The main bottleneck in applying cisgenesis and genome editing in grapevine is plant transformation and regeneration. Many genotypes show indeed recalcitrance to tissue culture and transformation, as well as to regeneration from protoplasts, limiting efforts to use biotechnological approaches for grapevine genetic improvement or functional genomics studies. Grapevine embryogenic calli, induced in tissue cultures by means of growth regulators, are used for Agrobacterium-mediated transformation, since they are tissues harbouring totipotent cells and able to regenerate transformed plants. Protoplasts obtained from embryogenic calli provide additional advantages for genome editing purposes, including delivery of multiple plasmids for cotransformation and high frequency transformation. Most importantly, by use of polyethylene glycol or electroporation the protoplast system allows the direct delivery of the genome editing machinery, such as preassembled Cas9-gRNA ribonucleoproteins, rather than plasmids encoding these components, removing the likelihood of inserting recombinant DNA in the host genome. The machinery is needed to trigger DNA repair and incorporate modifications but it is degraded rapidly after transfection, reducing the frequency of off-target effects in regenerated plants. In the present contribution we show the setup of a protocol to regenerate Vitis vinifera cultivars starting from embryogenic-calli derived protoplasts, first step on the way to the generation of DNA-free genome edited grapevine.
Genome editing in grapevine: plant regeneration from embryogenic-calli derived protoplasts
Perrone I;Nerva L;Pagliarani C;Boccacci P;Chitarra W;Gambino G
2018
Abstract
The main bottleneck in applying cisgenesis and genome editing in grapevine is plant transformation and regeneration. Many genotypes show indeed recalcitrance to tissue culture and transformation, as well as to regeneration from protoplasts, limiting efforts to use biotechnological approaches for grapevine genetic improvement or functional genomics studies. Grapevine embryogenic calli, induced in tissue cultures by means of growth regulators, are used for Agrobacterium-mediated transformation, since they are tissues harbouring totipotent cells and able to regenerate transformed plants. Protoplasts obtained from embryogenic calli provide additional advantages for genome editing purposes, including delivery of multiple plasmids for cotransformation and high frequency transformation. Most importantly, by use of polyethylene glycol or electroporation the protoplast system allows the direct delivery of the genome editing machinery, such as preassembled Cas9-gRNA ribonucleoproteins, rather than plasmids encoding these components, removing the likelihood of inserting recombinant DNA in the host genome. The machinery is needed to trigger DNA repair and incorporate modifications but it is degraded rapidly after transfection, reducing the frequency of off-target effects in regenerated plants. In the present contribution we show the setup of a protocol to regenerate Vitis vinifera cultivars starting from embryogenic-calli derived protoplasts, first step on the way to the generation of DNA-free genome edited grapevine.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
