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Plant Science 228 (2014) 150–158

Contents lists available at ScienceDirect

Plant Science

j ourna l ho me pa g e: www.elsev ier .com/ locate /p lantsc i

enetic transformation of Ornithogalum via particle bombardmentnd generation of Pectobacterium carotovorum-resistant plants

lexander Lipsky, Avner Cohen, Aurel Ion, Iris Yedidia ∗

epartment of Ornamental Horticulture, ARO, The Volcani Center, Derech Hamacabim 20, P.O. Box 6, Bet Dagan 50250, Israel

r t i c l e i n f o

rticle history:vailable online 12 February 2014

eywords:lower bulbsrnithogalumarticle bombardmentectobacterium carotovorum subsp.arotovorumoft rot

a b s t r a c t

Bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the mostdevastating diseases of Ornithogalum species. No effective control measures are currently available touse against this pathogen; thus, introduction of resistant genes via genetic transformation into this cropis a promising approach. Tachyplesin I, an antimicrobial peptide, has been shown to effectively controlnumerous pathogenic bacteria, including Pcc. In this study, liquid-grown cell clusters of Ornithogalumdubium and Ornithogalum thyrsoides were bombarded with a pCAMBIA2301 vector containing a celI leadersequence fused to a gene encoding tachyplesin I, a neomycin phosphotransferase (nptII) gene that servedas a selectable marker and a �-glucuronidase (GUS) gene that served as a reporter. Selection was carriedout in the dark in liquid medium containing 80 mg/L kanamycin. Regeneration was executed in thelight after 6–14 months depending on the cultivar. Hundreds of transgenic plantlets were produced andtheir identity was confirmed through GUS activity assays. PCR and RT-PCR were used to confirm thepresence of the target, reporter and selection genes in the divergent lines of plantlets. The resistance of

the O. dubium plants to Pcc was evaluated in vitro, following infection with a highly virulent isolate fromcalla lily. Although control plantlets were completely macerated within a week, 87 putative transgenicsubclones displayed varying levels of disease resistance. During three growing seasons in the greenhouse,the transgenic O. dubium lines grew poorly, whereas the transgenic O. thyrsoides plants grew similarly tonon-transgenic plants.

© 2014 Elsevier Ireland Ltd. All rights reserved.

ntroduction

Genetic transformation of flower bulb crops allows the incorpo-ation of a limited number of valuable traits, such as resistance toarious pathogens, into elite cultivars lacking such traits. Moreover,ue to increasing concerns regarding the use and spread of pesti-ides in the environment, many formulations have been bannedrom agricultural use. In light of these limitations, biotechnologicalpproaches to disease control have become increasingly attractive.s most flower bulb crops are monocots, which are not readily

nfected by Agrobacterium, transformation mediated by particleombardment has been considered a preferred method [1]. Thisechnique has been used to transform several monocotyledonousrnamental bulb crops, including tulip [2,3], gladiolus [4,5], lily

6,7] and Ornithogalum [6,8,9].

We have focused on the biolistic transformation ofrnithogalum, a genus of bulbous perennial plants belonging

∗ Corresponding author. Tel.: +972 3 9683387; fax: +972 3 9669583.E-mail address: irisy@volcani.agri.gov.il (I. Yedidia).

ttp://dx.doi.org/10.1016/j.plantsci.2014.02.002168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.

to the subclass Monocotyledons and classified in the familyHyacinthaceae. Recently, new taxonomic categories have beenproposed for the group, resulting in the classifications of theOrnithogaloideae as a tribe in the family Asparagaceae andOrnithogalum as one of four genera in that tribe [10–12]. The genusis found through Europe, southwest Asia and Africa and includesabout 180 species [13]. Only a few species of Ornithogalum areexploited by the floriculture industry, but, over the last 20 years,those few species have become commercially important as bothcut flowers and potted plants [14,15].

Two species in particular, O. dubium and O. thyrsoides, havebecome important products of the flower bulb industries of Israel,South Africa and the Netherlands, with Israel being the major pro-ducer of propagation material. This thriving industry is severelylimited by the plant’s susceptibility to bacterial soft rot causedby Pectobacterium carotovorum (Pcc) (previously Erwinia caro-tovora subsp. carotovora). This pathogen is not only a problem for

Ornithogalum, it is also a major threat to other bulbous crops suchas Zantedeschia spp. and Hyacinthus [16]. The pathogen is an oppor-tunistic necrotroph with a wide host range. It penetrates the plantsthrough wounds or natural openings such as stomata, spreads

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hrough the apoplast and secretes cell-wall degrading enzymes,ncluding pectinases, cellulases and proteases [17–20]. Thesenzymes break down plant cell walls and eventually macerate thentire plant, allowing subsequent infection of neighboring plants.he disease may infect the crop at different stages, in the green-ouse, during storage or during transport [16,18,19]. There are noffective control measures for use against this pathogen, makingtrict sanitation measures and the use of uncontaminated propaga-ion material the only ways to control the spread of the disease. Forhis reason, there is particular interest in the use of transformationechnologies to introduce resistant genes into the affected crops.

