Construction of an efficient editing system for forest tree genomes based on a visual screening marker in Eucalyptus urophylla × Eucalyptus grandis

Abstract

In this study, The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology for genome editing was used to develop an efficient gene editing system for Eucalyptus urophylla × Eucalyptus grandis and generate a new Eucalyptus germplasm with reduced lignin contents in the pulp for environmental sustainability in papermaking. By targeting the Cinnamate-4-hydroxylase (C4H) gene in E. urophylla × E. grandis, the recombinant plasmid pHEE401E-35S-RUBY-EuC4H was constructed through homologous recombination. This plasmid was then transformed into E. urophylla × E. grandis callus tissue. Using the RUBY gene as a marker, positive transformants were screened based on the callus tissue phenotype. Subsequent polymerase chain reaction and sequencing confirmed the successful creation of mutants with a significantly edited EuC4H gene. The method used in this study for identifying positive transformants with a visually screened marker gene offers a valuable framework and guidance for genetically improving and establishing an efficient gene editing system in Eucalyptus.