Abstract
Scutellaria orientalis and S. araxensis are perennial species of genus Scutellaria (Lamiaceae) which produce some important pharmaceutical secondary metabolites. Four strains of Agrobacterium rhizogenes (A4, A13, ATCC15834 and MSU440), three types of explant (stem, petiole and leaf), six concentrations of acetosyringone (25–150 μM), two different co-cultivation methods (light and dark condition) and two co-cultivation media (full and half-strength MS) were used for optimization of A. rhizogenes mediated transformation efficiency. Moreover, two inoculation approaches including immersion and injection as well as three infection period (5, 7 and 10 min) were examined. The molecular confirmation analysis of putative transgenic roots was performed by PCR using specific primers of the rol B gene. The results revealed that the transformation efficiency was affected by bacterial strain, co-cultivation medium and type of explants. The highest transformation frequency of 93.3% was achieved on stem explants of S. orientalis with ATCC15834 strain, followed by MSU440, A13 and A4 with the frequency of 63.3%, 46.6% and 30%, respectively. In S. araxensis, the best transformation frequency of 76.6% and 43.3% were obtained on stem and petiole explants with MSU440 strain. 5 and 7 min for infection period significantly improved the transformation efficiency in S. orientalis and S. araxensis respectively. Transformation rates of stem explants induced by ATCC15834, A4 and A13 strains were 53.3%, 36.6% and 20% respectively. Transformation frequency was significantly higher in half-strength MS, regardless of A. rhizogenes strains.
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Abbreviations
- ANOVA:
-
Analysis of variance
- HR:
-
Hairy root
- B5:
-
Gamborg B5
- HPLC:
-
High performance liquid chromatography
- LB:
-
Luria-Bertani
- MS:
-
Murashige and Skoog
- T-DNA:
-
Transfer DNA
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The authors would like to thank the authority of the University of Zanjan and school of pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran for their support. This paper is derived from Zahra Gharari’s Ph.D thesis with the grant number of 21147.
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Gharari, Z., Bagheri, K. & Sharafi, A. Establishment of in vitro genetically engineered cultures in Scutellaria orientalis and S. araxensis. Biologia 75, 2383–2393 (2020). https://doi.org/10.2478/s11756-020-00540-9
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DOI: https://doi.org/10.2478/s11756-020-00540-9