ORIGINAL ARTICLE

An efficient plant regeneration and Agrobacterium-mediatedgenetic transformation of Tagetes erecta

Vijayta Gupta & Laiq ur Rahman

Received: 20 March 2014 /Accepted: 27 November 2014# Springer-Verlag Wien 2014

Abstract Tagetes erecta, L. an asteraceous plant of industrialand medicinal value, contains important compounds like py-rethrins, thiophenes and lutein, possessing immense potentialfor insecticidal, nematicidal and nutraceutical activities.Considering the importance and demand for these naturalcompounds, genetic manipulation of this crop for better pro-ductivity of secondary metabolites holds great significance. Arapid and reproducible direct regeneration and genetic trans-formation system is the prerequisite for genetic manipulationof any crop. This paper elucidates the establishment of anefficient direct regeneration and transformation protocol ofT. erecta using Agrobacterium tumefaciens. Investigation ofthe effects of different types of explants (Hypocotyls, cotyle-donary leaves, rachis and leaf sections) and different BAP andGA3 combinations on the regeneration frequency of T. erectasuggested that the best regeneration frequency (66 %) with anaverage of 5.08±0.09 shoot buds/explant was observed fromhypocotyl explants cultured on media containing 1.5 mg/lBAP and 5 mg/l GA3. The transformation protocol wasestablished using A. tumefaciens strain LBA4404, containingthe binary vector pBI121, along with the gusA reporter genewith intron under the transcriptional control of the CauliflowerMosaic Virus (CaMV) 35S promoter and the neomycin phos-photransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization ofkanamycin concentration (200 mg/l) for selection, standardi-zation of cocultivation time (45 min) and acetosyringoneconcentration (150 μM) for obtaining higher transformationfrequency were established using hypocotyl explants. The

selected putative transgenic shoots were subsequently rootedon the Murashige and Skoog medium and transferred to thegreen house successfully. The plants were characterised byanalysing the gus expression, amplification of 600 bp npt IIfragment and Southern blot hybridization using the PCR-amplified gusA fragment as probe. The standardised protocolestablished during the study will open new vistas for geneticmanipulation and introduction of desired genes for geneticimprovement of T. erecta.

Keywords Agrobacterium tumefaciens . Genetictransformation . Hypocotyls . Tagetes erecta

Introduction

Tagetes erecta (family; Asteraceae) also known as MexicanMarigold or Aztec Marigold, is native to Mexico and CentralAmerica. Since prehistoric times, this plant has been used forornamental and medicinal purposes (Hamel and Mary 1975;Gutiérrez et al. 2006), and presently, it is used to cure variousdiseases such as parasites, diarrhea, liver ailments, vomiting,indigestion, toothache, among other infirmities (Balangcodand Balangcod 2011). The leaves and flowers of this plantcontain several bioactive compounds that exhibit nematicidal,fungicidal and insecticidal activities (Vasudevan et al. 1997).The crude extract of the plant is found to be effective against abroad spectrum of bacteria in vitro (Das and Mishra 2012).The essential oil of the flower contains antioxidants whichmay also be added to perfumes to infuse an apple scent (Aliand Hassan 2013). Previously, Native Americans and Asiansused it as a skin wash and for yellow dye; however, now-adays, it is grown to extract a carotenoid “lutein”, a commonyellow/orange food colour (Delgado–Vargas et al. 2000).Carotenoids are synthesised through the non-mevalonatepathway (Dubey et al. 2003), which could be manipulated

Handling Editor: Burkhard Becker

V. Gupta : L. ur Rahman (*)Plant Biotechnology Division, Central Institute of Medicinal andAromatic Plants (CSIR-CIMAP), Kukrail Picnic Spot Road, P.O.CIMAP, Lucknow 226015, Indiae-mail: l.rahman@cimap.res.in

ProtoplasmaDOI 10.1007/s00709-014-0740-y

by genetic engineering techniques to increase lutein accu-mulation or to produce new form of carotenoids. Studieshave also shown that a natural phytochemical “thiophene”that includes sulfur-containing rings is also an active ingre-dient of this plant (Marotti et al. 2010), possessing tremen-dous medicinal properties.

The current scenario of the demand of naturally ac-tive ingredients has paved the way to the application ofvarious biotechnological approaches for the manipula-tion of metabolic pathway or content of particular sec-ondary metabolites. For any kind of genetic manipula-tion or introduction of regulatory or structural genes, agenetic transformation with good regeneration system isa major pre-requisite to understand the molecular basisand regulation of secondary metabolism in plants and toengineer them for specific metabolites.

Numerous methods for transformation of agriculturallyimportant crops and utility of Agrobacterium tumefaciensand Agrobacterium rhizogenes in genetic transformation ofmedicinal and aromatic plants (Gomez-Galera et al. 2007;Khan et al. 2009; Naqvi et al. 2010; Yuan et al. 2011) havealready been reported and are well established, but very fewmedicinal plants have been targeted for genetic manipulation(Gomez-Galera et al. 2007; Wang et al. 2008). From severalearlier reports on medicinal plants, it was observed that plantswith highly expressed secondary pathways are reported to berecalcitrant for regeneration and are difficult for genetic trans-formation studies, as evident from the case of Plumbagozeylanica (Wei et al . 2006) , Euphorbia nivul ia(Sunandakumari et al. 2005), Mentha citrata (Spencer et al.1990), and Coleus forskohlii (Mukherjee et al. 2000). Thesestudies indicated that the screening of explants for genetictransformation and regeneration is mandatory. For genetictransformation studies in medicinal and aromatic plants, dif-ferent explants were used by different workers (Tripathi andTripathi 2003; Asghari et al. 2012).

