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Zheng et al. BMC Plant Biology (2019) 19:1 https://doi.org/10.1186/s12870-018-1600-2 RESEARCH ARTICLE Open Access Overexpression of SrDXS1 and SrKAH enhances steviol glycosides content in transgenic Stevia plants Junshi Zheng1,2, Yan Zhuang1, Hui-Zhu Mao1 and In-Cheol Jang1,2* Abstract Background: Stevia 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. Results: Here, 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 SrDXS1and 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. Conclusion: This 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. Keywords: 1-deoxy-d-xylulose-5-phosphate synthase 1, Kaurenoic acid hydroxylase, Metabolic engineering, Stevia transformation, Steviol glycosides, Transgenic Stevia Background Stevia rebaudiana is a perennial shrub that belongs to the Asteraceae family. It produces steviol glycosides (SGs) that range from 150 to 300 times as sweet as sucrose, making it unique among plants [1]. SGs are mainly accumulated in the leaves of Stevia, accounting for around 4–20% of leaf dry weight [2]. In Paraguay where Stevia is native to, people have long been using it to sweeten their teas and medicine [3]. In recent times, the value of Stevia leaf extracts or specific SG, like Rebaudioside A (Reb A), as a zero calorie natural sweetener has also gained recognition beyond its native * Correspondence: jangi@tll.org.sg 1 Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore 2 Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore country, leading to the introduction of Stevia as a commercial crop in many other countries [1]. SGs are a group of diterpenoids with varying levels of sweetness depending on the different number and types of sugar moieties (glucose, rhamnose, or xylose) substituted on its aglycone, steviol [4]. Steviol is synthesized through the methylerythritol phosphate (MEP) pathway in the chloroplast [5]. The first step in the MEP pathway involves the condensation of pyruvate and d-glyceraldehyde-3-phosphate into 1-deoxy-d-xylulose-5-phosphate (DXP) by DXP synthase (DXS) [6]. After six more steps of conversion, the final enzyme 4-hydroxy-3-methylbut-2-enyl pyrophosphate reductase converts (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate into isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are the basic five-carbon precursors for the formation of all terpenoids. For the production of SGs and other diterpenoids, two intermediates, IPP and DMAPP, undergo consecutive © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zheng et al. BMC Plant Biology (2019) 19:1 Page 2 of 16 condensation to form C20 geranylgeranyl pyrophosphate (GGPP). GGPP is then further cyclized to (−)-kaurene and subsequently oxidized to kaurenoic acid [7, 8]. All steps leading to the formation of kaurenoic acid are also common to gibberellic acid (GA) biosynthesis [9]. However, the hydroxylation of kaurenoic acid at C-13 position by kaurenoic acid hydroxylase (KAH) diverts it towards SG biosynthesis [9]. Finally, UDP-glycosyltransferases (UGTs) add sugar moieties at the C-13 or C-19 position of steviol to produce a variety of SGs [10]. Many Stevia genes uncovered from the next-generation sequencing are now publicly available [11, 12]. However, a reliable Stevia transformation technology remains to be developed for the functional genomics of Stevia and the generation of new Stevia with improved traits such as greater sweetness and resistance towards pests and diseases. Although Agrobacterium-mediated transformation of Stevia using β-glucuronidase (GUS) reporter gene was introduced [13], no further transgenic Stevia has been reported so far, which may result from the absence of a reliable transformation method. Tobacco plants have been routinely transformed using Agrobacterium and its protocol could be conveniently adapted to plants of Solanaceae family [14–17]. However, the transformation of other important crops such