Tachyplesin I (TPNI) is a small antimicrobial peptide isolatedrom hemocytes of the Southeast Asian horseshoe crab Tachy-leus tridentatus [21,22]. It is a strongly basic, 17 amino-acid-long2.3 kD) peptide that has been reported to inhibit the growth ofoth gram-negative and gram-positive bacteria by creating pores

n the bacterial cell membrane, as well as damaging cell wallsnd breaking down the DNA of the bacterium [21,23]. Naturalnalogs of tachyplesin have been isolated and their amino acid anducleotide sequences have been determined [23]. The 3-D structuref tachyplesin, a �-sheet that is held together by double disulfideonds, was found to be necessary for its destructive activity [23].PNI was found to extend the vase-life of cut roses by destroyingdentified bacterial species in the vase [24]. Moreover, transgeniclants expressing similar cationic peptides exhibit broad-spectrumesistance to phytopathogens [25]. This was demonstrated morepecifically against soft rot disease in potatoes (Solanum tuberosum)ollowing transformation with the TPNI gene [26]. The transgenicotatoes were found to be more resistant to Pcc, but expression lev-ls of the peptide were low and the peptide could not be detectedn the tubers.

Our aim was to introduce soft rot resistance into Ornithogalumlants by particle bombardment-mediated transformation withhe TPNI gene. Since this is a monocot bulb crop, special effortas made to demonstrate the expression of the transgene in the

ulbs. Here, we report the successful genetic transformation of twornithogalum species, O. dubium and O. thyrsoides, resulting in sta-le transgenic plants that exhibited variable levels of resistanceo Pcc in specifically designed challenge infection bioassays. Theresence of the transgene was visibly demonstrated in GUS activ-

ty assays of all parts of the plant, including the bulb, and by PCRnd RT-PCR analyses of the target, selection and reporter genes.

aterials and methods

lant material and the establishment of tissue cultures in liquidedia

Chemicals were purchased from Sigma–Aldrich (St. Louis, MO,SA) unless specified otherwise. Media used for the tissue cul-

ure were purchased from Duchefa (Haarlem, The Netherlands)nless specified otherwise. Ornithogalum dubium Houtt. (breeding

ine #95/49/60), which is known to be highly susceptible to soft rotisease, and O. thyrsoides Jacq. (breeding line #00/36/1) were usedor this study. Calli were initiated from basal parts of axenic leafegments grown on agar-solidified Murashige and Skoog mediumMS, [27]) supplemented with 0.537 �M 1-naphthaleneacetic acidNAA), 8.8 �M 6-benzylaminopurine (BAP) and 3% W/V sucrose (M-06). Initial highly morphogenic cell clusters of both plant speciesere generated from the calli by gently separating each callus into

ragments of ∼2–4 mm. The fragments (1 g FW) were placed in

0 mL of liquid M-206 in 120-mL, wide-mouth Erlenmeyer flasks.he pH of the media was adjusted to 5.6–5.7 before autoclaving at21 ◦C for 20 min [6]. Nearly homogenous liquid grown cell clusterseveloped and were cultivated on a rotary shaker (100 rpm) that

228 (2014) 150–158 151

was kept in the dark at 25 ◦C. Subculturing was performed everytwo and four weeks for O. dubium and O. thyrzoides, respectively.Two shorter growth cycles of (10 and 20 days, respectively) wereexecuted before bombardment.

Transformation vectors and particle bombardment

A chimeric gene with the 5′ leader sequence of a celI signal pep-tide from Arabidopsis thaliana [NCBI accession no. Q9CAC1(1–24)]fused to an in-frame sequence encoding the amino-acid sequenceof the original tachyplesin I peptide [38] (accession no. P14213)was synthesized by Entelechon GmbH (Regensburg, Germany).The sequence was optimized according to the codon usage of Lil-ium, a close relative of Ornithogalum. Two restriction sites wereadded, BamHI at the 5′ end of the chimeric gene and KpnI at the3′ end [28]. The following promoters were used: the Arabidopsisconstitutive polyubiquitin promoter (UBQ3) and the constitutivestrawberry vein-banding virus-deleted promoter (�SVB, accessionno. AF331666).

The promoters and target gene were cloned into a pCAM-BIA2301 vector containing the neomycin phosphotransferase gene(nptII), which confers kanamycin resistance, and a �-glucuronidasegene (uidA, GUS) to serve as a reporter, both driven by theCauliflower mosaic virus CaMV35S promoter. A nopaline synthase(NOS) terminator was first cloned into pCAMBIA 2301 KpnI-EcoRIsites to create pCAMBIA-2301-NOS. The �SVB or UBQ3 promoterwas then cloned into the HindIII-BamHI sites of the pCAM-BIA 2301-NOS to create pCAMBIA2301-�SVB-NOS and pCAMBIA2301-UBQ3-NOS. Finally, the chimeric celI-tpnI gene was clonedinto the BamHI-KpnI sites of pCaMBIA 2301-�SVB-NOS or pCaM-BIA 2301-UBQ3-NOS to create pCaMBIA 2301-�SVB-cel1-tpnI andpCaMBIA 2301-UBQ3-celI-tpnI, respectively. Standard procedureswere employed for DNA restriction, sub-cloning and propagationin Escherichia coli. The constructs were verified by restriction anal-ysis. The restriction enzyme cleavage sites and the relevant portionof the transformation plasmid pCAMBIA 2301 containing the celI-tpnI gene are shown in Fig. 1. The plasmid DNA for the particlebombardment was isolated from E. coli strain DH5�, purified usinga plasmid Midi-Kit (Qiagen, Valencia, CA, USA) and concentrated to1 �g/�L prior to bombardment. A ratio of 1 �g DNA to 0.7 mg goldparticles in 10 �L ethanol was used for each shot.

