AUTHOR=Movahedi Ali , Wei Hui , Kadkhodaei Saeid , Sun Weibo , Zhuge Qiang , Yang Liming , Xu Chen 

TITLE=CRISPR-mediated genome editing in poplar issued by efficient transformation

JOURNAL=Frontiers in Plant Science

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1159615

DOI=10.3389/fpls.2023.1159615

ISSN=1664-462X

ABSTRACT=<sec><title>Background</title><p>CRISPR has been increasingly used for plant genetic improvements because of its high efficiency and precision. Recently, the authors have reported the possibility of homology-directed repair (HDR) using CRISPR/Cas9 through woody plants such as poplar. HDR often replaces nucleotides with one donor DNA template (DDT), including homologous sequences.</p></sec><sec><title>Methods</title><p>CRISPR–Cas9 was recruited, and three variables, Agrobacteria inoculator concentration, pDDT/pgRNA ratio, and homologous arm length, were designed to integrate <italic>nptII</italic> and 2XCamV 35S into the <italic>MKK2</italic> promoter zone.</p></sec><sec><title>Results</title><p>Here, we showed that recovered poplars on kanamycin-supplemented media exhibited enhanced expression of <italic>MKK2</italic> affected by the precise integration of 2XcamV 35S and <italic>nptII</italic>, improving biochemical and phenotypic properties. Our findings confirmed that <italic>Agrobacterium</italic> inoculator OD<sub>600</sub> = 2.5, increased DDT numbers during cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused efficient HDR and increased <italic>MKK2</italic> expression.</p></sec><sec><title>Conclusion</title><p>Efficient transformations resulted from optimized variables, directly affecting the HDR efficiency through woody plants such as poplar.</p></sec>