as soybean and corn required further optimization of their specific regeneration strategies [18]. For Stevia, although there are a few protocols describing shoot regeneration from leaf explants, there has been a lack of consensus on the conditions used [19–21]. Therefore, the development of a new and efficient method for regeneration and genetic transformation of Stevia would be required for a broad range of biotechnological applications as well as functional genomic studies of Stevia. Here we describe an efficient and reliable method for the Agrobacterium-mediated transformation of Stevia and demonstrate that using this method, we could obtain transgenic Stevia plants expressing the green fluorescent protein (GFP) from leaf explants. As a further demonstration of the efficacy of our transformation method, we transformed SrDXS1 and SrKAH into Stevia. By SrDXS1 overexpression, we successfully increased the total SGs content in the transgenic lines compared to control by up to 54%. Moreover, SrKAH overexpression in Stevia resulted in an even higher increase in total SGs content of up to 88%. Despite the increase in SGs content, the normal growth and development of Stevia were not compromised for both SrDXS1- and SrKAH-overexpression lines. root regeneration, but all these steps require optimization to suit individual plants. To establish a standard transformation method for Stevia, we investigated the effects of different hormone combinations on callus induction and shoot regeneration by modifying existing procedures for tobacco transformation (Table 1) [15]. We chose the second and third leaves of in vitro cultured Stevia plants as the explant source (Fig. 1a). Plant growth regulators most frequently supplemented for shoot regeneration from Stevia leaf explants include 6-benzylaminopurine (BA) as the cytokinin and 1-naphthaleneacetic acid (NAA), or 3-indoleacetic acid (IAA) as the auxin [19–21]. When explants were placed on BA with either NAA or IAA under long day photoperiod (LD, 16 h Light/ 8 h Dark), calli were induced on both media but with a different appearance (Additional file 1: Figure S1a, b). Shoot regeneration could also be observed from the calli on the BA + IAA media after 6 weeks but its frequency would be insufficient for successful transformation (Additional file 1: Figure S1b). It has been shown that prolonged dark incubation promotes somatic embryogenesis from callus cultures of Stevia [22]. Interestingly, we found drastic improvements in shoot regeneration from calli induced in the dark (Additional file 1: Figure S1c). Therefore, we subsequently incubated the explants under darkness during callus induction and shoot regeneration. To compare the efficiency of BA with IAA or NAA on callus induction and shoot regeneration, four combinations (Conditions A-D in Table 1) with different concentrations of NAA or IAA were designed. The difference in callus induction rates on four different callus induction media (CIM; Conditions A-D in Table 1) was not observed to be statistically significant (P-value: 0.099; Table 2). However, calli on CIM containing NAA (Conditions A and B) appeared friable while those on media containing IAA appeared compact (Conditions C and D; Table 2). Subsequently, calli maintained on NAA (Conditions A and B) had lower shoot regeneration rates than Table 1 Cytokinin and auxin combinations tested for callus induction and shoot regeneration from Stevia leaf explants Condition CCM (mg/L) CIM (mg/L) SIM (mg/L) A – BA 1 + NAA 2 BA 1 + NAA 2 B – BA 1 + NAA 0.5 BA 1 + NAA 0.5 C – BA 1 + IAA 2 BA 1 + IAA 2 D – BA 1 + IAA 0.5 BA 1 + IAA 0.5 E 2,4-D 0.25 BA 1 + IAA 0.5 BA 1 + IAA 0.5 Results F 2,4-D 0.25 BA 1 + IAA 0.5 Callus induction and shoot regeneration from Stevia leaf explants F-light Plant transformation involves a few major steps namely, co-cultivation, callus induction, shoot regeneration and 2,4-D 0.25 BA 1 + IAA 0.5 BA 2 + IAA 0.25 a BA 2 + IAA 0.25a CCM co-cultivation