One-Step Regeneration of Hairy Roots to Induce High Tanshinone Plants in Salvia miltiorrhiza

Abstract

Salvia miltiorrhiza is a traditional Chinese medicinal plant of Labiatae, which has been widely utilized to treat a variety of cardiovascular and cerebrovascular diseases. However, due to the long growth cycle, low content of active ingredients, and serious quality deterioration of S. miltiorrhiza, the use of biotechnology to improve S. miltiorrhiza to meet the growing demand for clinical applications has become a research hotspot. In this study, a novel one-step hairy root regeneration method was developed, which could rapidly obtain hairy roots and regenerated plants with high tanshinone content. By optimizing the parameters of Agrobacterium rhizogenes transformation in S. miltiorrhiza, it was finally established that the explants were infected in Ar.qual (OD600 = 0.6) for 10 min, co-cultured for 3 days, and then screened on the screening medium containing 7.5 mg/l hygromycin, the maximum transformation frequency can reach 73.85%. GFP and PCR detection yielded a total of 9 positive transgenic hairy root lines and 11 positive transgenic regenerated plants. SmGGPPS1 was successfully overexpressed in positive transgenic regenerated plants, according to the results of qRT-PCR. The content of tanshinone IIA and cryptotanshinone were dramatically enhanced in transgenic regenerated plants and hairy roots by Ultra Performance Liquid Chromatography analysis. Based on the Agrobacterium-mediated transformation of S. miltiorrhiza, this study developed a new method for regenerating plants with transgenic hairy roots. This method provides a foundation for the breeding of S. miltiorrhiza and the sustainable development of medicinal plant resources, as well as provides a useful reference for the application of other species.

Induction, Flavonoids Contents, and Bioactivities Analysis of Hairy Roots and True Roots of Tetrastigma hemsleyanum Diels et Gilg

“…Agrobacterium rhizogenes ( A. rhizogenes ) C58C1 was activated and expanded under the culture conditions described by Naeini et al [ 32 ]. The A. rhizogenes Ar Qual and A. rhizogenes Ar Qual harboring the binary vector pCAMBIA 1381Z (CAMBIA)—which contains the GUS open reading frame (ORF) and the selective marker gene kanamycin resistance (KAN) under the control of CaMV35S promoter—were activated and expanded under the culture conditions described by Su et al [ 33 ]. Negative controls were set under the same conditions.…”

Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY

“…Where, HR is the percentage of hairy root induction, N hr is the number of cotyledons/seedlings with hairy roots (lengths ≥ 1 cm), N T is the total number of inoculated cotyledons/infected seedlings, TE is transformation efficiency, N RUBY is the number of cotyledons/ seedlings with at least one hairy root expressing the RUBY gene, and N I is the number of inoculated cotyledons/infected seedlings with hairy roots (length ≥ 1 cm) (Melito et al, 2010;Su et al, 2022).…”

Mother Transformer: A High-Throughput, Cost-Effective in Planta Hairy Root Transformation Method for Cannabis

“…HR (%) = HR: Percentage of hairy root induction; N hr : Number of seedlings with hairy roots (≥ 1 cm); N T : Total number of inoculate seedlings. (2) TE (%) = TE: Transformation e ciency; N RUBY : Number of seedlings with at least one hairy root expressing the RUBY gene; N I : Number of inoculated seedlings with hairy roots (≥ 1 cm)[24,25] …”