EMBRYO CULTURE

Agrobacterium -mediated transformation of Medicagotruncatula cell suspension culture provides a systemfor functional analysis

Anelia Iantcheva & Miglena Revalska & Grigor Zehirov &

Valya Vassileva

Received: 18 March 2013 /Accepted: 19 August 2013 /Published online: 31 August 2013 / Editor: J. Forster# The Society for In Vitro Biology 2013

Abstract Over the past decade,Medicago truncatula has beenadopted as a model legume species for a range of “omic”studies. The availability of different transformation techniqueshas greatly advanced functional genomic studies in this species.In the present work, an efficient procedure for Agrobacterium-mediated transformation ofM. truncatula cv. “Jemalong 2HA”through a cell suspension culture was developed. This proce-dure resulted in transformed single cells or cell clusters, givingrise to stable transgenic plants within 4 mo. Transformationexperiments were performed with a vector carrying two markergenes: β-glucuronidase (GUS) and green fluorescent protein(GFP) under the control of endogenous gene promoters fromLIKE AUX1 3 (LAX3) and GRAS transcription factor (namedafter GD3BERELLIC ACID INSENSITIVE [GAI ],REPRESSOR OF GA1 [RGA], and SCARECROW [SCR]),as well as with a binary destination vector for overexpressingthe cyclin-like F-box gene fused to GFP. Maximum transfor-mation efficiency was achieved under the following experi-mental conditions: acetosyringone at a concentration of25 μM, bacterial suspension with an optical density of 0.3 at600 nm, inoculation under agitation at 100 rpm for 24 h, co-cultivation periods of 48 h, and an uninterrupted selection with50 mg/L kanamycin. Selection of positive transformationevents was imposed early in the regeneration stage (after 48 hco-cultivation), following a large-scale screening for GFP ac-tivity. Histochemical GUS and GFP reporter activity was de-tected in single cells, embryogenic zones, emerging embryos,

in vitro plantlets, and T1 progeny seedlings. The transgenicnature of transformed plants was further confirmed by nptII-specific PCR amplification of T0 and T1 plant lines. Thetransgenic plants grown under standard greenhouse con-ditions displayed a wild-type phenotype and the obtainedprogeny segregated in a classical Mendelian manner. Thefundamental steps in the transformation procedure areoutlined and discussed.

Keywords Cell suspension culture . Agrobacteriumtransformation . Transcriptional reporters .β-Glucuronidase .

Green fluorescent protein . Transformation efficiency

Introduction

Unlike the model flowering plant Arabidopsis thaliana , thelegume Medicago truncatula establishes symbiotic relation-ships with nitrogen-fixing bacteria (rhizobia). This feature,together with the small, diploid and almost sequenced ge-nome, self-fertility, short life cycle, and high levels of naturaldiversity makes it an excellent model for legume “omic”studies (Young et al. 2011). Furthermore, the genome of itsmicrosymbiont Sinorhizobium meliloti has been sequencedand well studied (Galibert et al. 2001). In addition, M.truncatula is phylogenetically closely related to eco-nomically valued legumes: clover, alfalfa, pea, fababean, chickpea, and lentils, which facilitates ready trans-fer of knowledge and research tools from the modelplant to legume crops. Therefore, molecular and geno-mic studies of M. truncatula are important for bothfundamental and applied issues. The availability of an effi-cient system for transformation and regeneration of M.truncatula has opened up opportunities for identificationand functional analysis of candidate genes associated withimportant developmental and agronomic traits.

A. Iantcheva (*) :M. RevalskaAgroBioInstitute, Bul. Dragan Tzankov 8, Sofia 1164, Bulgariae-mail: aneliaiancheva@abi.bg

G. Zehirov :V. VassilevaInstitute of Plant Physiology and Genetics, Bulgarian Academyof Sciences, Academik Georgi Bonchev Str., Block 21,Sofia 1113, Bulgaria