In the past three decades, significant achievements havebeen obtained with genetically transformed hairy root culturesand production of thiophenes from T. erecta and T. patula(Mukundan and Hjortso 1990a, b; Giri and Narasu 2000)while only one report of transient transformation of Tageteswith A. tumefaciens is available (Godoy-Hernandez et al.2006). Direct differentiation of shoot buds in leaf segmentsof white marigold (Misra and Datta 2001) and induction offriable embyogenic callus and somatic embryo formationfrom cotyledonary explants of African marigold (T. erecta)(Bespalhok and Hattori 1998) have been reported, but genetictransformation was not attempted in these cases. To ourknowledge, no reports are available on the stable transforma-tion in marigold explants, and this is the first report highlight-ing stable genetic transformation in T. erecta. Here, we give anaccount for the successful regeneration and stable genetictransformation system of T. erecta for further manipulation

and production of genetically transformed plant cultures forenhanced expression of secondary metabolites.

Materials and methods

Direct regeneration of T. erecta using various explants

African marigold (T. erecta L.) seeds were surface sterilizedwith 0.1 % mercuric chloride for 90 seconds and eventuallywashed three times with autoclaved triple distilled water for10 min each. The disinfected seeds were then kept for germi-nation on hormone-free Murashige and Skoog’s (Murashigeand Skoog 1962) medium.

Leaves, hypocotyls, cotyledonary leaves and rachis fromin vitro grown achenes were used as explants for regeneration.All the above explants were transferred to Murashige andSkoog (MS) medium supplemented with BAP (0.5 mg/l to5.0 mg/l) in combination with GA3 (5.0–10 mg/l) and BAP(1.0–4.0 mg/l) in combination with IAA (1.0 mg/l) and wereincubated under cool fluorescent light (3,000 lux) with 16 L:8D-h photoperiods.

The proliferated shoots were transferred to hormone-freeMS medium for elongation and root induction. The rootedshoots were first acclimatized in Hoagland’s medium for1 week and then transferred to the sterilized potted soil. Datawere presented as mean (±SE) from 30 explants for eachtreatment and then recorded after 4 weeks of culture. Theexperiment was repeated three times. Out of all the aboveexplants, hypocotyls were used for further genetic transfor-mation studies since they gave the best regeneration responseamong all explants used.

Determination of kanamycin concentration for selectionof transformants

To determine the optimal kanamycin concentration for theselection of putative transformants, sensitivity test for shootdevelopment was studied in T. erecta by using hypocotyls asexplants to investigate the optimum concentration of kanamy-cin for the selection of transformants. The hypocotyl explantswere placed on modified MS medium supplemented with1.5 mg/l BAP, 5.0 mg/l GA3 (best suited for direct regenera-tion) with different concentrations of kanamycin (0–400 mg/l)for 4–6 weeks, and the observations were recorded.

Bacterial strains and plasmid vectors

A. tumefaciens strain LBA4404 containing the binary vectorpBI121 (Fig. 1) was used for genetic transformation. The T-DNA of pBI121 harboured the neomycin phosphotransferase(nptII) selection marker gene and gusA reporter geneinterrupted by a modified castor bean catalase intron. The

V. Gupta, L. ur Rahman

nptII gene is under the control of Pnos (nopaline synthasepromoter) and gusA gene is under the control of cauliflowermosaic virus (CaMV) 35S promoter (Ohta et al. 1990).

Genetic transformation with A. tumefaciens

A. tumefaciens strain LBA4404 was grown in YEP minimalmedium (Lichtenstein and Draper 1986) in the presence of50 mg/l kanamycin, 250 mg/l streptomycin and 100 mg/lrifampicin at 28 °C for 20–22 h at 200 rpm on incubatorshaker. A. tumefaciens cells were harvested in log phase atOD600=0.6 by centrifugation and re-suspended in liquid MSmedium. The explants were wounded by pricking to exposecut surfaces for infection, and the wounded explants wereimmersed in A. tumefaciens suspension with addition ofacetosyringone (50–250 μM) and kept at 28 °C on shaker(100 rpm) for 15–60 min. The explants were blotted dry onsterile filter paper and placed on co-cultivation mediumconsisting of MS salts, B5 vitamins, 100 mg/l myoinositoland 30 g/1 sucrose, and kept in the dark for 3 days. Theexplants were transferred onto the MS medium fortified with1.5 mg/l BAP, 5 mg/l GA3, 250 mg/l cefotaxime and 200 mg/lkanamycin for direct regeneration (as reported above) underdark conditions.

Media composition and culture conditions

The pH of all the tissue culture media was adjusted to 5.8 priorto the addition of agar and autoclaving. All the media werefortified with 0.8 % agar. Cultures were incubated at 25±2 °Cunder 16L/8D period. Light at 60 μmol photon m ̄ 2s 1̄ wassupplied by fluorescent tube lights fitted on the culture racks.All biochemicals and media constituents of molecular biologygrade were procured from Sigma Chemical Company (St.Louis, MO), unless otherwise stated.

