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=Volume 14 - 2023

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=Background: 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 is often used for replacing nucleotides with one donor DNA template (DDT), including homologous sequences. Methods: CRISPR-Cas9 has been recruited, and three variables, Agrobacteria inoculator concentration, the ratio of pDDT/pgRNA, and homologous arm length, have been designed to integrate nptII and 2XCamV35S into the MKK2 promoter zone. Results: Here, we showed that recovered poplars on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by precise integrated 2XCamV35S and nptII, improving biochemical and phenotypic properties. Our findings confirmed that Agrobacterium inoculator OD600 = 2.5, increased DDT numbers during cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused efficient HDR and increased MKK2 expression. Conclusion: Efficient transformations resulted from optimized variables, directly affecting the HDR efficiency through woody plants such as poplar.