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866 http://journals.tubitak.gov.tr/biology/ Turkish Journal of Biology Turk J Biol (2016) 40: 866-877 © TÜBİTAK doi:10.3906/biy-1509-83 Direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation of Solanum trilobatum L. Jayabalan SHILPHA, Manoharan JAYASHRE, Muthiah JOE VIRGIN LARGIA, Manikandan RAMESH* Department of Biotechnology, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India * Correspondence: [email protected] 1. Introduction Solanum trilobatum L. (Solanaceae) has been considered a medicinal treasure of the Indian Ayurveda and Siddha medicine for several decades due to its immense medicinal properties. It is a thorny creeper, commonly known as the purple fruited pea eggplant, and is widely distributed in the Indo-Malaysian region. All parts of this plant are medicinal and are useful in the treatment of respiratory, cardiac, and liver disorders and several kinds of leprosy (Purushothaman et al., 1969; Govindan et al., 1999). Besides therapeutic usages, the leaves are consumed as a nutraceutical vegetable as they are rich in minerals, carbohydrates, and crude fiber (Jawahar et al., 2004). e plant possesses steroid alkaloids such as solasodine, sobatum, β-solamarine, solanine, tomatidine, and diosgenin (Vijaimohan et al., 2010), which are responsible for its exemplary therapeutic actions. Direct shoot organogenesis of S. trilobatum using node and shoot tip explants has been reported earlier (Jawahar et al., 2004). Our group has established a reproducible protocol for the production of genetically uniform plants of S. trilobatum from meristematic tissues of nodal and shoots tip explants (Shilpha et al., 2014). Attempts were also made for indirect shoot organogenesis by exploiting internode (Arockiasamy et al., 2002), stem, and mature leaf explants (Alagumanian et al., 2004). Cotyledonary leaf explants were used for shoot organogenesis via somatic embryogenesis (Dhavala et al., 2009). However, there is no report on direct shoot organogenesis from in vitro derived leaf explants of S. trilobatum. e major benefits of using in vitro grown leaves are the large availability of explants without any negative effect on bioresources, axenic nature with minimum risk of contamination, and amenability for genetic transformation (Chaudhuri et al., 2008). Agrobacterium mediated transformation is the most preferred technique for plant transformation due to its propensity to introduce a low copy number of transgenes with minimum rearrangement, ability to transfer larger DNA fragments, and selective integration into transcriptionally active regions of the chromosome (Ingelbrecht et al., 1991; Hamilton et al., 1997; Patel et al., 2013). e transfer of T-DNA and its integration into the plant genome is influenced by several important factors that affect the transformation efficiency such as the culture media and its components, Agrobacterium density, inoculation duration, cocultivation time, Abstract: Solanum trilobatum L. is one of the most valued medicinal plants of Indian Ayurveda and Siddha medicine. An efficient protocol for direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation using in vitro derived leaf explants of S. trilobatum was established for the first time. High frequency direct shoot regeneration (89%) was achieved in MS medium containing 2 mg/L 6-benzylaminopurine and 0.25 mg/L indole-3-acetic acid. e Agrobacterium strain EHA 105 harboring the binary vector pCAMBIA 1301, which contained hygromycin phosphotransferase (hpt) as a selectable marker gene and β-glucuronidase (gus) as a reporter gene, was used for the transformation. e factors that influence the transformation frequency were optimized as follows: age of explants (40 days old), acetosyringone concentration (150 µM), duration of preculture (2 days), bacterial optical density (O.D. 0.4), Tween 20 concentration (0.01%), infection time (15 min), pH of cocultivation medium (5.4), and cocultivation duration (3 days). e putative transformants were screened on selection medium containing 15 mg/L hygromycin. e transgenic plants were confirmed by histochemical gus analysis, PCR, and RT-PCR analysis. A stable transformation efficiency of 64% was achieved under optimized transformation conditions. us, the protocol can be used for genetic improvement of this plant in the future. Key words: Acetosyringone, Agrobacterium tumefaciens, direct shoot organogenesis, genetic transformation, Solanum trilobatum Received: 25.09.2015 Accepted/Published Online: 16.12.2015 Final Version: 21.06.2016 Research Article

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866

http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2016) 40: 866-877© TÜBİTAKdoi:10.3906/biy-1509-83

Direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation of Solanum trilobatum L.

