Optimization of regeneration and Agrobacterium-mediated transformation of Stevia (Stevia rebaudiana Bertoni): a commercially important natural sweetener plant

Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2–25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l−1) + naphthalene acetic acid (0.5 mg l−1), gibberellic acid (1.0 mg l−1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l−1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

Prior to autoclaving (at 121 °C for 15 min), the pH of MS medium was adjusted to 5.7. After autoclaving, the sterile medium was dispensed into sterile jam bottles. All the cultures were incubated at 22 ± 2 °C under white light (PFD: ~ 52 μmol m −2 s −1 ) for a photo-period of 16/8 h light/darkness. All experiments were repeated at least three times. Callus formation, shoot regeneration, shoot number and root number, shootlength and root length, were recorded at regular intervals and the cultures were maintained under aseptic conditions. Rooted plantlets were washed with autoclaved Milli-Q water to remove phytagel media from roots. The plantlets were planted in small plastic pots containing sterile soilrite (soil-conditioning mixture) and regularly irrigated with Hoagland solution. The pots were shielded with transparent perforated polythene sheets to maintain moisture (80-90%) and incubated in a plant growth chamber (Conviron, USA) set at 22 ± 2 °C and 16 light/8 h dark photo-period with PFD of 80 µmol m -2 s -1 . After 22-25 days of acclimatization, polythene bags were removed from the plants.
Agrobacterium strains and vector construct. The plant expression vector pPZP200 35sde-bar-loxP harbouring herbicide tolerant bar gene driven by DECaMv35s promoter and loxP terminator, was used in the study (Fig. 5A). The enzyme phosphinothricin acetyl transferase (PAT) encoded by the bar gene inactivates phosphinothricin. The aadA2 gene encodes an antibiotic (streptomycin-spectinomycin) resistant protein. The construct was used to transform Agrobacterium tumefaciens strain GV1301. The active ingredient of the herbicide bialaphos (glufosinate ammonium) was used as selection agent.

Agrobacterium-mediated transformation and explant regeneration. Nodal sections and leaf
originated callus from in vitro regenerated plants ( Fig. 2A) were dissected and incubated for 2 days on preincubation media containing MS salts + BAP (2 mg l −1 ). These acclimatized explants were used for Agrobacterium-mediated transformation with pPZP200-bar-loxP constructs. Explant wounding was done with sterile needle to enhance the transformation efficiency. Different parameters such as type of explants, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration and cocultivation duration were optimized meticulously ( Fig. 1A-F). After agroinoculation, the explants were incubated on co-cultivation medium (Fig. 2B) containing MS salts + BAP (2 mg l −1 ) + acetosyringone (As), at 22 °C, in dark for 3 days. Thereafter, the explants were washed twotimes with liquid MS media fortified with cefotaxime (250 mg l −1 ), followed by incubation on MS media containing 250 mg l −1 cefotaxime. After 8-10 days, explants were subjected to first selection media (SIM-1) having MS salts + BAP (1 mg l −1 ) + NAA (0.5 mg l −1 ), cefotaxime www.nature.com/scientificreports/ (250 mg l −1 ) + glufosinate ammonium (2 mg l −1 ) for 22-30 days. The regenerated shoots with a pair of vegetative leaves were identified, excised into segments and were placed on the second selection medium (SIM-2) (Fig. 2C) having the same constituents as SIM-1, except 4 mg l −1 of glufosinate ammonium, and incubated for 22-30 days. The independent shoots that regenerated on SIM-2 were transferred to culture-tubes containing elongation medium (SEM) having MS salts + GA 3 (1 mg l −1 ), and incubated for 15-20 days (Fig. 2D). The elongated shoots were incubated on rooting medium (RIM) containing ½ strength MS medium, for 15-20 days (Fig. 2E). The rooted plantlets were transferred to plastic containers filled with sterile soilrite (soil conditioning mixture) and irrigated with Hoagland solution. The containers were bagged with perforated polythene bags and incubated in a plant growth chamber (Conviron, USA) set at 80% relative humidity, for 15 days of acclimatization (hardening) in soilrite (Fig. 2F). The acclimatized plantlets were planted in plastic pots (12 in.) filled with soil:sand:farmyard manure (3:1:1) and transferred to glasshouse maintained at 24 ± 1 °C under natural light, for normal vegetative and reproductive growth phases (Fig. 2G). The Fig. 4 represents an outline of the optimized protocol for stevia transformation. The percentage transformation efficiency using nodal segments and calluses was determined by the formula as shown below Molecular characterization of transgenic plants. Genomic   www.nature.com/scientificreports/ stringent washings. The blots were exposed to Fuji screen for forty eight hours and then analyzed on a phosphoimager (Typhoon Trio + , Sweden).

