Plant Science 228 (2014) 150158

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Genetic transformation of Ornithogalum via particle bombardment

and generation of Pectobacterium carotovorum-resistant plants
Alexander Lipsky, Avner Cohen, Aurel Ion, Iris Yedidia
Department of Ornamental Horticulture, ARO, The Volcani Center, Derech Hamacabim 20, P.O. Box 6, Bet Dagan 50250, Israel

a r t i c l e

i n f o

Article history:
Available online 12 February 2014
Keywords:
Flower bulbs
Ornithogalum
Particle bombardment
Pectobacterium carotovorum subsp.
carotovorum
Soft rot

a b s t r a c t
Bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum (Pcc) is one of the most
devastating diseases of Ornithogalum species. No effective control measures are currently available to
use against this pathogen; thus, introduction of resistant genes via genetic transformation into this crop
is a promising approach. Tachyplesin I, an antimicrobial peptide, has been shown to effectively control
numerous pathogenic bacteria, including Pcc. In this study, liquid-grown cell clusters of Ornithogalum
dubium and Ornithogalum thyrsoides were bombarded with a pCAMBIA2301 vector containing a celI leader
sequence fused to a gene encoding tachyplesin I, a neomycin phosphotransferase (nptII) gene that served
as a selectable marker and a -glucuronidase (GUS) gene that served as a reporter. Selection was carried
out in the dark in liquid medium containing 80 mg/L kanamycin. Regeneration was executed in the
light after 614 months depending on the cultivar. Hundreds of transgenic plantlets were produced and
their identity was conrmed through GUS activity assays. PCR and RT-PCR were used to conrm the
presence 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 from
calla lily. Although control plantlets were completely macerated within a week, 87 putative transgenic
subclones 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 to
non-transgenic plants.
2014 Elsevier Ireland Ltd. All rights reserved.

Introduction
Genetic transformation of ower bulb crops allows the incorporation of a limited number of valuable traits, such as resistance to
various pathogens, into elite cultivars lacking such traits. Moreover,
due to increasing concerns regarding the use and spread of pesticides in the environment, many formulations have been banned
from agricultural use. In light of these limitations, biotechnological
approaches to disease control have become increasingly attractive.
As most ower bulb crops are monocots, which are not readily
infected by Agrobacterium, transformation mediated by particle
bombardment has been considered a preferred method [1]. This
technique has been used to transform several monocotyledonous
ornamental bulb crops, including tulip [2,3], gladiolus [4,5], lily
[6,7] and Ornithogalum [6,8,9].
We have focused on the biolistic transformation of
Ornithogalum, 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).
http://dx.doi.org/10.1016/j.plantsci.2014.02.002
0168-9452/ 2014 Elsevier Ireland Ltd. All rights reserved.

to the subclass Monocotyledons and classied in the family

Hyacinthaceae. Recently, new taxonomic categories have been
proposed for the group, resulting in the classications of the
Ornithogaloideae as a tribe in the family Asparagaceae and
Ornithogalum as one of four genera in that tribe [1012]. The genus
is found through Europe, southwest Asia and Africa and includes
about 180 species [13]. Only a few species of Ornithogalum are
exploited by the oriculture industry, but, over the last 20 years,
those few species have become commercially important as both
cut owers and potted plants [14,15].
Two species in particular, O. dubium and O. thyrsoides, have
become important products of the ower bulb industries of Israel,
South Africa and the Netherlands, with Israel being the major producer of propagation material. This thriving industry is severely
limited by the plants susceptibility to bacterial soft rot caused
by Pectobacterium carotovorum (Pcc) (previously Erwinia carotovora subsp. carotovora). This pathogen is not only a problem for
Ornithogalum, it is also a major threat to other bulbous crops such
as Zantedeschia spp. and Hyacinthus [16]. The pathogen is an opportunistic necrotroph with a wide host range. It penetrates the plants
through wounds or natural openings such as stomata, spreads

