Abstract 

CRISPR/Cas technology for specific and precise modification of genomes has transformed molecular biology. However, quick elimination of the transgene remains a challenge in plant biotechnology after genome edition, especially for crops due to their long life cycle and multiploidy, not only to avoid transgene position effects and to minimize the probability of off-target mutation appearance, but also to deliver end users with edited plants free of the recombinant gene editing machinery. Counter selection based on resistance marker genes are inconvenient in the case of CRISPR/Cas applications because plants lacking the transgene cannot survive the selection, and thus two more generations must be screened to evaluate the presence of the transgene. In the case of some crops, generations can last between 6 months and a few years, and the workload may be a limiting factor because transgene detection by PCR requires germination of seeds, and selected plants must be grown until a new generation can be harvested. The expression of fluorescent proteins as selective marker has been successfully used in Arabidopsis thaliana as a fast method for transgene presence detection prior to seed germination, but it has not been tested in other species yet, because of the special requirements of in vitro transformation protocols. Using the modular cloning system Golden Braid we have adapted fluorescence-dependent monitoring of transgene to genome editing approaches in tomato and rice. We demonstrate how this strategy allows obtaining transgene-free homozygous edited crop plants in one generation after in vitro transformation.

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