Reverse transcriptase pcR (Rt-pcR) analysis. RNA from leaves of transgenic (T 0 ) plants was extracted
with TRI reagent (Cat #T9424; Sigma, USA) according to manufacturer's instructions. For RNA quantification, ND-1000 spectrophotometer (NanoDrop Technologies Inc., USA) was used. DNase (Cat #AMPD1; Sigma, USA) treatment was given to each RNA sample to remove any DNA contamination. cDNA was synthesized from RNA using Power ScriptRT (Cat #RR037B; Takara, Japan) using 5 µg of plant total RNA. Reverse transcription reaction consisted of pre-treatment of 25 μl reaction mixture at 50 °C (10 min), initial denaturationat 95 °C (5 min), denaturation at 95 °C (10 s), followed by and annealing and extension at 60 °C (30 s). Amplification of cDNA fragments was done using specific primers that amplifies 200 bp region of bar gene. These amplified gene fragments were then electrophoresed on agarose gel (1%) and analyzed using a gel-documentation system (Bio-Rad, USA).
Quantitative real-time pcR (qRt-pcR). SYBR green premix was used to perform qRT-PCR on synthe- Herbicide tolerance assay. Herbicide tolerance (glufosinate) assay was performed to check the efficacy of transgenic stevia plants for herbicide tolerance as compared to non-transgenic plants. To perform this assay, the wild type (non-transgenic control) stevia plants were divided into five groups, each having five plants (4 test + 1 control). Each group was sprayed (using hand sprayer) with different concentrations of glufosinate (50 ml of 2, 4, 6, and 8 mg l −1 ) under green-house conditions to find the minimum lethal dose for stevia plants. It was observed that 8 mg l −1 of glufosinate was the minimum lethal dose for stevia plants. Thereafter, five healthy T 0 transgenic and non-transgenic control (wild type) stevia plants each were sprayed separately with 8 mg l −1 glufosinate in green-house. The experiment was repeated three times and the observations were recorded after 12 days of spray.

Residual phytotoxic effect.
Residual phytotoxic effect of glufosinate on soil was studied by spraying the potted soil with three different concentration of glufosinate i.e. 0.25, 0.50, and 1.0% (v/v) under greenhouse condition. After five days of spray, ten seeds each of indicator plants i.e. corn and cucumber were sown into these pots. The experiment was performed in triplicates and the soil of control plant was sprayed with water as an experimental control. Seed germination percentage was recorded after 10 days of sowing. The plantlet height was recorded with the help of a measuring scale after 20 days of sowing.
Statistical analysis. Different parameters (percentage of callus formation, number of shoots and roots, were examined using three replicates for each treatment. The values given in tables were expressed as mean ± SD of three replicates i.e. n = 3. Mean and standard deviation were calculated using SPSS software, version 14.0 (SPSS Inc. USA).