A. Lipsky et al. / Plant Science 228 (2014) 150158

through the apoplast and secretes cell-wall degrading enzymes,

including pectinases, cellulases and proteases [1720]. These
enzymes break down plant cell walls and eventually macerate the
entire plant, allowing subsequent infection of neighboring plants.
The disease may infect the crop at different stages, in the greenhouse, during storage or during transport [16,18,19]. There are no
effective control measures for use against this pathogen, making
strict sanitation measures and the use of uncontaminated propagation material the only ways to control the spread of the disease. For
this reason, there is particular interest in the use of transformation
technologies to introduce resistant genes into the affected crops.
Tachyplesin I (TPNI) is a small antimicrobial peptide isolated
from hemocytes of the Southeast Asian horseshoe crab Tachypleus tridentatus [21,22]. It is a strongly basic, 17 amino-acid-long
(2.3 kD) peptide that has been reported to inhibit the growth of
both gram-negative and gram-positive bacteria by creating pores
in the bacterial cell membrane, as well as damaging cell walls
and breaking down the DNA of the bacterium [21,23]. Natural
analogs of tachyplesin have been isolated and their amino acid and
nucleotide sequences have been determined [23]. The 3-D structure
of tachyplesin, a -sheet that is held together by double disulde
bonds, was found to be necessary for its destructive activity [23].
TPNI was found to extend the vase-life of cut roses by destroying
identied bacterial species in the vase [24]. Moreover, transgenic
plants expressing similar cationic peptides exhibit broad-spectrum
resistance to phytopathogens [25]. This was demonstrated more
specically against soft rot disease in potatoes (Solanum tuberosum)
following transformation with the TPNI gene [26]. The transgenic
potatoes were found to be more resistant to Pcc, but expression levels of the peptide were low and the peptide could not be detected
in the tubers.
Our aim was to introduce soft rot resistance into Ornithogalum
plants by particle bombardment-mediated transformation with
the TPNI gene. Since this is a monocot bulb crop, special effort
was made to demonstrate the expression of the transgene in the
bulbs. Here, we report the successful genetic transformation of two
Ornithogalum species, O. dubium and O. thyrsoides, resulting in stable transgenic plants that exhibited variable levels of resistance
to Pcc in specically designed challenge infection bioassays. The
presence of the transgene was visibly demonstrated in GUS activity assays of all parts of the plant, including the bulb, and by PCR
and RT-PCR analyses of the target, selection and reporter genes.

Materials and methods

Plant material and the establishment of tissue cultures in liquid
media
Chemicals were purchased from SigmaAldrich (St. Louis, MO,
USA) unless specied otherwise. Media used for the tissue culture were purchased from Duchefa (Haarlem, The Netherlands)
unless specied otherwise. Ornithogalum dubium Houtt. (breeding
line #95/49/60), which is known to be highly susceptible to soft rot
disease, and O. thyrsoides Jacq. (breeding line #00/36/1) were used
for this study. Calli were initiated from basal parts of axenic leaf
segments grown on agar-solidied Murashige and Skoog medium
(MS, [27]) supplemented with 0.537 M 1-naphthaleneacetic acid
(NAA), 8.8 M 6-benzylaminopurine (BAP) and 3% W/V sucrose (M206). Initial highly morphogenic cell clusters of both plant species
were generated from the calli by gently separating each callus into
fragments of 24 mm. The fragments (1 g FW) were placed in
20 mL of liquid M-206 in 120-mL, wide-mouth Erlenmeyer asks.
The pH of the media was adjusted to 5.65.7 before autoclaving at
121 C for 20 min [6]. Nearly homogenous liquid grown cell clusters
developed and were cultivated on a rotary shaker (100 rpm) that

151

was kept in the dark at 25 C. Subculturing was performed every

two and four weeks for O. dubium and O. thyrzoides, respectively.
Two shorter growth cycles of (10 and 20 days, respectively) were
executed before bombardment.
Transformation vectors and particle bombardment
A chimeric gene with the 5 leader sequence of a celI signal peptide from Arabidopsis thaliana [NCBI accession no. Q9CAC1(124)]
fused to an in-frame sequence encoding the amino-acid sequence
of 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 Lilium, a close relative of Ornithogalum. Two restriction sites were
added, BamHI at the 5 end of the chimeric gene and KpnI at the
3 end [28]. The following promoters were used: the Arabidopsis
constitutive polyubiquitin promoter (UBQ3) and the constitutive
strawberry vein-banding virus-deleted promoter (SVB, accession
no. AF331666).
The promoters and target gene were cloned into a pCAMBIA2301 vector containing the neomycin phosphotransferase gene
(nptII), which confers kanamycin resistance, and a -glucuronidase
gene (uidA, GUS) to serve as a reporter, both driven by the
Cauliower mosaic virus CaMV35S promoter. A nopaline synthase
(NOS) terminator was rst cloned into pCAMBIA 2301 KpnI-EcoRI
sites to create pCAMBIA-2301-NOS. The SVB or UBQ3 promoter
was then cloned into the HindIII-BamHI sites of the pCAMBIA 2301-NOS to create pCAMBIA2301-SVB-NOS and pCAMBIA
2301-UBQ3-NOS. Finally, the chimeric celI-tpnI gene was cloned
into the BamHI-KpnI sites of pCaMBIA 2301-SVB-NOS or pCaMBIA 2301-UBQ3-NOS to create pCaMBIA 2301-SVB-cel1-tpnI and
pCaMBIA 2301-UBQ3-celI-tpnI, respectively. Standard procedures
were employed for DNA restriction, sub-cloning and propagation
in Escherichia coli. The constructs were veried by restriction analysis. The restriction enzyme cleavage sites and the relevant portion
of the transformation plasmid pCAMBIA 2301 containing the celItpnI gene are shown in Fig. 1. The plasmid DNA for the particle
bombardment was isolated from E. coli strain DH5, puried using
a plasmid Midi-Kit (Qiagen, Valencia, CA, USA) and concentrated to
1 g/L prior to bombardment. A ratio of 1 g DNA to 0.7 mg gold
particles in 10 L ethanol was used for each shot.
Liquid cultures of O. dubium and O. thyrsoides were ltered and
competent cell clusters (23 g) were placed on wet lter paper
discs in 55-mm Petri dishes. Bombardment was carried out using
a particle in-ow gun under a partial vacuum of about 12.6 psi
(650 mmHg) that was assembled following the model developed
by Finer [29], with adaptations as described by Gray [30] and additional modications for increased safety. Gold particles (1.53 m)
were sterilized and coated with the appropriate constructs and then
used as DNA microcarriers. The particles were directed toward the
cells with helium at a pressure of 80 psi from a target distance of
18 cm. Three samples (three biological repeats) of each plant type
and construct were taken for bombardment and each of these samples was bombarded twice and then divided into four subcultures
(replicates). After bombardment, the paper discs with the clusters
were placed on agar-solidied M-206 medium without any selective agent and kept there for 72 h. After that period, the cell clusters
were harvested from the discs and transferred to the selective liquid medium. Non-bombarded cell clusters of the two Ornithogalum
species 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 liquid
proliferation medium supplemented with 80 mg/L kanamycin.

