New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for plant improvement to biotic and abiotic stresses satisfying the European Policies requirements that promote chemical input reduction and a more sustainable agriculture. We decided to apply genome editing (via CRISPR/Cas9) focusing on susceptibility genes to control powdery mildew: we chosen to knock-out two genes belonging to MLO (Mildew Locus O) family: VvMLO7 and VvMLO6. The same approach was used to cope with abiotic stresses, in specifc drought, performing a knock-out of four genes, two belonging to GST (Glutathione S-Transferase) and two to PME (Pectin Methyl Esterase) gene families. In parallel to genome editing, we also applied cisgenesis to move the resistance locus RPV3-1 (Resistance to Plasmopara viticola) into economically important cultivars. This locus is formed by two different genes that were inserted individually and in combination to evaluate their effects. One of the drawbacks linked to classical Agrobacterium tumefaciens mediated transformation is the insertion of unrelated transgene (e.g., antibiotic resistance). These markers are required for transgenic plants selection, but undesirable to be retained in commercial plants due to possible toxicity or allergenicity to humans and animals, in addition to their potential hazards for the environment. To overcome these limits, we exploit an inducible excision system based on a Cre-lox recombinase technology controlled by a heat-shock inducible promoter that will be activated once the transformation event(s) will be confrmed. Embryogenic calli of Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese, were used in stable transformation with A. tumefaciens carrying the genome editing construct with the MLO-guideRNAs and the cisgenic construct carrying the two RPV3-1 genes. Embryogenic calli of rootstocks 110 Richter and SO4 were transformed with genome editing construct carrying GST and PME guideRNAs in two independent transformations. Regenerated embryos from all the transformation events are now under evaluation.

NPBTs FOR SUSTAINABLE VITICULTURE MANAGEMENT TO BIOTIC AND ABIOTIC STRESS

Loredana Moffa
Primo
Writing – Original Draft Preparation
;
2021

Abstract

New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for plant improvement to biotic and abiotic stresses satisfying the European Policies requirements that promote chemical input reduction and a more sustainable agriculture. We decided to apply genome editing (via CRISPR/Cas9) focusing on susceptibility genes to control powdery mildew: we chosen to knock-out two genes belonging to MLO (Mildew Locus O) family: VvMLO7 and VvMLO6. The same approach was used to cope with abiotic stresses, in specifc drought, performing a knock-out of four genes, two belonging to GST (Glutathione S-Transferase) and two to PME (Pectin Methyl Esterase) gene families. In parallel to genome editing, we also applied cisgenesis to move the resistance locus RPV3-1 (Resistance to Plasmopara viticola) into economically important cultivars. This locus is formed by two different genes that were inserted individually and in combination to evaluate their effects. One of the drawbacks linked to classical Agrobacterium tumefaciens mediated transformation is the insertion of unrelated transgene (e.g., antibiotic resistance). These markers are required for transgenic plants selection, but undesirable to be retained in commercial plants due to possible toxicity or allergenicity to humans and animals, in addition to their potential hazards for the environment. To overcome these limits, we exploit an inducible excision system based on a Cre-lox recombinase technology controlled by a heat-shock inducible promoter that will be activated once the transformation event(s) will be confrmed. Embryogenic calli of Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese, were used in stable transformation with A. tumefaciens carrying the genome editing construct with the MLO-guideRNAs and the cisgenic construct carrying the two RPV3-1 genes. Embryogenic calli of rootstocks 110 Richter and SO4 were transformed with genome editing construct carrying GST and PME guideRNAs in two independent transformations. Regenerated embryos from all the transformation events are now under evaluation.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11390/1210004
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