Jayabalan SHILPHA, Manoharan JAYASHRE, Muthiah JOE VIRGIN LARGIA, Manikandan RAMESH*Department of Biotechnology, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India

* Correspondence: [email protected]

1. IntroductionSolanum trilobatum L. (Solanaceae) has been considered a medicinal treasure of the Indian Ayurveda and Siddha medicine for several decades due to its immense medicinal properties. It is a thorny creeper, commonly known as the purple fruited pea eggplant, and is widely distributed in the Indo-Malaysian region. All parts of this plant are medicinal and are useful in the treatment of respiratory, cardiac, and liver disorders and several kinds of leprosy (Purushothaman et al., 1969; Govindan et al., 1999). Besides therapeutic usages, the leaves are consumed as a nutraceutical vegetable as they are rich in minerals, carbohydrates, and crude fiber (Jawahar et al., 2004). The plant possesses steroid alkaloids such as solasodine, sobatum, β-solamarine, solanine, tomatidine, and diosgenin (Vijaimohan et al., 2010), which are responsible for its exemplary therapeutic actions.

Direct shoot organogenesis of S. trilobatum using node and shoot tip explants has been reported earlier (Jawahar et al., 2004). Our group has established a reproducible protocol for the production of genetically uniform plants of S. trilobatum from meristematic tissues of nodal and shoots tip explants (Shilpha et al., 2014). Attempts were

also made for indirect shoot organogenesis by exploiting internode (Arockiasamy et al., 2002), stem, and mature leaf explants (Alagumanian et al., 2004). Cotyledonary leaf explants were used for shoot organogenesis via somatic embryogenesis (Dhavala et al., 2009). However, there is no report on direct shoot organogenesis from in vitro derived leaf explants of S. trilobatum. The major benefits of using in vitro grown leaves are the large availability of explants without any negative effect on bioresources, axenic nature with minimum risk of contamination, and amenability for genetic transformation (Chaudhuri et al., 2008).

Agrobacterium mediated transformation is the most preferred technique for plant transformation due to its propensity to introduce a low copy number of transgenes with minimum rearrangement, ability to transfer larger DNA fragments, and selective integration into transcriptionally active regions of the chromosome (Ingelbrecht et al., 1991; Hamilton et al., 1997; Patel et al., 2013). The transfer of T-DNA and its integration into the plant genome is influenced by several important factors that affect the transformation efficiency such as the culture media and its components, Agrobacterium density, inoculation duration, cocultivation time,

Abstract: Solanum trilobatum L. is one of the most valued medicinal plants of Indian Ayurveda and Siddha medicine. An efficient protocol for direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation using in vitro derived leaf explants of S. trilobatum was established for the first time. High frequency direct shoot regeneration (89%) was achieved in MS medium containing 2 mg/L 6-benzylaminopurine and 0.25 mg/L indole-3-acetic acid. The Agrobacterium strain EHA 105 harboring the binary vector pCAMBIA 1301, which contained hygromycin phosphotransferase (hpt) as a selectable marker gene and β-glucuronidase (gus) as a reporter gene, was used for the transformation. The factors that influence the transformation frequency were optimized as follows: age of explants (40 days old), acetosyringone concentration (150 µM), duration of preculture (2 days), bacterial optical density (O.D. 0.4), Tween 20 concentration (0.01%), infection time (15 min), pH of cocultivation medium (5.4), and cocultivation duration (3 days). The putative transformants were screened on selection medium containing 15 mg/L hygromycin. The transgenic plants were confirmed by histochemical gus analysis, PCR, and RT-PCR analysis. A stable transformation efficiency of 64% was achieved under optimized transformation conditions. Thus, the protocol can be used for genetic improvement of this plant in the future.

Key words: Acetosyringone, Agrobacterium tumefaciens, direct shoot organogenesis, genetic transformation, Solanum trilobatum

Received: 25.09.2015 Accepted/Published Online: 16.12.2015 Final Version: 21.06.2016

Research Article

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preselection period, temperature treatment, concentration of acetosyringone or other antibiotics in the media, explant type, and genotypes (Opabode, 2006; Wu et al., 2006). Recently, a number of successful genetic transformations have been reported for medicinal plants (Li et al., 2014; Sivanandhan et al., 2015). So far, there is no report on genetic transformation of S. trilobatum using Agrobacterium tumefaciens. Thus, the present investigation has been carried out with the objective of establishing an efficient direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation using leaf explants of S. trilobatum L.