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A. Lipsky et al. / Plant Science 228 (2014) 150158

SVB
UBQ3
Promoter

T-Border (L)
nptII

CaMV35S

HindIII

cel1-tpn1

BamHI

NOS

KpnI

CaMV35S

GUS

T-Border (R)
NOS polyA

EcoRI

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 the
identity of their promoter sequences.) The left (L) and (R) borders are indicated. nptII, the coding sequence of the neomycin phosphotransferase II gene; CaMV 35S, the
CaMV 35S promoter sequence; SVB, the strawberry vein-banding virus-deleted promoter (accession no. AF1666); UBQ3, the Arabidopsis thaliana polyubiquitin promoter;
celI-tpnI, the 5 leader sequence of a celI signal peptide from Arabidopsis [NCBI accession no. Q9CAC1(124)] and an in-frame sequence of tachyplesin I peptide ([38], accession
no. P14213); GUS, the -glucuronidase GUS reporter (uidA) gene; NOS polyA, the terminator region of the nopaline synthase gene from Agrobacterium tumefaciens. Enzymatic
cleavage sites are also indicated.

The cultures were placed on a rotary shaker (100 rpm) for prolonged selection in darkness. Control samples were cultured in
the same media with and without kanamycin (negative and
positive control, respectively). Subculturing into fresh media
was carried out at approx. 2-week intervals. Transformed O.
dubium and O. thyrsoides aggregates were kept in the proliferation medium for 23 or 912 months, respectively, to allow for
the development of critical biomass prior to the transfer of the
aggregates to regeneration medium exposed to light. Each cluster contained dividing transgenic cells, as well as non-transgenic
cells.
Following the extended selection period, the dark-grown
cultures were transferred to the same medium for shootregeneration. The cultures were kept in the light (16-h photoperiod,
60 mol m2 s1 provided by uorescent light bulbs) on a rotary
shaker (100 rpm) at 25 C for 23 months. In the nal stage, the
shoot clusters were transferred to Petri dishes containing agarsolidied M-206 or MS medium with 3% sucrose and 80 mg/L
kanamycin for continued shoot selection, growth and development.
Developed shoots were transferred to baby food jars containing MS
medium with 3% sucrose supplemented with 50 mg/L kanamycin
or without the antibiotic for the controls.
Assessment of soft-rot bacteria resistance of putative transgenic
plants
Pectobacterium isolate Pcc13 from calla lily (Zantedeschia
aethiopica) was isolated using crystal violet polypectate (CVP)
medium, as described [31]. We chose to use this bacterial isolate for
the resistance experiments, since it was isolated from a monocot
plant and displayed a level of virulence toward O. dubium that was
high, but not as high as that of the extremely virulent Pc1 isolated
from Ornithogalum, which completely macerated all plantlets [20].
Single colonies were isolated on Luria-Bertani (LB) medium and
stored in 20% glycerol at 80 C. Bacteria were cultured overnight
in LB medium, centrifuged, washed and re-suspended in PBS buffer
(pH 7.2) to a concentration of 1.5 107 cfu/mL. Of these, 2-mL
aliquots were mixed with 1 mL of 2% warm agar. The warm liquid
mixture was poured over 90-mm Petri dishes containing halfstrength solid MS medium to fully cover each plate. Uniform
2-cm-long leaf segments were excised from putative and control
(wild-type) plantlets and immersed in the agar at a 45 angle (upper
part of the leaf on top), 10 segments per plate. The plates were then
transferred to a growth chamber, where they were kept for up to
30 days at 24 C with a 16/8-h (day/night) photoperiod and monitored every few days. In some cases, to reconrm bacterial virulence
following 21 days in the growth chamber, fresh control leaf segments were added to the original plates containing the surviving
putative TPNI leaf segments. Overall, we evaluated the level of Pcc
resistance in 87 different putative transgenic plantlets (subclones)
derived from the 20 original transgenic lines. We also evaluated the

level of Pcc resistance among 20 non-transformed control plantlets.

