal or contractual obligations, or technological restrictions. The user undertakes that the material shall be used or conserved only for the purposes of research, breeding and training for food and agriculture.
The user shall not claim any intellectual property or other rights that limit the facilitated access to the material, or its genetic parts or components.
In the case that the user commercializes a product that is a plant genetic resource for food and agriculture and that incorporates our material, and where such product is not available without restriction to others for further research and breeding, the user shall pay a fixed percentage of the sales of the commercialized product.
Training stages: Within the project will be organized a training stage with young researchers, thus encouraging the exchange of experience in international mobility, to an research institution abroad (BRC-Hungary) in order to acquire practical and theoretical knowledge in the field of molecular biology and vegetable biochemistry.
Deliverables (brief description and month of delivery)
In project there will be published or accepted for publication scientific papers (described above) with a sum of relative impact factor of at least 4.
It will also develop a training course of one month with two young scientists at a research institute abroad.