Liquid cultures of O. dubium and O. thyrsoides were filtered andcompetent cell clusters (2–3 g) were placed on wet filter paperdiscs in 55-mm Petri dishes. Bombardment was carried out usinga particle in-flow gun under a partial vacuum of about 12.6 psi(650 mmHg) that was assembled following the model developedby Finer [29], with adaptations as described by Gray [30] and addi-tional modifications for increased safety. Gold particles (1.5–3 �m)were sterilized and coated with the appropriate constructs and thenused as DNA microcarriers. The particles were directed toward thecells with helium at a pressure of 80 psi from a target distance of18 cm. Three samples (three biological repeats) of each plant typeand construct were taken for bombardment and each of these sam-ples was bombarded twice and then divided into four subcultures(replicates). After bombardment, the paper discs with the clusterswere placed on agar-solidified M-206 medium without any selec-tive agent and kept there for 72 h. After that period, the cell clusterswere harvested from the discs and transferred to the selective liq-uid medium. Non-bombarded cell clusters of the two Ornithogalumspecies served as controls.

Selection in kanamycin liquid medium, growth and regeneration

Seventy-two hours after bombardment with the plasmid DNA,the cell aggregates were re-cultured in 20 mL of M-206 liquidproliferation medium supplemented with 80 mg/L kanamycin.

152 A. Lipsky et al. / Plant Science 228 (2014) 150–158

GUS

HindIII

CaMV35S nptII cel1-tpn1 NOST-Border (L)

CaMV35ST-Border (R)

BamHI KpnI EcoRI

NOS polyA

ΔSVB UBQ 3

Promoter

Fig. 1. Map of the relevant portions of the transformation plasmids, pCaMBIA2301-�SVB-celI-tpnI and pCaMBIA2301-UBQ3-cel1-tpn1. (The two plasmids differ only in theidentity of their promoter sequences.) The left (L) and (R) borders are indicated. nptII, the coding sequence of the neomycin phosphotransferase II gene; CaMV 35S, theCaMV 35S promoter sequence; �SVB, the strawberry vein-banding virus-deleted promoter (accession no. AF1666); UBQ3, the Arabidopsis thaliana polyubiquitin promoter;c ion non minatc

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Ap

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elI-tpnI, the 5′ leader sequence of a celI signal peptide from Arabidopsis [NCBI accesso. P14213); GUS, the �-glucuronidase GUS reporter (uidA) gene; NOS polyA, the terleavage sites are also indicated.

he cultures were placed on a rotary shaker (100 rpm) for pro-onged selection in darkness. Control samples were cultured inhe same media with and without kanamycin (negative andositive control, respectively). Subculturing into fresh mediaas carried out at approx. 2-week intervals. Transformed O.

ubium and O. thyrsoides aggregates were kept in the prolifera-ion medium for 2–3 or 9–12 months, respectively, to allow forhe development of critical biomass prior to the transfer of theggregates to regeneration medium exposed to light. Each clus-er contained dividing transgenic cells, as well as non-transgenicells.

Following the extended selection period, the dark-grownultures were transferred to the same medium for shoot-egeneration. The cultures were kept in the light (16-h photoperiod,0 �mol m−2 s−1 provided by fluorescent light bulbs) on a rotaryhaker (100 rpm) at 25 ◦C for 2–3 months. In the final stage, thehoot clusters were transferred to Petri dishes containing agar-olidified M-206 or MS medium with 3% sucrose and 80 mg/Lanamycin for continued shoot selection, growth and development.eveloped shoots were transferred to baby food jars containing MSedium with 3% sucrose supplemented with 50 mg/L kanamycin

r without the antibiotic for the controls.

ssessment of soft-rot bacteria resistance of putative transgeniclants

Pectobacterium isolate Pcc13 from calla lily (Zantedeschiaethiopica) was isolated using crystal violet polypectate (CVP)edium, as described [31]. We chose to use this bacterial isolate for

he resistance experiments, since it was isolated from a monocotlant and displayed a level of virulence toward O. dubium that wasigh, but not as high as that of the extremely virulent Pc1 isolated

rom Ornithogalum, which completely macerated all plantlets [20].ingle colonies were isolated on Luria-Bertani (LB) medium andtored in 20% glycerol at −80 ◦C. Bacteria were cultured overnightn LB medium, centrifuged, washed and re-suspended in PBS bufferpH 7.2) to a concentration of 1.5 × 107 cfu/mL. Of these, 2-mLliquots were mixed with 1 mL of 2% warm agar. The warm liquidixture was poured over 90-mm Petri dishes containing half-

trength solid MS medium to fully cover each plate. Uniform-cm-long leaf segments were excised from putative and controlwild-type) plantlets and immersed in the agar at a 45◦ angle (upperart of the leaf on top), 10 segments per plate. The plates were thenransferred to a growth chamber, where they were kept for up to0 days at 24 ◦C with a 16/8-h (day/night) photoperiod and moni-ored every few days. In some cases, to reconfirm bacterial virulenceollowing 21 days in the growth chamber, fresh control leaf seg-

ents were added to the original plates containing the survivingutative TPNI leaf segments. Overall, we evaluated the level of Pccesistance in 87 different putative transgenic plantlets (subclones)erived from the 20 original transgenic lines. We also evaluated the

