The combined availability of whole genome sequences and genome editing tools is set to revolutionise the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialisation of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene (S-gene) in order to increase resistance to powdery mildew (PM) in grape cultivar and DIPM-1, DIPM-2 and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimised for each grape and apple cultivar. The targeted mutagenesis insertion and deletion (indel) rate was analysed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.

Microbial volatile organic compounds (MVOCs) are produced by a wide array of microorganisms ranging from bacteria to fungi. A growing body of evidence indicates that MVOCs are ecofriendly and can be exploited as a cost-effective sustainable strategy for use in agricultural practice as agents that enhance plant growth, productivity and disease resistance. As naturally occurring chemicals, MVOCs have potential as possible alternatives to harmful pesticides, fungicides and bactericides as well as genetic modification. Recent studies performed under open field conditions demonstrate that efficiently adopting MVOCs may contribute to sustainable crop protection and production. We review here the chemical diversity of MVOCs and their potential physiological effects on crops and analyze potential and actual limitations for MVOC use as a sustainable strategy for improving productivity and reducing pesticide use.

Current agricultural practice depends on a wide use of pesticides, bactericides, and fungicides. Increased demand for organic products indicates consumer preference for reduced chemical use. Therefore, there is a need to develop novel sustainable strategies for crop protection and enhancement that do not rely on genetic modification and/or harmful chemicals. An increasing body of evidence indicates that bacterial and fungal microbial volatile organic compounds (MVOCs) might provide an alternative to the use of chemicals to protect plants from pathogens and provide a setting for better crop welfare. It is well known that MVOCs can modulate the physiology of plants and microorganisms and in this Opinion we propose that MVOCs can be exploited as an ecofriendly, cost-effective, and sustainable strategy for agricultural practices.

The availability of genome sequences for many fruit crops has redefined the boundaries of genetic engineering and genetically modified (GM) crop plants. However commercialization of GM crops is hindered by numerous regulatory and social hurdles. Here, we focus on recently developed genome-editing tools for fruit crop improvement and their importance from the consumer perspective. Challenges and opportunities for the deployment of new genome-editing tools for fruit plants are also discussed.