Improved rapid and efficient hairy root transformation using Rhizobium rhizogenes in legume crops.

Hairy root transformation mediated by Rhizobium rhizogenes is a widely used tool for molecular analysis and root material for secondary metabolite production. However, this method is time-intensive, technically demanding, and exhibits low transformation efficiency. To address these limitations, we developed a rapid and efficient hairy root transformation system for legume crops, optimizing protocols with the soybean (Glycine max L. Merrill) cultivar Fukuyutaka. Sterilizing seeds with vapor of 5% sodium hypochlorite and germinating them in a double-tier container resulted in over 90% healthy, straight seedlings ideal for transformation, with 3- to 5-day-old seedlings showing the highest transformation rates. Exposing the plant shoot during co-cultivation by covering only the injection area, combined with low nitrogen levels in the hydroponic solution, significantly enhanced hairy root production, yielding up to 16 transgenic hairy roots per plant. Additionally, low nitrogen concentrations were crucial for promoting nodule formation in transgenic hairy roots. These optimized conditions were validated across 12 soybean, 1 cowpea, and 1 mungbean cultivars. The protocol's effectiveness was confirmed through the induction of symbiotic gene expression of GmEnod40a and GmErn1b using a promoter β-glucuronidase (GUS) reporter system in transgenic hairy roots. Expression of these genes was detected in both premature and mature nodules, while GmErn1b expression was also observed in epidermal cells during early nodulation. This optimized hairy root transformation protocol, requiring under 22 days from seed sterilization to transgenic root induction and 61 days to expression analysis, offers a promising approach for efficient gene function studies in legume crops.