Release of chimeras and efficient selection of editing mutants by CRISPR/Cas9-mediated gene editing in apple

Gene editing, a powerful tool, can provide unprecedented opportunities for the genetic improvement of apples. However, in the application of apple gene editing, chimeras with coexisting mutations of various types occur frequently. Therefore, it is crucial to establish an efficient gene editing system and an editing variant selection method. For the work described herein, the visual marker gene PDS encoding phytoene desaturase in Malus x domestica cultivar "Greensleeves" was targeted for gene editing using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Transformants have been subcultured for over two years, with albinos and chimeras of variegated and pale green shoots emerging at different times. These plants contained individual PDS mutations. Furthermore, chimera mutants were converted to albinos after 2-3 subcultures. "Additional regeneration" of green transformants revealed that additional mutations were generated, simultaneously leading to albino in-dividuals. These protocols can be combined to obtain homozygous mutants efficiently in apples.