BIOTECHNOLOGY

Stable germ line transformation of a leafy vegetablecrop amaranth (Amaranthus tricolor L.) mediatedby Agrobacterium tumefaciens

Ajantaa Pal & Swasti S. Swain & Anath B. Das &

Arup K. Mukherjee & Pradeep K. Chand

Received: 1 August 2012 /Accepted: 10 January 2013 / Editor: J. Forster# The Society for In Vitro Biology 2013

Abstract We have optimized a procedure for genetic trans-formation of a major leafy vegetable crop, Amaranthustricolor L., using epicotyl explant co-cultivation withAgrobacterium tumefaciens. Two disarmed A. tumefaciensstrains EHA 105 and LBA 4404, both carrying the binaryplasmid p35SGUSINT harboring the neomycin phospho-transferase II gene (nptII) and the β-glucuronidase gene(gus), were evaluated as vector systems. The former dis-played a higher transforming efficiency. Several key factorsinfluencing the transformation events were optimized. Thehighest percentage of transformed shoots (24.24%) wasachieved using hand-pricked epicotyl explants, a 10-mininfection period, with 100 μM acetosyringone-pretreatedAgrobacterium culture corresponding to OD600≅0.6 anddiluted to 109 cells ml−1, followed by 4 d co-cultivation inthe regeneration medium. Putative transformed explantscapable of forming shoots were selected on medium supple-mented with 75 μgml−1 kanamycin, and transient as well asstable glucuronidase expression was determined by histo-chemical analysis. From a total of 48 selected shoot lines

derived from independent transformation events with epi-cotyl explants co-cultivated with EHA 105, 32 showedpositive PCR amplification for both the nptII and gus genes.Germ line transformation and transgene stability were evi-dent in progeny of primary transformed plants (T0). AmongT1 seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in aratio typical of the Mendelian monohybrid pattern (3:1) asverified by the chi-square (χ2) test. Southern hybridization ofgenomic DNA from kanamycin-resistant T1 transgenic segre-gants to an nptII probe substantiated stable integration of thetransgene. Neomycin phosphotransferase (NPTII) activitywas detected in leaf protein extracts of selected T1 transgenicplants, thereby confirming stable expression of the nptII gene.

Keywords Amaranthus tricolor . Agrobacteriumtumefaciens . gus-intron . NPTII assay . Transgene stability

Introduction

The amaranths (Amaranthus spp.) comprise an economical-ly important crop group valued for their nutritional andhorticultural significance. While “grain amaranths” are amajor source of protein that complements cereal diets,“green amaranths” are savored as a major leafy vegetablein tropical Asian countries. Some species are also valued fortheir ornamental, forage, or pharmaceutical properties (TheWealth of India 1948). Leafy amaranths, native to SouthEast Asia, with axillary determinate inflorescences, consti-tute one of the most widely consumed tropical vegetablecrops. A majority of these Amaranthus species, especiallyAmaranthus tricolor (Chinese spinach), are cultivated asgreen vegetables similar to spinach, broccoli, and cabbage.The plant species make a wholesome diet, especially forpoor, rural communities. The mucilaginous leaves are richin vitamins and minerals, and they also contain other pro-

A. Pal : S. S. Swain : P. K. Chand (*)Plant Cell & Tissue Culture Facility, Post-Graduate Department ofBotany, Utkal University, Bhubaneswar 751 004, Orissa, Indiae-mail: pkchanduubot@yahoo.co.in

A. Pal :A. B. Das :A. K. MukherjeeDivision of Plant Biotechnology, Regional Plant Resource Centre,Bhubaneswar 751 015, Orissa, India

Present Address:A. B. DasDepartment of Agricultural Biotechnology, Orissa University ofAgriculture and Technology, Bhubaneswar 751 003, Orissa, India

Present Address:A. K. MukherjeeDivision of Crop Protection, Central Institute for Cotton Research,Shankarnagar,Nagpur 440010, India

In Vitro Cell.Dev.Biol.—PlantDOI 10.1007/s11627-013-9489-9

health compounds such as the antioxidants squalene andcarotenoids (Kumari and Prakash 2005). In addition, A.tricolor is endowed with C4-type photosynthetic efficiencyand high yields coupled with minimal cultivation constraintsand moderate tolerance to drought and heat make it a pop-ular choice for cultivation.

Unfortunately, a majority of amaranths are susceptible tofungal pathogens such as Fusarium, Aspergillus, andPenicillium species (Bresler et al. 1991). Several Amaranthusspecies, including A. tricolor, are also known to be challengedby foliar insects such as leaf miners, leaf rollers, cutworms,aphids, flea beetles, and mites. Blister beetles and alfalfawebworm are the two leaf feeders that are known to causesubstantial loss (Metcalf and Metcalf 1993). Besides, amar-anths are also susceptible to Amaranthus leaf mottle virus andAmaranthus mosaic virus (Taiwo and Owolabi 2004).

In view of the lack of documentary evidence relating tosuccess in the genetic improvement of amaranths, eitherthrough conventional or modern biotechnological methods, itis imperative to strengthen investigations aimed at geneticengineering of this under-exploited crop. Success inAgrobacterium tumefaciens-mediated transgenic manipulationof amaranths is limited only to the grain-type amaranths, suchas Amaranthus hypochondriacus, for the expression of a light-harvesting chlorophyll a/b-binding protein gene promoter(Jofre-Garfias et al. 1997). To date, there are only two reportson heterologous gene transfer to more important vegetableamaranths, i.e., those which demonstrated integration of roland opine synthase genes (native to Ri plasmid borne T-DNAof Agrobacterium rhizogenes) in plants regenerated from hairyroot cultures of A. tricolor (Swain et al. 2010) and Amaranthusspinosus (Pal et al. 2013). The present work, therefore, aimedto explore the potential of Agrobacterium-mediated genetictransformation of A. tricolor introducing Ti plasmid-basedconstructs harboring transgenes that confer resistance to bioticstresses due to pests, fungal pathogens, or viruses. A necessaryprerequisite was to develop a transformation protocol and todemonstrate stable inheritance in the expression of a provenselectable marker and a reporter transgene coding for amino-glycoside tolerance and β-glucuronidase (GUS), respectively.

Materials and Methods

Plant material. Seeds of A. tricolor L. (NBPGR accessionno. IC-447684) were obtained from the National Bureau ofPlant Genetic Resources, Government of India, New Delhi.Seeds were treated in a 50-ml beaker with a net cover andplaced under running tap water for 30 min followed by8 min in an aqueous solution of 7% (v/v) sodium hypochlo-rite (Merck Specialities, Mumbai, India) plus 5% (v/v)Teepol (liquid detergent; Reckitt's Colman, Kolkata,India). Then, the seeds were rinsed with autoclaved tap

water (five to six times). After this, they were surface dis-infected with 0.1% (w/v) aqueous solution of mercuric chlo-ride (Hi-Media, Mumbai) for 5 min followed by fiverinses in autoclaved double-distilled water. The surface-disinfected seeds were inoculated in 300 ml screw-cappedglass jars (Excel Corporation, Alleppey, India) or 150 mlErlenmeyer flasks (Borosil, Mumbai) containing half-strength MS (Murashige and Skoog 1962) salts and vitaminswith 30 gl−1 sucrose (1/2 MS). The seeds were allowed togerminate at 25±1°C with 35 μmolm−2s−1 photon fluxdensity (PFD) provided by cool white fluorescent tubes(Philips, Mumbai) and 60% relative humidity. For raising invitro shoot cultures, green epicotyl or hypocotyl segments(ca. 1.0–2.0 cm lengths), excised from 7-d-old seedlings,were inoculated in a shoot regeneration medium comprisingMS salts and 30 gl−1 sucrose augmented with a range ofconcentrations of N6-benzyladenine (BA; 4.4–13.2 μM)plus naphthaleneacetic acid (NAA; 0.54–2.7 μM). Allshoot-forming cultures were maintained at 25±1°C undera 16-h photoperiod with light provided by cool white fluo-rescent tubes at a PFD of 50 μmolm−2s−1.