Results and discussion
callus induction. In this study, we used different concentrations of 2,4-D (1-3 mg l −1 ), KIN (1-2 mg l −1 ) and BAP (1-3 mg l −1 ) to obtain callus from different explants viz. leaf, nodes and shoot tips of in vitro raised Stevia plants. Callus was initiated from leaf-discs after 4-5 weeks on culture media while, the other explants responded after 6-7 weeks. Hence, leaf discs were most efficient in callus formation and maximum callus induction was achieved on MS2 medium [2,4-D (2 mg l −1 ) and KIN (1 mg l −1 )] (Supplementary Table 1 Shoot regeneration. In this study it has been found that less number of shoots was produced from callus in comparison to direct shoot regeneration from explants (Supplementary Table 2) (Fig. 3). Although, leaf explants derived-callus cultured on MS3 media [BAP (1 mg l −1 ) and NAA (0.5 mg l −1 )] showed maximum shoot regeneration (4 ± 1.00), the nodal sections cultured on MS6 media [BAP (1 mg l −1 ) and NAA (0.5 mg l −1 )] exhibited maximum direct shoot regeneration (25 ± 3.2). Direct regeneration of shoots, without any callusing stage is more effective as compared to indirect regeneration. Shoots regenerated through callus are generally asynchronous while directly regenerated shoots are homogeneous, diploid and true-to-type. Significantly higher numbers of shoots were obtained directly from nodal sections cultured on MS4, MS5 and MS6 media as compared to rest of the explants. In a study, a combination of 1.5 mg l −1 BAP with 0.5 mg l −1 KIN was reported to efficiently induce multiple shoot regeneration from nodal explants 28 . In a study by Debnath, maximum shoot formation was observed with nodal sections and shoot tips cultured on MS media fortified with IAA (1.13 mg l −1 ) and BAP (2.0 mg l −1 ) 31 . In a report by Singh and Dwivedi, they mentioned that the nodal explant showed maximum shoot regeneration (98%) response as compared to shoot tips (55%) and inter-nodal segments (15%). Bud regeneration was reported earlier (in 5.50 days) using nodal section than other explants 32  Shoot elongation and rooting. The regenerated shoots were cut and further sub-cultured on SEM containing MS media supplemented with various concentrations of GA 3 (0.5-3.0 mg l −1 ). It has been observed that  Fig. 1). According to Sreedhar et al., MS medium fortified with IBA 4.92 µM and 30 g sucrose was most efficient for shoot elongation 35 . GA 3 was used for shoot elongation in stevia regeneration by Giridhar et al. 36 . It has been reported in their study that 0.05 µM of GA 3 was most efficient for maximum shoot elongation 36 . Sivaram and Mukundan reported that rooting medium (MS media fortified with 4.90 µM IBA) also acted as shoot elongation medium 37 . The elongated shoots (~ 2 cm) were transferred to root-induction medium (RIM). In our study, maximum number of roots (9 ± 2.0) was reported from shoots (5-7 cm) regenerated from nodal section, cultured on halfstrength MS media devoid of PGR (Supplementary Table 3). No. of roots and root length was found significantly higher in directly regenerated shoots from nodal sections cultured on MS5 and ½ MS media. A comparison between number of shoots and roots originated from directly regenerated shoots and callus regenerated shoots is presented in Supplementary table 2  optimization of Agrobacterium-mediated transformation. To find the effect of explants type on genetic transformation of stevia, two types of explants (nodal sections and callus) were subjected to Agro-inoculation (O.D 600 = 0.6). Young nodal sections (0.5 cm) exhibited a high regeneration response of 69.92%, in comparison to a low response of 31.43% with callus explants (Fig. 1A). Different concentrations (50, 100, 200 and 300 µM) of acetosyringone (As) were used to find their effect on transformation efficiency. Supplementation of 100 µm acetosyringone (As) gradually increased the percentage of responding explants to 72.5% while, a lower or a higher concentration (than 100 µM) resulted in reduction of percentage regeneration response (Fig. 1C). Different cell densities (0.2, 0.4, 0.6 and 0.8 O.D 600 of Agrobacterium culture) were used to evaluate their effect on transformation efficiency Maximum regeneration response observed at O.D 600 was 0.6, while at higher O.D 600, Agrobacterium contamination was observed on explants (Fig. 1D). Maximum regeneration response (62%) was achieved with a pre-incubation duration of 2 days (Fig. 1B), Agro-inoculation duration of 20 min (55% regeneration response) (Fig. 1E) and co-cultivation duration of 2 days (55% regeneration response) (Fig. 1F). Incubation with Agrobacterium enhances the transformation process due to active cell division and formation of vir-inducing compounds which enhance the binding of Agrobacterium cells on the surface of newly synthesized cell wall 38 .
