Key message The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Abstract Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot—a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome. Multiplexing CRISPR/Cas9 vectors expressing two single-guide RNA (gRNAs) targeting the carrot flavanone-3-hydroxylase (F3H) gene were tested for blockage of the anthocyanin biosynthesis in a model purple-colored callus using Agrobacterium-mediated genetic transformation. This approach allowed fast and visual comparison of three codon-optimized Cas9 genes and revealed that the most efficient one in generating F3H mutants was the Arabidopsis codon-optimized AteCas9 gene with up to 90% efficiency. Knockout of F3H gene resulted in the discoloration of calli, validating the functional role of this gene in the anthocyanin biosynthesis in carrot as well as providing a visual marker for screening successfully edited events. Most resulting mutations were small Indels, but long chromosome fragment deletions of 116–119 nt were also generated with simultaneous cleavage mediated by two gRNAs. The results demonstrate successful site-directed mutagenesis in carrot with CRISPR/Cas9 and the usefulness of a model callus culture to validate genome editing systems. Given that the carrot genome has been sequenced recently, our timely study sheds light on the promising application of genome editing tools for boosting basic and translational research in this important vegetable crop.

In this study we report the development of effective in vitro systems for a medicinal plant Nigella damascena L. comprising: (1) callus induction, (2) somatic embryogenesis in callus cultures with subsequent plant regeneration, and (3) isolation and regeneration of callus-derived protoplasts. Callus development was achieved on 83–100% of hypocotyl and cotyledon explants, whereby Murashige and Skoog medium (MS) supplemented with 3 mg L⁻¹ 6-benzylaminopurine and 0.5 mg L⁻¹α-naphthaleneacetic acid (NAA; BN medium) was more advantageous than MS with kinetin and NAA (KN medium). Histological observations of calli revealed the presence of embryogenic zones from which somatic embryos developed on the hormone-free medium. Plant regeneration was observed on 76–95% of calli. A high capacity to form somatic embryos and regeneration was maintained in long-lasting cultures, i.e. even in 2 year old callus.The obtained callus was also a good source tissue for protoplast isolation. By applying a mixture of cellulase and pectolyase, the acceptable yield of viable protoplasts was achieved, especially from hypocotyl-derived callus maintained on BN medium. Protoplasts embedded in an alginate matrix and cultured in modified Kao and Michayluk media re-constructed their cell wall and re-entered mitotic divisions. About 30% of small cell aggregates formed microcalli, which, after the release from alginate, proliferated continuously on KN and BN media, irrespective of the tissue variant used as the protoplast source. Somatic embryo formation and plant regeneration were successful on hormone-free media. An effective plant regeneration system of N. damascena protoplast cultures has been developed and is being reported for the first time.

The new model orange callus line, similar to carrot root, was rich in carotenoids due to altered expression of some carotenogenesis-associated genes and possessed unique diversity of chromoplast ultrastructure.

In this review, we present genetically modified (GM) horticultural events that have passed the regulatory process and have been approved for cultivation or food use in different countries. The first authorization or deregulation of a GM horticultural plant issued 25 years ago initiated a fast expansion of GM organisms (GMO) engineered by using gene transfer technology. The list of GM horticultural species comprises representatives of vegetables, fruit plants and ornamentals. We describe their unique characteristics, often not achievable by conventional breeding, and how they were developed, and the approval process. Information on the adoption of GM horticultural cultivars and sale is accessed if commercialization has occurred. The review comprises, among others, Flavr Savr ^TM and other tomato cultivars with delayed ripening and improved shelf-life, insect-resistant eggplant (or brinjal), as well as virus-resistant squash, melon and the common bean, and also fruit trees, plum and papaya. Cultivation of the latter was particularly valuable to farmers in Hawaii as it ensured restoration of papaya production devastated earlier by the Papaya ringspot virus (PRSV). In contrast, a plum resistant to sharka ( Plum pox virus ; PPV) deregulated in the USA is still awaiting commercialization. GM events with improved quality include the recently marketed non-browning apple and high-lycopene pineapple. We also present orange petunia, blue ‘Applause’ rose and Moon-series carnations with a modified purple and violet flower colour. Finally, we discuss prospects of GM horticultural plants, including their development using promising new breeding technologies relying on genome editing and considered as an alternative to the transgenic approach.