2. Materials and methods2.1. Shoot regeneration via leaf explantsLeaves of 1-month-old in vitro grown shoots (Shilpha et al., 2014) were used as explants for direct shoot organogenesis. Leaves of the size 1.5–2 cm were aseptically dissected out and inoculated on Murashige and Skoog (MS) medium supplemented with 3% (w/v) sucrose, 0.7% (w/v) agar, and various concentrations of the cytokinin 6-benzylaminopurine (BAP) alone (1–3 mg/L) or in combination with auxins like indole-3-acetic acid (IAA) (0.1–0.5 mg/L) and α-naphthalene acetic acid (NAA) (0.1–0.5 mg/L) (Table 1). All culture media were adjusted to pH 5.8 and autoclaved at 121 °C (1.06 kg cm–2 pressure) for 20

min, and the cultures were incubated at 25 ± 2 °C under a 16/8 h photoperiod provided by cool, white fluorescent tubes (Phillips, India) with a photon flux density of 50 µmol m–2 s–1.2.2. Shoot elongation, rooting, and acclimatization of regenerated plantsFor shoot elongation, 4-week-old multiple shoot buds of all hormonal treatments (Figures 1a–1m) were transferred to MS medium containing 2 mg/L BAP, 0.1 mg/L IAA, and 0.5 mg/L gibberellic acid (GA3). The elongated shoots (Figure 1n) were rooted in MS medium containing 2 mg/L indole-3-butyric acid (IBA) (Shilpha et al., 2014). The in vitro rooted shoots (Figure 1o) were transferred to small plastic cups containing equal proportions of sterile soil rite (Keltech, Bengaluru, India), garden soil, and peat (1:1:1, v/v) and incubated in a growth chamber (Sanyo versatile environmental test chamber, Japan, model MLR 351H) with 16/8 h light/dark and 75%/50% relative humidity, respectively, at 26 ± 2 °C for 1 month (Figure 1p). The plantlets were initially maintained in a shade house for 2–3 weeks and then finally acclimatized in the field.2.3. Agrobacterium strain, plasmid vector, and culture conditionsAgrobacterium tumefaciens strain EHA 105 harboring the binary plasmid pCAMBIA 1301 was used in the

Table 1. Effect of plant growth regulators on shoot organogenesis from leaf explants of S. trilobatum L.

Plant growth regulators (mg/L)

BAP IAA NAA Regeneration frequency (%) Morphogenic response No. of shoots per

explantAverage shootlength (cm)

1 - - 42.0 ± 3.8f Shoot buds 2.6 ± 0.5ef 2.3 ± 0.2bc

2 - - 63.6 ± 4.9b Shoot buds 6.0 ± 1.7d 3.0 ± 0.8b

3 - - 54.0 ± 4.7cd Shoot buds 3.6 ± 0.6e 2.1 ± 0.1bc

2 0.1 - 66.0 ± 3.2b Shoot buds 10.0 ± 0.9c 2.2 ± 0.1bc

2 0.25 - 89.0 ± 3.5a Shoot buds 26.0 ± 0.8a 4.3 ± 0.2a

2 0.5 - 58.3 ± 2.8bc Shoot buds 14.4 ± 0.6b 2.0 ± 0.8cd

3 0.1 - 62.6 ± 3.7b Shoot buds 11.6 ± 1.1c 2.5 ± 0.4bc

3 0.25 - 50.6 ± 3.3de Shoot buds 10.3 ± 2.7c 2.0 ± 0.2cd

3 0.5 - 40.0 ± 3.3f Shoot buds 7.3 ± 0.4d 1.9 ± 0.2cd

2 - 0.1 44.6 ± 4.0ef Callus + shoot buds 1.3 ± 0.3fg 1.6 ± 0.1cd

2 - 0.25 30.0 ± 4.0g Callus + shoot buds 0.7 ± 0.2fg 1.1 ± 0.2d

2 - 0.5 21.6 ± 2.7h Green compact callus 0 ± 0 g 00 ± 0e

Mean values within a column having different letters are statistically significant at P < 0.05 according to Duncan’s multiple range test.

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present study. Strain EHA 105 was kindly offered by Dr K Veluthambi, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India. The plasmid pCAMBIA 1301 (11.8 kb) contains hpt as the selectable marker gene and gus as the reporter gene (Karthikeyan et al., 2012) (Figure 2). The Agrobacterium strain was maintained at 28 °C on yeast extract peptone medium containing 10 mg/L rifampicin and 100 mg/L kanamycin.2.4. Determination of hygromycin sensitivityIn order to determine the effective tolerance limit to hygromycin, the untransformed leaf explants were subjected to different concentrations of hygromycin (0, 5, 10, 15, 20, and 25 mg/L). The maximum concentration of hygromycin above which the explants failed to survive and regenerate was considered as the optimum concentration of the selective agent for the selection of transformants (Figure 3).