For each transgenic plant line, at least 20 leaf segments from ve
different plantlets were tested. The results are presented as the percentage of leaf segments surviving in the presence of the bacterium
following 14 days of incubation.
Histochemical staining of GUS activity
Following the transformation, fresh plant tissue samples were
assayed at each developmental stage (i.e., cell clusters, embryos, tissue culture plantlets and mature plants grown in the greenhouse).
Transient GUS expression was evaluated 48 h after bombardment
by staining 50 mg of tissue (0.1% of the bombarded tissue) and analyzing blue GUS-expressing foci. Blue spots were counted in the
tissue samples using a binocular microscope and calculated per
gram of cell culture for each of three biological repeats of the two
promoters and the two Ornithogalum species. GUS activity was also
assayed in different plant tissues, including leaves, roots and bulbs.
Similar non-transformed tissues were used as controls. During the
selection period, GUS activity was monitored every 23 weeks in
samples from the liquid-grown cultures, 810 cell clusters in total
and 56 initial embryos of O. dubium from the three biological
repeats. Since O. thyrsoides developed more slowly during the selection period, only ve samples were taken for each biological repeat
in that species. Once both O. dubium and O. thyrsoides plantlets had
been regenerated, about 12 plantlets were sampled for GUS reaction from each of the three biological repeats. Only two to four
of those plantlets were tested for GUS reaction in bulb tissue. The
different samples were briey washed in GUS reaction buffer prepared by mixing equal amounts of two solutions: (a) 75 mM sodium
phosphate buffer, pH 7.0; 50 M potassium ferrocyanide; 50 M
potassium ferricyanide; 0.1% Triton X-100, 20% methanol and (b)
2 mM X-Gluc (5-bromo-4-chloro-3-indole--d-glucuronide) dissolved in 1.5% DMSO. The tissues were then incubated in the GUS
reaction buffer at 37 C overnight. After incubation, the samples
were washed with ethanol:acetic acid (3:1) on a shaker, to clear
the chlorophyll. Finally, the samples were rinsed with 70% ethanol,
photographed and stored at 4 C.
Verication of transformation by PCR and RT-PCR
Putative transgenic plants were chosen for PCR and RT-PCR
analysis according to their divergence during the transformation
procedure. Overall, 12 genetically distinct O. dubium lines and 8
lines of O. thyrsoides were tested. These lines were cloned and
subcloned for further analyses. Total DNA was isolated from leaf
segments of putative transgenic plantlets and control plantlets
regenerated from non-bombarded explants. This DNA was subjected to PCR analysis carried out according to the protocol
presented by Fulton et al. [32]. Approximately 500 ng of DNA were
used as template in a total volume of 20 L reaction mixture, which

A. Lipsky et al. / Plant Science 228 (2014) 150158

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 cultures
of aggregated cell clusters in liquid selective medium containing 80 mg/L kanamycin 3 months after bombardment. (C and D) Final regeneration of putative transgenic shoots
in selective medium, following exposure to light. (E and F) Putative transgenic plantlets on solid MS selective medium containing 50 mg/L kanamycin. (G and H) Transgenic
O. dubium and O. thyrsoides plants in the greenhouse during the second and the third growing seasons, respectively.

contained 1 L of each primer (5 M), 0.8 L of dNTP mix (5 mM),

2.0 L buffer (10) with MgCl2 (15 mM), 1.5 L DMSO and 0.15 L
Taq DNA polymerase (5 u/L, JMR Holding, London). The cycling
program began with an initial hot start at 95 C for 2 min, followed
by 35 cycles of denaturation (94 C, 1 min), annealing (57 C, 45 s)
and extension (72 C, 1.5 min) and a nal extension at 72 C for
5 min. PCR products were separated on 1% agarose gel and visualized by staining with ethidium bromide.
The presence of the nptII gene was conrmed by PCR using nptIIspecic primer sequences: forward (5 3 ) GCC CCT GAT GCT CTT
CGT CCA GAT C and reverse TCG GCT ATG ACT GGG CAC AAC AGA C,
which are expected to yield a 434-bp PCR product. The presence of
the uidA gene (GUS) was conrmed using specic primers: forward
(5 3 ) GAC GGC CTG TGG GCA TTC AGT CTG G and reverse GTG TAG
AGC ATT ACG CTG CGA TGG A, which are expected to yield a 487bp PCR product. The presence of UBQ3 promoter and the signal
peptide target gene celI-tpnI were conrmed by the combination
of F1 (5 3 ) CTC TTA CGC CTC TTG ATT TGG and R1-GCG GAT AAC
AAT TTC ACA CAG G, which is expected to yield a 547-bp product.
The presence of the SVB-promoter celI-tpnI was conrmed with
a specic F2 upstream primer, (5 3 ) AGT GGT CCA CAA GAC GCA
CTC AG, and the same reverse primer (R1), resulting in a 723-bp
product (including part of the SVB sequence). The combination of
F3 (5 3 ) CAT GAC TAT CGG GAT GCG CTC AG and the same reverse
primer (R1) yielded a 392-bp product common to both constructs.
The primers were synthesized by IDT, Inc. (Coralville, IA, USA).
For the RT-PCR analysis, total RNA was isolated from leaf
segments (100 mg) of different putative transgenic and control plantlets and plants, using a ZR Plant RNA MiniPrep kit
(Zymo Research, Orange, CA, USA) according to the manufacturers instructions. Crude RNA was treated with RNase-free DNase
(Invitrogen, Carlsbad, CA, USA) to remove any residual DNA and
further cleaned using a ZR column. For cDNA synthesis, total RNA
(1 g) was reverse-transcribed in a 20-L reaction mixture using

