Key message Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. Abstract Site-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2–12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1–10 bp, but also vector DNA inserts of 34–236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.

The technology to produce genetically engineered (GE) plants is celebrating its 30th anniversary and one of the major achievements has been the development of GE crops. The safety of GE crops is crucial for their adoption and has been the object of intense research work often ignored in the public debate. We have reviewed the scientific literature on GE crop safety during the last 10 years, built a classified and manageable list of scientific papers, and analyzed the distribution and composition of the published literature. We selected original research papers, reviews, relevant opinions and reports addressing all the major issues that emerged in the debate on GE crops, trying to catch the scientific consensus that has matured since GE plants became widely cultivated worldwide. The scientific research conducted so far has not detected any significant hazards directly connected with the use of GE crops; however, the debate is still intense. An improvement in the efficacy of scientific communication could have a significant impact on the future of agricultural GE. Our collection of scientific records is available to researchers, communicators and teachers at all levels to help create an informed, balanced public perception on the important issue of GE use in agriculture.

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

Climate change, associated with global warming, extreme weather events, and increasing incidence of weeds, pests and pathogens, is strongly influencing major cropping systems. In this challenging scenario, miscellaneous strategies are needed to expedite the rate of genetic gains with the purpose of developing novel varieties. Large plant breeding populations, efficient high-throughput technologies, big data management tools, and downstream biotechnology and molecular techniques are the pillars on which next generation breeding is based. In this review, we describe the toolbox the breeder has to face the challenges imposed by climate change, remark on the key role bioinformatics plays in the analysis and interpretation of big “omics” data, and acknowledge all the benefits that have been introduced into breeding strategies with the biotechnological and digital revolution.