. Q9CAC1(1–24)] and an in-frame sequence of tachyplesin I peptide ([38], accessionor region of the nopaline synthase gene from Agrobacterium tumefaciens. Enzymatic

level of Pcc resistance among 20 non-transformed control plantlets.For each transgenic plant line, at least 20 leaf segments from fivedifferent plantlets were tested. The results are presented as the per-centage of leaf segments surviving in the presence of the bacteriumfollowing 14 days of incubation.

Histochemical staining of GUS activity

Following the transformation, fresh plant tissue samples wereassayed at each developmental stage (i.e., cell clusters, embryos, tis-sue culture plantlets and mature plants grown in the greenhouse).Transient GUS expression was evaluated 48 h after bombardmentby staining 50 mg of tissue (0.1% of the bombarded tissue) and ana-lyzing blue GUS-expressing foci. Blue spots were counted in thetissue samples using a binocular microscope and calculated pergram of cell culture for each of three biological repeats of the twopromoters and the two Ornithogalum species. GUS activity was alsoassayed in different plant tissues, including leaves, roots and bulbs.Similar non-transformed tissues were used as controls. During theselection period, GUS activity was monitored every 2–3 weeks insamples from the liquid-grown cultures, 8–10 cell clusters in totaland 5–6 initial embryos of O. dubium from the three biologicalrepeats. Since O. thyrsoides developed more slowly during the selec-tion period, only five samples were taken for each biological repeatin that species. Once both O. dubium and O. thyrsoides plantlets hadbeen regenerated, about 12 plantlets were sampled for GUS reac-tion from each of the three biological repeats. Only two to fourof those plantlets were tested for GUS reaction in bulb tissue. Thedifferent samples were briefly washed in GUS reaction buffer pre-pared by mixing equal amounts of two solutions: (a) 75 mM sodiumphosphate buffer, pH 7.0; 50 �M potassium ferrocyanide; 50 �Mpotassium ferricyanide; 0.1% Triton X-100, 20% methanol and (b)2 mM X-Gluc (5-bromo-4-chloro-3-indole-�-d-glucuronide) dis-solved in 1.5% DMSO. The tissues were then incubated in the GUSreaction buffer at 37 ◦C overnight. After incubation, the sampleswere washed with ethanol:acetic acid (3:1) on a shaker, to clearthe chlorophyll. Finally, the samples were rinsed with 70% ethanol,photographed and stored at 4 ◦C.

Verification of transformation by PCR and RT-PCR

Putative transgenic plants were chosen for PCR and RT-PCRanalysis according to their divergence during the transformationprocedure. Overall, 12 genetically distinct O. dubium lines and 8lines of O. thyrsoides were tested. These lines were cloned andsubcloned for further analyses. Total DNA was isolated from leafsegments of putative transgenic plantlets and control plantlets

regenerated from non-bombarded explants. This DNA was sub-jected to PCR analysis carried out according to the protocolpresented by Fulton et al. [32]. Approximately 500 ng of DNA wereused as template in a total volume of 20 �L reaction mixture, which

A. Lipsky et al. / Plant Science 228 (2014) 150–158 153

Fig. 2. Development of putative transgenic O. dubium (A, C and E) and O. thyrsoides (B, D and F) plants via bombardment with pCAMBIA2301 vector. (A and B) Tissue cultureso 3 moi tlets oO d grow

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f aggregated cell clusters in liquid selective medium containing 80 mg/L kanamycinn selective medium, following exposure to light. (E and F) Putative transgenic plan. dubium and O. thyrsoides plants in the greenhouse during the second and the thir

ontained 1 �L of each primer (5 �M), 0.8 �L of dNTP mix (5 mM),.0 �L buffer (10×) with MgCl2 (15 mM), 1.5 �L DMSO and 0.15 �Laq DNA polymerase (5 u/�L, JMR Holding, London). The cyclingrogram began with an initial hot start at 95 ◦C for 2 min, followedy 35 cycles of denaturation (94 ◦C, 1 min), annealing (57 ◦C, 45 s)nd extension (72 ◦C, 1.5 min) and a final extension at 72 ◦C for

min. PCR products were separated on 1% agarose gel and visu-lized by staining with ethidium bromide.