the Fermentas RevertAid rst-strand cDNA synthesis kit (Fermentas, Burlington, Ottawa, Canada) according to the manufacturers
instructions. A 1.0-L cDNA sample from the reverse-transcription
reaction was used for the PCR analysis. The same primer combinations were used for the PCR and the RT-PCR analyses. The following
cycling program was used: one cycle of 95 C for 3 min, followed by
30 cycles of 94 C for 30 s, 57 C for 30 s, 72 C for 1 min and a nal
extension at 72 C for 10 min. The RT-PCR products were separated
by electrophoresis on a 1.5% agarose gel stained with ethidium bromide 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 cultures following the particle acceleration-mediated transformation
resulted in thousands of cells per gram of cell culture showing transient GUS expression 48 h after bombardment for both promoters
and both Ornithogalum species (data not shown). Stable expression
was detected after 6 days. Two constitutive promoters were tested
in both O. thyrsoides and O. dubium, the Arabidopsis polyubiquitin
(UBQ3) and the SVB promoter that controls the expression of the
chimeric celI-tpnI gene (Fig. 1).
The key to successful and efcient transformation was the production of competent tissue with a high regeneration capacity
following bombardment (Fig. 2A). The highly regenerable tissue
was obtained from calli induced on leaf explants, which were grown
in liquid M-206 media in darkness to form competent cell clusters
prior to bombardment. Two shorter growth cycles before bombardment increased the rate of cell division and the rate of regenerability
(data not shown). The two species, O. dubium and O. thyrsoides,

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A. Lipsky et al. / Plant Science 228 (2014) 150158

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 cell
cluster 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 with
GUS at 6 months after transformation. (D) Plantlets expressing GUS activity. (E) Transverse sections of putative transformed O. dubium bulbs. Leaf segments from transgenic
O. 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 and E) or at the bottom (photos F and G). Bars, 5 mm.

differed in their ability to produce competent cultures. While O.

dubium required only 2 months to produce uniform competent cell
aggregates with small clamps of de-differentiated cells (Fig. 2A), it
took O. thyrsoides 6 months in the same liquid culture to form cell
clumps suitable for bombardment and those clumps were bigger
and less amorphous than those of O. dubium (Fig. 2B). Following the
bombardment, an extended 2- to 6-month selection on kanamycin
was essential for the development of transgenic meristematic centers and a gradual elimination of non-transgenic cells. O. dubium
exhibited better and earlier regeneration, with large quantities of

embryos observed as early as 1.52 months after the transfer to

light (Fig. 2C). In contrast, O. thyrsoides did not respond to the
differentiation 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 putative
transgenic O. dubium plants grew poorly, as compared to the nontransgenic plants. In contrast, the putative transgenic O. thyrsoides

A. Lipsky et al. / Plant Science 228 (2014) 150158

155

Table 1
PCR 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 carotovorum
subsp. 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 the
original line.
Treatment

O.dubium
Control

Putative transgenic

O. thyrsoides
Control
Putative transgenic

Line

PCR

RT-PCR

GUS reaction

Pcc maceration
(%)

nptII

uidA

tpnI target
gene

nptII

uidA

tpnI target
gene

Odnt1
Odnt2
Odnt3

nv

nv

nv

80
90
100

E1d11-42/3 (19)
E1d21-31/2 (32)
E2d10-61/6 (57)
E2d11-31/5 (68)
E2d20-2/8 (81)
E3d10-24/8 (94)
E3d21-31/4 (129)
E1u11-33/1
E1u20-52/2 (168)
E2u20-41/2 (210)
E3u11-22/8 (245)
E3u21-1/3 (250)

+
+
+
+
+
+
+
+
+
+
+
+

+
+

+
+
+
+

+
+
+
+

+
+

+
+
+
+
+
+
+
+
+

+
+
nv
+
nv
+
+
nv
+
+
+
+

+
+
nv
+
nv
+
+
nv
+
+
+
+

+
+
nv
+
+
+
+
nv
+
+
+
+

+
+

+
+
+

40
25
80
25
25
20
60
40
0
35
40
15

Otnt1
Otnt2

Et1d11/1 (18)
Et1d20 (19)
Et2d20/1 (20)
Et3d11/1 (21)
Et1u11 (22)
Et1u20 (23)
Et2u2c (24)
Et3u2 (25)

