Institute of agricultural biology and biotechnology (IBBA)

Research activities

The research activities carried out in the institute are presented pooled into four major projects.

Project title: AG.P01.001 / MOLECULAR, PHYSIOLOGICAL AND CELLULAR BASES OF PLANT PRODUCTION.

The resarch activities, carried out in the sections of Milano and Pisa, aim at the molecular characterization of the genetic, cellular, biochemical and physiological mechanisms that determine the productivity of cultivated plants. Major topics are: stress response, protein synthesis and accumulation, and regulation of gene expression and metabolism. Expertise of the research groups includes: molecular genetics, protein biochemistry, plant cell biology, plant-environment interactions and plant-rhizosphere interactions.

The reserch activities are within the general frame of plant molecular biology, and regard: 1) response to biotic and abiotic stresses; 2) mycorrhizal symbiosis, including morphological and functional characterizations; 3) protein folding, assembly, intracellular sorting and stability; 4) regulation of gene expression by transcription factors and epigenetic mechanisms.

Methods of investigation are: genetic and genomic analysis, plant growth in different conditions followed by biochemical and physiological studies, protein expression in protoplasts and trangenic plants followed by analysis of gene expression, synthesized products and resulting phenotypes.

Techniques include: recombinant DNA, macroarrays, gene chips, Real-Time PCR, chromatin immunoprecipitation, transient expression in protoplasts, in vitro plant tissue culture, plant transformation, protein engineering, fluorescence microscopy, metabolite analys by HPLC, various techniques of protein purification and analysis.

Goals are the identification and characterization of genes, metabolic pathaways and simple and complex (systems biology) molecular interactions responsible for the following biological processes: i) plant-environment interactions, with particular focus on abiotic stress response and the characterization of mycorrhizal fungi in relationship with plant nutrition; ii) protein synthesis and accumulation, with particular focus on the mechanisms of structural maturation and intracellular targeting and sorting; iii) regulation of gene expression by transcription factors and epigenetic mechanisms.

Scheduled activity:
Plant-environment interactions: characterization of drought and salt stress response of the low phytic acid (lpa) P. vulgaris mutant. Analysis of T2 transgenic rice plants expressing Ubi1-Osmyb4 and of the expression of other members of the Osmyb4 sub-family under normal and stress conditions. Biological and molecular characterization of Mediterranean arbuscular mycorrhizal strains isolated from trap cultures, and selection based on the features of extraradical mycelial networks.
Cell biology: Comparison of the assembly properties of different low molecular weigth (LMW) glutenin subunits. Development of an assay to measure the assembly of HMW glutenins. Characterization of the requirements for the assembly of gamma-zein. Studies on the intracellular traffic of ribosome inactivating proteins and their mechanisms of toxicity. Identification of tonoplast sorting mechanisms of tail-anchored and trans-membrane proteins and determination of the role played by the Golgi complex.
Molecular genetics: macroarray transcriptional analysis of seed development in the lpa P.vulgaris mutant. Studies on the regulation of genes differentially expressed in rice coleoptiles grown under aerobic or anaerobic conditions. Transformation of Citrus with Osmyb7 under the Ubi1 promoter.


Project title: AG.P01.003 / GENETIC, PHYSIOLOGICAL AND MOLECULAR BASIS OF DEVELOPMENT AND DIFFERENTIATION OF MODEL AND CROP SPECIES OF INTEREST TO AGRO-FOOD IN RESPONSE TO ENDOGENOUS AND ENVIRONMENTAL CUES

Scientific research is carried out by the IBBA Operative Units of Roma and Pisa. The project aims at defining the genetic, physiological and environmental basis that control cell determination and the activity of meristems for plant genetic improvement and human health, that through a biotechnological and multidisciplinary approach. Expertise of the groups involved in the project includes: functional genomics of proteins involved in the morphogenesis and differentiation of higher plants, characterisation of transcription factors, genetics of model and crop species, selection and production of transgenic plants, plant physiology, biochemistry of antioxidant system, cytology, histology and immunocytochemistry, intracellular localisation of proteins, mutagenesis and antimutagenesis in yeast, bioinformatics and gene silencing.

