Effect of two key treatments on the transformation efficiency of fenugreek hairy roots

Plant hairy roots are an important platform for the synthesis of valuable secondary metabolites and for the functional analysis of natural product biosynthetic pathways. Diosgenin is a precursor of more than three hundred sterol drugs, but knowledge regarding how it is biosynthesized in plants remains limited. Fenugreek (Trigonella foenum-graecum L.) is a plant with high diosgenin content. In this study, we used a fenugreek variety from Shanxi Province in China to investigate the effects of two key factors on fenugreek hairy root formation. The two key factors were the density of Agrobacterium rhizogenes and co-incubation supplemented with ultrasonic treatment. Hairy root number and its transformation frequency were improved under increasing A. rhizogenes density, with OD₆₀₀ values from 1.1 to 4.4. Compared with that measured at the OD₆₀₀ value of 1.1, the incubation at 2.2 and 4.4 led to better performance, with hairy root numbers increasing 2.58 and 7.33 times, respectively, and root transformation frequency improving 3.90 and 4.32 times, respectively. During the co-incubation of A. rhizogenes with fenugreek explants, 30 s ultrasonic treatment (operating frequency 40 KHz, ultrasonic power 180 W) slightly decreased the efficiency of hairy root induction, whereas 60 s treatment increased the efficiency by 2.48 times compared with incubation without ultrasonic treatment. The established fenugreek hairy root system provides a platform for the synthesis of diosgenin and a strong basis for the analysis of diosgenin biosynthesis pathways in the future.