Objectives: Detailed keypoint objectives of the reporting period were: 1. Molecular characterisation of GM events in the various genotypes engineered with the defence genes under constitutive and inducible promoters; 2. T1 analyses and T2-T3 seed multiplication; 3. Evaluation of the resistance to leaf and neck blast the level of mature plants of the parental genotypes in field conditions with various experimental design with/without artificial inoculation; 4. Evaluation of grain quality traits related to the disease; 6. Phytopathological assays for studies of the interaction plant/pathogen (rice/Magnaporthe) for attesting the properties of fungal-inducible promoters under study with stable transgenic lines; 7. Assessment of the validity and choice of the adeguate inducible promoter; 8. Detailed molecular characterisation of selected transgenic events in cv.Senia engineered with native and synthetic afp gene; 11. First test of GM-plants with gene b32 under constitutive promoters for neck blast; 12. Studies of environmental control for gene flow from GM rice and both cultivated rice and wild red rice.

Results and Milestones:Referring to the listed Objectives, the results obtained can be summarised as follows. Final vectors carrying the defence genes under selected inducible promoters (L and S versions of the maize ZmPR4 promoter) have been prepared. Plants engineered with the native and synthetic afp gene are being analysed; gene CecA and trimer of cecA were discarded as plants are stunted and sterile; plants with gene b32 under constitutive promoters are fully fertile. Molecular analyses of the single and double contructed Maratelli plants are underway, in order to verify the gene pyramiding strategy. Tests for characterising response to Magnaporthe grisea and Sclerotium are underway. Experimental designs to be adopted in the field for a reliable evaluation of leaf and neck blast resistance have been performed: the contiguous design and honeycomb design with artificial inoculation resulted the best for evaluation; the contiguous design was used for the first trial in controlled field conditions (B/IT/02/06 and B/IT/02/07) for neck blast evaluation. Ubi1-b32 engineered plants resulted the most promising. Evaluation of grain quality traits related to the disease effect have been accomplished in order to verify beneficial effects ofthe engineered genes. Phytopathological assays for studies of the interaction plant/pathogen (rice/Magnaporthe) for attesting the properties of fungal-inducible promoters have been completed attesting validity of the BH6-12 promoter as wounding inducible promoter, with GUS-engineered stable transgenic lines. A corresponding BH6-12 gene in rice has been identified from public database and its promoter is cloned and analysed, in order to make available a rice-inducible promoter, for refinement of the transformation strategies. Experiments aimed at verifying gene flow from the engineered plants and cultivated rice and the wild red rice have been performed and seeds collected for evaluation in both cases. Basta resistance has been taken as selecteble marker for screening. Safety of the strategies adopted for conduction of GM-field trial respect to gene flow, has been attested.

Benefits and Beneficiaries: The results obtained and deliverables released are inputs for all partners in undertaking the next phase of the project related to: 1. Creation of  the rice genotypes engineered with the defence genes under inducible promoters; 2. Evaluation of the defence response of the rice/Magnaporthe interaction in various GM-rice lines produced; 3. T2-T3 seed amplification for pathogenicity assays; 5. Completion of the study of gene flow red rice/cultivated rice. Social benefits from the project have to be identified in the constant presence of the Partners in scientific as well as divulgative meetings/seminars for dissemination of the project activities and active participation in the GM-crops dispute at the national level.