The optimizations for Agrobacterium-mediated transformation and regeneration of stevia are (1) preferred explant type: nodal sections; (2) acetosyringone (As) concentration: 100 μm; preincubation duration: 2 days;  (Table 1) as compared to 27.94 ± 5.75% with the callus explants ( Table 2). The parameters (for shoot regeneration, elongation and rooting) that were optimized for in vitro regeneration of stevia were in consonance with the regeneration after transformation. Figure 4 represent the outline of optimized protocol for stevia transformation using nodal/callus explants. www.nature.com/scientificreports/ Molecular characterization of putative transformants. Genomic DNA and total RNA from randomly selected nine putative transformants (TR1-TR9) were used for bar gene integration and expression analyses by PCR, RT-PCR, Southern-blot hybridization and qRT-PCR. PCR result of the nine putative transformants showed amplification of anticipated 146 bp region of bar gene which was similar to positive control (plasmid DNA developed with gene-specific primers) (Fig. 5A,B). However, no such amplification was observed with untransformed control plantlets. RT-PCR analysis of the nine promising transformants also revealed amplification of the expected fragment of 200 and 150 bp, which verify the presence of bar and actin gene transcript in the transgenic plants (Fig. 5C,D). However, the band intensities of the c-DNA amplification product differed in each plant. The TR1 exhibited highest band-intensity while the TR9 exhibited the lowest. The nine T0 transformants were also subjected to qRT-PCR analysis.  www.nature.com/scientificreports/ bar gene was calculated in terms of 2 −ΔΔCT method and plotted on graph 39 . Figure 5E shows ~ 47-fold increase in bar gene expression in TR1 transgenic stevia plant as compared to the control (TR9). Southern hybridization analysis of six highly expressing T 0 transgenic events revealed the transgene copy number. Genomic DNA from non-transgenic and T 0 transgenic plants (TR1, TR2, TR3, TR4, TR6 and TR7) was digested with EcoRI and subsequently hybridized with 552 bp of bar-gene-probe. The hybridization pattern of six T 0 transgenic plants revealed single and double copy integration that ranged in sizes from 3.5 to 20.5 kb, but the non-transgenic plant (control) did not show hybridization with the gene probe (Fig. 5F).
comparison of morphological characters and chlorophyll content. The transgenic plants did not exhibit any significant difference with the control in terms of morphological characters and chlorophyll content (Supplementary Table 4). Average plant height observed in wild type was 73 cm while it was 71 cm in transgenic plants. The leaf count in wild type and transgenic plants was 215 and 211, respectively. Moreover, average chlorophyll content was 7.85 mg g −1 and 7.32 mg g −1 in wild type and transgenic plants, respectively.
Herbicide tolerance assay. Herbicide tolerance assay was performed by spraying the wild type stevia (non-transformed) and T 0 transgenic plants with 8 mg l −1 glufosinate ammonium, in green-house. The wild type stevia plants showed symptoms of chlorosis (Fig. 6A), phytotoxicity and defoliation after 4th day of herbicide spray and even death after 12th day. On the other hand the transgenic plants did not show such symptoms and remained healthy (Fig. 6B-E).
