Botrytis cinerea-resistant marker-free Petunia hybrida produced using the MAT vector system

ORIGINAL PAPER

Botrytis cinerea-resistant marker-free Petunia hybrida producedusing the MAT vector system

Raham Sher Khan • Syed Sartaj Alam •

Iqbal Munir • Pejman Azadi • Ikuo Nakamura •

Masahiro Mii

Received: 21 July 2010 / Accepted: 15 November 2010

� Springer Science+Business Media B.V. 2010

Abstract The presence of marker genes conferring anti-

biotic or herbicide resistance in transgenic plants has been

a controversial issue and a serious problem for their public

acceptance and commercialization. The MAT (multi-auto-

transformation) vector system has been one of the strate-

gies developed to excise the selection marker gene and

produce marker-free transgenic plants. In an attempt to

produce transgenic marker-free Petunia hybrida plants

resistant to Botrytis cinerea (gray mold), we used the ipt

gene as a selectable marker gene and the wasabi defensin

(WD) gene, isolated from Wasabia japonica (a Japanese

horseradish which has been a potential source of antimi-

crobial proteins), as a gene of interest. The WD gene was

cloned from the binary vector, pEKH-WD, to an ipt-type

MAT vector, pMAT21, by gateway cloning technology and

transferred to Agrobacterium tumefaciens strain EHA105.

Infected leaf explants of P. hybrida were cultured on

hormone- and antibiotic-free MS medium. Extreme shooty

phenotype (ESP)/ipt shoots were produced by the explants

infected with the pMAT21-WD. The same antibiotic- and

hormone-free MS medium was used in subcultures of the

ipt shoots. Ipt shoots subsequently produced morphologi-

cally normal shoots. Molecular analyses of genomic DNA

from the transgenic plants confirmed the integration of the

gene of interest and excision of the selection marker.

Expression of the WD gene was confirmed by northern blot

and western blot analyses. A disease resistance assay of the

marker-free transgenic plants exhibited enhanced resis-

tance against B. cinerea strain 40 isolated from P. hybrida.

Keywords MAT vector � ipt gene � Wasabi defensin

gene � Agrobacterium tumefaciens � Petunia hybrida

Introduction

Antibiotic- and/or herbicide-resistant genes are widely used

as selectable markers in plant transformation (Bevan et al.

1983; Waldron et al. 1984; Akama et al. 1995). Once the

transgenic plants have been developed, however, these

marker genes are redundant, although still incorporated into

the genome, and this continuous existence in the genome of

the transgenic plants raises issues of environmental and

ecological concerns (Dale 1992; Gressel 1992; Nap et al.

1992; Hill and Sendashonga 2006). Moreover, it is difficult

to pyramid transgenes (gene stacking) with the same

selectable marker. In addition, the existence of marker

genes in transgenic crops—and their gene products—needs

to be evaluated by additional and lengthy risk assessment

process for their public use (Goldstein et al. 2005).

Several strategies have been tested to produce transgenic

plants free from selection marker genes (Yoder and

Goldsbrough 1994; Ebinuma et al. 1997a; Puchta 2000;

Jaiwal et al. 2002; Hare and Chua 2002), such as the

introduction of only the gene of interest and laboriously

screening the regenerants using PCR analyses (Vetten et al.

2003; Ballester et al. 2010), site-specific recombination

(Gleave et al. 1999; Zubko et al. 2000; Roy et al. 2008;

R. S. Khan (&) � S. S. Alam � P. Azadi � I. Nakamura �M. Mii (&)

Graduate School of Horticulture, Chiba University,

648 Matsudo, Matsudo, Chiba 271-8510, Japan

e-mail: [email protected]

M. Mii

e-mail: [email protected]

R. S. Khan � I. Munir

Institute of Biotechnology and Genetic Engineering,

Khyber Pakhtunkhwa Agricultural University,

Peshawar, Pakistan

123

Plant Cell Tiss Organ Cult

DOI 10.1007/s11240-010-9888-0

Zhang et al. 2009; Khan et al. 2010a), and cotransforma-

tion (Komari et al. 1996; Lu et al. 2009) [reviewed in Hohn

et al. (2001) and Darbani et al. (2007)].