+
+
+
+
+
+
+
+

+
+
+
+

+
+
+
+

+
+

+
+
+
nv
nv
+
+
+

+
+
+
nv
nv
+
+
+

+
+
+
nv
nv
+
+

+
+

plants displayed vigorous growth and homogenous owering, similar to the non-transgenic control plants (Fig. 2G and H).
GUS analysis at different stages of plant development
The identity of putative transgenic individuals was conrmed
rst using GUS activity assays and later through molecular characterization. GUS analysis was performed at all stages of plant
development and in different plant tissues (Fig. 3). Stable GUS
expression was observed in selective liquid culture 6 days after
bombardment (Fig. 3A) and in 70-day-old embryogenic clusters
(Fig. 3B). The embryos that grew from the clusters exhibited GUS
staining in all of their parts after 6 months and this staining was
also observed in plantlets and in transverse sections of bulbs ready
for planting in the greenhouse (Fig. 3C and E).
About 50% of all of the putative transgenic plantlets expressed
GUS activity. A positive GUS reaction in the bulbs of developed
plants conrmed the presence of the transgene in the most vulnerable tissue of the plant (i.e., the storage organ). Stable GUS expression
was also observed in the leaves of mature plants grown in the
greenhouse during the second growing season (Fig. 3F and G). No
GUS activity was observed in any plant tissue that had not been
bombarded.
PCR and RT-PCR verication of transgenic Ornithogalum
Putative transformants derived from kanamycin-resistant culture lines were veried by PCR and RT-PCR. PCR was performed
with gene-specic primers for nptII, uidA and the target gene celItpnI under the control of the UBQ3 or SVB promoters. The analysis
yielded 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 amplication of
these products conrmed the presence of the celI-tpnI target gene.
Interestingly, out of the 12 putative transgenic O. dubium lines and
the 8 putative transgenic O. thyrsoides lines that were analyzed, only
10 of the O. dubium lines and 5 of the O. thyrsoides lines expressed all
three of the analyzed genes (Table 1). In both species, two putative
transgenic lines were GUS-negative and one O. dubium line and two
O. thyrsoides lines lacked the target gene. The presence of the nptII
gene was conrmed in both Ornithogalum species and in all of the
putative transgenic lines analyzed, indicating the efciency of the
selection procedure (Fig. 4A and B). No band of any of the enhanced
genes was present in the non-transformed control plants.
To further conrm the expression of the transformed genes at
the RNA level, the same primer combinations for each of the two
genes, nptII and uidA, were used for RT-PCR analysis of putative
transgenic lines and the non-transgenic controls (Fig. 5A and B). The
expression of the target gene celI-tpnI at the RNA level was tested
with the primer combination F3 and R1, which yielded a 392-bp
product common to the two constructs used for the transformation.
Six putative transgenic O. dubium and 6 O. thyrsoides plants were
analyzed and tested positive for all three transgenes at the RNA
level, except for one of the putative O. thyrsoides plants, which was
negative for the celI-tpnI. No RNA product was recovered from the
non-transformed control plants.
Resistance to bacterial soft rot in the transgenic plants
Putative transgenic plantlets were challenged with Pcc during
different stages of growth in tissue culture. Leaf segments from
putative transgenic plants (10 cm) were immersed in Petri dishes

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A. Lipsky et al. / Plant Science 228 (2014) 150158

Macerated leaves (%)

100

80

60
*

40

*
*

*
*

20

95
E1 /49/
6
E1 d10 0 C
d -2 1
E1 11- /9 (
d 23 3)
E1 20- /3 (
d 1/2 7)
E2 21- (2
1 2
d
E2 10 /6 ( )
d1 -3/ 34
3 )
E2 1-3 (4
1 6
E3 d20 /2 ( )
d -2 67
E3 10- /5 ( )
2 8
E3 d11 4/3 0)
d -1 (9
E1 21- /2 2)
u1 31/ (10
E1 1-2 2 (1 0)
u 3/ 28
E1 20- 1 (1 )
u2 52/ 51
2 )
E2 1-3 (1
u 4/2 68
E2 10- (1 )
1
u /5 74
E3 20- (1 )
1 8
E3 u10 /1 ( 3)
u1 -1/ 20
1 4
E3 1-2 (2 )
u2 1/6 25
1 ( )
E1 -1/ 241
d1 3 ( )
S 25
E1 (19 0)
u /12
(1 )
9/
05
)

Putative transgenic plantlets

Fig. 4. Molecular characterization of transgenic Ornithogalum. (A) PCR analysis of

the nptII gene, uidA gene and celI-tpnI target gene and assay of GUS activity in those
same 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 target
gene driven by SVB promoter, pdt; non-transgenic control, C; putative transgenic
lines, lanes 48; plasmid control with target gene driven by the UBQ3 promoter,
put.

