A simple widely applicable hairy root transformation method for gene function studies in medicinal plants

Genetic transformation is a fundamental tool in molecular biology research of medicinal plants. Tailoring transgenic technologies to each distinct medicinal plant would necessitate a substantial investment of time and effort. Here, we present a simple hairy root transformation method that does not require sterile conditions, utilizing Agrobacterium rhizogenes strain K599 and the visible RUBY reporter system. Transgenic hairy roots were obtained for six tested medicinal plant species, roots or rhizomes of which have recognized medicinal value, spanning four botanical families and six genera ( Platycodon grandiflorus , Atractylodes macrocephala , Scutellaria baicalensis , Codonopsis pilosula , Astragalus membranaceus , and Glycyrrhiza uralensis ). Furthermore, two previously identified Glycyrrhiza uralensis UGTs that convert liquiritigenin into liquiritin in heterologous systems were studied in planta using the method. Our results indicate that overexpression of GuUGT1 but not GuUGT10 and Cas9-mediated knockout of GuUGT1 profoundly influenced the accumulation of liquiritin and isoliquiritin in licorice roots. Therefore, the method described here represents a simple, rapid and widely applicable hairy root transformation method that enables fast gene functional study in medicinal plants. A simple widely applicable hairy root transformation method using Agrobacterium rhizogenes K599 and the RUBY visual reporter, successfully applying it to six medicinal plant species across four families. Functional analysis in licorice revealed that GuUGT1 overexpression and CRISPR-mediated knockout significantly regulate liquiritin/isoliquiritin accumulation.