Generation of transfer_DNA-free base-edited citrus plants

To recover transgenic citrus plants in the most efficient manner, the use of selection
marker genes is essential. In this work, it was shown that the mutated forms of
the acetolactate synthase (ALS) gene in combination with the herbicide selection
agent imazapyr (IMZ) added to the selection medium may be used to achieve this
goal. This approach enables the development of cisgenic regenerants, namely, plants
without the incorporation of those bacterial genes currently employed for transgenic
selection, and additionally it allows the generation of edited, non-transgenic plants
with altered endogenous ALS genes leading to IMZ resistance. In this work, the citrus
mutants, in which ALS has been converted into IMZ-resistant forms using a base
editor system, were recovered after cocultivation of the explants with Agrobacterium
tumefaciens carrying a cytidine deaminase fused to nSpCas9 in the T-DNA and selecting
regenerants in the culture medium supplemented with IMZ. Analysis of transgene-free
plants indicated that the transient expression of the T-DNA genes was sufficient to
induce ALS mutations and thus generate IMZ-resistant shoots at 11.7% frequency.
To our knowledge, this is the first report of T-DNA-free edited citrus plants. Although
further optimization is required to increase edition efficiency, this methodology will allow
generating new citrus varieties with improved organoleptic/agronomic features without
the need to use foreign genes.