media, CIM callus induction media, SIM shoot induction media, BA 6-benzylaminopurine, NAA 1-naphthaleneacetic acid, IAA 3indoleacetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid a Explants were incubated under light with 16 h L/ 8 h D photoperiod Zheng et al. BMC Plant Biology (2019) 19:1 Page 3 of 16 a b c d e f g h Fig. 1 (See legend on next page.) Zheng et al. BMC Plant Biology (2019) 19:1 Page 4 of 16 (See figure on previous page.) Fig. 1 Agrobacterium-mediated transformation of Stevia using Condition F. a The red arrows indicate the second and third leaves that were used as the explant source. b Leaf explants on CCM. c Induced callus on CIM. d Transformed callus showing GFP fluorescence under a fluorescence stereomicroscope. e Shoots regenerated from calli on SIM. f Shoot regenerated from transformed calli showing GFP fluorescence under a fluorescence stereomicroscope. g Regenerated shoots on RM. h Rooting of regenerated shoots on RM. Scale bars = 1 cm for (a-c, e, g and h); 1 mm for (d) and (f). CCM, co-cultivation media; CIM, callus induction media; SIM, shoot induction media; RM, rooting media those on IAA (Conditions C and D; Table 2). Furthermore, we found that a higher BA to IAA ratio (Condition D) was more efficient for promoting shoot regeneration (Table 2). 2,4-dichlorophenoxyacetic acid (2,4-D) is commonly used for the dedifferentiation of somatic cells [23]. Therefore, to further enhance regeneration rates under Condition D, we designed Condition E with an additional 3 d incubation on 0.25 mg/L 2,4-D (Table 1), which can also be used as the co-cultivation media (CCM) for Agrobacterium-mediated transformation. Although regeneration rates for Conditions E were similar to Condition D, the regenerated shoots were healthier (Table 2 and Additional file 2: Figure S2a, b). In general, a higher cytokinin to auxin ratio promotes shoot formation [24]. We further optimized Condition E by doubling the cytokinin concentration of the shoot induction media (SIM) to 2 mg/L and reducing the auxin concentration from 0.5 mg/L to 0.25 mg/L to form Condition F (Table 1). Under Condition F, rates for callus formation and shoot regeneration as well as the shoot condition were comparable to those under Condition E (Table 2), but the number of regenerated shoots per callus clump seemed to be higher (Additional file 2: Figure S2c). Next, we tested Condition F simultaneously under LD condition after the explants were transferred onto CIM (Condition F-light; Table 1) to verify the enhancement of shoot regeneration in the dark. Certainly, the percentage of explants with regenerated shoots was 1.8 times higher under Condition F (Table 2), confirming that dark incubation greatly promotes shoot regeneration. Therefore, we subsequently applied Condition F for Stevia transformation. Stevia transformation To investigate the feasibility of adapting condition F for transformation, we co-cultivated Stevia leaf explants on the CCM media containing acetosyringone with Agrobacterium harboring the pK7WG2D vector [25], which contains a neomycin phosphotransferase (nptII) gene and an enhanced GFP gene fused to an endoplasmic reticulum targeting signal (EgfpER) to allow concurrent selection (Fig. 1b). Figure 1 outlines the overall procedures for Agrobacterium-mediated transformation of Stevia. The appearance of the calli and regenerated shoots on media are shown in Fig. 1c and e, respectively. Incubation in the dark resulted in their etiolated appearance. GFP signals from transgenic calli or regenerated shoots were monitored and selected under a fluorescence stereomicroscope (Fig. 1d, f ). With reduced autofluorescence from chlorophyll, GFP signals could easily be visualized. For rooting, transgenic shoots were transferred onto rooting media (RM) and exposed to light for approximately 1 month (Fig. 1g, h). Using this approach, we were able to efficiently produce transgenic Stevia plants expressing GFP. Transformation