Abstract
Hypericum sinaicum L. is an endangered Egyptian medicinal plant of high importance due to the presence of naphthodianthrones (hypericins), which have photodynamic properties and pharmaceutical potential. We sought to assess H. sinaicum ability to develop hairy roots that could be cultured in contained conditions in vitro and used as a source for hypericin production. We used four A. rhizogenes strains differing in their plasmids and chromosomal backgrounds to inoculate excised H. sinaicum root, stem and leaf explants to induce hairy root development. Additionally, inoculum was applied to shoots held in Rockwool cubes supporting their stand after removal of the root system. All explant types were susceptible to A. rhizogenes although stem explants responded more frequently (over 90%) than other explant types. The A4 and A4T A. rhizogenes strains were highly, and equally effective in hairy root induction on 66-72% of explants while the LBA1334 strain was the most effective in transformation of shoots. Sonication applied to explants during inoculation enhanced the frequency of hairy root development, the most effective was 60 s treatment doubling the percentage of explants with hairy roots. However, shoot transformation was the most effective approach as shoots developed hairy roots within 10 days after inoculation. Molecular analyses confirmed that the established hairy root cultures in vitro were indeed obtained due to a horizontal gene transfer from bacteria. These cultures grew fast and the hypericin content in hairy roots was about two fold higher than in H. sinaicum plants as determined by HPLC.
Highlights
The genus Hypericum belonging to the Hypericeae family comprises approximately 450 species of trees, shrubs and herbs found in different temperate regions in the world (Mártonfiová et al, 2014)
In this work we show for the first time the successful A. rhizogenes-mediated transformation of H. sinaicum and the ability of the obtained hairy roots to biosynthesize hypericin, a plant metabolite of pharmaceutical significance
The results obtained indicate that different bacterial strains can be used for hairy root induction in H. sinaicum with various effectiveness depending on the applied method and explant type
Summary
The genus Hypericum belonging to the Hypericeae family comprises approximately 450 species of trees, shrubs and herbs found in different temperate regions in the world (Mártonfiová et al, 2014). Hairy roots excised from the source tissue retain their vigorous growth and exhibit extensive lateral branching when cultured on growth regulator-free media, and can be maintained in in vitro culture using either a solid or liquid medium (Li et al, 2008) They show potential for stable production of various bioactive compounds that can be extracted if desired (Tusevski et al, 2013). A. rhizogenes strain A4-mediated transformation system applied to H. perforatum led to the development of hairy root cultures amenable for subsequent shoot regeneration These transgenic shoots produced naphtodianthrone compounds, and the concentration of hypericin and pseudohypericin was about 12-fold higher than in not transgenic control (Bivadi et al, 2014). The developed method can be helpful in studying regulatory aspects involved in the biosynthesis of important bioactive metabolites in this species
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