In Vitro Cell.Dev.Biol.—Plant (2014) 50:149–157DOI 10.1007/s11627-013-9554-4

During the last 15 yr, a number of Agrobacteriumtumefaciens-based transformation protocols have been devel-oped. In general, they rely on indirect somatic embryogenesisof highly regenerable genotypes using different explants types,such as leaves (Chabaud et al. 1996; Hoffmann et al. 1997;Trinh et al. 1998; de Susa Araújo et al. 2004), roots (Crane et al.2006), clusters of embryos (Iantcheva et al. 2005), and floralorgans (Kamate et al. 2000). Rapid genetic transformationprotocols via organogenesis and in planta transformation ofM. truncatula have been also reported (Trieu and Harrison1996; Trieu et al. 2000). The limiting steps are generallythe moderate regeneration efficiency of transformedcells into transgenic plants and the long time requiredfor the regeneration phase.

Another important tool in root and symbiotic biology ishairy root transformation via Agrobacterium rhizogenes ; afast and efficient system for producing composite plants withtransgenic hairy roots and nontransgenic shoots (Boisson-Dernier et al. 2001). Such composite plants are an ideal systemto study gene function of plants in association with other typesof organisms. Also, transformed roots are able to regeneratewhole viable plants with high genetic stability during subse-quent subculturing and plant regeneration. An effective sys-tem for producing M. truncatula (genotype R108) transgenicplants from hairy roots has been also developed (Crane et al.2006). However, currently no transformation system has beendeveloped using cell suspension cultures suitable for studyinggene function in M. truncatula .

This study is part of an ongoing project (Integrated func-tional and comparative genomic studies of the model legumeM. truncatula and Lotus japonicus , IFCOSMO), that aims touse forward and reverse genetics to explore the contribution ofunknown genes of M. truncatula based on Tnt1 insertionalmutant collection (Vassileva et al. 2010; Revalska et al. 2011).The large collection of Tnt1-tagged lines was created duringthe course of the European Grain Legumes Integrated Project“GLIP” (the Sixth Framework Programme, FP6, FOOD-CT-2004-506223). The Bulgarian team contributed greatly indefining conditions for efficient Tnt1 transposition, resultingin lines with a high number of inserts (Iantcheva et al. 2009),which served as a basis to start functional genomics studieswithin the frame of the IFCOSMO project.

An important tool for legume researchers is the establish-ment of a transformation system using single or few cells,which is particularly suitable for obtaining overexpression,reduction of function/silencing, or β-glucuronidase (GUS)/green fluorescent protein (GFP) transcriptional reporter lines.Such a system reduces or eliminates production of “escapes”(untransformed plants) and gives an opportunity to investigategene function and genetic interactions via high-throughputmethods of cell biology.

The present study focuses on development of an efficientprotocol for Agrobacterium-mediated transformation of cell

suspension cultures, leading to production of stablytransformed M. truncatula plants with normal phenotypeand fertility. We demonstrate the applicability of this transfor-mation system to analyzing specific functions of importantgenes involved in plant development.

Material and Methods

Plant material and growth conditions. Seeds from the highlyregenerable genotype M. truncatula cv. “Jemalong 2HA”(Nolan et al. 1989) were surface sterilized with 6% (v/v )solution of sodium hypochlorite (commercial bleach) for15 min, rinsed at least three times with sterile distilled water,and germinated on Murashige and Skoog (MS) basal medium(Murashige and Skoog 1962). The germinated seedlings werethen propagated by cuttings. Leaf explants collected from 30-d-old in vitro plants were used to initiate callus tissue. In vitroplant materials were grown in Magenta boxes (60×60×96 mm; Sigma) in a growth chamber at 24°C, with a 16-hphotoperiod and light intensity of 30 μmol m−2 s−1.

The putative transgenic rooted plantlets were cultivated onselective MS basal medium supplemented with 50 mg/Lkanamycin (Km). The transgenic regenerated shoots, capableof developing roots under selective pressure, were maintainedin vitro; after sampling for analyses, they were transferred tosoil and grown in a greenhouse.