In the study reported here, we employed the multi-auto-

transformation (MAT) vector system to develop disease-

resistant marker-free Petunia hybrida without a sexual

crossing step. The MAT vector system, which was devel-

oped by Ebinuma and co-workers (Ebinuma et al. 1997a;

Sugita et al. 1999; Ebinuma and Komamine 2001), is a

novel transformation system in which transgenic tissues are

visually selected by their morphological changes caused by

oncogenes [isopentenyltransferase (ipt) gene] or rhizogenes

(or the rol gene) of Agrobacterium tumefaciens.

The MAT vectors are equipped with the yeast site-specific

recombination R/RS system to mediate the excision of the

DNA fragment and the ipt gene located between two directly

oriented recombination sites (Araki et al. 1987). The ipt gene

codes for isopentenyltransferase, which catalyzes the for-

mation of isopentenyl AMP, a precursor of several cytoki-

nins. Following transformation, overexpression of the ipt

gene results in an increase in endogenous cytokinins and,

consequently, the production of extreme shooty phenotype/

ipt shoots that are characterized by the loss of apical domi-

nance, short internodes, abnormal morphological changes,

and lack of rooting ability. As a result, the ipt shoots can be

visually selected and subcultured. The excision of the ipt

gene by site-specific recombination induced by recombinase

of the R/RS system during subculturing produces morpho-

logically normal marker-free transgenic plants.

The MAT vector system has been evaluated in tobacco

(Ebinuma et al. 1997a; Sugita et al. 2000), Antirrhinum

majus (Cui et al. 2000, 2001; Ebinuma and Komamine

2001), hybrid aspens (Matsunaga et al. 2002), rice (Endo

et al. 2002), Nierembergia (Khan et al. 2006a), white

poplar (Zelasco et al. 2007), Citrus (Ballester et al. 2007),

Cassava (Saelim et al. 2009), Medicago truncatula (Sca-

ramelli et al. 2009), Kalanchoe blossfeldiana (Thiruk-

kumaran et al. 2009) and Petunia hybrida (Khan et al.

2010a, b). Most of these studies have evaluated the appli-

cability of the MAT vector system using mostly the b-

glucuronidase (GUS) gene as a model gene of interest.

Petunia hybrida, an important floral plant, is susceptible

to a number of pathogenic bacteria and fungi that affect

both its growth and market value. As a useful strategy to

confer resistance to wide range of pathogens, genes for

cysteine-rich antimicrobial peptides (AMPs) have been

introduced in different plant species. Antimicrobial pep-

tides (also called host defence peptides) are an evolution-

arily conserved component of the innate immune response

and found among all living organisms. These AMPs have

been shown to play a role in plant defense against a wide

range of pathogens, including bacteria and fungi (Broekaert

et al. 1995, 1997; Cammue et al. 1992, 1994).

The antimicrobial protein gene WjAMP-1 isolated from

leaves of Wasabia japonica was found to inhibit fungal and

bacterial growth when expressed in Nicotiana benthamiana

(Saito et al. 2001; Kiba et al. 2003). Transgenic tobacco

plants overexpressing the wasabi defensin gene have also

been reported to be resistant against Botrytis cinerea (gray

mold; Nishihara, unpublished). Similarly, the growth of

blast fungus was inhibited in transgenic rice carrying

wasabi defensin (Kanzaki et al. 2002). In an earlier study,

we produced transgenic potato plants carrying wasabi de-

fensin that exhibited antifungal activity against B. cinerea

(Khan et al. 2006b). Transgenic Phalaenopsis plants

expressing wasabi defensin protein (WD) showing resis-

tance to Erwinia caratovora have also been produced

(Sjahril et al. 2006). Transgenic ‘Egusi’ melon (Colocyn-

this citrullus) was produced recently that exhibited resis-

tance against Aletnaria leaf spot and Fusarium wilt (Ntui

et al. 2010).

Previous work by our group, using the GUS gene as a

model gene of interest, showed that marker-free P. hybrida

can be produced using the MAT vector system (Khan et al.