The presence of the nptII gene was confirmed by PCR using nptII-pecific primer sequences: forward (5′–3′) GCC CCT GAT GCT CTTGT CCA GAT C and reverse TCG GCT ATG ACT GGG CAC AAC AGA C,hich are expected to yield a 434-bp PCR product. The presence of

he uidA gene (GUS) was confirmed using specific primers: forward5′–3′) GAC GGC CTG TGG GCA TTC AGT CTG G and reverse GTG TAGGC ATT ACG CTG CGA TGG A, which are expected to yield a 487-p PCR product. The presence of UBQ3 promoter and the signaleptide – target gene celI-tpnI were confirmed by the combinationf F1 – (5′–3′) CTC TTA CGC CTC TTG ATT TGG and R1-GCG GAT AACAT TTC ACA CAG G, which is expected to yield a 547-bp product.he presence of the �SVB-promoter celI-tpnI was confirmed with

specific F2 upstream primer, (5′–3′) AGT GGT CCA CAA GAC GCATC AG, and the same reverse primer (R1), resulting in a 723-bproduct (including part of the �SVB sequence). The combination of3 (5′–3′) CAT GAC TAT CGG GAT GCG CTC AG and the same reverserimer (R1) yielded a 392-bp product common to both constructs.he primers were synthesized by IDT, Inc. (Coralville, IA, USA).

For the RT-PCR analysis, total RNA was isolated from leafegments (100 mg) of different putative transgenic and con-rol plantlets and plants, using a ZR Plant RNA MiniPrep kitZymo Research, Orange, CA, USA) according to the manufac-

urer’s instructions. Crude RNA was treated with RNase-free DNaseInvitrogen, Carlsbad, CA, USA) to remove any residual DNA andurther cleaned using a ZR column. For cDNA synthesis, total RNA1 �g) was reverse-transcribed in a 20-�L reaction mixture using

nths after bombardment. (C and D) Final regeneration of putative transgenic shootsn solid MS selective medium containing 50 mg/L kanamycin. (G and H) Transgenic

ing seasons, respectively.

the Fermentas RevertAid first-strand cDNA synthesis kit (Fermen-tas, Burlington, Ottawa, Canada) according to the manufacturer’sinstructions. A 1.0-�L cDNA sample from the reverse-transcriptionreaction was used for the PCR analysis. The same primer combina-tions were used for the PCR and the RT-PCR analyses. The followingcycling program was used: one cycle of 95 ◦C for 3 min, followed by30 cycles of 94 ◦C for 30 s, 57 ◦C for 30 s, 72 ◦C for 1 min and a finalextension at 72 ◦C for 10 min. The RT-PCR products were separatedby electrophoresis on a 1.5% agarose gel stained with ethidium bro-mide and photographed using a MiniBIS Pro Bio-Imaging System(DNR Bio-Imaging Systems, Jerusalem, Israel).

Results

Transformation and selection on kanamycin media

The analysis of GUS-expressing foci in competent cell cul-tures following the particle acceleration-mediated transformationresulted in thousands of cells per gram of cell culture showing tran-sient GUS expression 48 h after bombardment for both promotersand both Ornithogalum species (data not shown). Stable expressionwas detected after 6 days. Two constitutive promoters were testedin both O. thyrsoides and O. dubium, the Arabidopsis polyubiquitin(UBQ3) and the �SVB promoter that controls the expression of thechimeric celI-tpnI gene (Fig. 1).

The key to successful and efficient transformation was the pro-duction of competent tissue with a high regeneration capacityfollowing bombardment (Fig. 2A). The highly regenerable tissuewas obtained from calli induced on leaf explants, which were grown

in liquid M-206 media in darkness to form competent cell clustersprior to bombardment. Two shorter growth cycles before bombard-ment increased the rate of cell division and the rate of regenerability(data not shown). The two species, O. dubium and O. thyrsoides,

154 A. Lipsky et al. / Plant Science 228 (2014) 150–158

Fig. 3. GUS expression in transgenic O. dubium plants at different developmental stages. (A) Cell clusters 6 days after bombardment. (B) 70-day-old pro-embryogenic cellcluster developed in liquid selective medium containing 80 mg/L kanamycin in the dark. (C) Embryos initiated from liquid cultures of O. dubium cell clusters, stained withGUS at 6 months after transformation. (D) Plantlets expressing GUS activity. (E) Transverse sections of putative transformed O. dubium bulbs. Leaf segments from transgenicO. dubium (F) and O. thyrsoides, (G) plants grown in the greenhouse during the second growing season. Negative, non-transformed controls are shown in the right (photos A,D

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and E) or at the bottom (photos F and G). Bars, 5 mm.

iffered in their ability to produce competent cultures. While O.ubium required only 2 months to produce uniform competent cellggregates with small clamps of de-differentiated cells (Fig. 2A), itook O. thyrsoides 6 months in the same liquid culture to form celllumps suitable for bombardment and those clumps were biggernd less amorphous than those of O. dubium (Fig. 2B). Following the

ombardment, an extended 2- to 6-month selection on kanamycinas essential for the development of transgenic meristematic cen-

ers and a gradual elimination of non-transgenic cells. O. dubiumxhibited better and earlier regeneration, with large quantities of

embryos observed as early as 1.5–2 months after the transfer tolight (Fig. 2C). In contrast, O. thyrsoides did not respond to thedifferentiation conditions before 9 months after being transferred(Fig. 2D).

Once they had developed in M-206 medium (Fig. 2E and F),the transgenic plantlets of the two species were transferred to

the greenhouse, where their further development was observed.Observations over three growing seasons revealed that the putativetransgenic O. dubium plants grew poorly, as compared to the non-transgenic plants. In contrast, the putative transgenic O. thyrsoides

A. Lipsky et al. / Plant Science 228 (2014) 150–158 155

Table 1PCR and RT-PCR analyses of the presence and expression of the transgenes, respectively, GUS reactions of control non-transformed O. dubium (Odnt), putative transgenic O.dubium lines, control non-transformed O. thyrsoides (Otnt) and putative transgenic O. thyrsoides lines, non-validated (nv), maceration caused by Pectobacterium carotovorumsubsp. carotovorum in O. dubium lines, as measured in a leaf-segment assay. Infection data for each transgenic line include an average of two subclones cultured from theoriginal line.