Gus histochemical assay

Transient gus expression was analysed in the treated explantsjust after infection, while stable gus expression analysis wascarried out with kanamycin-resistant regenerated transgenicplants. Gus staining was done using 5-bromo-4-chloro- 3-

indolyl glucuronide (X-gluc, Biosynth) supplemented with20 % methanol, as described by Kosugi et al. (1990). Thisassay gives blue colour in the presence of the β-glucuronidaseenzyme. Explants were tested by incubating overnight withthe substrate (1 %w/v in phosphate buffer, pH 7.4, supple-mented with 0.01 % Triton X-100) at 37 °C. Stained tissueswere rinsed extensively in 70% ethanol to remove the residualchlorophyll.

Gus activity assay

The standard protocol of Jefferson et al. (1987) was followedfor assaying gus enzyme activity. Fresh leaf tissue (100 mg)was ground with 1 ml extraction buffer (50 mM Na2HPO4

pH 7.0, 10 mM DTT, 0.1 % sodium lauryl sarcosine (w/v),Triton X-100, 10 mM Na2EDTA) and mixed well to obtain ahomogeneous mixture. The mixture was centrifuged at13,000 rpm, at 4 °C, for 5–10 min. Supernatant (90 μl) wasadded with 10 μl of 10 mM MUG (4 methyl-umbelliferyl-β-D- glucuronide). The mixture was then incubated at 50–55 °Cfor 25 min, and 25 μl methanol was added in the dark. Thesamples were incubated at 37 °C for 2 h, and the reaction wasstopped with a tenfold volume of 0.2 M Na2CO3. The proteinconcentration was determined as described by Bradford(1976), using bovine serum albumin as a standard.

Molecular analysis of transgenic plants PCR amplificationof nptII and gusA gene in putative transformants

Plant genomic DNAwas isolated using DNeasy Plant Minikit,QIAGEN. The transformants were examined by polymerasechain reaction (PCR) amplification of nptII and gusA genes.The oligonucleotide primers used for nptII gene amplificationwere ‘forward’: 5’CTG AAT GAA CTG CAG GAC GAG G3’ and ‘reverse’: 5’GCC AAC GCT ATG TCC TGATAG C3’ and those for gusA gene were ‘forward’: 5’GAT CTGAGGGTA AAT TTC TAG TTT TTC TC 3’ and ‘reverse’: 5’TGGATT CCG GCA TAG TTA AAG AAA T 3’. Amplificationwas carried out at 94 °C for 5 min, 94 °C for 1 min, 58 °C (fornptII) and 60 °C (for gusA containing intron) for 1 min asannealing, 72 °C for 1 min as elongation and 72 °C for 5 min

Fig. 1 Construct map: T-DNA construct of binary vector pBI121 show-ing restriction sites and gusA with intron used for transformation ofT. erecta. RB right border, Pnos nopaline synthase promoter, nptII

neomycin phosphotransferase gene, Tnos nopaline synthase terminator,P35SCaMV Cauliflower Mosaic Virus 35S promoter, gusA intron con-taining b-glucuronidase gene and LB left border of T-DNA

An efficient plant regeneration and Agrobacterium-mediated genetic

as final extension. Amplicons were electrophoresed on 1.0 %(w/v) agarose gel.

Southern blot analysis

For Southern blot analysis, 10 μg of genomic DNA isolatedfrom the putative transgenic plants and non-transformed con-trol plant was digested with restriction endonuclease enzymeHindIII, fractionated on a 0.8 % agarose gel in 0.5× Tris–acetate–EDTA (TAE) buffer (pH 8.0) and blotted onto a nylonmembrane. As there was no HindIII cleavage site within theselected gusA probe, the number of hybridizing fragmentsindicated the number of insertion events. The membranewas prehybridized for 1 h at 65 °C in a solution containing0.25M sodium phosphate (pH 7.4), 1 mMEDTA, 1 % bovineserum albumin, 7 % sodium dodecyl sulphate (SDS) andhybridized in the same solution with radiolabeled probe pre-pared by PCR of a part of the gusA gene.

The membrane was then washed twice with 2× standardsaline citrate (SSC) solution at room temperature and oncewith 0.1×SSC solution containing 0.1 % SDS at 60 °C for15 min each and exposed to autoradiographic film for 24 h.The cassette was stored in the dark at room temperature. Theautoradiogram was scanned on a phospho-imager (MolecularImager FX, Bio-Rad).

Results

Plant regeneration from different explants of T. erecta

The various explants such as hypocotyls, cotyledonary leaf,rachis and leaves, when placed on MS media containing BAPin combination with GA3 and IAA, showed regeneration ofshoots (Fig. 2). However, the frequency and number of shoots/explants varied with the combinations of media and explantsused for regeneration. Maximum regeneration efficiency of66 % was observed with hypocotyls in modified MS mediumcontaining 1.5 mg/l BAP and 5.0 mg/l GA3 (Table 1). In thisoptimum treatment, an average of 5.08±0.09, shoot buds weredifferentiated within 4 weeks of incubation. These shootswere healthy and properly elongated. It was observed thatthe lower concentration of GA3 (5.0 mg/l} promoted shootregeneration in hypocotyl explants followed by rachis. Thecotyledonary leaves showed regeneration of only one to twoshoots in most of the media used. When GA3 was replaced byIAA (1.0 mg/l), shoots were regenerated within 4–5 weeks.Maximum five to seven shoots per explant from hypocotylsand rachis were developed on MS media containing 3.0 mg/lBAP and 1.0 mg/l IAA (Table 2), but these shoots were hyper-hydrated and appeared abnormal in shape. Elongated differ-entiated shoots were separated after 5 weeks of culture and

transferred to MS media for rooting. Root initiation wasobserved within 10 days and became profuse after 4 weeksof culture. These rooted plants showed 70 % survival in pots.

