eYGFPuv-Assisted Transgenic Selection in WV94 and Multiplex Genome Editing in Protoplasts of × Clone '52-225'.

Although CRISPR/Cas-based genome editing has been widely used for plant genetic engineering, its application in the genetic improvement of trees has been limited, partly because of challenges in -mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood () clone WV94 can be transformed by . , but several challenges remain unresolved, including the relatively low transformation efficiency and the relatively high rate of false positives from antibiotic-based selection of transgenic events. Moreover, the efficacy of CRISPR-Cas system has not been explored in . yet. Here, we first optimized the protocol for -mediated stable transformation in . WV94 and applied a UV-visible reporter called eYGFPuv in transformation. Our results showed that the transgenic events in the early stage of transformation could be easily recognized and counted in a non-invasive manner to narrow down the number of regenerated shoots for further molecular characterization (at the DNA or mRNA level) using PCR. We found that approximately 8.7% of explants regenerated transgenic shoots with green fluorescence within two months. Next, we examined the efficacy of multiplex CRISPR-based genome editing in the protoplasts derived from . WV94 and hybrid poplar clone '52-225' ( × clone '52-225'). The two constructs expressing the Trex2-Cas9 system resulted in mutation efficiency ranging from 31% to 57% in hybrid poplar clone 52-225, but no editing events were observed in . WV94 transient assay. The eYGFPuv-assisted plant transformation and genome editing approach demonstrated in this study has great potential for accelerating the genome editing-based breeding process in poplar and other non-model plants species and point to the need for additional CRISPR work in .