Transgene‐free genome editing in poplar

Summary Precise gene‐editing methods are valuable tools to enhance genetic traits. Gene editing is commonly achieved via stable integration of a gene‐editing cassette in the plant's genome. However, this technique is unfavorable for field applications, especially in vegetatively propagated plants, such as many commercial tree species, where the gene‐editing cassette cannot be segregated away without breaking the genetic constitution of the elite variety. Here, we describe an efficient method for generating gene‐edited Populus tremula × P. alba (poplar) trees without incorporating foreign DNA into its genome. Using Agrobacterium tumefaciens , we expressed a base‐editing construct targeting CCoAOMT1 along with the ALS genes for positive selection on a chlorsulfuron‐containing medium. About 50% of the regenerated shoots were derived from transient transformation and were free of T‐DNA. Overall, 7% of the chlorsulfuron‐resistant shoots were T‐DNA free, edited in the CCoAOMT1 gene and nonchimeric. Long‐read whole‐genome sequencing confirmed the absence of any foreign DNA in the tested gene‐edited lines. Additionally, we evaluated the CodA gene as a negative selection marker to eliminate lines that stably incorporated the T‐DNA into their genome. Although the latter negative selection is not essential for selecting transgene‐free, gene‐edited Populus tremula × P. alba shoots, it may prove valuable for other genotypes or varieties.