Determination of kanamycin concentration for the selectionof transformants

The hypocotyl explants showed regeneration of green shootsafter 5 weeks of culture on media containing 100 mg/l ofkanamycin, although the number of shoots was less than thecontrol explants which showed regeneration of 3.13±0.14shoots per explant (Table 3). Bleaching of shoots was ob-served as the kanamycin concentration increased to 200 mg/l.The explants showed complete bleaching and regeneration ofyellowish white shoots at higher concentration of kanamycin(300–400 mg/l). Based on the observation, we found 200 mg/lkanamycin to be the best for the selection of transformants.

Genetic transformation with A. tumefaciens

The genetic transformation of T. erecta was established usingA. tumefaciens strain LBA4404, containing the binary vectorpBI121. Different parameters influencing the transformation-like optimization of kanamycin concentration for selection,standardization of co-cultivation time and acetosyringoneconcentration for getting higher transformation frequencywere established using hypocotyl explants.

The optimum infection time for getting highest transienttransformation efficiency (TE) was found to be 45 min withhypocotyl explants (Fig. 3a), while 15 min treatment showedlowest transient expression of gus gene. With longer treatmenttime, the transformation frequency decreased drastically (up to50 %). The explants also became necrotic and turned brown.The present transient gus expression also varied with theconcentration of acetosyringone (AS) used during co-cultivation period. It was observed that the expression per-centage kept on increasing till 150 μMof AS. Further increasein the concentration decreases the transformation event(Fig. 3b).

Gus expression analysis

Transient Gus assay carried out with hypocotyls explants afterco-cultivation showed distinct blue spots. The putative select-ed plants analysed histochemically for Gus assay indicated theexpression of blue colour in various parts of the plants. Theexpression was maximum in the mid rib and distal part of theleaves (Fig. 4). The embryo of the seed obtained fromkanamycin-resistant plants also showed gus expression. Thepresence of an intron in the gusA gene guards against the false-positives that may result from the expression of the gene inA. tumefaciens. Therefore, initially, histochemical gus activity,Gus assay test, PCR analysis using gusA primers in the

V. Gupta, L. ur Rahman

progeny and finally Southern hybridization showed integra-tion of gusA gene into the plant’s genomic DNA.

The optimized conditions for transformation, i.e. infectiontime 45 min, MS medium with BAP (1.5 mg/l) and GA3

(5 mg/l) and AS concentration (150 μM) were used to infectthe hypocotyl explants. Transgenic shoot buds were continu-ously multiplied and maintained on the selection mediumcontaining 200 mg/l kanamycin. Putative transformed shoots

took nearly 2 months to develop healthy lateral roots on theMS medium without any hormone. Acclimatized in vitrorooted plants showed 40% survival rate after being transferredto the glass-house. The plants grew normally and producedflowers within 60 days.

In subsequent generations, the transgenic plants were mor-phologically similar to the non-transformed control plants andperformed equally well. The histochemical gus expression

Fig. 2 Direct regeneration fromdifferent explants of T. erecta ashoot regeneration fromhypocotyl, b shoot regenerationfrom cotyledonary leaf, c shootregeneration from leaf, d shootregeneration from rachis

Table 1 Effect of BAP and GA3 concentrations on direct shoot regeneration from different explants of T. erecta

Media composition (MS) Explant used

Hypocotyls Cotyledonary Leaf Rachis Leaves

No. of shoots/explant

Response(%)

No. of shoots/explants

Response(%)

No. of shoots/explants

Response(%)

No. of shoots/explants

Response(%)

0.5 BAP mg/l+5 mg/lGA3 4.04±0.10 53.3±5.77 1.22±0.06 10.0±10 2.83±.459 46.67±7.2 1.21±.0327 6.67±5.77

0.5 BAP mg/l+10 mg/lGA3 1.67±0.09 13.33±5.77 1.67±0.3 4.67±0.3 1.72±0.16 9.31±.63 1.43±.24 3.59±.072

1.0 BAP mg/l+5 mg/lGA3 4.00±0.4 56.6±5.77 1.32±0.03 10.0±0 2.39±.163 46.67±5.5 1.37±.22 6.67±5.77

1.0 BAP mg/l+10 mg/lGA3 1.51±0.06 13.33±5.77 1.22±0.06 3.00±0.5 1.21±.0327 10.72±5.47 1.17±.20 2.413±.156

1.5 BAP mg/l+5 mg/lGA3 5.08±0.09 66.6±5.77 2.34±0.23 23.3±5.77 3.70±.4382 56.70±2.7 2.01±.044 14.54±0.5

1.5 BAP mg/l+10 mg/lGA3 2.01±0.04 26.67±15.2 1.22±0.06 4.67±0.3 1.22±.066 16.67±5.77 1.16±.49 3.769±.22

2.0 BAP mg/l+5 mg/lGA3 4.21±0.5 40.0±10 1.21±0.03 16.66±5.77 2.5803±.16 34.7±1.9 1.22±.066 10±0

2.0 BAP mg/l+10 mg/lGA3 1.21±0.03 10.00±0.00 1.62±0.05 3.00±0.5 1.77±.0476 10.32±.88 1.35±.062 2.534±.39

5.0 BAP mg/l+5 mg/lGA3 4.04±0.10 46.6±11.54 1.32±0.03 20.0±10 2.133±.13 36.6±3.34 1.24±.12 13.33±5.77

5.0 BAP mg/l+10 mg/lGA3 1.66±0.1 16.67±5.77 1.21±0.03 4.00±0.4 1.67±0.5 13.3±3.34 1.22±.066 2.81±.18

An efficient plant regeneration and Agrobacterium-mediated genetic

was observed as dark blue colour in the transformed embryo(Fig. 4) while embryo of non-transformed (control) seeds didnot show the expression of gus.

