Single cell targeted boosting of plant regeneration and genome editing in maize

Abstract

Transformation and regeneration of plant cells are the major bottleneck technologies for plant genome editing. To obtain an edited plant, the two events must fall on the same cell. Meanwhile, genome editing efficiency is largely influenced by the status of targeted cells. Phytohormones and plant morphogenic genes play important roles in regulating cell condition and regeneration. Accumulated evidence has suggested that epigenetically regulating chemicals modulate chromatin structure and thus may impact DNA accessibility. Our results reveal that plant genome editing can be dramatically improved by single cell targeted co-delivery strategy. Specifically, co-delivery of certain phytohormones promotes transient transformation of the co-transformed gene. The co-delivery of an epigenetically regulating chemical promotes transient transformation of the co-delivered gene, as well as genome editing when the editing components are co-transformed. Transient genome editing creates site-specific edits of targets without DNA integration at any points of process, which provide a highly accurate, safe, and powerful plant breeding tool for plant improvement. Our results further demonstrate that regeneration boost protein enhances cell proliferation and genome editing when transiently co-transformed and are adequate for rapid regeneration of single-edited cells directly from recalcitrant maize elites. The co-delivery strategy with the above-mentioned boosting factors allows for efficient site-specific knock-out or knock-in of target genes, as well as near genotype-independent regeneration of single edited cells in maize. The edited plants are transgene-free and fertile, while the edits are fully inheritable.