Overexpression of SrDXS1 and SrKAH enhances steviol glycosides content in transgenic Stevia plants

Abstract

BackgroundStevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking.ResultsHere, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42–54% and 67–88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants.ConclusionThis study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1600-2) contains supplementary material, which is available to authorized users.

Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

“…After confirmation by sequencing, the expression vector was transformed into the Agrobacterium strain AGL2. Transformation of Stevia using this Agrobacterium was well described (Zheng et al, 2018). Briefly, the leaf explants were co-cultivated with Agrobacterium on co-cultivation media (0.25 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) + 100 lM acetosyringone) for 3 days and transferred onto callus induction media (1 mg/L 6-benzylaminopurine (BA) + 0.5 mg/L 3-indoleacetic acid (IAA) + 125 mg/L cefotaxime + 50 mg/L kanamycin) after washing.…”

“…gDNAs extracted from the SrUGT76G1-OE lines were digested with HindIII and resolved on a 0.8% agarose gel together with the DIG-labelled DNA molecular weight marker II (Roche, Mannheim, Germany). The agarose gel was then treated for the transfer of fragmented gDNAs onto a positively charged nylon membrane (Hybond-N+) as mentioned previously (Zheng et al, 2018). Following DIG-based Southern blot hybridization (Roche), chemiluminescence from the membrane was detected using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA).…”

“…Previously, we described a method for the Agrobacteriummediated transformation of Stevia and successfully increased the SGs content in the leaves through the overexpression of two pathway genes, SrDXS1 and SrKAH (Zheng et al, 2018). Here, we generated transgenic Stevia plants overexpressing SrUGT76G1.…”

An Efficient Stevia rebaudiana Transformation System and In vitro Enzyme Assays Reveal Novel Insights into UGT76G1 Function

“…Subsequent induction of callus and the maintenance of callus under Hyg selection was also conducted in the dark until regeneration. Very recently, researchers reported using tissue cultured leaves as the explant and successfully obtained stable transgenic Stevia plants 9,26 ; this suggests that both types of originating explant are suitable for efficient transformation. Similar to our results, this same research group also concluded that a prolonged period of dark incubation was critical for obtaining high efficiency Stevia transformation.…”

“…Furthermore, successful recovery of transgenic Stevia callus, but not whole plants, was documented using a GUS-bar construct delivered via particle bombardment 25 . Very recently, a research group from the University of Singapore described the stable transformation of Stevia using kanamycin as the selectable marker and GFP as a reporter gene 9,26 .…”