Cloning and construction of expression vectors fortransformation. All the recombinant plasmids wereobtained through the GATEWAY™ system (Invitrogen LifeTechnologies). Briefly, three Entry Clones were constructedby inserting theM. truncatula promoter sequences of an auxininflux carrier, LAX3 (LIKE AUX1 3 ; MT3G072870, PLAZA2.5) and GRAS (GD3BERELLIC ACID INSENSITIVE[GAI ], REPRESSOR OF GA1 [RGA], and SCARECROW[SCR ]) transcription factor after (MT2G026250, PLAZA2.5) into the pDONRP4P1R donor vector, and the cyclin-like F-box gene (MT2G007220, PLAZA 2.5) coding se-quence into the pDONR221 donor vector. Eight expressionclones were generated by recombining the Entry Clones car-rying the promoter fragments into the pEX-K7SNFm14GW(promoter-NLS-GUS/GFP) destination vector harboring theneomycin phosphotransferase (nptII ) gene as a plant select-able marker (De Rybel et al. 2010). The cyclin-like F-boxEntry Clone fragment was transferred into pK7FWG2 desti-nation vector (a C-terminal translational GFP fusion) underthe control of CaMV 35S promoter and nptII gene for plantselection (Karimi et al. 2007). The resulting constructs wereintroduced into A. tumefaciens strain C58C1, which wasmaintained on agar (1.5%; w/v ) solidified YEB nutrient me-dium supplemented with 100 mg/L rifampicin, 100 mg/Lspectinomycin, and 50 mg/L gentamicin. Prior to inoculation,

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the Agrobacterium cultures were incubated in YEB for 24 hunder constant agitation at 250 rpm and 28°C. Bacterialsuspensions at an optical density of OD600=0.3 were usedfor inoculation.

Composition of culture media. Callus-inducing medium(CIMN): B5 (Gamborg et al. 1968) liquid and solid mediumcontaining 18.1μM2,4-dichlorophenoxyacetic acid, 3.72μMkinetin, 5.43 μM adenine (6-aminopurine), 500 mg/L caseinhydrolysate, 500 mg/L myo-inositol, and 3% (w/v ) sucrose.For preparation of solid medium, 7 g/L agar was addedbefore autoclaving at a temperature of 120°C and a pressure of1 atm for 1 h.

Embryo-inducingmedium (EIMN):MS supplementedwith3.99 μM 6-benzylaminopurine (BAP), 1.61 μM α-naphthaleneacetic acid, 3.0% (w/v) sucrose, and solidifiedwith 0.7% (w/v) agar. Km at a concentration of 50 mg/Lwas used as a selective agent.

Embryo development medium (EDMN):MS supplementedwith 0.22 μMBAP, 250 mg/L casein hydrolysate, 0.7% (w/v)agar, and 50 mg/L Km.

The pH of all culture media was adjusted to 5.8 by adding1 N NaOH.

Initiation of cell suspension culture. On solid CIMN, leafexplants with an average length of 0.5 cm detached from 30-d-old in vitro-grown seedlings produced white to yellow, soft,and very friable callus. Over 30–45 d, about 80% of theexplants formed callus tissue, which was primarily observedaround the wounded edges. At day 45, the whole leaflet resem-bled a round globular structure consisting of friable callus.

Friable calli from three to four leaflets were suspended in15 mL of liquid CIMN. This medium was used for initiation,maintenance, and replenishment of the cell suspension cul-tures. After incubation for 2 weeks on a rotary shaker(120 rpm), a fine cell suspension fraction could be observed.This fraction was transferred to new 250 mL flasks andmaintained as a cell suspension culture. During the experi-mental work, a 1:1 dilution of mother culture to fresh CIMN

liquid medium was defined. The mother suspension culture,refreshed with liquid CIMN every 10 d, could be maintainedon a rotary shaker for a period of 3 mo, and used for subse-quent transformation experiments. More details on mainte-nance and refreshment of suspension cultures can be foundin Iantcheva et al. (2006).

Genetic transformation. Fifteen milliliters of the fine cellfractionwere diluted 1:1with 15mL liquid CIMN supplementedwith 0, 25, or 50 μM acetosyringone (3′,5′-dimethoxy-4′-hydroxyacetophenone). After 24-h incubation, 4 mL of suspen-sion culture was transferred to a plastic Petri dish (Ø 40 mm),then inoculated with 400 μL of bacterial suspension (OD600=0.3, 0.5, or 0.7) and grown on an orbital shaker (100 rpm) for

24 h. The transformed suspension culture was gently pipettedonto solid CIMN and co-cultivated for a further 48 h, thentransferred to solid CIMN containing Km (50 mg/L) andcarbenicillin (400 mg/L) for selection of the transformed cellsand cell clusters, and removal of theAgrobacteria . The selectivemedium was refreshed every 20 d.