Molecular analysis of transgenic plants

The PCR analysis of genomic DNA of gusA T0 transgenicplants obtained as independent transformation eventsshowed amplification of the predicted 800 bp gusA aswell as 600 bp nptII fragments of transgenes (Fig. 5aand b). The positive control (PCR with plasmid pBI121)also gave similar size amplicons. The bacterial vir C gene-specific primers did not show amplification of the 730 bpamplicon (Fig. 5c). This was performed to eliminate thedetection of false-positives due to persistent contaminationof Agrobacterium cells if any in T0 plants. Southern blothybridization confirmed genomic integration of gusA genein four T0 transgenic plants. Different sizes of the insertestablished that the transgenic insertion was at differentloci in different transgenic plants (Fig. 5d).

Discussion

Success of Agrobacterium-mediated transformation andexpression of any gene depends upon callusing and

regeneration frequency, which helps in recovering alarge number of transformants. Efficient regenerationand transformation are the major prerequisites for thedevelopment of suitable expression system. Therefore,prior to an experiment, it would be appropriate to havea standardized protocol to maximize the results. Thehypocotyls explants have shown best response for re-generation. Hypocotyl explants have been utilized suc-cessfully for direct regeneration in many plant species(Kulkarni et al. 2000; Cui et al. 2004; Rashid and Singh2010). The hormonal combination of 1.5 mg/l BAP and5.0 mg/l GA3 was found to be the best for regenerationthrough hypocotyl explants. Previously, differentiationof shoots in leaf segments of white marigold wasachieved in a combination of BA (5 mg/l) + IAA(3 mg/l), but that was associated with callus (Misraand Datta 2001). Belarmino et al. (1992) used differentexplants (hypocotyls, stem sections and leaf portions)for regeneration study. They also found that the hypo-cotyl explants were the best source for plantlet produc-tion using NAA and BAP as growth regulators andfound 50 % of plant regeneration in Tagetes. On in-creasing the concentration of BAP, it became supra-optimal for the explants to differentiate, as they showedhyperhydricity without any further increase in the num-ber of shoots as has been observed previously by manyworkers (Ziv 1991; Debergh et al. 1992; Chiruvellaet al. 2014). On lowering the concentration of eitherBAP or IAA, differentiation could not be achievedproperly. GA3 played a very significant role for theinduction of shoot buds. In the present experiment,GA3 (5.0 mg/l) in combination with BAP was foundto induce shoot bud differentiation in different explantsused. The differentiation of shoot buds was direct with-out any associated callus. On further increasing theconcentration of GA3, the explants turned yellowishbrown in colour without any differentiation of shoots.GA3 has been found conducive to in vitro shoot regen-e ra t ion in f lo re t exp lan ts o f Chrysanthemum(Chakrabarty et al. 2000). Sekioka and Tanaka (1981)

Table 2 Effect of BAP and IAA concentrations on direct shoot regeneration from different explants of T. erecta

Media composition(MS)

Rachis Leaves Hypocotyl Cotyledonary leaf

No. of shoots/explant

Response(%)

No. of shoots/explant

Response(%)

No. of shoots/explant

Response(%)

No. of shoots/explant

Response(%)

1 mg/l BAP+1 mg/l IAA 2.38±0.10 23.3±5.77 1.22±0.06 6.66±5.77 2.802±.74 20±10 1.18±.330 8.20±1.06

2 mg/l BAP+1 mg/l IAA 2.71±0.03 26.6±5.77 1.21±0.03 6.66±5.77 2.996±.37 36.6±3.34 1.22±.762 8.9±6.088

3 mg/lBAP+1 mg/l IAA 5.67±0.2 60.0±10 1.32±0.03 16.66±11.54 6.458±.69 62.5±8.1 1.50±.53 26.67±15.28

4 mg/l BAP+1 mg/l IAAa 4.04±.10 33.3±11.54 1.22±0.06 10.0±10 4.523±.52 43±3.34 1.4766±.34 10.72±5.47

a Callus was observed at the base of rachis

Table 3 Shoot regeneration from hypocotyl explants of T. erecta ondifferent concentration of kanamycin

Kanamycin(mg/l)

No. of shoots/explant(2 weeks)

No. of shoots/explant(4 weeks)

0 2.13±0.08 3.13±0.14

100 1.83±0.08 2.11±0.10

200 1.54±0.11 1.66±0.10

300 1.50±0.12 1.62±.11

400 1.48±0.11 1.57±0.11

V. Gupta, L. ur Rahman

reported that GA3 can act as a replacement for auxin inshoot induction, and thus a ratio of cytokinin–GA3 maybe decisive for differentiation in certain plant tissues(Laboney et al. 2013; Kumar et al. 2013).

