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

Guoliang Yuan; Yang Liu; Tao Yao; Wellington Muchero; Jin-Gui Chen; Gerald A Tuskan; Xiaohan Yang

doi:10.3390/plants12081657

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

Plants (Basel). 2023 Apr 14;12(8):1657. doi: 10.3390/plants12081657.

Authors

Guoliang Yuan^{1

2

3}, Yang Liu¹, Tao Yao^{1

2}, Wellington Muchero^{1

2}, Jin-Gui Chen^{1

2}, Gerald A Tuskan^{1

2}, Xiaohan Yang^{1

2}

Affiliations

¹ Oak Ridge National Laboratory, Biosciences Division, Oak Ridge, TN 37831, USA.
² The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
³ Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA 99352, USA.

Abstract

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 Agrobacterium-mediated transformation. As an important model for poplar genomics and biotechnology research, eastern cottonwood (Populus deltoides) clone WV94 can be transformed by A. tumefaciens, 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 P. deltoides yet. Here, we first optimized the protocol for Agrobacterium-mediated stable transformation in P. deltoides 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 P. deltoides WV94 and hybrid poplar clone '52-225' (P. trichocarpa × P. deltoides 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 P. deltoides 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 P. deltoides.

Keywords: CRISPR-Cas9; eYGFPuv; gene editing; protoplasts; transformant screening; transient transformation.

Grants and funding

The work was supported by the Center for Bioenergy Innovation, a U.S. Department of Energy (DOE) Bioenergy Research Center supported by the Biological and Environmental Research (BER) program. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. Department of Energy under Contract Number DE-AC05-00OR22725.