Research activities: isolation, functional characterisation and biotechnological use of plant homeobox genes that control meristem (plant stem cells) formation, identity and activity in model and crop species for plant genetic improvement and human health. Stress response and development: identification of regulatory mechanisms that utilise signal transduction pathways that are common to stress response and developmental processes. Molecular and cytophysiological basis of meristematic and embryogenic competence, epigenetic processes, somaclonal variation and meiosis for in vitro selection of elite genotypes in crop species. Development of new protocols for crop genetic transformation that exploit cellular totipotency and adaptative responses to stress. Use of yeast systems to support plant post-genomics and for studies of mutagenesis and antimutagenesis in eukaryotic cells.

Methodologies: analyses and molecular characterisation of genes and proteins involved in the acquisition of meristematic competence through bioinformatics (in silico analyses); in vitro analyses by means of molecular synthesis, expression in heterologous systems (bacteria and yeast), macromolecules interaction studies by yeast Two-Hybrid assays, EMSA (Electrophoretic Mobility Shift Assay) and ChIP (Chromatin Immuno Precipitation); in vivo analyses of transient and/or stable genetic transformed plants; genetic, physiological and molecular analyses of plant and yeast mutants and/or transgenic lines in response to endogenous and environmental cues through gene and trait segregation; phenotypical analyses through the registration of morphology variations and/or physiological responses by cytologycal, histological and immunocytochemical techniques, optical and fluorescence microscopy; gene expression analyses by Northern blotting, RT-PCR, in situ hybridisation; biochemical analyses of products through electrophoresis techniques, HPLC or enzymatic assays.

Techniques used: in vitro plant cell and tissue culture, recombinant DNA technologies, gene expression analyses by Northern blotting, RT-PCR and in situ hybridisation; gene transfer for transient or stable expression in bacteria, yeast and plants for the production and purification of heterologous proteins; fluorescent microscopy of proteins fused to fluorescent molecules for protein tissue and intracellular localisation; biological assays for mutagenesis and antimutagenesis of substances of interest to agro-food, cytology, histology and immunocytochemistry for the characterisation of plant cells and tissues; protein-protein and protein-DNA interaction technologies (yeast Two-Hybrid, EMSA, ChIP); advanced programs of bioinformatics for the in silico analyses of nucleotide or amino acid sequences; gene silencing techniques (RNA interference, VIGS).

Goals: Plant homeobox genes and their applications: identification of target genes and protein complexes of transcription attivation/repression, identification of hormone signal transduction and metabolic pathways through which homeobox factors regulate cell determination, developmental processes and plant architecture, identification of endogenous and environmental cues that regulate their activity. Cellular totipotency: definition of molecular and cytophysiological basis of the meristematic and embryogenic competence and applications in biotechnology. Stress response and development: identification of regulatory mechanisms that utilise signal transduction pathways that are common to stress response and developmental processes and their use for the selection of improved crop varieties. Development of new tools for the genetic transformation of plants on interest to agriculture and biotechnology, set up of yeast systems to support plant post-genomics and studies of mutagenesis and antimutagenesis in eukaryotic cells.

Scheduled activity:
Plant homeobox genes and their applications: isolation and functional characterisation of homeobox genes in crop and tree species through: analyses of gene expression in response to endogenous and environmental cues, protein intracellular localisation, study and production of plants with altered levels of gene expression, identification of target genes and protein partners. Biotechnological application of homeobox genes for in vitro plant propagation, assisted crop genetic improvement and for human health.
Cellular totipotency: definition and control of meristematic and embryogenic competency, of epigenetic processes and somaclonal variation.
Stress response and development: identification of regulatory mechanisms that utilise signal transduction pathways that are common to stress response and developmental processes.
Systems development: development of new tools for the genetic transformation of plants on interest to agriculture and biotechnology. Set up of yeast systems to support plant post-genomics and studies of mutagenesis and antimutagenesis in eukaryotic cells.