of Stevia with SrDXS1 and SrKAH DXS has been reported to play a rate-limiting role in the MEP pathway [26–28], while Stevia KAH acts on kaurenoic acid as the committed step to SGs biosynthesis [9]. Thus, we hypothesized that their overexpression would lead to an increase in the flux towards SGs production. Four Stevia DXS homologs (SrDXS1–4) were identified from the RNA-seq data of Stevia leaves [12]. To investigate if all four SrDXSs were functionally active, we carried out a complementation assay using a dxs-deficient Table 2 Callus induction and shoot regeneration rates under the different cytokinin and auxin combinations listed in Table 1 Condition Explants with callus formation (%) Callus condition Explants with regeneration (%) Shoot Condition A 87.4 ± 2.5 Friable 5.0 ± 1.4 + B 99.2 ± 0.8 Friable 22.8 ± 2.6 ++ C 89.1 ± 5.1 Compact 29.4 ± 2.9 +++++ D 98.3 ± 0.8 Compact 65.8 ± 3.6 ++++ E 95.0 ± 3.8 Compact 53.3 ± 5.1 +++++ F 96.7 ± 3.3 Compact 53.3 ± 5.8 +++++ F-light 95.8 ± 1.7 Compact 29.5 ± 7.7 ++++ Values are mean ± SE of technical triplicates with n = 40 The shoot condition was scored based on their appearance (+: Most shoots appear watery, browning or deformed, + + + + +: Most shoots appear strong and healthy) Zheng et al. BMC Plant Biology (2019) 19:1 Escherichia coli. Figure 2a shows that dxs− E. coli transformed with all SrDXSs except SrDXS3 were able to grow on selection media, similar to the Arabidopsis DXS1 (AtDXS1) positive control, indicating their functionality. Among the 4 SrDXS homologs, only SrDXS1 was suggested to be involved in SG biosynthesis based on the correlation between its expression pattern and the site of SGs biosynthesis [12]. Transient expression of the yellow fluorescent protein (YFP)-fused SrDXS1 in Nicotiana benthamiana leaves showed that it localizes to the chloroplast (Fig. 2b). Therefore, we selected SrDXS1 for Stevia transformation. Unlike SrDXS1, the activity of SrKAH in converting kaurenoic acid to steviol has previously been demonstrated in E. coli [29]. Additionally, its overexpression in Arabidopsis had led to the production of steviol that was otherwise not detected [30]. Being a cytochrome P450 enzyme, SrKAH is expected to be localized to the endoplasmic reticulum (ER) similar to that of kaurene oxidase which acts upstream of it [9]. We confirmed this by Page 5 of 16 transiently co-expressing YFP-fused SrKAH and cyan fluorescent protein (CFP)-fused HDEL, an ER marker, in Nicotiana benthamiana leaves. Figure 2c shows the co-localization of SrKAH-YFP with CFP-HDEL, demonstrating that SrKAH indeed localizes to the ER. Next, we cloned the full-length open reading frame (ORFs) of SrDXS1 and SrKAH into pK7WG2D under the control of the cauliflower mosaic virus (CaMV 35S) promoter for Stevia transformation (Fig. 3a). Using our transformation protocol, we produced 13 and 9 lines of transgenic Stevia plants overexpressing SrDXS1 (SrDXS1-OE) and SrKAH (SrKAH-OE), respectively. Because of the GFP visual marker, we were able to efficiently select the transgenic Stevia plants emitting GFP signals from leaf and root tissues of SrDXS1-OE and SrKAH-OE lines under a fluorescence stereomicroscope and a confocal laser scanning microscope (CLSM; Fig. 3b, c). GFP expressions in leaves of each transgenic Stevia lines were also confirmed by immunoblot analysis (Additional file 3: Figure S3). a b c Fig. 2 Characterization of SrDXSs and SrKAH. a Complementation assay of Stevia DXSs using E. coli DXS deficient mutant (dxs−). Transformed cells were grown on LB plates containing either with 0.5 mM mevalonate (+ MVA) or without mevalonate (− MVA). E. coli dxs− with pDEST17 (empty vector) and AtDXS1 served as negative and positive controls, respectively. b Subcellular localization of SrDXS1. Auto, chlorophyll autofluorescence; Light, light microscope image; Merged, merged image between Auto and YFP channels. Scale bar = 10 μm. c Subcellular localization of SrKAH. Coexpression of