containing solid MS covered with a mixture of virulent Pcc and soft

agar. The data are presented as the percentage of macerated leaf
segments out of a total of 10 leaf segments per plate. Twenty control
plantlets and 87 putative transgenic plantlets were assessed for Pcc
resistance in the leaf-segment assay. Different levels of resistance to
the pathogen were observed, from highly resistant to susceptible.
The average maceration of the control was 84%, while the putative transgenic lines exhibited 16.770% maceration. Nonetheless,
in most cases, the putative transgenic plants were more resistant
than the non-transformed controls (Fig. 6). While control plants
were completely macerated after 7 days in the assay plates (Fig. 7A
and B), a few transgenic lines (#32, #94 and #168) displayed high
levels of resistance to the pathogen even 21 days after inoculation
(Fig. 7C and D). A test to conrm the virulence of the bacterium
was performed following 21 days in the assay plates. In this test,

Fig. 5. (A) RT-PCR analysis of the nptII gene, uidA gene and celI-tpnI target gene in
putative transgenic O. dubium plants. (B) RT-PCR analysis of those same genes in
putative transgenic O. thyrsoides plants. Lanes (left to right): 100-bp DNA ladder, M;
non-transgenic control, C; putative transgenic lines, lanes 38.

Fig. 6. Survival of leaf segments harvested from control (C1 on the left) or putative transgenic O. dubium plantlets following 14 days of an infection assay with
P. carotovorum subsp. carotovorum. Bars represent the percentage of infected leaf
segments + SE, as determined by a one-way analysis of variance with post hoc pairwise multiple comparisons (Dunnetts test). Asterisks indicate signicantly different
group means at an alpha level of 0.05.

fresh, non-transformed leaf segments (controls) were introduced

into the infection plates. The control leaf segments were completely
macerated within 2 days, while the putative transgenic segments
remained green (not shown).
Discussion
Control of plant pathogens such as soft rot bacteria is one of the
most challenging problems facing modern agriculture. Losses may
occur during production or storage. Bulb and tuber crops, including highly valuable ornamentals and food crops such as potato,
are particularly susceptible to soft rots, due to their specialized
underground storage organs. Bacteria from the genus Pectobacterium are found worldwide and have an extremely broad host
range, reecting their temperature tolerance and different virulence mechanisms [18,33]. As with most bacterial necrotrophs, no
effective control measure is currently available for use against bacterial soft rots and the only way to manage these diseases is through
strict sanitation measures and the use of clean propagation material
[19,34].
The aim of the present study was to employ a biotechnological
approach to reduce pathogen pressure in a highly sensitive ower
bulb crop. Previous studies have reported the potential abilities
of several cationic antimicrobial peptides, including TPNI, to confer broad-spectrum resistance against bacterial phytopathogens
[25]. TPNI small antimicrobial peptide was originally isolated from
hemocytes of the Japanese horseshoe crab (T. tridentatus) and has
proven effective against a variety of bacteria in vitro [22]. TPNI
has been described as an effective antimicrobial compound for use
against various Erwinia (Pectobacterium) species. More specically,
its expression in transgenic potatoes conferred resistance against
soft rot [26]. For this reason, TPNI was chosen as the target gene to
be introduced into two Ornithogalum species by particle bombardment.
The particle acceleration-mediated transformation was based
on a procedure described for the transformation of Lilium [35] and
Ornithogalum to confer resistance to Ornithogalum mosaic virus
[6,8]. Following selection in kanamycin-containing media, a relatively high proportion of transformed plants was obtained, more
than 50% of the putative transgenic plantlets exhibited GUS activity
and all were nptII-positive (as determined by PCR amplication of

A. Lipsky et al. / Plant Science 228 (2014) 150158

157

Fig. 7. Disease resistance assay of O. dubium leaf segments following challenge with P. carotovorum subsp. carotovorum (Pcc). (A) Non-transgenic control infected with Pcc
at time zero post-inoculation (dpi). (B) Non-transgenic control infected with Pcc, 11 dpi. (C) Putative transgenic clone #94, in which the transgene is controlled by the SVB
promoter, infected with Pcc, 21 dpi. (D) Putative transgenic clone #168, in which the transgene is controlled by the UBQ3 promoter, 21 dpi.