All plant materials were cultivated in growth chambers atconstant conditions of temperature 24°C, 70% relative humid-ity, white fluorescent light (30 μmol m−2 s−1) and 16/8 h light/dark photoperiod.

GUS activity assay. Histochemical assays for detection ofGUS activity were performed as described by Jefferson et al.(1987) with minor modifications. Different tissues (includingsingle cells and cell clusters, callus with emerging embryos,clusters of developed embryos, cotyledonary embryos, androots and leaves of regenerated [T0] plantlets or T1 seedlings)were incubated for between 2 and 24 h 37°C in X-glucsolution (10 mL contained 5 mg 5-bromo-4-chloro-3-indolyl-β -d-glucuronide dissolved in 50 μL formamide,5 mL 100 mM sodium phosphate buffer [pH 7.0], 200 μL0.5 M Na2EDTA, 10 μL Triton X-100, 1 mL 1 mMK4Fe(CN)6.3H2O, 1 mL 1 mM K3Fe(CN)6, 2 mL methanol,and 740 μL H2O).

GFP screening and imaging. Large-scale screening and de-tection of GFP was performed using a Typhoon Trio variablemode imager (GE Healthcare). The Petri dish containingtransformed callus tissues was scanned using a 520-mmbandpass filter, a photomultiplier tube (PMT) voltage 470 V,and a pixel size of 200 μm. Microscopic detection of GFPsignals in roots and leaves was carried out with a ZeissAxio Imager upright microscope equipped with a camera at40× objective.

Molecular analysis of transformed plants. Genomic DNAwas extracted from transgenic plants using DNeasy PlantMini Kit (Qiagen) according to manufacturer's instructions.PCR amplification was performed in a total volume of 25 μLwith puRE Taq Ready-To-Go PCR beads (GEHealthcare). Thefollowing amplification conditions were used: 94°C for 3 min,followed by 35 cycles, consisting of 94°C for 30 s, 55°C for45 s and 72°C for 45 s; and a final 5-min elongation step at72°C. PCR products were separated by gel electrophoresis in1.0% (w/v) agarose gels. Primers specific to the nptII sequencewere used to amplify a product with an expected size of 550 bpwere forward 5′-GAACAAGATGGATTGCACGC-3′ and re-verse 5′-GAAGAACTCGTCAAGAAGGC-3′.

Statistical analysis. All experiments were repeated at leastthree times and triplicate assays were performed for each ex-perimental sample. The data were analyzed using Statisticalsoftware package (Version 7, StatSoft Inc.). Differences were

MEDICAGO CELL SUSPENSION TRANSFORMATION 151

assessed by analysis of variance (ANOVA) or multiple-comparison ANOVA (Fisher's LSD test). Results are expressedas means±standard deviation (SD). Statistically significant dif-ferences were considered when P <0.05.

Results

Maintenance of cell suspension culture. The obtained finecell suspension of M. truncatula was composed of a numberof single cells, clusters of three to five cells, and small (up to20 cells) and large (between 20 and 40 cells) aggregates(Fig. 1A). Single cells and cells in clusters of a variety ofshapes (spherical, oval, or elongated) and with dense cyto-plasm could be clearly visualized. The mother suspensionculture, refreshed with liquid CIMN every 10 d, could bemaintained on a rotary shaker for a period of 3 mo, and usedfor subsequent transformation experiments. After the follow-ing transformation, such single cells and cell clusters could beused for confirming GUS and GFP activities (Fig. 1B,C).