2010a). The aim of the study reported here was to develop

transgenic marker-free P. hybrida plants resistant to B.

cinerea (gray mold) through the introduction of the wasabi

defensin (WD) gene using an ipt-type MAT vector,

pMAT21-WD.

Materials and methods

Vector construct

Escherichia coli strain Top10 was used as the host for

recombinant vector constructions. The ipt-type MAT vec-

tor, pMAT21 (Ebinuma et al. 1997b), which has the LacZ

(located outside the ‘hit and run’ cassette), GUS, ipt, and

recombinase (R) genes [located between directly oriented

recombination site (RS) sequences], was used as the des-

tination vector.

The binary vector construct used in this study was

constructed by Gateway LR clonase reactions (Fig. 1;

Invitrogen, CA, USA). The WD gene was isolated from the

binary vector pEKH by digestion with HindIII and cloned

in the pCR8/GW/TOPO TA vector (Invitrogen) by TA

cloning to produce the entry vector PCR8/WD. The desti-

nation vector was constructed by inserting the ccdB gene (a

highly lethal gene whose protein blocks growth of E. coli

wild-type gyrA ? strain) in Sma1 site of LacZ in pMAT21

(Fig. 1a). WD was integrated into the destination vector by

LR clonase (Invitrogen) to produce pMAT21-WD, an ipt-

type MAT vector harboring the WD gene and transferred to

A. tumefaciens strain EHA105 by the freeze/thaw method

(Weigel and Glazebrook 2006).


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A. tumefaciens harboring pMAT21-WD was grown

overnight in a reciprocal shaker (120 cycles min-1) at 28�C

in LB medium (10 g l-1 tryptone, 5 g l-1 yeast extract,

10 g l-1 NaCl, pH 7.2) containing 50 mg l-1 kanamycin

(Wako Pure Chemical Industries, Osaka, Japan) and

25 mg l-1 chloramphenicol (Sigma–Aldrich Chemie,

Steinheim, Germany). The bacterial culture was centri-

fuged (3,000 g) for 10 min and the bacterial pellet resus-

pended in hormone-free MS (Murashige and Skoog 1962)

medium [bacterial suspension:MS medium, 1:2 (v:v)]

containing 100 lM acetosyringone (3,5-dimethoxy-4-

hydroxy-acetophenone; Sigma-Aldrich) and 30 g l-1

sucrose to a final density of OD600 = 0.6.

Transformation

Leaf explants of in vitro-grown P. hybrida ‘Dainty Lady’

were infected with the overnight-grown bacterial suspen-

sion (OD600 = 0.6) for 5 min, blotted dry with sterilized

filter paper to remove excess bacteria, and incubated on

plant growth regulator (PGR)-free MS medium supple-

mented with 30 g l-1 sucrose, 0.25% gellan gum (Gelrite;

Kelco, Division of Merck, San Diego, CA), and 100 lM

acetosyringone, for 3 days under the dark condition for co-

cultivation. The infected explants were then washed with

liquid PGR-free MS medium supplemented with 10 mg l-1

meropenem (Meropen; Sumitomo Pharmaceuticals, Osaka,

Japan) and transferred to PGR- and antibiotic-free MS

medium containing 20 mg l-1 meropenem. The infected

and the uninfected control explants were kept under normal

growth conditions. The regenerated shoots were subcul-

tured on the same MS medium.

Histochemical GUS assay

Histochemical GUS assays of leaves from ESP (extreme

shooty phenotype) shoots and normal transgenic P. hybrida

were carried out by soaking the tissues in X-Gluc (5-

bromo-4-chloro-3-indolyl-beta-D-glucuronic acid) solution

(Jefferson 1987). After an overnight (15–16 h) incubation

at 37�C, chlorophyll was removed by soaking the tissues

for several hours in 70% ethanol.

