A dual sgRNA-directed CRISPR/Cas9 construct for editing the fruit-specific ß-cyclase 2 gene in pigmented citrus fruits.

CRISPR/Cas9 genome editing is a modern biotechnological approach used to improve plant varieties, modifying only one or a few traits of a specific variety. However, this technology cannot be easily used to improve fruit quality traits in citrus, due to the lack of knowledge of key genes, long juvenile stage, and the difficulty regenerating whole plants of specific varieties. Here, we introduce a genome editing approach with the aim of producing citrus plantlets whose fruits contain both lycopene and anthocyanins. Our method employs a dual single guide RNA (sgRNA)-directed genome editing approach to knockout the fruit-specific ß-cyclase 2 gene, responsible for the conversion of lycopene to beta-carotene. The gene is targeted by two sgRNAs simultaneously to create a large deletion, as well as to induce point mutations in both sgRNA targets. The EHA105 strain of Agrobacterium tumefaciens was used to transform five different anthocyanin-pigmented sweet oranges, belonging to the Tarocco and Sanguigno varietal groups, and 'Carrizo' citrange, a citrus rootstock as a model for citrus transformation. Among 58 plantlets sequenced in the target region, 86% of them were successfully edited. The most frequent mutations were deletions (from -1 to -74 nucleotides) and insertions (+1 nucleotide). Moreover, a novel event was identified in six plantlets, consisting of the inversion of the region between the two sgRNAs. For 20 plantlets in which a single mutation occurred, we excluded chimeric events. Plantlets did not show an altered phenotype in vegetative tissues. To the best of our knowledge, this work represents the first example of the use of a genome editing approach to potentially improve qualitative traits of citrus fruit.

New Plant Breeding Techniques in Citrus for the Improvement of Important Agronomic Traits. A Review.

New plant breeding techniques (NPBTs) aim to overcome traditional breeding limits for fruit tree species, in order to obtain new varieties with improved organoleptic traits and resistance to biotic and abiotic stress, and to maintain fruit quality achieved over centuries by (clonal) selection. Knowledge on the gene(s) controlling a specific trait is essential for the use of NPBTs, such as genome editing and cisgenesis. In the framework of the international scientific community working on fruit tree species, including citrus, NPBTs have mainly been applied to address pathogen threats. Citrus could take advantage of NPBTs because of its complex species biology (seedlessness, apomixis, high heterozygosity, and long juvenility phase) and aptitude for in vitro manipulation. To our knowledge, genome editing in citrus via transgenesis has successful for induced resistance to Citrus bacterial canker in sweet orange and grapefruit using the resistance gene CsLOB1. In the future, NPBTs will also be used to improve fruit traits, making them healthier. The regeneration of plants following the application of NPBTs is a bottleneck, making it necessary to optimize the efficiency of current protocols. The strengths and weaknesses of using explants from young in vitro plantlets, and from mature plants, will be discussed. Other major issues addressed in this review are related to the requirement for marker-free systems and shortening the long juvenility phase. This review aims to summarize methods and approaches available in the literature that are suitable to citrus, focusing on the principles observed before the use of NPBTs.

Cynara cardunculus L. as a Multipurpose Crop for Plant Secondary Metabolites Production in Marginal Stressed Lands.

Cardoon (Cynara cardunculus L.) is a Mediterranean crop, member of the Asteraceae family, characterized by high production of biomass and secondary metabolites and by a good adaptation to climate change, usable in green chemistry, nutraceutical, and pharmaceutical sectors. Recent studies demonstrated the ability of cardoon to grow up in a stressful environment, which is associated with enhanced biosynthesis of biologically active compounds in these plants, and this effect is increased by abiotic stresses (salt, heat, pollution, and drought stress) that characterize many world marginal areas, affected by the climate changes. The plant response to these stresses consists in implementing different processes that modify some plant biological functions, such as alleviating both cellular hyperosmolarity and ion disequilibrium or synthesizing antioxidant molecules. The aim of this work was to investigate different cardoon response mechanisms to abiotic stresses and to evaluate their influence on the biologically active compounds biosynthesis. Following this purpose, we analyzed the ability of cardoon seeds to germinate under different salt stress conditions, and on the sprouts obtained, we measured the total phenol content and the antioxidant activity. Moreover, the growth of cardoon seedlings grown under heavy metals stress conditions was monitored, and the expression levels of heavy metal transport-associated genes were analyzed. The results showed the ability of cardoon plants to tolerate abiotic stress, thanks to different defense mechanisms and the possibility to obtain biomass with high content of biologically active molecules by exploiting the natural tolerance of this species for abiotic stresses. Moreover, we identified some important genes encoding for metal transportation that may be involved in arsenic and cadmium uptake and translocation in C. cardunculus. Then, this species can be considered as a promising crop for green chemistry and energy in marginal lands.