2.5. Bacterial infection and cocultivationPreceding transformation, leaf explants of various ages (15–75 days) (Figure 4a) were manually wounded and precultured on MS medium containing 2 mg/L BAP and various concentrations of acetosyringone (0 to 250 µM) (Figure 4b) for different time intervals (0–4 days) (Figure 4c). Bacterial suspensions of various growth O.D.s (0.2 to 1.0 O.D.) were used (Figure 4d). In order to check the role of surfactant in improving transformation, Tween 20 was added to the bacterial suspension at different concentrations (0%–0.5%) (Figure 4e). Infection was carried out by immersing the explants in the bacterial suspension with gentle shaking for different time periods (5–25 min) (Figure 4f). To observe the effect of media pH on transformation efficiency, cocultivation media with various pH values (5–5.8) (Figure 4g) were tested. Additionally, to determine the optimum cocultivation

Figure 1. Morphogenic response of various plant growth regulators on shoot induction and stages of plant regeneration from in vitro leaf explants of S. trilobatum L. Effect of various concentrations of BAP alone and in combination with IAA and NAA on shoot induction from leaf explants after 4 weeks of culture. a) MS + 1 mg/L BAP, b) MS + 2 mg/L BAP, c) MS + 3 mg/L BAP, d) MS + 2 mg/L BAP + 0.1 mg/L IAA, e) MS + 2 mg/L BAP + 0.25 mg/L IAA, f) 2 mg/L BAP + 0.5 mg/L IAA, g) 3 mg/L BAP + 0.1 mg/L IAA, h) 3 mg/L BAP + 0.25 mg/L IAA, i) MS + 3 mg/L BAP + 0.5 mg/L IAA, j) MS + 2 mg/L BAP + 0.1 mg/L NAA, k) MS + 2 mg/L BAP + 0.25 mg/L NAA, l) MS + 2 mg/L BAP + 0.5 mg/L NAA, m) optimum multiple shoot induction from leaf explants after 4 weeks of culture in MS medium containing 2 mg/L BAP + 0.25 mg/L IAA, n) elongation of multiple shoot buds in MS medium containing 2 mg/L BAP + 0.1 mg/L IAA + 0.5 mg/L GA3 after 6 weeks of culture, o) in vitro rooting of elongated shoots in MS medium containing 2 mg/L IBA, p) rooted shoots established in soil and maintained in growth chamber at 26 ± 2 °C.

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period, the explants were cocultivated for different durations starting from 1 to 5 days (Figure 4h).2.6. Selection, regeneration, and acclimatization of transgenic plantsAfter cocultivation, the explants were washed in MS basal liquid medium containing 500 mg/L cefotaxime, blot-dried, and transferred to hygromycin selection medium I (HSM I) (Figure 5a). The types of media used for transformation and their compositions are listed in Table 2. Hygromycin resistant cultures were transferred to fresh HSM I medium at 15-day intervals (Figures 5b–5g). Further, the emerging shoots were elongated in hygromycin selection medium II (HSM II) (Figure 5h). Putative transgenic shoots were rooted in cefotaxime free hygromycin selection medium

III (HSM III) (Figure 5i). In vitro rooted hygromycin resistant plantlets were transferred to soil and acclimatized in the shade house as described above (Figure 5j). The stable transformation efficiency was calculated as the ratio of the number of explants regenerated under hygromycin selection to the number of explants cocultivated (Table 3).2.7. Histochemical assay for gus expressionHistochemical analysis of transient gus expression in control (Figure 5k) and cocultivated leaf explants (Figure 5l) and stable gus expression in putative transgenic shoots (Figures 5m–5o) was carried out according to Jefferson et al. (1987) using the substrate 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc-Duchefa, the Netherlands). The frequency of gus expression was estimated as the

Figure 2. Schematic representation of the T-DNA region of the binary vector pCAMBIA 1301 showing the position of the gus and hpt genes. CaMV 35S: Cauliflower Mosaic Virus 35S promoter, CaMV 35S poly A: Cauliflower Mosaic Virus 35S terminator, NOS poly A: nopaline synthase terminator.

Figure 3. Determination of hygromycin concentration for selection of putative transformants.

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Figure 4. Effect of various parameters on transient gus expression in leaf explants of S. trilobatum L: a) age of explants, b) concentration of acetosyringone, c) duration of preculture, d) optical density, e) concentration of Tween 20, f) infection time, g) pH of cocultivation medium, h) cocultivation duration.