PCR analysis

For the PCR analysis, genomic DNA was extracted from

ipt and morphologically normal shoots and from control

Petunia plants following the CTAB (cetyl trimethyl

ammonium bromide) method with slight modifications

(Rogers and Bendich 1988). PCR cycling was carried out

using the oligonucleotide primers (Bex, Japan) for the

GUS, ipt, and WD genes. The sequences of the oligonu-

cleotide PCR primers were: GUS1, 50-GGTGGGAAAGC

GCGTTACAAG-30; GUS2, 50-TTTACGCGTTGCTTCCG

CCA-30; IPT1, 50-CTTGCACAGGAAAGACGTCG-30;IPT2, 50- AATGAAGACAGGTGTGACGC-30; WD1, 50-TT

TGCTTCTATCATCGCTCTTC-30; WD2, 50-TTATTAGT

ACAACAAACCAACA-30.

Southern blot hybridization

For Southern blot hybridization, genomic DNA (10 lg)

from ipt and morphologically normal shoots and non-

transformed control P. hybrida plants were digested with

Fig. 1 Schematic representation of the T-DNA region of the

isopentenyltransferase (ipt)-type multi-auto-transformation (MAT)

vector, pMAT21-WD. a The MAT vector with a ‘hit and run’ cassette

in which the chimeric ipt, b-glucuronidase (GUS), and recombinase

(R) genes are inserted into the R/RS system between two directly

oriented recombination sites (RS). The wasabi defensin (WD) gene is

located outside the ‘hit and run’ cassette. RB Right border sequence of

a T-DNA, LB left border sequence of a T-DNA. The cauliflower

mosaic virus 35S promoter (35SP) drives the R and WD genes. The

GUS and ipt genes are driven by the nopaline synthase (NosP) and

native ipt (IptP) promoters, respectively. The terminators (T) of the

ipt, R, GUS, and WD genes are derived from nopaline synthase. b T-

DNA region after excision of the ‘hit and run’ cassette. Primer

positions and length of PCR products are indicated by double arrows.

Recognition sites of restriction enzymes (HindIII, Pst1, EcoRI,

BamHI) are also indicated


123

HindIII. The digested samples were separated on a 0.8%

(w/v) agarose gel, blotted to a positively charged nylon

membrane (Hybond-N?; Amersham Pharmacia Biotech,

Amersham, UK), and hybridized with a digoxigenin (DIG)-

labeled probe of the WD gene. The probe DNA fragment,

corresponding to a part of the WD gene, was labeled by

PCR using DIG-dUTP, following the supplier’s instruc-

tions (Boehringer Ingelheim, Ingelheim am Rhein, Ger-

many). Hybridization, washing, and detection were

performed using the DIG Easy Hyb (hybridization solu-

tion) and DIG Wash and Block Buffer set following the

supplier’s instructions (Boehringer Ingelheim). Hybridiza-

tion with the DIG-labeled probe was performed for 16 h at

41�C, and the hybridization patterns were detected with the

chemiluminescent substrate CDP-Star (Roche Molecular

Biochemicals, Mannheim, Germany) and anti-DIG-AP

(anti-DIG antibody linked to alkaline phosphatase). The

hybridized blot was exposed to Hyperfilm TM-MP X-ray

film (Amersham Pharmacia Biotech) for 15–20 min at

room temperature.

RNA extraction and northern blot hybridization

For northern blot analysis, total RNA was extracted using a

one-step acid phenol–guanidine isothiocyanate–chloroform

method (Sambrook and Russell 2001) to detect the

expression of the WD at the mRNA level. A 20-lg sample

of total RNA was fractionated by electrophoresis on a 1.5%

agarose-formaldehyde gel in 19 morphpropane sulphonic

acid (MOPS) and transferred overnight by capillary action

to a Hybond-N? nylon membrane (Amersham Biosci-

ences, Arlington Heights, IL) using 209 SSC (3 M NaCl,

0.3 M sodium citrate). The RNA on the membrane was

fixed by UV-crosslinking for 2–3 min. Northern blot

hybridization was carried out using the DIG-labeled DNA

probe of the WD gene following the same procedure as that

described for Southern blot analysis, with slight modifica-

tion where necessary.