Agrobacterium strains and plasmid vectors. Two disarmedA. tumefaciens strains LBA 4404 and EHA 105, each har-boring the plasmid construct p35SGUSINT (ca. 14.5 kb;Vancanneyt et al. 1990), were employed as vector systemsfor transformation. The binary vector p35SGUSINT wasderived from pBIN19 (Bevan 1984) containing a selectablemarker neomycin phosphotransferase gene (nptII) undernopaline synthase (nos) promoter and GUS-intron gene(gus-int) driven by CaMV35S promoter and the left border(LB) sequence of T-DNA from pTi 37 (Fig. 1). TheAgrobacterium strains were maintained in the dark at 28°Cin agar (1.5%, w/v)—solidified yeast mannitol broth (YMB)augmented with 50 μgml−1 kanamycin monosulfate (Sigma,St. Louis, MO).

Determination of phytotoxic levels of selective and bacteri-cidal antibiotics. Epicotyl or hypocotyl explants and in vitroregenerated shoots cultured in MS medium were tested forcytotoxicity using an increasing range of kanamycin mono-sulfate concentrations (25, 50, 75, 100, 125, and 150 μgml−1). The relative phytotoxicity of kanamycin concentra-tions was also tested to ascertain their effect on germinationof surface-disinfected seeds. The experiments were repeatedthree times, and subculturing was carried out in the samemedium every 15 d. Observations were recorded between 4and 8 wk, noting any morphological changes such asbleaching (partial/full) or necrosis. In a separate experiment,the bactericidal antibiotic cefotaxime (Sigma) was assessedat different levels (250, 500, 750, and 1,000 μgml−1) inshoot regeneration medium. The antibiotic was filter steril-ized (0.22 μm membrane filters; Sartorius, Germany) and

PAL ET AL.

added to the autoclaved medium after the latter had beencooled to 40°C prior to solidification.

Transformation and selection of putative transformedplants. Epicotyl or hypocotyl segments (ca. 1.0–2.0 cm)from axenic seedlings of A. tricolor were pre-cultured inshoot regeneration medium (MS+13.2 μM BA+1.08 μMNAA) for 1–6 d. A single bacterial cell colony was used toinoculate 20 ml liquid YMB medium containing 50 μgml−1

kanamycin and grown in dark (16–18 h, 28°C, 120 rpm).Bacterial cells corresponding to OD600≅0.6 were pelletedby centrifugation (6,000 rpm, 10 min) followed by twowashes with liquid YMB. The final cell density was adjust-ed to 109 cells ml−1 with liquid YMB. Pre-cultured explants,followed by wounding, were immersed in the bacterialsuspension for 10 min, blotted dry on autoclaved filterpaper/tissue towel, and co-cultivated on 0.8% (w/v) agar-gelled shoot regeneration medium without selection.

Two different methods were adopted for co-cultivatingexplants following infection. In the first method, infected andblotted explants were directly placed on the co-cultivationmedium prepared in 10 cm diameter TPX Petri dishes. In thesecond method (filter paper sandwich), blotted explants fol-lowing infection were placed on sterile Whatman #1 filterpaper discs and were overlaid on the co-cultivation mediumcontained in similar Petri dishes. The explants were coveredwith another sterile filter paper of the same dimension. Petridishes containing cultures from either co-cultivation methodswere placed under diffused light (10 μmol m−2s−1 PFD).

After co-cultivation for different durations, explants werewashed twice with autoclaved distilled water and blot dried.The explants were then transferred to 0.8% agar-solidifiedshoot regeneration medium (MS+13.2 μM BA+1.08 μMNAA) supplemented with the bactericidal antibiotic (500 μgml−1 cefotaxime). After a 2-wk culture period, explants weresubcultured on fresh regeneration medium with cefotaximeas well as the phytotoxic antibiotic kanamycin (75 μgml−1).Kanamycin-resistant shoots were subcultured at 15-d inter-vals. For elongation, shoots were transferred to MS medi-um+2.2 μM BA+75 μgml−1 kanamycin for 10–12 d andthereafter maintained as shoot cultures through periodicsubculture at 15–20 d intervals in MS medium lacking

BA, but stressed with kanamycin at a reduced concentration(50 μgml−1). Kanamycin-resistant shoots obtained fromeach single epicotyl explant derived from independent trans-formation experiments with EHA105 were indexed as puta-tive transgenic lines and maintained in culture; a single plantfrom each of which provided the tissue source for molecularanalysis. Plant control 1 (in vitro seed-grown shoot cultureswithout Agrobacterium treatment and without kanamycinstress) and plant control 2 (in vitro-grown shoot cultureswithout Agrobacterium treatment, but subjected to kanamy-cin stress) were established for comparison.

Evaluation of factors influencing transformation. A rangeof plant × Agrobacterium parameters was evaluated, and eachexperiment included four replicates using ten explants. Theexperiments were repeated at least four times each. Theparameters included the age of explants (harvested from 4-,7-, 10-, and 13-d-old seedlings), duration of explant pre-culture (0, 1, 2, 3, 4, 5, or 6 d) in shoot regeneration mediumprior to infection, bacterial growth phase (OD600 0.3, 0.4, 0.6,0.8, or 1.0), bacterial cell density (0.5×109, 1.0×109, 1.5×109, or 2.0×109 cells ml−1), acetosyringone concentrations(50, 100, 150, or 200 μM) for bacterial culture pretreatment,methods of wounding of explants (abrasion with sterile sand/-glass wool or pricking with hypodermic needle), infectiontime (5, 10, 15, or 20 min), and the co-cultivation period (0,1, 2, 3, 4, 5, or 6 d). Acetosyringone (Sigma; 100 mM stocksolution in DMSO) was added to the culture medium forgrowing overnight suspension of Agrobacterium prior to in-fection. All parameters were evaluated and optimized on thebasis of demonstration of PCR positive amplification for bothtransgenes.

Data were analyzed using ANOVA for a completelyrandomized design. Duncan's new multiple range test(Gomez and Gomez 1984) was used to separate the meansfor significant differences.

Histochemical assay for β-glucuronidase activity. The his-tochemical assay for GUS activity in the presence of itsspecific substrate, X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide; Biosynth AG, Thal, Switzerland) was carriedout as per Jefferson et al. (1987) with modifications. Briefly,

Figure 1. Partial restrictionmap of the p35SGUSINT T-DNA cassette. LB/RB left/rightborder sequences; NOS-P/NOS-pA nopaline synthase promoter/terminator; CaMV 35S-P/CaMV-pA cauliflower mosaicvirus (CaMV) 35S promoter/terminator. The position of thenptII probe (1.6 kb PstIfragment) is indicated.

STABLE GERM LINE TRANSFORMATION OF A LEAFY VEGETABLE AMARANTH

after 4 wk on selection medium, in vitro shoots (regeneratedfrom approximately 10% of the surviving explants) weretested for GUS activity. Leafy shoots were taken and incubat-ed in GUS histochemical buffer (50 mM sodium phosphate,pH7.0; 50 mM EDTA, pH8.0; 0.5 mM K3Fe(CN)6; 0.5 mMK4Fe(CN)6; 0.1% Triton X-100; 1 mMX-gluc) at 37°C in thedark for up to 24 h, thereafter rinsed twice in 70% ethanol at1 h intervals to ensure background destaining.