SrKAH-YFP with CFP-HDEL in. Light, light microscope image; Merged, merged image between CFP and YFP channels. Scale bar = 20 μm Zheng et al. BMC Plant Biology a b c Fig. 3 (See legend on next page.) (2019) 19:1 Page 6 of 16 Zheng et al. BMC Plant Biology (2019) 19:1 Page 7 of 16 (See figure on previous page.) Fig. 3 Identification of transgenic Stevia plants overexpressing SrDXS1 (SrDXS1-OE) or SrKAH (SrKAH-OE). a Schematic maps of T-DNA region of pK7WG2D-SrDXS1 and pK7WG2D-SrKAH used for Stevia transformation. LB, left border; nptII, neomycin phosphotransferase marker gene under the terminator and promoter of nopaline synthase gene; T35S and P35S, terminator and promoter of the cauliflower mosaic virus gene respectively; attB2 and attB1, gene recombination sites; SrDXS1, Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1; SrKAH, Stevia kaurenoic acid hydroxylase gene; EgfpER, enhanced green-fluorescent protein gene fused to endoplasmic reticulum targeting signal; ProlD, rol root loci D promoter; XbaI and HindIII, sites digested by XbaI and HindIII, respectively, for Southern blot analysis; Probe, probe used for Southern blot analysis. b Images of GFP signals from leaves and roots of representative SrDXS1-OE #6 or SrKAH-OE #4 under a fluorescence stereomicroscope. WT, wild-type. Scale bar = 1 mm. c Confocal images of the leaf underside and roots of WT, representative SrDXS1-OE #6 or SrKAH-OE #4. Auto, chlorophyll autofluorescence; GFP, GFP channel image; Light, light microscope image; Merged, merged image between Auto and GFP channels. Scale bar = 5 μm Analysis of transgenic Stevia lines To verify if exogenous SrDXS1 or SrKAH was integrated into the Stevia genome, genomic PCR analysis of the transgene from each transgenic line was performed. Genomic DNA amplification corresponding to the expected size of each transgene was observed for all the SrDXS1-OE or SrKAH-OE lines and the respective positive control lanes, but not for wild-type (WT; Fig. 4a, b). After confirming the existence of full-length ORFs of each transgene in transgenic Stevia plants, we performed digoxygenin (DIG)-based Southern blot analysis to determine the number of transgene integration sites for each line with nptII-specific probe (Fig. 3a). Figure 4c and d show that all SrDXS1-OE and SrKAH-OE lines contained one or more transgene (nptII) integration site, demonstrating stable transgene integration into the Stevia genome. No bands were detected in the WT lanes. Then, we analyzed the expression levels of SrDXS1 and SrKAH in SrDXS1-OE and SrKAH-OE lines, respectively. Figure 4e shows up to 13-fold increase in the expression levels of SrDXS1 among the transgenic lines compared to control. However, the expression levels of SrDXS1 in SrDXS1-OE lines did not correlate with the number of transgene integration sites. Among the top 5 SrDXS1-OE lines, four of them had a single transgene integration site (Fig. 4c, e). For further analysis, we chose three lines, SrDXS1-OE #1, #3 and #5, each having one transgene integration site but different levels of SrDXS1 overexpression. Among SrKAH-OE lines that contained single transgene integration site, lines #1, #4 and #7 showed around 40–60 fold higher expression of SrKAH compared to that of WT while line #2 did not show SrKAH overexpression, and line #9 only had a small increase of around 4-fold (Fig. 4f ). For further analysis of the effects of SrKAH overexpression, we selected lines #1, #4, and #9 with varying expression levels, and included line #2 as an internal control. Steviol glycosides (SGs) content increased in transgenic Stevia plants It is known that Stevia is a self-incompatible plant and its self-pollination results in sterile seed set [31]. Under our environmental conditions, we were also unsuccessful in harvesting viable transgenic T1 seeds. Therefore, we propagated the in vitro transgenic lines by cutting method and monitored the GFP signals emitted. Transgenic Stevia plants showing GFP