85 putative transgenic clones). These results can be attributed to

two factors: (1) the successful development of highly regenerable,
competent Ornithogalum cell cultures and (2) a prolonged selection process under selective conditions in media supplemented
with 80 mg/L kanamycin and kept in the dark. This approach minimized the probability of the development of non-transgenic cells
that would yield non-transgenic embryos and plants.
The two Ornithogalum species in the present study displayed different levels of susceptibility to the soft rot bacterium, O. dubium
was more susceptible (unpublished data). The high susceptibility
of O. dubium was to a great extent compensated for by its better performance under tissue-culture conditions. While O. dubium
required less time (2 months) to produce consistent competent cell
cultures for bombardment, O. thyrsoides required at least 6 months
in liquid and the O. thyrsoides cultures were less homogeneous.
Following the bombardment and incubation under selective conditions, O. dubium produced large quantities of embryos as early
as 1.52 month after being transferred to an environment that
included light, while O. thyrsoides did not respond to the differentiation conditions before 69 months had passed.
Once developed, the putative transgenic lines of the two species
were planted in the greenhouse to evaluate their growth and phenotypes under greenhouse conditions. Unlike in tissue culture,
during three growing seasons, transgenic O. thyrsoides plants performed similarly to the non-transgenic control plants, exhibiting
vigorous growth and high owering potential, whereas O. dubium
transgenic plants grew poorly relative to the control.
The presence of the three genes, nptII, uidA and the chimeric
celI-tpnI target gene, was conrmed by PCR analysis of 12 O. dubium
plantlets and 8 O. thyrsoides plantlets. The presence of the nptII gene
was conrmed by PCR in both Ornithogalum species in all analyses, most likely due to the prolonged selection in a relatively high
concentration (80 mg/L) of the selective agent (kanamycin). The
selection procedure was also responsible for the high rate of putative transgenic plants that reacted positively to the presence of the
target gene (85%) and the uidA gene (80%). Although the presence

of the uidA gene was conrmed, GUS activity was observed in only
55% of these putative transgenic plants, implying that conrmation
by PCR does not always indicate the activity of the transgene and
that a lack of GUS activity does not guarantee that the plant is not
transgenic. The RT-PCR analysis of the putative transgenic plants
conrmed that not only was DNA successfully incorporated into the
plants genome, but the transgene was also expressed at the RNA
level. These results were in complete agreement with those of a
previous study in which cationic peptide chimeras were expressed
in potatoes to produce broad-spectrum resistance against bacterial
and fungal phytopathogens [25].
The resistance of the putative transgenic plants to bacterial soft
rot was tested by challenging the plants with Pcc. Several assays
have been developed to screen for resistance during different stages
of plant development [28,36]. In the present study, putative transgenic plants (87 clones derived from 20 lines) and non-transformed
controls (20 plants) were tested for both a GUS reaction and resistance to soft rot in a leaf segment bioassay carried out over 21
days.
No GUS activity was observed in any of the control plants,
whereas 64% of the putative transgenic plants displayed a positive GUS reaction. In some of these putative transgenic plants, the
GUS reaction was specically tested in the bulbs to conrm the
expression of the transgene in that critical plant tissue. In a study
involving potato, transgene expression was detected in leaves, but
not in underground storage organs (tubers) [26]. In contrast, in this
study, a positive GUS reaction was observed in the bulbs, suggesting
that the target gene could also be expressed in the storage organ.
In agreement with the varied results of the GUS assays, disease
resistance was also highly variable among the putative transgenic
plants. The different levels of resistance conferred to the transformed plants by the transgenes may be the result of a number
of factors, three of which may be of great importance. First, TPNI
has a cyclic antiparallel -sheet structure that is maintained by
two disulde bridges that are essential for its activity. The peptide
effectively increases the permeability of bacterial membranes, but

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A. Lipsky et al. / Plant Science 228 (2014) 150158

does so only in its cyclic form [23]. Second, in order to maximize

its antimicrobial potential, the peptide has to be secreted into the
extracellular space, where Pcc proliferates and activates most of
its virulence factors during infection. Third, the peptide has to be
translated at a sufcient level to allow for efcient antimicrobial
activity.
These issues were explored by fusing a signal peptide from Arabidopsis celI [37] to TPNI [38], to allow the peptide to be secreted
into the extracellular space and the natural folding of the peptide
into its active -sheet structure [25]. Some of the variability in the
resistance of the transgenic plants may be the result of peptide
folding and secretion rather than just the level of expression in the
transformed plants. To control the level of expression, each of the
two constructs we used, included a constitutive promoter to control the target gene: the Arabidopsis polyubiquitin promoter (UBQ3)
or the strawberry vein-banding virus-deleted promoter (SVB). In
our analysis of the disease resistance of putative transgenic lines
expressing one of these two promoters, the promoters did not seem
to differentially affect the degree of disease resistance among the
transgenic plants.
Disease development was monitored as the percentage of leaf
segments macerated by the pathogen at 7 days after infection. More
than 80% maceration was observed in the control plants. The putative transgenic plants displayed relatively lower levels of disease,
with an average maceration rate of less than 50% at 12 days after
infection. Five clones that were produced from a transgenic line
that lacked the target gene displayed relatively high sensitivity to
the pathogen, with an average maceration rate of 80%, similar to
that of the control. Resistant lines of the putative transgenic plants
were not macerated even 21 days after inoculation, suggesting that
the resistance afforded to these plants by the antimicrobial peptide
is consistent and strong even in the face of strong disease pressure.
In the absence of effective control measures for use against bacterial soft rot, the engineering of disease-resistant plants through the
use of naturally occurring cationic peptides appears to be an interesting possibility for the sustainable production of ornamental bulb
plants.

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