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.

The efficient,stable delivery of siRNA into cells,and the appropriate controls for non-specific off-target effects of siRNA are major limitations to functional studies using siRNA technology.To overcome these drawbacks,we have developed a single lentiviral vector that can concurrently deplete endogenous gene expression while expressing an epitope-tagged siRNA-resistant target gene in the same cell.To demonstrate the functional utility of this system,we performed RNAi-depleted α-actinin-1 (α-ACTN1) expression in human T cells,α-ACTN1 RNAi resulted in inhibited chemotaxis to SDF-1α,but it can be completely rescued by concurrent expression of RNAi-resistant α-ACTN1 (rr-α-ACTN1) in the same cell.The presence of a GFP tag on rr-α-ACTN1 allowed for detection of appropriate subcellular localization of rr-α-ACTN1.This system provides not only an internal control for RNAi off-target effects,but also the potential tool for rapid structure-function analyses and gene therapy.