expression in whole tissues were transferred into the soil for hardening and grown in the greenhouse for 3 weeks before analysis. Using this method, we were able to maintain each transgenic line for further analysis and obtain reproducible results. To investigate the effect of SrDXS1 or SrKAH overexpression on SGs production, we analyzed the leaf extracts of the transgenic lines. As leaf SGs content can differ according to their nodal position, leaves from the same position of each line were harvested. Each SG peak was identified by comparing their retention time with that of their authentic standards (Additional file 4: Figure S4). By summing up the concentration of the top 4 most abundant SGs (stevioside, Reb A, Reb C and dulcoside A) in each of the SrDXS1-OE lines, we found an increase in SGs content in the transgenic lines as compared to the controls (Fig. 5a). The total SGs content was the highest in SrDXS1-OE line #3 at 5.9% (w/w dry weight, DW), followed by 5.6% (w/w DW) in line #5 and lastly 5.1% (w/w DW) in line #1 (Fig. 5a), in agreement with their relative SrDXS1 expression levels (Fig. 4e). These total SGs content in the transgenic lines represent an increase of between 33-54% and 23–42% compared to the 3.8% (w/w DW) and 4.1% (w/w DW) total SGs content in the vector-only control line and WT, respectively (Fig. 5a). Stevioside, which is the most abundant SG in Stevia, had concentrations of between 3.7– 4.3% (w/w DW) in the overexpression lines, increasing up to 20–47% compared to controls (Fig. 5b). Similar patterns of SGs increase for Reb A, Reb C and dulcoside A were found in SrDXS1-OE lines (Fig. 5c and Additional file 5: Figure S5a). Furthermore, in SrDXS1-OE line #9 where SrDXS1 transcript levels were comparable to controls, the stevioside and Reb A contents were also similar (Fig. 4e and Additional file 6: Figure S6). These results suggest that the overexpression of SrDXS1 in Stevia leads to a proportional increase in each SG. In the SrKAH-OE lines, the total amount of SGs was able to reach up to 88% higher than that of WT (Fig. 5d). Corresponding to their expression levels, SrKAH-OE lines #1 and Zheng et al. BMC Plant Biology (2019) 19:1 Page 8 of 16 a b c d e f Fig. 4 Genomic and expression analysis of transgenic Stevia plants overexpressing SrDXS1 (SrDXS1-OE) or SrKAH (SrKAH-OE). a and b SrDXS1 (a) or SrKAH (b) amplified from the gDNA of each transgenic Stevia lines. M1, 2-Log DNA ladder. PC, positive control amplified from the respective vector constructs. c and d Southern blot analysis of SrDXS1-OE (c) or SrKAH-OE lines (d). WT, wild-type. M2, DIG-labelled DNA molecular weight marker II. e and f Relative fold change in SrDXS1 (e) and SrKAH (f) transcript levels among the transgenic Stevia lines overexpressing SrDXS1 (SrDXS1-OE) and SrKAH (SrKAH-OE), respectively. Expression levels of both genes were normalized to that of actin and compared to that of wild-type (WT). The values are expressed as mean ± SE (n = 3). Student’s t-test was used for the analysis of statistical significance (*: p < 0.05, **: p < 0.01) #4 accumulated the highest total amount of SGs at 4.5% (w/ w DW) and 6% (w/w DW), respectively (Figs. 4f and 5d). On the other hand, SrKAH-OE #9 with only a four-fold increase in SrKAH transcript had total SGs content of 3.9% (w/w DW), indicating a moderate increase of 8–22% from the controls (Figs. 4f and 5d). SrKAH-OE line #2, an internal control line that shows similar expression levels of SrKAH with WT, did not contain higher total SGs content, confirming that elevated SrKAH transcript levels resulted in higher SGs in transgenic Stevia plants (Fig. 5d). Taking a closer inspection at the individual SGs, stevioside was present in concentrations of up to 4% (w/w DW) among the overexpression lines, which was an increase of 57–71% compared to controls (Fig. 5e). For Reb A, a 133–200% increase compared to controls was observed in SrKAH-OE #4 (Fig. 5f). Apart from stevioside