Abstract
Veratrum dahuricum L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to produce the anticancer drug cyclopamine. An efficient transformation system of Veratrum dahuricum mediated with Agrobacterium tumefaciens is presented. Murashige and Skoog (MS) medium containing 8 mg/L picloram was used to induce embryogenic calli from immature embryos with 56% efficiency. A. tumefaciens LBA4404 carrying the bar gene driven by the cauliflower mosaic virus 35S promoter was employed for embryogenic callus inoculation. A. tumefaciens cell density OD660 = 0.8 for inoculation, half an hour infection period, and three days of co-culture duration were found to be optimal for callus transformation. Phosphinothricin (PPT, 16 mg/L) was used as the selectable agent, and a transformation efficiency of 15% (transgenic plants/100 infected calli) was obtained. The transgenic nature of the regenerated plants was confirmed by PCR and Southern blot analysis, and expression of the bar gene was detected by RT-PCR and Quick PAT/bar strips. The steroid alkaloids cyclopamine, jervine, and veratramine were detected in transgenic plants, in non-transformed and control plants collected from natural sites. The transformation system constitutes a prerequisite for the production of the pharmaceutically important anticancer drug cyclopamine by metabolic engineering of Veratrum.
Highlights
IntroductionVeratrum dahuricum (Liliaceae), often referred to as mountain corn, which was used as traditional
Veratrum dahuricum (Liliaceae), often referred to as mountain corn, which was used as traditionalChinese medicine in the treatment of stroke, dysentery, jaundice, headache, and chronic malaria, grows in the Changbai mountains of Northeast China [1]
In this paper we report a complete genetic transformation method for V. dahuricum, a species derivatives have shown promising antineoplastic activities against several cancers that arise from the producing the pharmaceutically important steroid alkaloids cyclopamine, jervine, and veratramine disruption of the sonic hedgehog (SHh) pathway [7], because several genes in the pathway have been associated with these (Figure 1a–c)
Summary
Veratrum dahuricum (Liliaceae), often referred to as mountain corn, which was used as traditional. In vitro culture of Veratrum has already been established [17], and several functional genes covering the early steps in the biosynthesis of Plants 2020, 9, 191 cyclopamine have been characterized [18]. In this paper we report a complete genetic transformation method for V. dahuricum, a species derivatives have shown promising antineoplastic activities against several cancers that arise from the producing the pharmaceutically important steroid alkaloids cyclopamine, jervine, and veratramine disruption of the SHh pathway [7], because several genes in the pathway have been associated with these (Figure 1a–c). After three to four months of culture,jervine, the embryogenic calli producing pharmaceutically important steroid alkaloids cyclopamine, and veratramine were transferred to AA medium [20], supplemented with mg/L of the phytohormone.
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