Elucidating the effects of kanamycin on the growth ofT. erecta at various concentrations is a preliminary task fortransformation research. The use of antibiotic resistance

selection marker (for example, Kanamycin resistance), includ-ed in the vector along with the desired genes, gives a conve-nient and efficient selection system. However, in several cropspecies, the sensitivity to antibiotic selection is very low.Kanamycin-based selection systems do not eliminate thenon-transformed cells completely (McKently et al. 1995;Cheng et al. 1996; Sharma and Anjaiah 2000; Dodo et al.

Fig. 3 Histochemical Gusexpression in respect to ainfection time, b acetosyringoneconcentration

Fig. 4 Histochemical Gusexpression in T0 transgenic plantsa—b: gus expression in leaf, c—d: gus expression shown intransformed embryo and non-transformed embryo

An efficient plant regeneration and Agrobacterium-mediated genetic

2008) and could produce escapes or chimeric plants. It hasbeen reported that sometimes kanamycin resistance developsgradually in plants (Rommens 2006), which may lead tofalse transgenics. However, in this report, transformedplants were successfully selected on kanamycin(200 mg/l) containing media without any escapes. Theuntransformed shoots showed complete bleaching onkanamycin concentration of 200 mg/l or more. Thekanamycin has successfully been used for the selectionof genetic transformants by Oliveira et al. 2011; Mishraet al. 2013; Alvarez and Ordás 2013.

Acetosyringone is a phenolic compound exuded fromplant wounds that has been reported to induce theexpression of Vir genes in Ti-plasmid (Jin et al. 2005;Turk et al. 1994). The use of AS during infection hasbeen shown to increase Agrobacterium-mediated trans-formation frequencies (Sheikholeslam and Weeks 1987;Owens and Smigocki 1988; Godwin et al. 1991;Subramanyam et al. 2013; Mishra et al. 2013). Theconcentration of AS used in different plant system var-ied from 20–200 μM depending upon the plant species.In case of T. erecta, AS at 150 μM concentration wasfound to give maximum transformation frequency whichhas also been reported by Boase et al. 1998. The Gus

assay test followed by the PCR analysis using gusAprimers in the progeny showed integration of gusA geneinto the plant genomic DNA. When non-transformedexplants from hypocotyls were maintained on MS me-dium without kanamycin, 100 % of the hypocotylsregenerated to produce shoots respectively. With in-creasing concentrations of kanamycin, the number ofshoots decreased dramatically. A kanamycin gradient of100–400 mg/L was set up out of which kanamycinconcentration of 200 mg/L was used for selectingtransformants.

In conclusion, an efficient protocol for plant regenerationand genetic transformation using hypocotyl explants ofT. erecta has been established, which can be used for thegenetic manipulation and successful introduction of the de-sired gene. It provides a powerful tool to study the function ofinsecticidal and nematicidal genes, unique to this medicinalplant, their role in the growth and development of plant andfor pathway engineering in T. erecta.

Acknowledgments The authors are thankful to the Director, CSIR-CIMAP, for encouragement and providing financial support.VG is thank-ful to Council of Scientific and Industrial research (CSIR) for seniorresearch fellowship.

M 1 2 3 4 5 6 7 8 9 10

800bp

500bp

aM 1 2 3 4 5 6

2kb1.4kb

b

700bp

c 23.1 Kb

9.4 Kb

6.6 Kb

4.3 Kb

2.3 Kb

2.0 Kb

0.8 Kb

1 2 3 4 5 6d

Fig. 5 Detection oftransformants in T. erecta. a PCRfor the detection of nptII gene(600 bp). M marker, 1–8 putativetransformants, 9 positive control,10- negative control. b PCR forthe detection of gus gene(2.0 kb).M marker, 1–4 putativetransformants, 5 positive control,6 negative control. c PCR for theamplification virC gene (730 bp).Mmarker, PC positive control, 1–5 putative transformants. dSouthern blot hybridization tocharacterize the insertion of gusAgene in transgenic. Lanes 3–6represent the transgenic linesT42-1, T42-2, T42-3 and T42-4.Transgenic Tagetes lines T42-2and T42-4 carry single copy, T42-1 carries two copies and T42-3carries three copies of the insert.Lane 1 represent the non-transformed line. Lane 2 repre-sents the positive control(plasmid)

V. Gupta, L. ur Rahman

References

Ali EF, Hassan FAS (2013) Impact of Foliar Application of CommercialAmino Acids Nutrition on the Growth and Flowering of Tageteserecta, L. Plant. Journal of Applied Sciences Research 9(1): 652–657

Alvarez JM, Ordás RJ (2013) Stable Agrobacterium-mediated transfor-mation of maritime pine based on kanamycin selection. World J2013:1–9

Asghari F, Hossieni B, Hassani A, Shirzad H (2012) Effects of explantssource and different hormonal combinations on direct regenerationof basil plants (Ocimum basilicum L.). Aust J Agric Eng 3(1):12–17

Balangcod TD, Balangcod AKD (2011) Ethnomedical knowledge of plantsand healthcare practices among the Kalanguya tribe in Tinoc, Ifugao,Luzon, Philippines. Indian J Tradit Knowl 10(2):227–238

Belarmino MM, Abe T, Sasahara T (1992) Callus induction and plantregeneration inAfricanmarigold (Tagetes erectaL.). Jpn J Breed 42:835–841

Bespalhok JC, Hattori K (1998) Friable embryogenic callus and somaticembryo formation from cotyledon explants of African marigold(Tagetes erecta L.). Plant Cell Rep 17:870–875

