Summary Genome editing via the homology‐directed repair (HDR) pathway in somatic plant cells is very inefficient compared with error‐prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR‐based genome editing efficiency approximately threefold compared with a Cas9‐based single‐replicon system via the use of de novo multi‐replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon‐free but stable HDR alleles. The efficiency of CRISPR/LbCpf1‐based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31 °C under a light/dark cycle after Agrobacterium‐mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single‐replicon system into a multi‐replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR‐based genome editing of a salt‐tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self‐pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mm NaCl. Our work may pave the way for transgene‐free editing of alleles of interest in asexually and sexually reproducing plants.

Continuing crop domestication/redomestication and modification is a key determinant of the adaptation and fulfillment of the food requirements of an exploding global population under increasingly challenging conditions such as climate change and the reduction in arable lands. Monocotyledonous crops are not only responsible for approximately 70% of total global crop production, indicating their important roles in human life, but also the first crops to be challenged with the abovementioned hurdles; hence, monocot crops should be the first to be engineered and/or de novo domesticated/redomesticated. A long time has passed since the first green revolution; the world is again facing the challenge of feeding a predicted 9.7 billion people in 2050, since the decline in world hunger was reversed in 2015. One of the major lessons learned from the first green revolution is the importance of novel and advanced trait-carrying crop varieties that are ideally adapted to new agricultural practices. New plant breeding techniques (NPBTs), such as genome editing, could help us succeed in this mission to create novel and advanced crops. Considering the importance of NPBTs in crop genetic improvement, we attempt to summarize and discuss the latest progress with major approaches, such as site-directed mutagenesis using molecular scissors, base editors and especially homology-directed gene targeting (HGT), a very challenging but potentially highly precise genome modification approach in plants. We therefore suggest potential approaches for the improvement of practical HGT, focusing on monocots, and discuss a potential approach for the regulation of genome-edited products.

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