Project title: AG.P01.004 / DEVELOPMENT OF AGRO-INDUSTRIAL BIOTECHNOLOGIES FOR GENETIC IMPROVEMENT, ENVIRONMENT SUSTENIBILITY, PRODUCTS SAFETY, PRODUCTION OF PROTEINS AND METABOLITES USEFUL AS FOOD/FEED, MEDICINE AND AGRONOMY

These studies are entirely carried out in the division of Milan. The aim is to develop a variety of biotechnology approaches that should eventually lead to the production of useful products in the fields of medicine, animal nutrition and agronomy useful products in the fields of medicine, animal nutrition and agronomy. The aim is often achieved through the development of new methods of investigation, some of which specifically tailored for agri-food identification and labelling. Of recent, two novel subject of investigation have been introduced within the frame of this workpackage. The first concerns the development of methods for molecular recognition carried out at nanoscale level. The second concerns studies on non-food oil plants useful for biofuel production. The people that carry out this researches have expertises in a wide range of disciplines including genetics, cytogenetics, biochemistry, physiology, molecular and cell biology, plant breeding and trasformation, chemical mutagenesis and in vitro cell culture, plant growth under controlled environmental conditions.

Research activities: Classical genetics and physiology applied to the study and the characterization of bean varieties that have been improved for their nutritional content. Development of methods for the fast, easy and reliable identification of the genetic profile of crops and of derived processed food. Plant genetic transformation carried out with new promoters and genes conferring multiple stress resistances. Identification and characterization of the secondary metabolites profile in plants that have been transformed with the regulatory Osmyb4 gene. Genetics, biochemistry and metabolite analysis performed on oil plants useful for biofuel production. Definition of the better experimental conditions for producing stable vaccines in plants with emphasis on the mechanisms that control intracellular trafficking and localization. Molecular genetics-mediated mutagenesis for the production of ipo-allergenic proteins. Development of expression systems and strategies for the production of recombinant immuno-toxins. Antibodies raised against plant transcriptional factors and histone modifications.

The methods of investigations are those typically associated to the different disciplines listed above. So, they belong to the different fields of cell biology, biochemistry and genetics of proteins and nucleic acids, physiology assays and secondary metabolites detection. Tecniques currently in use are : production of new plant varieties through breeding and trangenic procedures, engineering of constructs useful for plant transformation, antibodies production, production of pharmaceuticals through DNA recombination, new methods for the genetic characterization of plants and food, new methods for identifying and studying plant DNA polimorphisms, collection-propagation and chracterization at physiology-biochemistry-genetic level of non-food oil plants useful for biofuel production.

Goals: Crops genetic improvement achieved either by classical breeding or via transgenics. To provide new tools to characterize the genetic resources and to follow up the chain of food production. To development novel diagnostic assays and tools in the field of agri-food . To build up the required knowledge to properly use and manage oil plants potentially useful for biofuel production. To exploit plants and bugs for the production of molecules and proteins useful in the agri-pharmaceutical field. To develop vaccines and immunotoxins useful for human health.

Scheduled activity: Genetic profiling of different varieties of bean, rose and Jatropha ssp. Development of a new version of the TBP method (phase 3. Development of methods for the microprinting of proteins and/or nucleic acids to study molecular interactions. Analysis of the bean biosynthetic pathway that leads to the production of phytic acid. Genetic and biochemical characterization of bean mutants with a low phytic acid content. Intron mediated expression studies carried out in transgenic rice plants. Metabolites profiling in trangenic plants expressing the rice Osmyb4 gene. Osmyb4 expression studies in rice varieties that exhibit a different response to abiotic stresses. Expression in yeast of recombinant products generated by the fusion of antibody fragments and saporin, a ribosomal inactivating protein. Expression in Pichia Pastoris or tobacco BY2 cells of the Derp1 protein and mutagenized forms of it. Purification through chromatography of polyclonal antibodies raised against plant transcriptional factors and modified hystones.