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ratio between the number of explants showing positive expression (appearance of blue color) and the number of explants assayed. 2.8. PCR and RT-PCR analysisTo confirm the presence of transgenes (gus, hpt) in putative transformants, polymerase chain reaction (PCR) was performed (Figures 6a and 6b). Total genomic DNA of the control and the putatively transformed plants was extracted using the HiPurA DNA isolation kit (HiMedia, India). Detection of gus (1.2 kb) and hpt (0.95 kb) genes was carried out using the following specific primers: GUS-F-5’-GGT GGG AAA GCG CGT TAC AAG-3’ and GUS-R-5’-GTT TAC GCG TTG CTT CCG CCA-3’; HPT-F-5’-GCC TGA ACT CAC CGC GAC G-3’ and HPT-R-5’-CAG CCA TCG GTC CAG ACG-3’. PCR amplification

was performed in 25 µL reaction volumes containing 1.0 U of Taq DNA polymerase, 1X Taq buffer, 0.2 mM of each dNTP (Fermentas, India), 0.4 µM of each primer (Sigma, USA), and 50 ng of template DNA. Amplification was performed in a programmable thermal cycler (Eppendorf, Germany) under the following conditions: gus gene: initial denaturation at 94 °C for 5 min, 40 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min followed by a final extension for 10 min at 72 °C; hpt gene: initial denaturation at 95 °C for 7 min, 35 cycles of annealing at 95 °C for 1 min, annealing at 62 °C for 1 min, and extension at 72° C for 2 min followed by a final extension at 72 °C for 10 min. For reverse transcriptase polymerase chain reaction (RT-PCR), total RNA was isolated from 100 mg of putative

Figure 5. Regeneration and histochemical gus analysis of putative transgenic plants of S. trilobatum L. a) In vitro leaf explants inoculated in HSM I (MS medium containing 2 mg/L BAP, 0.25 mg/L IAA, and 15 mg/L hygromycin). b–d) Survival and induction of putative transgenic shoot buds after 5 weeks in HSM I. e–g) Regeneration of hygromycin resistant shoots after 8 weeks of culture. h) Elongation of putatively transformed shoots in HSM II (MS medium containing 2 mg/L BAP, 0.1 mg/L IAA, 0.5 mg/L GA3, and 15 mg/L hygromycin) after 6 weeks. i) Root induction in HSM III (MS medium containing 2 mg/L IBA and 15 mg/L hygromycin) after 6 weeks. j) Acclimatization of transgenic plants in the shade house. k) Histochemical gus expression analysis of a control leaf (nontransformant). l) Infected leaf showing transient gus expression after co-cultivation. m–o) Putative transgenic shoots showing stable gus expression under hygromycin selection.

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transgenic and nontransformed plants using the TRIzol (Sigma, USA) method according to the manufacturer’s instructions. One microgram of total RNA was used as a template for the synthesis of first-strand cDNA with oligo (dT)18 (Superscript III, Invitrogen Inc., USA). RT-PCR of the gus gene was carried out according to the conditions described above using cDNA as a template (Figure 6c). The actin gene was used as an internal control to check the expression of the gus gene. The primers used for the amplification of the actin gene were F-5’-TGGGTGATGAAGCTCAGTCA-3’ and R-5’-TTAAGGGGTGCCTCAGTCAG. The amplification conditions were as follows: initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 45 s, annealing at 58 °C for 45 s, and extension at 72 °C for 45 s; and a final extension for 7 min at 72 °C.2.9. Statistical analysisA completely randomized design was employed in all experiments. Regeneration experiments comprised 10 explants, the transformation experiment consisted of 25 explants in replicates, and each experiment was performed at least three times. All data were statistically analyzed using analysis of variance (ANOVA) and the means differing significantly were compared using Duncan’s multiple range test at 5% probability level. Variability around the mean was represented as ±standard deviation.

3. Results 3.1. Direct plant regeneration from leaf explantsFor direct shoot organogenesis from leaf explants, the effect of BAP alone and in combination with IAA or NAA was tested. A differential morphogenic response (Figures 1a–1l) was observed when leaf explants were cultured in different hormonal media. After 1 week, the size of the explants increased and cluster primordial formation was observed at the cut ends of leaves. Direct emergence of shoot buds occurred after 4 weeks of culture in all treatments except in BAP and NAA combinations. Out of two auxins tested, IAA was found to be beneficial as it directly induced shoot buds without any intermittent callus phase, whereas treatment with BAP in combination with NAA resulted in callus formation, hardly forming shoots, at all tested concentrations (Table 1). Among different concentrations of BAP tested, 2 mg/L BAP resulted in optimal regeneration response either alone or in combination with IAA or NAA. A high frequency of direct shoot regeneration (89%) was observed in MS medium supplemented with 2 mg/L BAP and 0.25 mg/L IAA. Particularly at this combination, a prompt response for multiple shoot primordial formation and subsequent regeneration was noticed within 4 weeks of culture (Figure 1m). A maximum of 26 shoots per explant were regenerated when these 4-week-old multiple shoot buds were transferred to elongation medium (MS

Table 2. Types of media and compositions used for transformation study in Solanum trilobatum L.