T0 plants, 2 wk following transfer to the glasshouse, werealso tested for retention of GUS activity. Leaf discs (ca.0.5 cm diameter) were punched from randomly chosenleaves from individual plants and subjected to histochemicalanalysis in 96-well microtiter plates (Hi-Media) each with100 μl of assay buffer and finally mounted on microscopeslides for observation.

To examine stable expression of the gus gene in wholeseedlings, 1–2-d-old T1 seedlings were treated in GUS buff-er in a similar manner. The possibility of endogenous GUSexpression was tested by subjecting uninfected shoots (con-trol 1) to the histochemical GUS assay.

Polymerase chain reaction. Genomic DNA was isolatedfrom leaf tissues of a single plant from each of the putativetransgenic lines following a standard procedure. DNA fromleaf tissue samples of non-transformed seed-grown plants inaxenic cultures (control 1), which had not been subjected toAgrobacterium treatment, served as a negative control. Theplasmid DNA construct (p35SGUSINT) served as a positivecontrol (50 pg/reaction). The forward and reverse primer(oligonucleotide) sequences (Bangalore Genei, Bangalore,India) used for PCR amplification of nptII gene were 5′-CCA TCG GCT GCT CTG ATG CCG CCG T-3′ (forward)and 5′-AAG CGA TAG AAG GCG ATG CGC TGC-3′(reverse) and gus-int 5′-GGT GGG AAA GCG CGT TACAAG-3′ (forward) and 5′-TGG ATC CCG GCA TAG TTAAA-3′ (reverse). The expected sizes of the amplification prod-ucts for nptII and gus genes were 693 and 650 bp, respective-ly. The optimum PCR mixture (25 μl) contained 1.5 U TaqDNA polymerase (Bangalore Genei), 1× Taq DNA polymer-ase buffer (20 mM Tris–HCl [pH9.0], 50 mM KCl, 1.5 mMMgCl2), 200 μM of each dNTP (Bangalore Genei), 1 μl ofeach forward and reverse primer (10 pM), and 50 ng templateDNA. PCR was carried out using a DNA Thermal Cycler(Applied Biosystems 9700, Foster City, CA) programmed asfollows: initial template denaturation at 94°C for 5 min,annealing at 58°C for 1 min, and extension at 72°C for1.5 min for the first cycle followed by 33 cycles each withdenaturation (94°C, 1 min), annealing (58°C, 1 min), andextension (72°C, 1.5 min) The final cycle involved an addi-tional step of extension at 72°C for 5 min prior to holding at 4°C. The amplification products were separated by 1% agarosegel electrophoresis in 1× TAE buffer (0.04 M Tris–acetate,1 mM EDTA, pH8.0) at 80 V for 45 min. The gel was stained

with ethidium bromide solution and photographed using theGel Documentation system (Bio-Rad, Hercules, CA).

Determination of transformation efficiency. The transfor-mation efficiency (TE%) was calculated as the percentageof PCR positive events, i.e., plants showing separate ampli-fication of both nptII and gus genes recovered from the totalnumber of explants infected with Agrobacterium.

Root induction, acclimatization, and field transfer. For ini-tiation of roots, putatively transformed shoots (kanamycinresistant, GUS positive, PCR tested) were placed in half-strength MS medium (1/2 MS) augmented with indole-3-acetic acid (IAA; 1.4–11.4 μM), indole-3-butyric acid (IBA;1.2–9.8 μM), or indole-3-propionic acid (IPA; 1.3–10.6 μM).All media were supplemented with 50 μgml−1 kanamycinsulfate and cultures were maintained at 25±1°C under 16 hphotoperiod with PFD of 35 μmolm−2s−1. Well-rooted trans-formed plantlets were removed from the culture medium androots were washed gently under running tap water to removethe adhering agar. The plantlets were then transferred to plasticpots (30×7.5 cm) containing autoclaved vermicompost(Ranjan Agrotech, Bhubaneswar, India) that had been moist-ened with autoclaved water. The potted plants, covered withpolyethylene bags, were maintained inside a plant growthchamber set at 25±1°C, 85–90% relative humidity, and16/8 h (light/dark) photoperiod using cool white fluorescentlights at an intensity of 50 μmol photons m−2 s−1. After 3 wk,primary transformed plants (T0) were transferred to largeearthenware pots (50×18 cm) containing garden soil (soil/-compost, 1:1) and kept in a glasshouse in agronet confinementand allowed to self-fertilize and set seed. Transgene segrega-tion patterns in T1 progeny plants were ascertained by germi-nating surface-disinfected seeds on 1/2MS0 supplementedwith 75 μgml−1 kanamycin sulfate in TPX Petri dishes(Tarsons, Kolkata, India; 20 seeds per 20 ml medium). At3 wk following germination, the kanamycin responsivenessof T1 seedlings was scored as resistant (with true green leavesor leaves which were mostly green or occasionally had limitedbleached areas) or sensitive (completely bleached andnecrotic).

Southern hybridization. Genomic DNA from selected sin-gle kanamycin-resistant T1 transgenic lines, as well as anon-transformed in vitro seed-grown plant (control 1), weresubjected to Southern blot analysis, which was carried outaccording to standard procedures. Specifically, 10 μg of leafgenomic DNA samples digested with PstI/HindIII was sep-arated on a 0.8% agarose gel (25 V, overnight) and thentransferred to a nylon membrane (HYbond N+; Amersham,Piscataway, NJ). Hybridization was performed with a DNAprobe, radiolabelled with α[32P]-dCTP (specific activity11.1×1013Bq/mmol; BARC, Mumbai, India) using the

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random primer labeling kit (Amersham). The probe was a1.6-kb PstI fragment from p35SGUSINT, containing the nptIIgene (Fig. 1). The blots were washed at 65°C in the followingsuccession: 2× saline-sodium citrate (SSC)+0.5% sodiumdodecyl sulfate (SDS) (5 min), 2× SSC+0.1% SDS (15 min),1× SSC+0.1% SDS (30 min), and 0.1× SSC+0.1% SDS(30 min). They were then autoradiographed with an intensify-ing screen at −85°C for 5 d.

NPTII enzyme assay. An assay method, modified from Reisset al. (1984), was adopted to demonstrate NPT II (APH [3′]type II) activity in leaf tissues of selected kanamycin-resistantT1 plants that had been confirmed positive by Southern blotanalysis. Leaf protein was extracted from 10 g tissue of trans-formed plants and non-transformed negative control (control1). Plant proteins were resolved in 1.5 mm non-denaturingpolyacrylamide gel (9%) by electrophoresis at 240 V for 5 h.An aminoglycoside phosphotransferase standard was provid-ed by a sonicated extract of Escherichia coli JA221 carryingnptII coding sequence from pKm2 (Beck et al. 1982). Thepolyacrylamide gel was equilibrated for 30 min in 100 ml ofreaction buffer (67 mM Tris maleate [pH7.1], 42 mMMgCl2,and 400 mM NH4Cl) and placed beneath a 1% Sea Plaqueagarose gel, made using the same buffer, and containing1.0 mgl−1 kanamycin sulfate (Sigma, Gillingham, UK) and250 μCi γ-[32P]-dATP (5,000 Cimmol−1, Amersham,Buckinghamshire, UK). The reaction proceeded for 30 minat room temperature. One sheet of P81 paper followed by twosheets of 3MM (both from Whatman) were placed on top ofthe gel sandwich. A 5-cm-thick layer of paper towels wassuperimposed and held in place by a glass plate and 500 g leadweight on top. Proteins were capillary blotted onto the P81paper for 3 h. The P81 paper was then kept in the incubationsolution (1% SDS and 1.0 mgl−1 proteinase K) for 30 min at60–80°C to stop the reaction. The P81 paper was washedthrice in 10 mM Na phosphate buffer (NaH2PO4+Na2HPO4,pH7.5) at 80°C, followed by six washes in distilled water atroom temperature. Thereafter, the paper was dried in a vacuumoven and then autoradiographed by exposing to KodakX-OmatS film with an intensifying screen in dark for 2–20 h.