Boase MR, Bradley JM, Borst NK (1998) An improved method fortransformation of regal pelargonium (Pelargonium 9 domesticumDubonnet) by Agrobacterium tumefaciens. Plant Sci 139:59–69

Bradford MM (1976) A rapid and sensitive method for quantitation ofmicrogram quantities of protein utilizing the principle of proteindye-binding. Ann Biochem 72:248–254

Chakrabarty D, Mandal AKA, Datta SK (2000) Retrieval of newcolouredChrysanthemum through organogenesis from sectorial chi-mera. Curr Sci 78:1060–1061

Cheng M, Jarret RL, Li Z, Xing A, Demski JW (1996) Production offertile transgenic peanut (Arachis hypogaea L.) plants usingAgrobacterium tumefaciens. Plant Cell Rep 15:653–657

Chiruvella K, Arifullah M, Ghanta RG (2014) Phenotypic aberrationsduring micropropagation of Soymida febrifuga (Roxb.) Adr. Juss.Not Sci Biol 6(1):99–104

Cui M, Ezura H, Nishimura S, Kamada H, Handa T (2004) A rapidAgrobacteriummediated transformation of Antirrhinum majus L byusing direct shoot regeneration from hypocotyls explants. Plant Sci166(4):873–879

Das B,Mishra PC (2012) Antibacterial analysis of crude extracts from theleaves of Tagetes erecta. Int J Environ Sci 2(3):1605–1609

Debergh P, Aitken-Christie J, Cohen D, Grout B, Von Arnold S,Zimmerman R, Ziv M (1992) Reconsideration of the term vitrifica-tion as used in micropropagation. Plant Cell Tissue Organ Cult 30:135–140

Delgado–Vargas F, Jiménez AR, Paredes–López O (2000) Natural pigments:carotenoids, anthocyanins, and betalains—characteristics, biosynthesis,processing and stability. Crit Rev Food Sci Nutr 40:173–289

Dodo HW, Konan KN, Chen FC, Egnin M, Viquez OM (2008)Alleviating peanut allergy using genetic engineering: the silencingof the immunodominant allergen Ara h 2 leads to its significantreduction and a decrease in peanut allergenicity. Plant Biotechnol J6:135–145

Dubey VS, Bhalla R, Luthra R (2003) An overview of the non-mevalonate pathway for terpenoid biosynthesis in plants. J Biosci28(5):637–46

Giri A, Narasu ML (2000) Transgenic hairy roots: recent trends andapplications. Biotechnol Adv 18:1–22

Godoy-Hernandez G, Aviles Berzunza E, CCastro Concha L, deMiranda-Ham ML (2006) Agrobacterium-mediated transient trans-formation ofmarigold (Tagetes erecta). Plant Cell Tissue Organ Cult84:365–368

Godwin I, Todd G, Ford-Lloyd B, Newbury HJ (1991) The effects ofacetosyringone and pH on Agrobacterium-mediated transformationvary according to plant species. Plant Cell Rep 9:671–675

Gomez-Galera S, Pelacho AM, Gene A (2007) The genetic manipulationof medicinal and aromatic plants. Plant Cell Rep 26:1689–1715

Gutiérrez RMP, Luna HH, Garrido SH (2006) Antioxidant activity ofTagetes erecta essential oil. J Chil Chem Soc 51:883–886

Hamel PB,Mary UC (1975) Cherokee Plants and Their Uses–A 400 YearHistory. Sylva, N.C. Herald Publishing Co. pp 44

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker inhigher plants. EMBO J 6:3901–3907

Jin S, ZhangX, Liang S, Nie Y, Guo X, Huang C (2005) Factors affectingtransformation efficiency of embryogenic callus of upland cotton(Gossypium hirsutum) with Agrobacterium tumefaciens. Plant CellTissue Organ Cult 81:229–237

Khan MY, Aliabbas S, Kumar V, Rajkumar S (2009) Recent advances inmedicinal plant biotechnology. Indian J Biotechnol 8:9–22

Kosugi S, Ohashi Y, Nakajima K, Arai Y (1990) An improved assay forb-glucuronidase in transformed cells: methanol almost completelysuppresses a putative endogenous b-glucuronidase activity. Plant Sci70:133–140

Kulkarni A, Thengane SR, Krishnamurthi KV (2000) Direct shoot re-generation from node, internode, hypocotyl and embryo explants ofWithania somnifera. Plant Cell Tissue Organ Cult 62:203–209

Kumar S, Mangal M, Dhawan AK, Singh N (2013) Callus induction andplant regeneration from leaf explants of jojoba [Simmondsiachinensis (Link) Schneider]. Indian J Biotechnol 12:544–547

Laboney UZ, Biswas GC, Shoeb MA, Miah A (2013) Callus inductionand regeneration of potato from shoot tip culture. Int J Agric Sci3(10):40–45

Lichtenstein C, Draper J (1986) Genetic engineering of plants. In: GloverDM (ed) DNA cloning: a practical approach. IRL Press, Oxford/Washington, pp 11–67

Marotti I, Marotti M, Piccaglia R, Nastri A, Grandi S, Dinelli G (2010)Thiophene occurrence in different Tagetes species: agricultural bio-masses as sources of biocidal substances. J Sci FoodAgric 90:1210–1217

McKently AH, Moore GA, Doostdar H, Niedz RP (1995)Agrobacterium-mediated transformation of peanut (Arachishypogea L.) embryo axes and the development of transgenic plants.Plant Cell Rep 14:699–703