Treatment Line PCR RT-PCR GUS reaction Pcc maceration(%)

nptII uidA tpnI targetgene

nptII uidA tpnI targetgene

O.dubiumControl Odnt1 − − − − − − − 80

Odnt2 − − − − − − − 90Odnt3 − − − nv nv nv − 100

Putative transgenic E1d11-42/3 (19) + + + + + + + 40E1d21-31/2 (32) + + + + + + + 25E2d10-61/6 (57) + − − nv nv nv − 80E2d11-31/5 (68) + + + + + + + 25E2d20-2/8 (81) + + + nv nv + − 25E3d10-24/8 (94) + + + + + + − 20E3d21-31/4 (129) + + + + + + − 60E1u11-33/1 + − + nv nv nv − 40E1u20-52/2 (168) + + + + + + − 0E2u20-41/2 (210) + + + + + + + 35E3u11-22/8 (245) + + + + + + + 40E3u21-1/3 (250) + + + + + + + 15

O. thyrsoidesControl Otnt1 − − − − − − −

Otnt2 − − − − − − −Putative transgenic Et1d11/1 (18) + + + + + + ±

Et1d20 (19) + + + + + + +Et2d20/1 (20) + + + + + + +Et3d11/1 (21) + + + nv nv nv −Et1u11 (22) + − − nv nv nv −

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Et1u20 (23) + + +

Et2u2c (24) + − +

Et3u2 (25) + + −

lants displayed vigorous growth and homogenous flowering, sim-lar to the non-transgenic control plants (Fig. 2G and H).

US analysis at different stages of plant development

The identity of putative transgenic individuals was confirmedrst using GUS activity assays and later through molecular char-cterization. GUS analysis was performed at all stages of plantevelopment and in different plant tissues (Fig. 3). Stable GUSxpression was observed in selective liquid culture 6 days afterombardment (Fig. 3A) and in 70-day-old embryogenic clustersFig. 3B). The embryos that grew from the clusters exhibited GUStaining in all of their parts after 6 months and this staining waslso observed in plantlets and in transverse sections of bulbs readyor planting in the greenhouse (Fig. 3C and E).

About 50% of all of the putative transgenic plantlets expressedUS activity. A positive GUS reaction in the bulbs of developedlants confirmed the presence of the transgene in the most vulnera-le tissue of the plant (i.e., the storage organ). Stable GUS expressionas also observed in the leaves of mature plants grown in the

reenhouse during the second growing season (Fig. 3F and G). NoUS activity was observed in any plant tissue that had not beenombarded.

CR and RT-PCR verification of transgenic Ornithogalum

Putative transformants derived from kanamycin-resistant cul-

ure lines were verified by PCR and RT-PCR. PCR was performedith gene-specific primers for nptII, uidA and the target gene celI-

pnI under the control of the UBQ3 or �SVB promoters. The analysisielded 434-bp and 487-bp products for the nptII and uidA genes

+ + + ++ + + −+ + − +

and 547-bp and 723-bp products corresponding to the UBQ3 or�SVB promoters and target gene, respectively. The amplification ofthese products confirmed the presence of the celI-tpnI target gene.Interestingly, out of the 12 putative transgenic O. dubium lines andthe 8 putative transgenic O. thyrsoides lines that were analyzed, only10 of the O. dubium lines and 5 of the O. thyrsoides lines expressed allthree of the analyzed genes (Table 1). In both species, two putativetransgenic lines were GUS-negative and one O. dubium line and twoO. thyrsoides lines lacked the target gene. The presence of the nptIIgene was confirmed in both Ornithogalum species and in all of theputative transgenic lines analyzed, indicating the efficiency of theselection procedure (Fig. 4A and B). No band of any of the enhancedgenes was present in the non-transformed control plants.

To further confirm the expression of the transformed genes atthe RNA level, the same primer combinations for each of the twogenes, nptII and uidA, were used for RT-PCR analysis of putativetransgenic lines and the non-transgenic controls (Fig. 5A and B). Theexpression of the target gene celI-tpnI at the RNA level was testedwith the primer combination F3 and R1, which yielded a 392-bpproduct common to the two constructs used for the transformation.Six putative transgenic O. dubium and 6 O. thyrsoides plants wereanalyzed and tested positive for all three transgenes at the RNAlevel, except for one of the putative O. thyrsoides plants, which wasnegative for the celI-tpnI. No RNA product was recovered from thenon-transformed control plants.