Preculture medium MS medium + 2 mg/L BAP +150 µM acetosyringone + 0.8% agar + 30 g/L sucrose, pH 5.8

Cocultivation medium MS medium + 150 µM acetosyringone + 0.8% agar + 30 g/L sucrose, pH 5.4

Hygromycin selection medium I(HSM I) (for shoot induction)

MS medium + 2 mg/L BAP + 0.25 mg/L IAA + 15 mg/L hygromycin + 300 mg/L cefotaxime + 0.8% agar + 30 g/L sucrose, pH 5.8

Hygromycin selection medium II(HSM II) (for shoot elongation)

MS medium + 2 mg/L BAP + 0.1 mg/L IAA + 0.5 mg/L GA3 + 15 mg/L hygromycin + 200 mg/L cefotaxime + 0.8% agar + 30 g/L sucrose, pH 5.8

Hygromycin selection medium III (HSM III) (for rooting) MS medium + 2 mg/L IBA + 15 mg/L hygromycin + 0.8% agar + 30 g/ L sucrose, pH 5.8

Table 3. Stable transformation efficiency of S. trilobatum L. leaf explants cocultivated with Agrobacterium tumefaciens strain EHA105 (pCAMBIA 1301).

S. no. No. of leaf explants cocultivated

No. of explants showing regeneration under hygromycin selection

No. of transgenicshoots per explant

Stable transformationfrequency (%)

1. 50 34 6.5 ± 1.7 682. 50 30 8.0 ± 2.3 603. 50 32 7.4 ± 0.4 64Mean 50 32 7.3 ± 0.6 64

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+ 2 mg/L BAP, 0.1 mg/L IAA, and 0.5 mg/L GA3). The maximum average shoot length attained after 6 weeks of culture in elongation medium was 4.3 cm (Table 1). When elongated shoots (Figure 1n) were transferred to rooting medium (MS medium with 2 mg/L IBA) profuse rooting was attained after 6 weeks of culture (Figure 1o). Shoots with extensively developed root systems were transferred to soil and maintained in a growth chamber at 26 ± 2 °C for 1 month (Figure 1p). Eventually the plantlets were gradually acclimatized from the shade house to the field with a 90% survival rate.3.2. Optimization of hygromycin concentration for selectionIn the present study, hygromycin was used as the selective agent (Figure 2) and various concentrations of hygromycin were tested to optimize the suitable concentration for selection of transformants. The untransformed leaves of S. trilobatum were highly sensitive to hygromycin in a dose-dependent manner. Upon increasing the concentration of hygromycin, the regeneration response of explants decreased considerably and necrosis occurred to varying degrees. On shoot induction media containing 15 mg/L hygromycin, only 13.3% of leaf explants showed a regeneration response producing an average of 2 shoots per explant, beyond which there was a complete inhibition

of shoot regeneration (Figure 3). Therefore, 15 mg/L hygromycin was considered the minimal concentration required for selection of putative transformants of S. trilobatum. 3.3. Optimization of factors influencing transformation In order to achieve a high transformation efficiency using leaf explants, we extensively and systematically examined various parameters that influence the genetic transformation such as the age of explants, acetosyringone concentration, duration of preculture, bacterial optical density, Tween 20 concentration, infection time, pH of the cocultivation medium, and cocultivation duration. The age of explants used for cocultivation had an influential role in transformation and the present investigation revealed that 45-day-old leaf explants of S. trilobatum are highly amenable for transformation by exhibiting greater transient gus expression (56.3%) (Figure 4a). Prior to cocultivation, the explants that were precultured in medium containing acetosyringone exhibited significantly higher transformation efficiency than control explants. The addition of 150 µM acetosyringone in both preculture medium and cocultivation medium (Table 2) drastically improved the percentage of transformation (59%) (Figure 4b). Among various preculture time periods tested, 2 days of preculture resulted in optimal gus expression

M +C –C T1 T2 T3 T4 T5

1.2 kb

M +C –C T1 T2 T3 T4 T5

0.95 kb

Actin

gus

-C T1 T2 T3 T4 T5 (C)

(A) (B)

Figure 6. Molecular analysis of transgenic plants. a) PCR amplification of 1.2 kb fragment of gus gene. b) PCR amplification of 0.95 kb fragment of hpt gene. +C: positive control (Plasmid DNA), -C: negative control (untransformed plant), T1–T5: five different transgenic plants. c) RT-PCR analysis of gus gene expression in transgenic plants.