Project title: AG.P02.002 /
CONSERVATION OF ANIMAL GENETIC RESOURCES AND VALORISATION OF THEIR PRODUCTS.

Researchers, located in Lodi and Milano, work on the management and conservation of animal genetic resources using a combination of standard phenotipic evaluation methods, molecular analysis (for the characterization of the animal and for the evaluation of the quality and typicalness of the products) together with advanced reproductive biotecnologies. Research have a strong relapse on meat and milk production and on breeds conservation.
The main skill of the researchers are: genetic, molecular and cell biology, bioinformatics.

Research activities:
- Molecular biology applied to animal production. 1) Animal functional genome analysis i) Early host-pathogen interactions during mastitis in goats ii) Identification of candidate genes affection meat quality in pig; 2) Development of molecular diagnostic tools i) Genotyping: Microarray analysis for the identification of bovine milk protein genetic polymorphisms; ii) Microbial diagnostics: identification of pathogens in milk and cheese by means of multiplex PCR and oligonucleotide microarrays.
- Reproductive biotechnology for animal genetic resources conservation. 1) Development of advanced methods for semen analysis i) Computer assisted semen analysis for the evaluation of kinetics parameters of spermatozoa, ii) Fluorescence microscopy for membrane and acrosomal integrity assessment, single sell sel slectrophoresis for the analysis of Sperm DNA integrity. 2) Study on alternative methods for conservation of animal germplasm i) Cryopreservation of epidydimal sperm
- Modelisation of conservation and farming systems. 1) Cryopreservation of Livestock Genetic Resources: i) Evaluation of the costs of cryoconservation, ii) Simulation of the combined use of embryos and semen for the creation of genebanks. 2) Implementation of information systems on livestock production: Information system for the exchange of research and technical information on Italian animal breeds

Methods of investigation and techniques: DNA e RNA Estraction and purification from different tissues, PCR, Retro Transcript-PCR, multiplex PCR, Pulse Field Gel Electrophoresis, DNA microarray, Fluorescence microscopy, Computerized analysis of sperm kinetic.

Scheduled activity: In the framework of the agreement between the DAA-Regione Lombardia it will be started the studies planned in Workpakage "Chacterisation and genetic management of Aniaml Genetic resources": i) high-throughput characterisation of cattle breeds for diagnostic and selection ii) creation of the Cryo Bank of Lombardia Animal Genetic Resources iii) development of selection schemes for goats local breeds.
CE project "Towards self-sustainable European regional cattle breeds (EURECA)": analysis of cryopreservation program of cattle breeds in Italy, recording of genetic material of local cattle breeds stored in Italy, comparison of crypreservation program in 5 european countries.
SUINO GARLASCO Project: storage of 192 semen doses collected from 8 boars.
PRIN "Optimisation of freezing procedures of chicken and guinea-fowl semen in pellets": evaluation of quality, biochemical, microbilogical modifications induced by the freezing procedure on the spermatozoa.
SELMOL Project "Marker assisted selection for the competitiveness of national livestock production": on 240 pigs 4 genes previuosly identified as candidate gene for meat quality will be tested. Phenotipic evaluation for meat quality will be recordered at slaughther and at the end of seasoning.
SAFEMILK Project "Diagnosi precoce di mastiti subcliniche per un miglioramento quali-quantitativo delle produzioni lattiero-casearie": in farms located in Lombardia molecular diagnostic kits, DNA chip based, will be evaluated for sanitary test of dairy cows.
IDENTILAT Project "IdentitĂ  del latte: caratterizzazione di parametri biochimici, genetici e microbiologici": analysis of DNA components in cows and goat milk according to feeding.