Results

Evaluation of natural antibiotic sensitivity of explants andin vitro shoots. Multiple shoots (averaging 7.8 per epicotylexplant or 5.1 per hypocotyl explant) with a mean length of4.8 cm were regenerated via adventitious organogenesis in78% of epicotyl and 67% hypocotyl explants from 7-d-old invitro seedlings grown on MS medium augmented with13.2 μM BA+1.08 μM NAA, but without kanamycin(Fig. 2A). The effect of increasing concentrations of theselective antibiotic kanamycin on seedling explants (epicotyl

or hypocotyl) and in vitro regenerated shoots was assessedseparately (Fig. 2B). Kanamycin was apparently ineffectiveat the minimum concentration tested (15 μgml−1). About85–88% of the explants and in vitro shoots were bleachedafter 30 d in culture medium containing 50 μgml−1 kanamy-cin. The phytotoxic effect was severe in 75 μgml−1 kanamy-cin as initiation of bleaching occurred for explants/shoots10–15 d after inoculation, leading to a total loss of chloro-phyll pigmentation by 30 d. There was an absolute arrest ofshoot bud initiation and all explants became necrotic at thisconcentration. Hence, subsequent selection of putative trans-formed explants/shoots used shoot regeneration mediumsupplemented with 75 μgml−1 kanamycin (Fig. 2C).

The bactericidal antibiotic cefotaxime at a range of con-centrations (250, 500, 750, and 1,000 μg ml−1), which wasused to reduce background Agrobacterium growth, had adetectably varied effect on shoot organogenesis. In the pres-ent study, cefotaxime at 500 μgml−1 was found to be mosteffective for bacterial elimination without adversely affect-ing explant morphogenesis. Higher concentrations werephytotoxic as most of the young shoots turned necroticand died.

Kanamycin tolerance of putative transformed shoots. Epicotylas well as hypocotyl explants, following infection and co-cultivation with Agrobacterium, regenerated two to threeshoots per explant in the selection medium supplementedwith 75μgml−1 kanamycin. In this medium, 36.3% of epi-cotyl segments and 30.0% of hypocotyl segments producedshoots (Table 1). Shoot regeneration was drastically reduced(ca. one shoot/explant) or completely inhibited in nutrientmedia containing 100 or 125 μgml−1 kanamycin, respec-tively. Of the two bacterial strains employed, EHA105 wassuperior to LBA 4404 in generating a larger number ofantibiotic-tolerant shoots (Table 1). Shoots from all controlexperiments, regardless of explant type, which were pro-duced without involving an Agrobacterium infection (con-trol 2) were absolutely bleached in the selection mediumfortified with 75 μgml−1 kanamycin.

Transient GUS expression of putative transformed shoots. Theenzymatic activity of GUS was substantiated by the positivehistochemical assay as evidenced by the observation ofcharacteristic blue coloration, which developed in the leavesand petioles of selected shoots subjected to the specificsubstrate X-gluc (Fig. 2D). Shoots obtained from ca. 88–92% of epicotyl explants, which survived the selection me-dium (75 μgml−1 kanamycin), were GUS expressive. Someof the shoots, which were selected with a lower concentra-tion of kanamycin (50 μgml−1), also exhibited GUS activity,but the activity was displayed by a limited number of shootsper explant. Leaf tissues from in vitro shoots withoutAgrobacterium infection did not show GUS activity.

STABLE GERM LINE TRANSFORMATION OF A LEAFY VEGETABLE AMARANTH

Detection of transgenes by PCR. DNA isolated from severalindependent transgenic lines capable of showing kanamycinresistance coupled with GUS expression revealed theexpected amplicons of 693 and 650 bp with nptII- and gusgene-specific primers, respectively (Fig. 3A, B). This indi-cated the presence of both marker transgenes nptII and gusin the transformed plant genomes. No amplification productwas detected in DNA from non-transformed plants whensubjected to PCR amplification with either of the two pri-mers. Of a total of 48 putative transformed shoot linesoriginating from independent transformation experimentswith epicotyl explants treated with EHA105 strain, sub-jected to PCR analysis for gus and nptII genes, 32 plants

showed positive amplification for both the genes. Theseresults indicate the stringency of kanamycin selection,which had enabled an effective restriction of false-positive(“escape”) selection.

Optimization of transformation parameters. Of the explantsharvested from seedlings of different ages, 7-d-old seedlingexplants were most amenable to transformation. Epicotylsegments were more responsive than hypocotyls and pro-duced a higher number of putative transformed shoots(Table 1) in all cases (irrespective of remaining plant ×bacterial factors). Epicotyls pre-cultured for 4 d had thehighest transformation efficiency (TE=24.24% with EHA

Figure 2. Transformation andplant regeneration of A. tricolor.A, An epicotyl segmentregenerating multiple shoots inMS medium+13.2 μM BA+1.08 μM NAA for 30 d(positive control). B, Antibioticselection of putativetransformed epicotyl segments(arrow) in regeneration medium(A) stressed with 75 μgml−1

kanamycin. C, Shootregeneration in a transformedepicotyl segment in theselection medium (B) 30 d afterinfection. D, Histochemicalassay showing GUS activity in4-wk-old in vitro regeneratedkanamycin-resistant leafyshoot. E, A 3-wk-old T1

transgenic plant (3A2)acclimated in vermicompostinside glasshouse. F, A matureT1 transgenic plant (3A2)established in garden soil insideglasshouse.

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105 and 15.15% with LBA 4404). Shorter durations of pre-culture (1–3 d) elicited a lower shoot organogenic responsewhile longer durations (5–6 d) resulted in more non-transformed shoots.

Bacterial strains and growth phase. Of the two differenttypes of disarmed A. tumefaciens strains, the transformationefficiency of EHA 105 was markedly higher than that ofLBA 4404 (Table 1) and produced a higher number ofshoots displaying kanamycin resistance and GUS expres-sion. To ascertain the optimum stage of Agrobacteriumgrowth for high efficiency transformation, four differentgrowth phases were studied. Maximum transformation wasachieved at late-log phase, corresponding to OD600≅0.6(Fig. 4A). In all experiments, the transformation efficiencyof EHA 105 remained higher than LBA 4404. Bacteria fromcultures experiencing either a higher or lower absorbancewere not suitable for transformation, while extensive tissuedamage occurred at OD600 values >1.0 because of bacterialovergrowth.

Bacterial cell density and infection time. A range of bacte-rial cell densities (0.5–2.0×109 cells ml−1) were evaluatedfor explant infection. Optimum results were obtained with adensity of 109 cells ml−1 following a 50% (1:1) dilution ofthe original culture at OD600≅0.6. Of a range of increasingdurations (5–20 min) tested for explant infection with dilut-ed Agrobacterium culture, maximum transformation effi-ciency was recorded for a 10-min treatment with abacterial cell density of 109 cells ml−1 (Table 2). The opti-mum concentration of the bacterial cell density and infectiontime held good for both explant types: cotyledons as well ashypocotyls.