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frequency of 60.3% (Figure 4c). When various ranges of bacterial optical densities were investigated, 0.4 O.D. showed the most efficient transformation frequency (60.6%) (Figure 4d). Beyond 0.4 O.D. there was a vast decline in transformation rate due to necrosis of explants caused by excessive bacterial density. Along with bacterial suspension, when a surfactant like Tween 20 was incorporated, it increased the percentage of explants showing gus expression (62%) at 0.01% (Figure 4e). However, higher concentrations of Tween 20 (0.1% and 0.5%) caused browning and eventual death of explants in selection medium after cocultivation. Similarly, the optimum infection time to attain a maximum rate of gus expression (62.3%) was found to be 15 min (Figure 4f). Our results indicated that the pH of the cocultivation media had a profound effect on the T-DNA delivery process. It was observed that the level of transient gus expression was highest (63%) when the pH of the cocultivation medium was 5.4 (Figure 4g). After optimizing all conditions, the duration of cocultivation was evaluated by testing various time intervals (1–5 days). We found that cocultivation of explants with Agrobacterium for 3 days under optimized conditions remarkably elevated the transformation rate (64.6%) (Figure 4h).3.4. Regeneration and stable gus expression of transgenic plantsAfter cocultivation, the infected leaf explants were subjected to selection in HSM I medium containing 15 mg/L hygromycin (Figure 5a). The regeneration of putatively transformed shoot buds occurred after 5 weeks of culture in HSM I medium (Figures 5b–5d), while untransformed explants turned brown and ultimately died within 6–7 weeks of culture. The hygromycin resistant shoot buds remained green and continued proliferation upon frequent subculture in HSM I medium (Figures 5e–5g). After 8–10 weeks, the actively proliferating shoot buds were transferred to elongation medium (HSM II) for elongation (Table 2) and the shoots were elongated after 6 weeks of culture in HSM II (Figure 5h). Subsequently, the elongated shoots were rooted in rooting medium (HSM III) (Figure 5i) and acclimatized in the shade house (Figure 5j). Transient gus expression was observed soon after cocultivation (Figures 5k and 5l), whereas stable gus expression was detected in emerging shoots (Figures 5m–5o) after 5–10 weeks under hygromycin selection. Thus, a stable transformation frequency of 64% was achieved in the present study (Table 3).3.5. Molecular analysis of transgenic plantsTo confirm the transgenic nature of regenerants, genomic DNA was extracted from five different putative transformants and untransformed plants. PCR analysis was carried out to detect the presence of transgenes (gus and hpt) in gus positive, hygromycin resistant shoots. The

PCR analysis detected the presence of the expected 1.2 kb amplified fragment corresponding to the gus gene (Figure 6a) and the 0.95 kb amplified product corresponding to the hpt gene (Figure 6b) in all five transformants, whereas no amplification was detected in untransformed plants (control). Furthermore, to assess the stable expression of the gus gene in transformed plants, RT-PCR analysis was carried out using gus specific primers. The expression of the gus gene was detected in all five transformants examined, while the expression was not observed in untransformed plants (control) (Figure 6c).

4. DiscussionIn the present study, we have described an effective protocol for direct shoot organogenesis through leaf explants and it was further employed for genetic transformation via Agrobacterium tumefaciens using strain EHA 101. Among various concentrations and combinations of plant growth regulators tested, a combination of 2 mg/L BAP and 0.25 mg/L IAA was critical for successful direct plant regeneration from in vitro derived leaf explants of S. trilobatum. The synergistic effect of BAP and IAA on direct shoot regeneration from leaf explants has been reported earlier in other medicinal plants such as Salvia nemorosa (Skała and Wysokińska, 2004), Spilanthes acmella (Saritha and Naidu, 2008), and Tylophora indica (Haque and Ghosh, 2013).

In this study, we have investigated various factors that increase the competence for genetic transformation of S. trilobatum. For the selection of transformants, the selective agent hygromycin was used at the concentration of 15 mg/L, at which the least frequency of regeneration (13.3%) occurred, while above this concentration shoot regeneration was completely arrested. Similarly, 15 mg/L hygromycin was used for the selection of transformants in Scoparia dulcis L. (Aileni et al., 2011). Explants taken from 45-day-old in vitro grown plants showed optimum results whereas the tissues of older explants were not competent enough to receive T-DNA and had significantly reduced transformation efficiency. These results indicate that physiological maturity of the starting material is an important factor in assuring successful transformation. The addition of a transformation induction agent such as acetosyringone is essential for efficient T-DNA delivery, as it activates the transcription of Agrobacterium virulence genes (Cheng et al., 1997). In our study, incorporation of 150 µM acetosyringone in both preculture and coculture media largely improved the transformation rate (59%). Earlier, the use of 150  µM  acetosyringone dramatically increased the transformation efficiency (100-fold) of almond (Costa et al., 2006) and Withania somnifera (L.) Dunal (Sivanandhan et al., 2015). Apart from using acetosyringone, recently Özcan et al. (2015) reported that

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phenolic compounds of squirting cucumber fruit juice can be used to significantly enhance the transformation efficiency of tobacco and potato.