Genetic transformation of cell suspension cultures. The mainsteps of regeneration after Agrobacterium-mediated genetictransformation of cell suspension cultures are shown inFig. 2. For each transformation experiment, aliquots (4 mL) of

cell suspension culture pretreated with 0, 25, or 50 μMacetosyringone for 24 h were inoculated with 400 μL bacterialsuspension harboring the promoter constructs of the auxininflux carrier LAX3, GRAS transcription factor and cyclin-like F-box and carrying GUS/GFP as reporter genes, andincubated for a further 24 h on an orbital shaker. In the presenttransformation procedure, the effect of acetosyringone ontransformation efficiency was evaluated by the number ofGFP-positive structures. The highest transformation efficien-cy was recorded with 25 μM acetosyringone (Fig. 3). Thisefficiency was approximately 5.5 times higher than that foundwithout acetosyringone and approximately two times higherthan when 50 μM acetosyringone was used.

Three different optical densities of the bacterial suspensionwere tested. Bacterial culture with OD600=0.3 was found to beoptimal for transformation of cell culture. The other two testedconcentrations (OD600=0.5 and OD600=0.7) led to a severeAgrobacterium infection, which was detrimental to cell viabil-ity. An inoculation period of 24 h under 100 rpm agitation wasfound to favor cellular transformation, as compared to staticconditions in a Petri dish or a 48-h inoculation period (data notshown). Under agitation, the cells and cell clusters assembledwith Agrobacteria in common dense aggregates. These aggre-gates were gently transferred for co-cultivation to solid CIMN.After 48 h co-cultivation, the suspension culture was transferred

Figure 1. Fractions from cellsuspension culture of M.truncatula cv. ‘Jemalong 2HA’(A) before transformation, (B)GUS-expressing cell suspensionafter transformation, (C) GFP-expressing cell suspension aftertransformation.

Figure 2. Key steps ofAgrobacterium-mediatedtransformation of M. truncatulacell suspension culture; (A) firstpassage callus structures onselective CIMN, (B) callusstructures with detected Cyclin-like F-box GFP signal, (C ,D)embryogenic zones and embryoappearance, (E ,F) embryoelongation and development.Scale bars A, B, D–F, 1 cm; scalebar C , 0.2 cm.

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to selective CIMN, giving rise only to the transformed cells orcell clusters over a period of 15–20 d, which formed into smallround clumps (Fig. 2A). Further, only large callus structureswith a size of 1–2 mm (approximately 65% of total clusterstructures), which survived the first round of selection weretransferred to a second round of selection (another 20 d) on thesame medium (CIMN; Fig. 2B). Large-scale screening of callusstructures (1–4 mm) for the presence of green fluorescence wasthen performed (Table 1). Only calli with strong GFP signalswere cultured further for embryo induction and development.Figure 4A shows a representative example of GFP-positivecallus structures after transformation with the destination vectorpK7FWG2 (containing eGFP) for creating cyclin-like F-boxoverexpression plant lines. Nontransformed suspension culturedid not form callus on selective medium and GFP signals werenot observed (Fig. 4B).

Embryo induction and development. Subsequent develop-ment of the GFP-positive callus structures was carried outon EIMN. During the first 10–15 d on this medium, the clumpsenlarged and started to form green patches on the calli. Therelationship between auxin and cytokinin levels in the medi-um played an essential role in this process (Fig. 2C,D).Embryo development was performed on EDMN in the pres-ence of 0.22 μM BAP and 250 mg/L casein hydrolysate,which stimulated embryo elongation and formation of bipolarstructures (Fig. 2E,F). Despite the presence of the selectiveagent Km during the growth of differentiating structures, theirtransgenic nature was confirmed by analyses of GUS activity.Figure 5 shows representative examples of GUS expressionregulated by LAX3 (auxin influx carrier protein) in structures

Figure 3. Effect of different concentrations (0, 25, and 50 μM) ofacetosyringone on the transformation efficiency of M. truncatula cellsuspension culture. AS acetosyringone. Vertical bars indicate the stan-dard deviation from three experiments. Different letters indicate signifi-cant differences at P<0.05. T

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MEDICAGO CELL SUSPENSION TRANSFORMATION 153

induced after transformation of cell suspension culture. Theplant hormone auxin acts as a trigger for embryo patterningand development and further plant growth. The expressionwas visualized in embryogenic zones that formed after trans-fer of round callus clumps to EIMN (Fig. 5A). Very strongpLAX3 -GUS staining was observed in developed embryostructures (Fig. 5B). Subsequently, this expression was detect-ed in cotyledonary leaves of well-formed embryos (Fig. 5C).

