Abstract
The present study demonstrates the development of an Agrobacterium-mediated genetic transformation method for species of the Sedum genus, which includes the Cd/Zn hyperaccumulator Sedum plumbizincicola and the non-hyperaccumulating ecotype of S. alfredii. Multiple shoots were induced from stem nodes of two Sedum plants using Murashige and Skoog (MS) medium containing 0.1 mg/L cytokinin 6-benzyladenine (6-BA) and 1.0 mg/L auxin 1-naphthaleneacetic acid (NAA). The shoot primordia were used as direct targets for Agrobacterium infection. Selection on hygromycin was highly effective in generating Agrobacterium-transformed explants. This callus-free procedure allowed us to obtain transgenic plantlets after rooting hygromycin-resistant shoots on phytohormone-free MS medium containing the antibiotic. The presence and expression of the reporter genes gusA and GFP in transgenic plants were confirmed by a real-time polymerase chain reaction, histochemical GUS assays, and confocal microscopy. This reliable method for genetic transformation of Sedum plants will help us to understand gene functions and the molecular mechanisms underlying Cd hypertolerance and hyperaccumulation in these species.
Highlights
The genus Sedum L. is the most species-rich member of the family Crassulaceae (Nikulina et al, 2016)
Shoot buds can be induced from stem internode-derived callus in S. alfredii using shoot-induction medium containing 6-BA and naphthaleneacetic acid (NAA) (Zhao et al, 2009)
To optimize conditions for producing multiple shoots in S. alfredii and to evaluate the effect of combining different concentrations of phytohormones on the number of shoots regenerated, stem node explants were cultured on Murashige and Skoog (MS) media containing various concentrations of 6-BA (0.1, 0.5, and 1 mg/L) and NAA (0.1, 0.5, 1, and 1.5 mg/L)
Summary
The genus Sedum L. is the most species-rich member of the family Crassulaceae (Nikulina et al, 2016). There are about 420 species (Thiede and Eggli, 2007), most of which can endure harsh environments ranging from very cold to hot temperatures and dry conditions. In response to conditions of drought stress, many Sedum species evolve crassulacean acid metabolism (CAM) system to take up inorganic carbon in the night (Castillo, 1996; Smirnoff, 1996; Winter and Holtum, 2014). Sedums are usually cultivated as ornamental plants due to their interesting appearance, diversified phenotypes and extensive adaptability. Some sedums, such as Sedum sarmentosum, Genetic Transformation of Sedum Species. S. plumbizincicola and population of S. alfredii, native to cadmium (Cd)/zinc (Zn) mining areas in Southeast China, were remarkable Zn and Cd hyperaccumulators that have high capacity to accumulate, translocate, and tolerate high concentrations of heavy metals (Yang et al, 2004; Wu et al, 2013)
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