The effect of different strains of Agrobacterium rhizogenes in the Hypericum perforatum hairy root induction

Abstract
Background and Objective:
Hypericum perforatum L. (St. John's wort) is an endangered medicinal plant valued for its diverse secondary metabolites. The commercial extraction of these compounds from wild plants presents challenges, making in vitro hairy root culture an effective alternative for their production. Induction of hairy roots using Agrobacterium rhizogenesis a practical method for studying the biosynthetic pathway of secondary metabolites. Various studies indicate that among genes of rolA, rolB, rolC, and rolD in A. rhizogenes, the rolB gene plays a major role in inducing the hairy root than the other genes. The present study aimed to investigate the effect of different strains and explant types (leaf, stem, and root) on hairy root induction in H. perforatum."
Materials and Methods:
Seeds of H. perforatum were obtained from the Natural Resources Gene Bank at the Research Institute of Forests and Rangelands (RIFR), Tehran, Iran. A factorial experiment was conducted with factor A included three explant types (leaf, stem, and root), while factor B included five A. rhizogenes strains (1724, ATCC, Mus, A4, and R1000) using a completely randomized design with three replications in 2022 in RIFR. Fifteen-day-old plantlets were subcultured twice in full-strength MS medium. The explants were then transferred to a growth medium and inoculated with A. rhizogenes strains for hairy root induction. Morphological traits, including primary and secondary root length, number of hairy roots, fresh and dry weight, and induction percentage were evaluated over two months. Since the rolB gene plays a major role in hairy root induction, Genomic DNA was extracted using the CTAB method, and the presence of the rolB gene was verified via PCR to confirm transgenic hairy root formation. Positive and negative control wells containing inoculated hairy roots were included in the bioassay. DNA fragments of different lengths were separated using electrophoresis. Data were statistically analyzed and means were compared via the Duncan multiple range test. Statistical analyses were conducted using SPSS, and graphs were generated in Excel.
Results:
In comparison between explants, the highest hairy root induction rate (24.44%), fresh weight (7.54 g), and dry weight (4.46 g) were observed in leaf explants inoculated with the R1000 strain, which was significantly higher than that other A. rhizogenes strains. The lowest induction rate (17.77%) was observed in leaf explants treated with strain 1724, while stem explants inoculated with strain ATCC showed the lowest overall induction rate (8.88%). For root explants, only strains A4 and Mus successfully induced hairy roots, while the other strains failed. Transgenic hairy root lines were confirmed through PCR amplification of the rolB gene using gene-specific primers.
Conclusion:
The results showed that the type of explant, type of A. rhizogenes strains, and the type of culture medium are effective in production of transgenic hairy roots. The R1000 strain demonstrated superior performance in hairy root induction, biomass accumulation, and overall growth in leaf and stem explants, making it the most effective strain for in vitro hairy root production in H. perforatum. In root explants, the A4 strain had the highest impact on the induction rate. The strong response of leaf explants to the R1000 strain suggests enhanced interaction with the plant biosynthetic pathway, leading to improved hairy root formation and growth in Hypericum perforatum