and Reb A, statistically significant increases of Reb C and dulcoside A content were also found in the two SrKAH high expressers, SrKAH-OE lines #1 and #4, with patterns of increase similar to that of the total SGs content (Additional file 5: Figure S5b). Phenotype of transgenic Stevia plants To determine if the overexpression of SrDXS1 and SrKAH would result in other changes in the Stevia plant, Zheng et al. BMC Plant Biology (2019) 19:1 Page 9 of 16 a d b e c f Fig. 5 Analysis of steviol glycosides (SGs) content in transgenic Stevia plants. a-f Total SGs (a and d), stevioside (b and e) and Reb A (c and f) content in the transgenic Stevia lines overexpressing either SrDXS1 (SrDXS1-OE) or SrKAH (SrKAH-OE). Data are presented as mean ± SE. Statistical analysis was carried out using Student’s t-test relative to wild-type (WT) (n = 5, *: p < 0.05, **: p < 0.01) we examined their phenotype. SrDXS1-OE lines did not display any morphological differences from controls. The height of the plants, size of the leaves and the internode length among the 2 month-old Stevia plants were comparable (Fig. 6a, c, e). Figure 6b, d and f show that SrKAH-OE lines also did not exhibit any obvious differences in growth. The leaf size and color and internode length were indistinguishable from the controls. Apart from morphology, we also determined the relative concentration of chlorophyll a, chlorophyll b and total carotenoids because these compounds are also derived from the MEP pathway [6]. Figure 6g-j shows that except for SrDXS1-OE #1, there were no significant changes in chlorophylls and carotenoids content in both SrDXS1-OE and SrKAH-OE lines when compared to WT. Furthermore, compared to Vector-only control, chlorophyll a and total carotenoid content in SrDXS1-OE #1 were also not significantly different. Additionally, we measured the concentration of a few monoterpenes that were present in the Stevia leaf tissues since monoterpenes can also be synthesized from the MEP pathway [12]. Using gas chromatography–mass spectrometry (GC-MS) analysis, the relative amount of linalool, α-pinene and β-pinene were determined (Additional file 7: Figure S7). There were no statistically significant changes to the amount of monoterpenes in the leaves of SrDXS1-OE lines compared to those of controls. Hence, our results show that both SrDXS1 and SrKAH overexpression could increase SGs content in transgenic Stevia without changing the abundance of other metabolites or having any detrimental effects on their growth and development. Zheng et al. BMC Plant Biology (2019) 19:1 Page 10 of 16 a b c d e g i f h j Fig. 6 Phenotypic analysis of transgenic Stevia plants. a and b Representative transgenic Stevia plants overexpressing SrDXS1 (SrDXS1-OE) (a) or SrKAH (SrKAH-OE) (b) one week after hardening in the soil. c and d Representative leaf harvested from third node position of SrDXS1-OE lines (c) or SrKAH-OE lines (d) one month after being transferred to the soil. e and f Average length of the third and fourth internodes in the SrDXS1-OE (e) or SrKAH-OE (f) lines one month after being transferred to the soil. Wild-type (WT) and vector-only line were included as a control. Scale bar = 1 cm. g-j Relative chlorophylls content and total carotenoids content in the transgenic Stevia plants overexpressing SrDXS1 (SrDXS1-OE) (g and i) or SrKAH (SrKAH-OE) compared to wild-type (WT) (h and j). All measurements are expressed as mean ± SE (n = 5) and statistical analysis was carried out using Student’s t-test relative to wild-type (WT) for SrKAH-OE lines (n = 5, *: p < 0.05) Discussion Since the whole transcriptome of Stevia has been sequenced [11, 12], the transformation of Stevia is indispensable not only in functional genomics for elucidating crucial genes such as those involved in SGs biosynthesis and stress response, but also for metabolic engineering to fulfill commercial interests in producing SGs more efficiently. Here, we optimized conditions for shoot