We observed that preculture of explants with phytohormones for 2 days prior to incubation with Agrobacterium had a positive effect on transformation and also enhanced the regeneration of cocultivated explants. Phytohormone pretreatment triggers cell division and the formation of new and thin cell walls facilitates the efficient attachment of Agrobacterium to host cells (Chakravarty and Pruski, 2010). Preconditioning also stimulates the plant cells to enter into the regeneration pathway, resulting in improved transformation efficiency (Ainsley et al., 2001). Our results are in agreement with previous findings where high transformation frequency was achieved in 2-day precultured leaf explants of medicinally important Scoparia dulcis L. (Aileni et al., 2011) and in Pogostemon cablin (Blanco) Benth (Paul et al., 2012). The density of bacterial suspension used for infection of the explant was also found to be a key factor affecting the transformation frequency (Mishra et al., 2013). In our study, as the bacterial concentration increased, the transient gus expression increased up to 0.4 O.D. and then declined with further increase in bacterial density. This could be due to increased necrosis of the tissue caused by the release of toxic compounds at high bacterial density (Sonia et al., 2007). Such lower bacterial density (0.4 O.D.) was successfully employed in genetic transformation of other medicinal species such as Withania coagulans (Mishra et al., 2013) and Bacopa monnieri (L.) (Aggarwal et al., 2012).

Addition of surfactants to the bacterial suspension or inoculation medium has also been found to induce the transformation. Surfactants enhance T-DNA delivery by lowering the surface tension of host cells, which aids in the efficient attachment of Agrobacterium cells (Cheng et al., 1997). Tween 20 is a polysorbate surfactant used as a detergent and emulsifier in a number of biotechnological, industrial, and pharmacological applications. Our study revealed that treatment of bacterial suspension with Tween 20 at 0.01% increased the susceptibility of explants to Agrobacterium infection, resulting in a striking elevation of transient gus expression (62%). Tween 20 has been successfully used to enhance the transformation efficiency of plants by many researchers (Kim et al., 2009; Paul et al., 2012; Khan et al., 2015).

The pH of the cocultivation medium has been reported to be an equally important factor in assisting the transformation efficiency (Mondal et al., 2001). We

noticed that cocultivation medium with a lower pH of 5.4 was beneficial for improving the transformation frequency rather than a pH of 5.8. This was in accordance with earlier reports of Huang and Wei (2005) in maize and Rahman et al. (2011) in indica rice. The period of infection and cocultivation with bacterial culture is an important step in Agrobacterium mediated transformation (James et al., 1993). In the present study, immersion of explants in bacterial suspension for 15 min followed by cocultivation for 3 days was found to be optimally efficient in genetic transformation of S. trilobatum. We observed that a cocultivation period exceeding 3 days resulted in a lower transformation frequency. Cocultivation of explants with A. tumefaciens for a suitable duration improves the transformation frequency, but longer cocultivation often results in death of explants due to overgrowth of bacteria (Folta and Dhingra 2006; Li et al., 2007). Similar observations were recorded in strawberry (Zhang and Wang, 2005), Indian kino tree (Tippani et al., 2013), and Casuarina (Jiang et al., 2014). Thus, the current study was carried out with the optimized parameters resulting in a stable transformation frequency of 64%. Previously, various ranges of stable transformation frequencies were reported for several medicinal species including Salvia miltiorrhiza – 86.7% (Yan and Wang, 2007), Centella asiatica – 33.3% (Nanditha et al., 2008), Withania somnifera – 1.67% (Pandey et al., 2010), and Bacopa monnieri – 70.6% (Mahender et al., 2012). The presence of transgenes in putative transformants was confirmed by PCR and RT-PCR analysis revealed the stable expression of the gus gene in transformed plants. This is the first report on Agrobacterium tumefaciens mediated genetic transformation of S. trilobatum and thus the protocol described herein can be used for the successful introduction of genes involved in biosynthesis of steroid alkaloids of S. trilobatum in the future.

AcknowledgmentsThe first author thankfully acknowledges the CSIR (Council for Scientific and Industrial Research) for financial support in the form of a Senior Research Fellowship (CSIR file No. 09/688(0016)/2011-EMR-I; dated 22-01-2014). The authors also acknowledge the Computational and Bioinformatics Facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by the Department of Biotechnology, Government of India: Grant No. BT/BI/25/001/2006) for performing statistical analysis of the work.

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