Embryo conversion and rooting. Embryos with well-developed cotyledons were transferred to selective basal MSmedium for development into plantlets. Occasionally, whenclusters of cotyledonary embryos formed, they were separatedto allow root growth to occur. The timing of this processdepended on the vigor of embryos and typically lasted between20 and 40 d. Subculturing on this medium led to the formationof healthy plantlets that could be acclimatized to greenhouseconditions. Conversion rates of embryos across the experimentswith three different constructs varied from 55% to 65%(Table 1). All vigorous plants producing roots on selectivemedium were then analyzed for GUS activity. For the twopromoters studied,GUS expressionwas detected in roots, stems,leaves, secondary root branches, and occurred throughout plantdevelopment. Randomly selected GUS-positive plants(Fig. 6A,B), were tested for the integration of the selectablemarker gene nptII in T0 transformed plants carrying the promot-er region of the gene encoding GRAS transcription factor.Histochemical assays for GUS activity are presented inFig. 6A,B and the integration of nptII gene in Fig. 7A.

Transformation efficiency for each of the constructs is basedon GFP-positive callus structures, on the number of inducedembryos, the percentage of embryo conversion, and the numberof converted plantlets (Table 1). Confirmed transgenic plantswere transferred to soil and grown to maturity under greenhouseconditions to obtain seeds. The selected T0 plants possessed nomorphological abnormalities, had regular flowering time, andpod production. The transgenic T1 seeds were grown on selec-tive medium and segregated in the classical Mendelian manner(Table 1). Part of the T1 seedlings was sampled and used toverify GUS expression (Fig. 6C) and the presence of nptIIthrough gene-specific PCR amplification (Fig. 7B). Bright greenfluorescence was detected by conventional epifluorescence mi-croscopy in root and leaf epidermal cells (Fig. 6D).

Discussion

The availability of highly totipotent cells as a transformationtarget is the basic requirement for a successful and efficientgene transfer procedure. To date, protocols for gene transfer inM. truncatula have been mostly developed on the basis ofregenerable embryogenic and organogenic tissues (reviewedin Atif et al. 2013). Prior to this study, cell suspension cultureshave not been used as a source of totipotent cells targeted forgene transfer.

The presence of acetosyringone in the induction medium at25 μM was a key factor in maximizing the transformationefficiency, producing greater than 5.5-fold increase in the

Figure 4. Screening fordetection of green fluorescence:(A) An example of GFP positivecallus after transformation withthe vector pK7FWG2 carryingCyclin F-box-GFP after secondpassage on selective CIMN, (B)nontransformed suspensionculture. Scale bars 1 cm.

Figure 5. Expression of theGUS marker gene driven by theLAX3 promoter. (A) Induction ofGUS activity in embryogeniczones, (B) strong GUS signals indeveloped embryos, (C) GUSactivity in cotyledonary stageembryo. Scale bar 1 cm.

154 IANTCHEVA ETAL.

number of transformed callus structures for all three constructs,as compared to the experiments without acetosyringone,and around 2.0-fold increase, compared to supplementa-tion at 50 μM.

The stimulatory effect of acetosyringone on plant pathogenrecognition and transformation frequency has been well doc-umented in soybean (Olhoft et al. 2003; Paz et al. 2004), azukibean (Yamada et al. 2001), cowpea (Raveendar andIgnacimuthu 2010), and other plant species (Sheikholesmanand Weeks 1987; Raja et al. 2010). The efficiency ofAgrobacterium-mediated genetic transformation has been re-lated to the activity of virulence (vir ) genes in the Ti-plasmid

of A. tumefaciens . Generally, phenolic compounds likeacetosyringone markedly stimulate vir gene induction andare crucial for efficient T-DNA delivery (Lai et al. 2006).The concentration of acetosyringone in the inoculationand co-cultivation media needs to be optimized for eachexplant type and species. In this study, the highesttransformation efficiency (with 25 μM acetosyringone)generated a large number of GFP-positive callus struc-tures, resulting in 55–77 regenerated plantlets per 4 mLstarting culture.