Acetosyringone treatment. In the absence of acetosyringonetreatment, kanamycin-tolerant shoots developed, but only ata low frequency (2–3%) regardless of strain or explant type.In contrast, explants infected with bacterial suspension sup-plemented with acetosyringone showed a higher transfor-mation efficiency irrespective of the Agrobacterium strainemployed. Of the range of concentrations of acetosyringoneevaluated (50–200 μM), 100 μM resulted in highest trans-formation efficiency (Fig. 4B).

Table 1 Transformation efficiency of seedling explants of A. tricolor

A. tumefaciens strain % explants forming shoots on selectionmedium

% kanamycin-resistant, GUS-expressiveexplants

TE%z

Epicotyl Hypocotyl Epicotyl Hypocotyl Epicotyl Hypocotyl

EHA 105 36.25 a 30.00 a 33.6 a 27.25 a 24.24 a 17.42 a

LBA 4404 28.75 b 18.25 b 25.25 b 16.50 b 15.15 b 6.06 b

Data were pooled from four separate experiments each with 20 flasks containing three to four explants per flask. Mean values within column with adifferent letter are significantly different at p<0.05 (Duncan's new multiple range test)

TE% transformation efficiencyz Calculated as percentage of PCR positive events (plants showing amplification for nptII and gus genes) recovered from the total number ofexplants infected

Figure 3. (A) PCR amplicon (693 bp) specifying nptII transgene inthe leaf genomic DNA of putative transgenic lines (T0). (B) PCRamplicon (650 bp) specifying gus-int transgene in the leaf genomicDNA of putative transgenic lines (T0). M1, DNA marker (1 kb ladder);M2, DNA marker (100 bp ladder). Lanes 1–6, DNA from putativetransgenic lines (3A2, 11B5, 5D4, 16E3, 6F4, and 2H7, respectively).C−, DNA from non-transformed plant (negative control), C+,p35SGUSINT plasmid DNA (positive control).

STABLE GERM LINE TRANSFORMATION OF A LEAFY VEGETABLE AMARANTH

Explant wounding. A maximum throughput of transformedshoots was scored by hand pricking (24.24%; Fig. 4C) irre-spective of the Agrobacterium strain used. Intact explants orthose wounded with sterile sand yielded a relatively lownumber of transformed shoots, 3.32% or 14.67% respectively,for strain EHA 105. The lowest number of transformedexplants (9.35%) coupled with a high rate of tissue browningwas noted in explants injured with glass wool.

Co-cultivation period. With a view to determining the suit-able period for co-cultivation, both explant types were

separately co-cultivated with different Agrobacteriumstrains for increasing lengths of 1–6 d (Fig. 4D). The max-imum transformation efficiency (24.24 %) was achievedafter 4 d co-cultivation. Explants co-cultivated for a shorterduration (1–3 d) produced shoots, but only a few of thesewere resistant to kanamycin. Durations longer than 4 d wereunsuitable on account of bacterial overgrowth.

Field establishment of transgenic plants and transgenesegregation. Root initiation in putative transformed shootsoccurred in 12–15 d in half-strength MS augmented with

Table 2 Effect of bacterial cell density and infection period on transformation efficiency (TE%)

Dilutionfactor

Agrobacterium celldensity

Duration of treatment (min)

5 10 15 20

EHA105

LBA4404

EHA105

LBA4404

EHA105

LBA4404

EHA105

LBA4404

1:3 (25%) 0.5×109 cells/ml 12.25 6.55 18.40 9.45 15.98 8.23 14.03 2.34

1:1 (50%) 1.0×109 cells/ml 20.14 11.14 24.24 15.15 22.27 13.26 17.24 6.54

3:1 (75 %) 1.5×109 cells/ml 18.46 11.08 23.79 14.35 20.12 8.75 11.39 4.28

1:0 (100%) 2.0×109 cells/ml 9.49 5.29 17.40 7.46 14.36 5.5 9.43 3.21

CD at 5%z 4.59 3.23 3.01 4.11 5.00 2.30 5.04 1.44

Values represent mean percentage of PCR positive events (plants showing amplification for nptII and gus genes) recovered from the total number ofepicotyl explants infected. Data were pooled from four experiments, each with 20 flasks containing three to four explants per flask

CD critical differencez Critical difference at p<0.05 (Duncan's new multiple range test)

Figure 4. Factors influencingtransformation efficiency usingepicotyl explants co-cultivatedwith A. tumefaciens strains(EHA105 and LBA4404). (A)Growth stage. (B)Acetosyringone treatment. (C)Infection method. (D) Co-cultivation period. Bars withdifferent letters are significantlydifferent at p<0.05 asdetermined by Duncan's newmultiple range test.

PAL ET AL.

IAA/IBA/IPA and supplemented with 50 μgml−1 kanamy-cin. Of a range of concentrations of auxins tested, IBA at4.9 μM was found to be the most effective for inducingrooting, with a maximum of 77.6% shoots with an averageof 5.1 roots/shoot and a root length of 4.2 cm. Rootingfrequency in MS+4.9 μM IBA without kanamycin was85–90% with six to seven roots per shoot. Root numberand root length of putative transformed plantlets increasedrapidly in subsequent cultures if denied kanamycin. Rootelongation required a further period of 10–12 d being fa-vored by a reduced IBA concentration (2.46 μM). In 21–24 d, well-rooted shoots in pots were acclimated in vermi-compost (Fig. 2E) and ca. 80% survived following transferto the potted soil inside the agronet-confined area of theglasshouse, where they were established in 30–40 d(Fig. 2F). A completely optimized protocol for genetictransformation of A. tricolor using a Ti plasmid-based vec-tor system of A. tumefaciens is presented in Fig. 7.

To establish the genome stability status of the primarytransformed plants, the inheritance pattern of the domi-nant selectable transgene (nptII) was analyzed in the T1

generation. Of a total of 32 PCR-tested transgenic plantlines (T0), derived from kanamycin resistant (GUS-posi-tive epicotyl explants transformed with EHA105 strain),12 were transplanted outdoors. All plants survived trans-plantation and seeds obtained from the self-fertilizedprimary transformed plants were germinated and the T1

plants were analyzed for the segregation of the transgene.Ratios of kanamycin resistant to sensitive T1 seedlings,raised from selected plants of nine different lines (3A2,4A7, 6B3, 11B5, 3D8, 5D4, 7E2, 16E3, 5F6, 2H7, and8H5), were exactly or close to the Mendelian monohy-brid segregation pattern (3:1) as revealed by the chi-square (χ2) test (Table 3).

Stable integration and expression of transgenes.Analysis of nptII gene integration—Kanamycin-resistant T1

segregant plants raised from seeds obtained via self-pollination of five randomly selected primary transformed(T0) lines (3A2, 11B5, 5D4, 16E3, and 2H7) were subjectedto Southern blot analysis to confirm integration of the trans-gene. A 1.6-kb PstI fragment of the T-DNA of the binaryvector p35SGUSINT that includes nptII was used as aprobe. PstI digestion of genomic DNA from each of thesix putative T1 transgenic lines generated an expected inter-nal transgene fragment of 1.6 kb that hybridized to the nptIIprobe, while no homology was detected for DNA from thenon-transformed plants (Fig. 5A). However, in one trans-genic line (11B5), the probe hybridized to an extra band ofca. 9 kb which indicated double copy integration in this line.To substantiate and confirm the stable integration of nptIIgene into A. tricolor genome and to detect junction frag-ments of T-DNA and plant DNA, genomic DNA fromtransformed T1 plant lines were digested with HindIII andprobed with 1.6 kb PstI fragment. Hybridization of theprobe to HindIII-digested genomic DNA revealed single-copy integration resulting in variable band sizes (>4.2 kb)among T1 plants (Fig. 5B). Interestingly, in the transgenicline 11B5, an additional band of ca. 3.8 kb was apparent.