Resistance to bacterial soft rot in the transgenic plants

Putative transgenic plantlets were challenged with Pcc duringdifferent stages of growth in tissue culture. Leaf segments fromputative transgenic plants (10 cm) were immersed in Petri dishes

156 A. Lipsky et al. / Plant Science 228 (2014) 150–158

Fig. 4. Molecular characterization of transgenic Ornithogalum. (A) PCR analysis ofthe nptII gene, uidA gene and celI-tpnI target gene and assay of GUS activity in thosesame O. dubium plantlets. (B) PCR analysis of those genes and GUS activity assay in O.thyrsoides plantlets. Lanes (left to right): 1-kb DNA ladder, M; plasmid control targetglp

casprtTtitwal(w

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Fig. 6. Survival of leaf segments harvested from control (C1 on the left) or puta-tive transgenic O. dubium plantlets following 14 days of an infection assay withP. carotovorum subsp. carotovorum. Bars represent the percentage of infected leafsegments + SE, as determined by a one-way analysis of variance with post hoc pair-

ene driven by �SVB promoter, pdt; non-transgenic control, C; putative transgenicines, lanes 4–8; plasmid control with target gene driven by the UBQ3 promoter,ut.

ontaining solid MS covered with a mixture of virulent Pcc and softgar. The data are presented as the percentage of macerated leafegments out of a total of 10 leaf segments per plate. Twenty controllantlets and 87 putative transgenic plantlets were assessed for Pccesistance in the leaf-segment assay. Different levels of resistance tohe pathogen were observed, from highly resistant to susceptible.he average maceration of the control was 84%, while the puta-ive transgenic lines exhibited 16.7–70% maceration. Nonetheless,n most cases, the putative transgenic plants were more resistanthan the non-transformed controls (Fig. 6). While control plantsere completely macerated after 7 days in the assay plates (Fig. 7A

nd B), a few transgenic lines (#32, #94 and #168) displayed high

evels of resistance to the pathogen even 21 days after inoculationFig. 7C and D). A test to confirm the virulence of the bacteriumas performed following 21 days in the assay plates. In this test,

ig. 5. (A) RT-PCR analysis of the nptII gene, uidA gene and celI-tpnI target gene inutative transgenic O. dubium plants. (B) RT-PCR analysis of those same genes inutative transgenic O. thyrsoides plants. Lanes (left to right): 100-bp DNA ladder, M;on-transgenic control, C; putative transgenic lines, lanes 3–8.

wise multiple comparisons (Dunnett’s test). Asterisks indicate significantly differentgroup means at an alpha level of 0.05.

fresh, non-transformed leaf segments (controls) were introducedinto the infection plates. The control leaf segments were completelymacerated within 2 days, while the putative transgenic segmentsremained green (not shown).

Discussion

Control of plant pathogens such as soft rot bacteria is one of themost challenging problems facing modern agriculture. Losses mayoccur during production or storage. Bulb and tuber crops, includ-ing highly valuable ornamentals and food crops such as potato,are particularly susceptible to soft rots, due to their specializedunderground storage organs. Bacteria from the genus Pectobac-terium are found worldwide and have an extremely broad hostrange, reflecting their temperature tolerance and different viru-lence mechanisms [18,33]. As with most bacterial necrotrophs, noeffective control measure is currently available for use against bac-terial soft rots and the only way to manage these diseases is throughstrict sanitation measures and the use of clean propagation material[19,34].

The aim of the present study was to employ a biotechnologicalapproach to reduce pathogen pressure in a highly sensitive flowerbulb crop. Previous studies have reported the potential abilitiesof several cationic antimicrobial peptides, including TPNI, to con-fer broad-spectrum resistance against bacterial phytopathogens[25]. TPNI small antimicrobial peptide was originally isolated fromhemocytes of the Japanese horseshoe crab (T. tridentatus) and hasproven effective against a variety of bacteria in vitro [22]. TPNIhas been described as an effective antimicrobial compound for useagainst various Erwinia (Pectobacterium) species. More specifically,its expression in transgenic potatoes conferred resistance againstsoft rot [26]. For this reason, TPNI was chosen as the target gene tobe introduced into two Ornithogalum species by particle bombard-ment.

The particle acceleration-mediated transformation was basedon a procedure described for the transformation of Lilium [35] andOrnithogalum to confer resistance to Ornithogalum mosaic virus

[6,8]. Following selection in kanamycin-containing media, a rel-atively high proportion of transformed plants was obtained, morethan 50% of the putative transgenic plantlets exhibited GUS activityand all were nptII-positive (as determined by PCR amplification of

A. Lipsky et al. / Plant Science 228 (2014) 150–158 157

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ig. 7. Disease resistance assay of O. dubium leaf segments following challenge witt time zero post-inoculation (dpi). (B) Non-transgenic control infected with Pcc, 11romoter, infected with Pcc, 21 dpi. (D) Putative transgenic clone #168, in which th

5 putative transgenic clones). These results can be attributed towo factors: (1) the successful development of highly regenerable,ompetent Ornithogalum cell cultures and (2) a prolonged selec-ion process under selective conditions in media supplementedith 80 mg/L kanamycin and kept in the dark. This approach min-

mized the probability of the development of non-transgenic cellshat would yield non-transgenic embryos and plants.

The two Ornithogalum species in the present study displayed dif-erent levels of susceptibility to the soft rot bacterium, O. dubiumas more susceptible (unpublished data). The high susceptibility

f O. dubium was to a great extent compensated for by its bet-er performance under tissue-culture conditions. While O. dubiumequired less time (2 months) to produce consistent competent cellultures for bombardment, O. thyrsoides required at least 6 monthsn liquid and the O. thyrsoides cultures were less homogeneous.ollowing the bombardment and incubation under selective con-itions, O. dubium produced large quantities of embryos as earlys 1.5–2 month after being transferred to an environment thatncluded light, while O. thyrsoides did not respond to the differ-ntiation conditions before 6–9 months had passed.