Another major factor, contributing to the improved transfor-mation efficiency, was the early and uninterrupted selection oftransformed cells and small cell clusters. The selective pressurewas imposed 48 h after inoculation of the suspension culturewith Agrobacteria . The application of the selective agentinhibited or precluded division and growth of nontransformedcells and clusters. Large-scale screening for GFP signals aftertwo subsequent subcultivations (40 d in total) on selectiveCIMN allowed visualization of the callus structures with strongfluorescence. Only positive, transformed cell clusters continuedto develop further. The number of callus structures with strongexpression of the reporter gene varied from 39 to 50 among thetransformation experiments (three independent experimentswith three repetitions). The output of every 4 mL transformedcell suspension culture was a high number of independenttransformation events. The study by Iantcheva et al. (2009)

Figure 6. In vitro plants and T1

progeny of transformed plantsexpressing GUS and GFP. (A–C)GUS activity under control of theGRAS transcription factorpromoter in roots (A), leaves andstems (B) of in vitro-derivedplants; (C) GUS activity in T1progeny seedling; (D) GFPlocalization in the nucleus of rootepidermal cells of T1 progenyseedling.

Figure 7. PCR screening of DNA samples from randomly selected T0

(A) and T1 (B) transgenic M. truncatula plants for the presence of nptIIgene. 1–8 in (A): positive T0 plants; 1–10 in (B): positive T1 plants; cnegative control; c+ positive control.

MEDICAGO CELL SUSPENSION TRANSFORMATION 155

indicates that each callus could be considered as an independenttransformation event and some of the regenerated plants de-rived from the same callus may possess different transpositionprofiles indicating separate transformation events. Although wewere able to regenerate plantlets from each GFP-positive callus,the conversion rate was about 55–65% vigorous plants formingroots on selective medium. Summarizing all data obtained, weconclude that the established transformation protocol can beconsidered highly efficient, producing 55–77 transgenic plantsper 4 mL suspension culture, thereby offering the possibility fora large-scale production of independent transformed plantletswithin a period of 4 mo (120 d).

Over the last decade, the trend in the development oftransformation protocols for legumes has tended to shortenor eliminate the tissue culture step. The present protocolrequires some experience in growing and maintaining cellsuspension cultures, but the transformation step itself is verystraightforward and rapid, which gives an opportunity totransform multiple constructs concurrently. Detailed descrip-tion of summarized transformation/regeneration protocols(Chabaud et al. 2007) offers procedures starting from leafexplants of three different genotypes of M. truncatula(Jemalong cultivars ‘2HA’, ‘M9-10a’, and ‘R108-1-c3’).Production of the transgenic plants took 4–5 mo and resultedin construction of several independent transgenic lines startingfrom at least 24 leaf explants. The transformation procedureproposed in the present work requires very small amount ofstarting suspension culture (4 mL) for production of a largenumber of stable transgenic plants. Furthermore, early selec-tion prevented production of nontransgenic plants enablingmore efficient use of resources. In addition, the cell suspen-sion can be easy maintained for long periods by regularsubculturing. Thus, this protocol appears to confer a signifi-cant advantage over the existing labor-intensive and moretime-consuming transformation protocols for M. truncatulaand could become a powerful tool for future functional geno-mics studies of this legume species.

In summary, we have developed an optimized protocol forefficient cell suspension transformation, resulting in stablytransformed, fertile M. truncatula plants. In general, cellsuspension culture is a valuable tool for examining a varietyof plant physiological, biochemical, cellular, and moleculartraits. The development and optimization of advancedfunctional genomic techniques for the model legume M.truncatula along with the improved cell suspension culture-mediated protocol for transformation provides opportunitiesto characterize specific gene functions and genetic interactionsthat control key steps in legume plant development. Anotheradvantage of the present study is that cell suspension culturescontain a relatively homogeneous cell population and thetransformed single cells and cell clusters allows combiningmolecular and cell biology approaches with high-throughputimaging methods.

Acknowledgments This study was supported by a grant from theMinistry of Education and Science of Republic Bulgaria (projectDo 02–268). The authors are grateful to Kety Krastanova for valuabletechnical assistance.

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