NPTII enzyme activity—Translationally active NPTII en-zyme activity was detected in leaf protein extracts of con-firmed T1 transgenic plant lines as shown by positive spotson the autoradiograph that clearly revealed aminoglycosidephosphorylation, i.e., the transfer of radiolabelled phosphate(32P) from the γ-PO4 position of ATP to kanamycin (Fig. 6).Protein extracts of non-transformed plants (control 1) failedto display NPT II activity.

Prolonged retention of GUS activity in T1 progeny—GUS-positive signals were detected in the leaf tissues from

Table 3 Segregation pattern of T1 transgenic progeny based on resistance of seedlings to kanamycin (75 μgml−1)

Transgenic lines Total no. of seeds inoculated Kan-resistant seedlings Kan-sensitive seedlings Chi-square χ2 Null hypothesis (H0)

3A2 120 90 30 0<χ20.05 3:1

4A7 90 63 27 1.2<χ20.05 3:1

6B3 100 72 28 0.48<χ20.05 3:1

11B5 100 69 31 2.44<χ20.05 3:1

3D8 80 55 25 1.667<χ20.05 3:1

5D4 110 81 29 0.108<χ20.05 3:1

7E2 90 64 26 0.725<χ20.05 3:1

16E3 80 80 0 ΤΝΑ ΤΝΑ

5F6 70 48 22 1.543<χ20.05 3:1

6F4 100 75 25 0<χ20.05 3:1

2H7 100 73 27 0.213<χ20.05 3:1

8H5 90 62 28 1.792<χ20.05 3:1

Tabulated χ2 value at p0.05=3.84 (df=1)

TNA χ2 test not applicable

STABLE GERM LINE TRANSFORMATION OF A LEAFY VEGETABLE AMARANTH

all the 12 putative T0 transformed plants established in theglasshouse. Leaf tissues from glasshouse grown plants orseedlings without exposure to Agrobacterium infectionfailed to show GUS activity. Interestingly, stable GUS ex-pression was also demonstrated by T1 seedlings raised fromall the putative transgenic lines, albeit with varying intensityamong individual lines.

Discussion

Success of crop genetic engineering involving Ti plasmid-based vectors lies in the optimization of factors influencingtransfer of a proven selection marker and/or reporter trans-gene via Agrobacterium to the plant cell. This is followed bydemonstration of the transgene integration in the recipientplant genome and subsequently its expression and stabilityin the resulting transgenic plants. Considering the well-proven edibility of an important vegetable amaranth A.tricolor (Chinese spinach), the present investigation was

undertaken with a view to identifying and optimizing sev-eral parameters for efficient Agrobacterium-mediated genet-ic transformation procedures, with the long-term objectiveto incorporate desirable traits such as tolerance to bioticstress, especially due to pathogens, pests, or viruses.

In vitro culture of explants in the optimized regenerationmedium prior to infection and co-cultivation withAgrobacterium is another important parameter for transfor-mation. In the present study, pre-culture was found to en-hance regeneration efficiency of transformed tissues of A.tricolor. Pre-culturing parameters were also beneficial forplant cell infection in vegetable crops such as broccoli (Metzet al. 1995) and cauliflower (Chakrabarty et al. 2002).Perhaps a short-duration culture in regeneration mediumprior to Agrobacterium treatment activates explants to enterinto an exponential phase of cell division and therebyenhances their competence to undergo transformation andtheir ability to regenerate shoots early in the culture period.A prolonged lag phase in culture is known to cause a declinein the morphogenic potential, an increase in the number ofpolyploid and aneuploid cells (Nehra et al. 1990), or hyper-activation of retrotransposons thereby increasing their copynumber leading to off-types (Hirochika et al. 1996), each ofwhich or together might reduce overall transformationthroughput.

Reports on a number of crop plants indicated thatAgrobacterium strains differ with respect to their capacity totransform tissues (Pal 2008). Of the two different types ofdisarmed binary A. tumefaciens strains, viz., EHA 105 andLBA 4404, employed in this study, the former was moreefficient resulting in a higher number of transformed A.

Figure 5. A Southern autoradiograph of PstI-digested leaf genomicDNA from T1 transgenic lines hybridized with 32P-labeled nptII probedemonstrating the internal transgene fragment (1.6 kb). B Southernautoradiograph of HindIII-digested leaf genomic DNA from T1 trans-genic lines hybridized with 32P-labeled nptII probe demonstrating

variable sites of integration. C+, HindIII-digested plasmid DNAp35SGUSINT (positive control); C−, HindIII-digested DNA fromnon-transformed plant (negative control). Lanes 1–5, DNA from trans-genic lines (3A2, 11B5, 5D4, 16E3, and 2H7, respectively).

Figure 6. Autoradiograph showing neomycin phosphotransferase(NPTII) activity in leaf extracts of T1 transgenic plants. C+, sonicatedextract of E. coli JA221 carrying pKm2 with nptII gene (positivecontrol); C−, protein extract from non-transformed plant (negativecontrol). Lanes 1–3, protein extract from transgenic lines 11B5, 5D4,and 2H7.

PAL ET AL.

tricolor lines displaying kanamycin resistance and GUS ex-pression. Genes borne on the Agrobacterium chromosomeand the virulence region are important internal factors influ-encing the infecting ability of A. tumefaciens (Wang and Fang1998). LBA4404 and EHA105 do not only have dissimilarchromosomal backgrounds, but also different vir-helper plas-mid with varying levels of activating potency (Hoekema et al.1983; Hood et al. 1993). EHA105was effective for many cropplants such as banana (Subramanyam et al. 2011) whilst LBA4404 was suitable for double haploid cabbage (Tsukazaki etal. 2002). Differences in relative virulence among strains werereported in two different breeding lines of spinach in whichthe supervirulent A. tumefaciens strain 1065 was able totransform root explants more effectively than strain 0065despite the fact that both strains carried the same binary vectorpMOG23 (Knoll et al. 1997). Between two different strainsused for lettuce transformation, Mohapatra et al. (1999) ob-served that strain 1310 harboring the supervirulent pToK47plasmid generated multiple T-DNA inserts, whereas the otherstrain 0310 favored single insertion events for the bar gene.Perhaps, additional vir genes present in pToK47 promotedefficient transfer of the T-DNA to plant cells thereby resultingin multiple integrations. High T-DNA copy number was beingconsidered as one of the major factors of gene silencing(Matzke et al. 1996); the use of the latter strain was consideredpreferable because transgenic plants with single-copy inser-tions would be advantageous in terms of predictable inheri-tance of transgenes in progeny plants. Novel disarmed Tiplasmids (“helper plasmids”), constructed by site-specific de-letion mutagenesis to yield replicons carrying the vir genes tocomplement binary vectors in trans, have been proven usefulfor plant transformation (Hood et al. 1993).

For A. tricolor, the most effective bacterial cell density,growth phase, and period for co-cultivation with A. tumefa-ciens were identified to be 109 cells ml−1, 0.6, and 4 d,respectively, regardless of the explant type, resulting in themaximum transformation efficiency. Optimal growth phase ofbacterial culture (OD600) was reportedly 0.5 for Chinese cab-bage (Ji-Hong et al. 2009) and 1.5 for lettuce (Ahmed et al.2007). A co-cultivation period for 1 d was the best for lettuce(Ahmed et al. 2007), 2 d for Chinese cabbage (Ji-Hong et al.2009) and cauliflower (Chakrabarty et al. 2002), and 3 d forspine gourd (Thiruvengadam and Chung 2011). Such varia-tions in the requirement for definite co-cultivation periodperhaps may be attributed to the plant tissue specificity forT-DNA transfer, choice of Agrobacterium strain, and its opti-mal cell density that allow infectivity without compromisingwith survival of infected plant cells (Pal 2008).