Once developed, the putative transgenic lines of the two speciesere planted in the greenhouse to evaluate their growth and phe-otypes under greenhouse conditions. Unlike in tissue culture,uring three growing seasons, transgenic O. thyrsoides plants per-ormed similarly to the non-transgenic control plants, exhibitingigorous growth and high flowering potential, whereas O. dubiumransgenic plants grew poorly relative to the control.

The presence of the three genes, nptII, uidA and the chimericelI-tpnI target gene, was confirmed by PCR analysis of 12 O. dubiumlantlets and 8 O. thyrsoides plantlets. The presence of the nptII geneas confirmed by PCR in both Ornithogalum species in all analy-

es, most likely due to the prolonged selection in a relatively high

oncentration (80 mg/L) of the selective agent (kanamycin). Theelection procedure was also responsible for the high rate of puta-ive transgenic plants that reacted positively to the presence of thearget gene (85%) and the uidA gene (80%). Although the presence

rotovorum subsp. carotovorum (Pcc). (A) Non-transgenic control infected with PccC) Putative transgenic clone #94, in which the transgene is controlled by the �SVBsgene is controlled by the UBQ3 promoter, 21 dpi.

of the uidA gene was confirmed, GUS activity was observed in only55% of these putative transgenic plants, implying that confirmationby PCR does not always indicate the activity of the transgene andthat a lack of GUS activity does not guarantee that the plant is nottransgenic. The RT-PCR analysis of the putative transgenic plantsconfirmed that not only was DNA successfully incorporated into theplant’s genome, but the transgene was also expressed at the RNAlevel. These results were in complete agreement with those of aprevious study in which cationic peptide chimeras were expressedin potatoes to produce broad-spectrum resistance against bacterialand fungal phytopathogens [25].

The resistance of the putative transgenic plants to bacterial softrot was tested by challenging the plants with Pcc. Several assayshave been developed to screen for resistance during different stagesof plant development [28,36]. In the present study, putative trans-genic plants (87 clones derived from 20 lines) and non-transformedcontrols (20 plants) were tested for both a GUS reaction and resis-tance to soft rot in a leaf segment bioassay carried out over 21days.

No GUS activity was observed in any of the control plants,whereas 64% of the putative transgenic plants displayed a posi-tive GUS reaction. In some of these putative transgenic plants, theGUS reaction was specifically tested in the bulbs to confirm theexpression of the transgene in that critical plant tissue. In a studyinvolving potato, transgene expression was detected in leaves, butnot in underground storage organs (tubers) [26]. In contrast, in thisstudy, a positive GUS reaction was observed in the bulbs, suggestingthat the target gene could also be expressed in the storage organ.

In agreement with the varied results of the GUS assays, diseaseresistance was also highly variable among the putative transgenicplants. The different levels of resistance conferred to the trans-formed plants by the transgenes may be the result of a number

of factors, three of which may be of great importance. First, TPNIhas a cyclic antiparallel �-sheet structure that is maintained bytwo disulfide bridges that are essential for its activity. The peptideeffectively increases the permeability of bacterial membranes, but

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oes so only in its cyclic form [23]. Second, in order to maximizets antimicrobial potential, the peptide has to be secreted into thextracellular space, where Pcc proliferates and activates most ofts virulence factors during infection. Third, the peptide has to beranslated at a sufficient level to allow for efficient antimicrobialctivity.

These issues were explored by fusing a signal peptide from Ara-idopsis celI [37] to TPNI [38], to allow the peptide to be secretednto the extracellular space and the natural folding of the peptidento its active �-sheet structure [25]. Some of the variability in theesistance of the transgenic plants may be the result of peptideolding and secretion rather than just the level of expression in theransformed plants. To control the level of expression, each of thewo constructs we used, included a constitutive promoter to con-rol the target gene: the Arabidopsis polyubiquitin promoter (UBQ3)r the strawberry vein-banding virus-deleted promoter (�SVB). Inur analysis of the disease resistance of putative transgenic linesxpressing one of these two promoters, the promoters did not seemo differentially affect the degree of disease resistance among theransgenic plants.

Disease development was monitored as the percentage of leafegments macerated by the pathogen at 7 days after infection. Morehan 80% maceration was observed in the control plants. The puta-ive transgenic plants displayed relatively lower levels of disease,ith an average maceration rate of less than 50% at 12 days after

nfection. Five clones that were produced from a transgenic linehat lacked the target gene displayed relatively high sensitivity tohe pathogen, with an average maceration rate of 80%, similar tohat of the control. Resistant lines of the putative transgenic plantsere not macerated even 21 days after inoculation, suggesting that

he resistance afforded to these plants by the antimicrobial peptides consistent and strong even in the face of strong disease pressure.n the absence of effective control measures for use against bacte-ial soft rot, the engineering of disease-resistant plants through these of naturally occurring cationic peptides appears to be an inter-sting possibility for the sustainable production of ornamental bulblants.

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