Mishra S, Sangwan RS, Bansal S, Sangwan NS (2013) Efficient genetictransformation of Withania coagulans (Stocks) Dunal mediated byAgrobacterium tumefaciens from leaf explants of in vitro multipleshoot culture. Protoplasma 250:451–458

Misra P, Datta SK (2001) Direct differentiation of shoot buds in leafsegments of white marigold (Tagetes erecta L). Vitro Cell Dev Biol(Plant) 37(4):466–470

Mukherjee S, Ghosh B, Jha S (2000) Establishment of forskolin yieldingtransformed cell suspension cultures of Coleus forskohlii as con-trolled by different factors. J Biotechnol 76:73–81

Mukundan U, Hjortso MA (1990a) Thiophene accumulation by hairyroot cultures of Tagetes patula in response for fungal elicitors.Biotechnol Lett 12(8):609–614

Mukundan U, Hjortso MA (1990b) Thiophene content in normal andtransformed root cultures of Tagetes erecta: a comparison withthiophene content in roots of intact plants. J Exp Bot 41(232):1497–1501

Murashige T, Skoog F (1962) A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiol Plant 15:473–497

Naqvi S, Farre G, Sanahuja G, Capell T, Zhu C, Christou P (2010) Whenmore is better: multigene engineering in plants. Trends Plant Sci15(1):48–56

Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expres-sion in tobacco of a b-glucuronidase (GUS) reporter gene containingan intronwithin the coding sequence. Plant Cell Physiol 31:805–813

Oliveira Y, Adamuchio L, Degenhardt-Goldbach J, Gerhardt I, BespalhokJ, Dibax R, Quoirin M (2011) Use of kanamycin for selection of

An efficient plant regeneration and Agrobacterium-mediated genetic

Eucalyptus saligna genetically transformed plants. BMC Proc 5(7):147

Owens LD, Smigocki AC (1988) Transformation of soybean cells usingmixed strains of Agrobacterium tumefaciens and phenolic com-pounds. Plant Physiol 88:570–573

Rashid A, Singh S (2010) Effect of hormones on direct shoot regenerationin hypocotyl explants of tomato. Not Sci Biol 2(1):70–73

Rommens CM (2006) Kanamycin resistance in plants: an unexpectedtrait controlled by a potentially multifaceted gene. Trends Plant Sci11:317–319

Sekioka TA, Tanaka JS (1981) Differentiation in callus culture of cucum-ber (Cucumis sativus L). Hortic Sci 16:451 (Abstr. 386)

Sharma KK, Anjaiah V (2000) An efficient method for the production oftransgenic plants of peanut (Arachis hypogea L.) throughAgrobacterium tumefaciens-mediated genetic transformation. PlantSci 159:7–19

Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes highefficiency transformation of Arabidopsis thaliana explants byAgrobacterium tumefaciens. Plant Mol Biol 8:291–298

Spencer A, Hamill JD, Rhodes MJ (1990) Production of terpenes bydifferentiated shoot cultures of Mentha citrata transformed withAgrobacterium tumefaciens T37. Plant Cell Rep 8:601–604

Subramanyam K, Rajesh M, Jaganath B, Amrithalingam V, Jeevaraj T,Elayaraja D, Sirabalan K, Manickavasangam M, Ganapathi A(2013) Assessment of factors influencing the Agrobacterium medi-ated in planta seed transformation of brinjal (Solanum melongenaL). Appl Biochem Biotechnol 171:450–468

Sunandakumari C, Zhang CL, Martin KP, Slater A, Madhusoodanan PV(2005) Effect of auxins on indirect in vitro morphogenesis and

expression of gusA transgene in a lectinaceous medicinal plant.Euphorbia nivulia Buch-Ham. In Vitro Cell Dev Biol (Plant) 41:695–699

Tripathi L, Tripathi JN (2003) Role of biotechnology in medicinal plants.Trop J Pharm Res 2(2):243–253

Turk SCHJ, van LangeRP, Regensburg-Tuϊnk TJG, Hooykaas PJJ (1994)Localization of the VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium tumefaciens. PlantMol Biol 25:899–907

Vasudevan P, Kashyap S, Sharma S (1997) Tagetes: A multipurposeplant. Bioresour Technol 62:29–35

Wang M, Huang LQ, Li MM (2008) Progress in research and applicationof gene engineering on medicinal plants. Zhongguo Zhong Yao ZaZhi 33:1365–1371, Review Chinese

Wei XP, Gou XP, Yuan T, Russell SD (2006) A highly efficientin vitro plant regeneration system and Agrobacterium-medi-ated transformation in Plumbago zeylanica. Plant Cell Rep25(6):513–521

Yuan D, Bassaie L, Sabalza M, Miralpeix B, Doshevskaya S, Farre G,Rivera SM, Banakar R, Bai C, Sanahuja G, Arjo G, Avilla E,Zorrilla-Lopez U, Ugidos-Damboriena N, Lopez A, AemacellasD, Zhu C, Capell T, Hahne G, Turyman RM, Christou P (2011)The potent ia l impact of plant biotechnology on theMilleniumDevelopment goals. Plant Cell Reports 30(3):249–265

Ziv M (1991) Vitrification: morphological and physiological disorders ofin vitro plants. In: Debergh PC, Zimmerman RH (eds)Micropropagation technology and application. Kluwer AcademicPublishers, Dordrecht, pp 45–69

V. Gupta, L. ur Rahman