The use of the plant phenolic inducer acetosyringone (AS)enhanced the transformation efficiency of A. tricolor. AS sup-plementation, at varying concentrations, to the Agrobacteriumculture media has been used for genetic transformation ofvegetable crops. Whilst 50 μM was ideal for cauliflower

(Chakrabarty et al. 2002) and banana (Subramanyam et al.2011), in others, higher concentrations, i.e., 100 μM (cabbage,Jin et al. 2000) and 200μM (spinach, Zhang and Zeevart 1999;spine gourd, Thiruvengadam and Chung 2011) were helpful.Perhaps AS forms a complex with the constitutively expressedchemoreceptor virA protein and consequently phosphorylatesvirG product which eventually induces the remaining batteryof genes of the vir regulon, such a signal transduction beingessential for T-DNA transfer (Gelvin 2003).

Various bacteriostatic and bactericidal antibiotics such asTimentin (carbenicillin), cefatoxin, cefotaxime, triacillin, andvancomycin are most commonly used to control the growth ofAgrobacterium during the shoot induction phase and are main-tained in all subsequent media following the co-cultivationperiod. Bactericidal antibiotics are to be so chosen as to haveminimal toxicity on plant tissues while being effective insuppression of bacterial growth. In the present study on A.tricolor, cefotaxime (500 μgml−1) was successful in complete-ly eliminating the adhering Agrobacterium after co-cultivationperiod without any noticeably adverse effect on shoot morpho-genesis. This is in conformity with observations in banana(Subramanyam et al. 2011). Although both carbenicillin andcefotaxime show plant hormone-like activity, little is knownabout the exact mode of action of these antibiotics.

Results indicate the useful stringency of kanamycin se-lection, which had imposed an effective restriction to false-positive (escape) throughput. The effectiveness of antibioticselection following co-cultivation is an important factor forpossible elimination of “escapes” and recovery of trans-formed plants. This seems to be dependent on factors liketissue type, explant size, antibiotic concentration, and timeof application for effective and efficient recovery of trans-formed plants (Birch 1997). The use of 75 μgml−1 of theaminoglycoside kanamycin as the selective threshold dos-age in the present study was more stringent compared toseveral other leafy vegetables in which 10 μgml−1 kanamy-cin (Chinese cabbage, Ji-Hong et al. 2009) or 50 μgml−1

kanamycin (spinach, Knoll et al. 1997; Zhang and Zeevart1999; lettuce, Ahmed et al. 2007) was used for selection.However, there are also a few reports claiming that 100 μgml−1 kanamycin proved effective for selection of trans-formed shoots as for lettuce cultivars (Valimareanu 2010)and spine gourd (Thiruvengadam and Chung 2011). Basedon our results, all selection used 75 μgml−1 kanamycinduring the initial stage during shoot regeneration and earlyshoot development and 50 μgml−1 in the subsequent stagesfor maintenance of shoot cultures and root initiation.Though the escape rate in our experiments on A. tricolorwas apparently high (Table 1), it could not have beenavoided because it was necessary to make a compromisewith survival per se of the putative transformed tissues.There was an excessive mortality of transformed shootsassociated with a selection pressure exerted by the selective

STABLE GERM LINE TRANSFORMATION OF A LEAFY VEGETABLE AMARANTH

antibiotic (100 μgml−1 kanamycin) beyond the thresholddosage (75 μgml−1). Thus, it was important to effectivelystrike a delicate balance between the net throughput oftransformed plants and the apprehended risk of false pos-itives (escapes).

Residual Agrobacterium cells adhering to non-transformedplants in culture might also, to some extent, account for GUSactivity in a standard histochemical assay and, thus, maycomplicate analysis of results during early events of transfor-mation. To avoid such a situation, a portable plant intron intothe coding sequence of β-glucuronidase gene (gus) has beenintroduced to interrupt the open reading frame (Vancanneyt etal. 1990). Since then, gus-intron chimeric gene constructs havebeen safely and successfully exploited in Agrobacterium-me-diated genetic transformation of several vegetable crops suchas spinach (Knoll et al. 1997), double haploid cabbage(Sparrow et al. 2004), spine gourd (Thiruvengadam andChung 2011), as well as in a green amaranth (Chinese spinach,A. tricolor) in the present investigation.

A transformation system can be considered efficient onlywhen tissue culture-induced phenotypic abnormalities in thetransgenic plants are kept to a minimum. In the presentstudy, the transgenic plants of A. tricolor appeared normaland the phenotype closely resembled that of regeneratedcontrol plants. All plants reached maturity and set viableseeds. To establish the stability status of the primary trans-formed plants, the inheritance pattern of the dominant se-lectable nptII transgene was analyzed in the T1 generation.The segregation pattern of kanamycin resistance among T1

offspring revealed an expected monogenic segregation ofnptII transgene following the characteristic Mendelianmonohybrid progeny ratio (3:1), thus substantiating germline inheritance and transgene stability (Table 3).

To demonstrate stable integration of the transgene in T1

plants, Southern blot analysis was performed with respect tonptII gene. While PstI cleavage generated a 1.6-kb internaltransgene fragment in all independent T1 lines, cleavage withHindIII demonstrated variable DNA fragment sizes depend-ing on the site of integration of T-DNA in the plant genome.Due to the presence ofPstI sites on both sides of the nptII gene(Fig. 1), the copy numbers or sites of integration of the T-DNAin plant genome could not be analyzed and in all tested linesbands of ca. 1.6 kb were detected specifying the nptII trans-gene; the molecular size corresponding to that of the PstIfragment from the plasmid DNA (positive control). The addi-tional DNA band of ca. 9 kb indicates the presence of anadditional T-DNA insert in which the PstI site close to theright border was perhaps either truncated (fully/partially de-leted) or methylated, thereby itself rendered non-recognizablefor endonucleolytic cleavage. This “restriction skipping”might have resulted in a longer fragment which included apart of the T-DNA towards the right border plus the plantDNA beyond the right border till the nearest PstI site.

Appearance of additional bands of unexpected sizes inSouthern blots, though intriguing, is not uncommon to mo-lecular analysis of putative transgenic plants, for instance, inlettuce (Mohapatra et al. 1999). Following HindIII digestion,the appearance of the unequal fragment sizes on the Southernblot autoradiograph obviously varied with the site of T-DNAintegration. The internal transgene fragment (2.8 kb) wouldnot have hybridized to the nptII gene probe and, therefore, wasnot detected. A larger fragment (ca. 11.7 kb) encompassingthe remainder of the plasmid minus the gus-int region predict-ably appeared for the positive control.

It was interesting to observe that two transformed lines,namely, 3A2 and 5D4, exhibited apparently similar sizebands (slightly above 5.0 kb), although they had been de-rived from two independent experiments and representedseparate PCR-indexed lines (Fig. 7). Albeit unexpected,the possibility of such coincidences in T-DNA integrationat analogous sites of the genomic DNA of independenttransgenic plant lines cannot be overruled and is, indeed,not exceptional to literature. Recently, Chugh et al. (2012)observed an additional band of ca. 4.2 kb that appeared infour different T1 transgenic lines of wheat Triticum aestivumbesides the expected band of 2.8 kb corresponding to thegus gene. The appearance of such supplementary signals

Figure 7. A flowchart representation of the completely optimizedprotocol for Agrobacterium-mediated genetic transformation of A.tricolor. Figures within parenthesis denote concentrations in micromo-lar for plant growth regulators used and in microgram per milliliter forkanamycin monosulfate used.