Various reports are available regarding the application of glufosinate on crop plants and weed species. Manickavasagam et al. performed herbicide resistance trials on transgenic (herbicide resistant) sugarcane cultivars Co92061 and Co671 40 . In this study in vitro regenerated non-transformed sugarcane plants were sprayed with different concentrations (0.5, 2.5 and 5.0 g l −1 ) of glufosinate ammonium to find the lethal dose of herbicide. Glufosinate @ 2.5 g l −1 with an average dose of 6.25 mg plant −1 was observed as lethal dose. This lethal dosage was then sprayed on transgenic plants under greenhouse conditions. Observations were recorded after 30 days to select the transgenic plants. Herbicide resistant sweet potato (Ipomoea batatas L.) cultivar "Yulmi" was sprayed and painted with 0.5% glufosinate herbicide (@ 900 mg l −1 ) under greenhouse condition to estimate their efficacy for herbicide resistance. It was found that control plants showed extensive leaf necrosis while transgenic plants remained green without any symptom of leaf necrosis 41 . In a study by Abdeen and Miki, it has been reported that glufosinate spray on Arabidopsis plants led to inhibition of photosynthesis and ultimately plant death, after 6-48 h of spray. While, the transgenic Arabidopsis harboring bar gene survived under the experimental conditions 42 . Two Chinese rice cultivars (HD297T-31, HD297T-523) were also transformed with bar gene through Agrobacterium mediated transformation, for making them resistant to glufosinate herbicide. Transgenic HD297T-31 exhibited almost 100% resistantce to glufosinate while, HD297T-523 showed moderate resistance 43 .  C TR1 TR2 TR3 M  -C TR4 TR5 TR6 TR7 TR8 TR9   B  C   +C -C TR1 TR2 TR3 TR4 TR6 TR7 23.1 9.4 6.  C TR1 TR2 TR3 TR4 TR5 TR6 -C TR7 TR8 TR9   0

Residual phytotoxic effect.
No harmful phyto-toxic effect of glufosinate was found on seed germination of both the indicator plants. The difference between parameters (seed germination and seedling length) of control and treated pots was found non-significant. The corn seed germination percentage of 92.78% was observed in water treated pots (control) and 91-92% in glufosinate treated pots. Similarly, with cucumber seed germination percentage of 93.28% was observed in water treated pots and 90-91% in glufosinate treated pots. The corn seedling length of 33.43 cm was recorded in water treated pots and 32-33 cm in glufosinate treated pots. The cucumber seedling length of 7.34 cm was recorded in water treated pots and 6-7 cm in glufosinate treated pots. Moreover, no phytotoxic effect was observed on seedlings of both the indicator plants (Supplementary Table 5).
conclusions Glufosinate (Basta) and glyphosate (Roundup) are two most commonly used broad-spectrum herbicides for weed control in agricultural fields. However, extensive use of a single herbicide induces the weeds to develop resistance against them. At present, nearly 38 weed species has developed resistance to glyphosate, which has been distributed to 37 countries 17 . These resistant weeds are a major challenge to efficient weed control strategies. However, these weeds can be controlled by the strategic application of glufosinate 45 . www.nature.com/scientificreports/ In addition to herbicide tolerance, bar gene has also been used as a selective marker in many plant genetic transformation experiments 46,47 . In a recent report, cotyledon explants from seven cultivars of soybean were used to introduce bar gene as a selective marker. With a transformation efficiency of 14.71%, transgenic soybean expressing bar gene was successfully developed 48 . In our study, much higher transformation efficiency (40.48 ± 0.72) was achieved with nodal sections of stevia plants. This suggests the feasibility of using them for high-frequency Agrobacterium-mediated transformation with novel genes of diverse origin.To the best of our knowledge, it is the first report on Agrobacterium-mediated nuclear transformation of stevia for herbicide resistance trait. This robust transformation protocol can be useful in stevia crop-improvement programs.