PAL ET AL.

was explained on the basis of additional restriction sites forPstI in the vector p35SGUSINT, which might have resultedin partial digestion of the genomic DNA (Chugh et al.2012). Interestingly, in T1 transformed plants of durumwheat (Triticum durum), an extra band of smaller size(3.5 kb) was detected in four different transgenic lines inaddition to the expected signal of 11.3 kb specifying the bargene in EcoR1 digest of the genomic DNA (Chugh et al.2012).

A major problem often encountered in plant transforma-tion is the non-predictability of the pattern of integration oftransgenes in the host genome and their expression. The keycauses of variability of transgene expression in plants havebeen ascribed to the copy number (Hobbs et al. 1993;Koprek et al. 2001; Kohli et al. 2003), tandem/invertedrepeat organization (Muskens et al. 2000; Wang andWaterhouse 2000), site of transgene integration into theplant genome (position effect), methylation of transgenes(Matzke et al. 1996; Meyer et al. 1996), and integration ofvector backbone sequences (Kononov et al. 1997; De Bucket al. 2000) among other reasons.

One obvious complication arises due to differences in thecopy number of transgenes present in independent transgen-ic plants. Analyses of transgenes in a large number of plantspecies have revealed that independent transgenic lines car-ry one to several random insertions of transgenes. Literatureis inconsistent in relating transgene copy number with theirexpression levels. No firm connection could be establishedbetween copy number and expression of transgenes in to-bacco or cauliflower (Hobbs et al. 1993; Chakrabarty et al.2002). A positive correlation between high copy numberand expression of transgenes was reported in potato, tobac-co, and rice (Gendloff et al. 1990; van der Hoeven et al.1994). Conversely, a negative correlation was documentedfor petunia, maize, and tobacco (Hobbs et al. 1990; Matzkeet al. 1996; Koprek et al. 2001; Kohli et al. 2003; Tenea andCucu 2006; Donnarumma et al. 2011). A very large numberof transgenic loci can be meiotically unstable thereby lead-ing to excision of loci and loss of transgene expression insubsequent generations (Stoger et al. 1998). Tenea and Cucu(2006) quantified the GUS activity by fluorometric assay of25 primary transformed plants of Atropa belladonna whichresulted from A. tumefaciens LBA 4404 (pBINplus)-medi-ated transformation and noted variable gene expressionwhich was inversely correlated with the T-DNA copy numb-ers using the gus probe in Southern hybridizations. Southernblot analysis of two independently transformed lines show-ing high GUS gene expression revealed single-copy insertsof the T-DNA, whereas three other lines with low GUSexpression revealed multiple copy integration patterns.

The transgene copy numbers were shown to have variedwith the method employed for transformation. Transgenicplants obtained by direct vector-less DNA transfer methods

(e.g., biolistics or electroporation) were found to contain alarge number of transgene copies (up to 100), whereasAgrobacterium-mediated transformation revealed the inser-tion of fewer transgene copies (<10) with a more frequentoccurrence of single-copy integrations (Koprek et al. 2001;Gelvin 2003; Reddy et al. 2003). Perhaps, direct methodspermit multiple insertions at random due to a greater chanceof illegitimate recombination events thus resulting in ahigher copy number in transgenic plants, while restrictionsto T-DNA integration could be due to its sequence homol-ogy, albeit to a limited extent (“microhomology”), with thatof the recipient plant nuclear genome (Tzfira et al. 2004).

Vital to efficient transformation is the development of anideal selection scheme in addition to methods of gene trans-fer. Though no direct relationship, to our knowledge, hasbeen established between kanamycin resistance and trans-gene copy number, selection based on herbicide tolerancewas shown to potentially lead to an increase in gene copynumber as a result of strong selection for high gene expres-sion (Shyr et al. 1992). In the stepwise selection for glyph-osate resistance with suspension cultures of carrot, there wasa general correspondence of glyphosate resistance, the ac-tivity of the target enzyme 5-enolpyruvylshikimate-3-phos-phate synthase (EPSPS), EPSPS mRNA levels, and theEPSPS gene copy number. The enhanced tolerance toglyphosate was thought to be due to stepwise amplificationof the EPSPS gene copy number. Li et al. (2003) developedprotoporphyrinogen oxidase (PPO), the key enzyme in thechlorophyll/heme biosynthetic pathway as an efficient se-lection marker for Agrobacterium-mediated transformationof maize. To optimize butafenacil (the herbicide targetingPPO) concentrations used for selection, three selectionschemes were compared for their ability to produce trans-formed plants. Although transformation variability was highamong the three transformation schemes, the obvious trendwas that the transformation efficiency was higher with in-creased butafenacil levels. Selection employing butafenacilper se did not alter transgene copy number with respect toother selectable marker systems; analysis revealed that themajority (71%) of PPO transgenic plants were single-copyevents. Further analysis was done for a subset of 46 highlytolerant pWCO38 events identified from about 2,500 trans-formed plants by a greenhouse spray assay. About 57% ofhighly tolerant events had a single copy of the PPO gene,indicating that multiple copies can only be negatively cor-related with high tolerance to butafenacil.

Considerable effort has been made towards reducing thevariability of transgene expression as a result of Agrobacterium-mediated genetic transformation, encompassing (a) the use ofmatrix-associated regions/scaffold attachment regions to shieldT-DNA inserts from the influence of adjoining chromatin (Allenet al. 1996; Butaye et al. 2004), (b) selection of appropriateexplants to reduce T-DNA copy number (Grevelding et al.

STABLE GERM LINE TRANSFORMATION OF A LEAFY VEGETABLE AMARANTH

1993), and (c) positioning the selectable marker gene proximalto the left T-DNA border to reduce selection of plants containingtruncated T-DNA inserts (Becker et al. 1992). Further researchshould focus on targeting foreign genes to a predetermined non-heterochromatic (transcriptionally active) chromosomal site ofthe recipient plant genome mediated by homologous recombi-nation. New technologies also need to be developed to increasethe frequency of single-copy transgene integration and to gen-erate elite transgenic plants carrying single transgene copies,which would be expectedly less prone to the natural defensemechanism of alien gene silencing compared to those withmultiple transgene copies. This will ensure that their segregationpattern through inheritance would be predictably simpler lead-ing to production of homozygous plants with high heritability.

The present investigation, examining and optimizing somesalient factors that influenced early events of transformation,has resulted in a reproducible protocol for Agrobacterium-mediated genetic transformation of A. tricolor—a leafy greenvegetable crop important for the low-income food-deficitcountries. Exhibition of single-copy insertions in most of thetested transformed plants indicates a minimum risk ofhomology-dependent gene silencing. Germ line transforma-tion substantiated by stable integration and translationallyactive expression of foreign genes is critically important forthe successful application of genetically engineered crops inagriculture. Thus, our protocol can be used to enable engi-neering resistance in this elite Amaranthus species againstspecific or broad-spectrum pathogens, viruses, or pests.

Acknowledgments We wish to thank Dr. Jatindra K. Nayak, Profes-sor of English, Utkal University, Bhubaneswar, India for criticallyreading the manuscript and making useful changes in the text language.

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STABLE GERM LINE TRANSFORMATION OF A LEAFY VEGETABLE AMARANTH