Reverse transcription-PCR

Reverse transcription (RT)–PCR was performed using a

SuperScript Transcriptase III kit (Life Technologies,

Carlsbad, CA). Total RNA (1.0 lg per 20 ll) with the

WD-specific primers was used for RT–PCR. cDNA was

synthesized at 55�C for 40 min and the reaction stopped

at 70�C for 15 min. The cDNA was denatured prior to

the PCR analysis at 94�C for 2 min. PCR amplification

was then performed in 30 cycles of 94�C for 30 s, 59�C

for 30 s, and 72�C for 1 min. The resultant PCR prod-

ucts were analyzed by electrophoresis on a 1.2% agarose

gel.

Western blot analysis

To evaluate the expression of the integrated WD in the

genomic DNA of transgenic plants, total proteins were

extracted from fresh leaves of 4- to 5-week-old marker-free

transgenic and non-transformed control in vitro petunia

plants (containing 5–7 mature leaves). The samples were

homogenized with extraction buffer [(62.5 mM Tris-HCl,

pH 6.8, 2% (v/v) sodium dodecyl sulphate (SDS), 10% (v/

v) glycerol)] and 0.2% b-mercaptoethanol after being

ground in liquid nitrogen. The homogenized samples were

centrifuged (20,000 g) for 5 min at 4�C, and the total

proteins in the supernatant from each sample were dena-

tured by boiling for 3 min followed by incubation on ice

for 2 min and separation by SDS–polyacrylamide gel

electrophoresis (PAGE 15%) using a Bio-Rad Mini elec-

trophoresis system per the manufacturer’s instructions

(Bio-Rad, Hercules, CA. The fractionated proteins were

electroblotted onto a polyvinylidine difluoride (PVDF)

membrane (Amersham Biosciences). The PVDF membrane

was blocked for 1 h in 5% bovine serum albumin (BSA) in

TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl). Detection

of the WD protein was performed using polyclonal antisera

raised in rabbit against the defensin protein (primary anti-

body, 1:1000, v/v) and subsequently goat-anti-rabbit IgG

(Amersham Biosciences, USA) conjugated to horseradish

peroxidase (HRP) as secondary antibody (1:100000, v/v)

with an enhanced chemiluminescence (ECL) western blot

detection system (Amersham Biosciences).

Fungal-resistance assays of transgenic plants

Antifungal activity of the WD protein in the marker-free

transgenic Petunia plants was tested against B. cinerea

strain 40 (isolated from P. hybrida). B. cinerea was grown

on potato dextrose agar (PDA) until the surface of agar in

the petri dish (20 9 90 mm) was covered with the fungal

mycelia. For inoculation, a block of the agar (with cultured

mycelia) was placed near the base of in vitro marker-free

transgenic and nontransformed control plants (2–3 weeks

old) grown in glass bottles and incubated in a growth

chamber maintained at 25�C and 100% relative humidity

under a 16/8-h (light/dark) photoregime. Pictures were

taken 2 weeks after inoculation (Fig. 7a).

Detached leaf assay

For the detached leaf assay, a spore suspension was pre-

pared by flooding the cultures of B. cinerea strain 40,

grown in petri dishes on PDA at 25�C for 7–8 days, with

10 ml of sterile distilled water and then rubbing the agar

surface gently with a sterilized loop to dislodge the spo-

rangia. The spore suspension was collected in Eppendorf


123

tubes with a sterile Pasteur pipette and vortexed vigorously

to induce the spores. The spores were counted on a

hemocytometer and the suspension concentration adjusted

to 1 9 106 spores per milliliter using sterile water. The

spore suspension was used immediately for inoculation.

The youngest fully expanded mature leaves from the

marker-free transgenic and control plants were harvested

and immediately placed, adaxial side up, on wet filter paper

in petri plates. The center of each leaf was wounded on

both sides of midrib, and 20 ll of spore suspension

(1 9 106 spores ml-1) was pipetted onto the wounded spot

(Fig. 7c, d). The petri plates were placed in a growth

chamber maintained at 25�C and 100% relative humidity

under a 16/8-h (light/dark) photoregime. The area (mm2) of

the necrotic lesions on the leaf-disks was measured 5 days

after inoculation.

Restriction of fungal colonization

To further confirm the anti-fungal activity of WD protein in

preventing the colonization of B. cinerea strain 40, we

challenged in vitro transgenic and control Petunia plants

with the fungal spores, as described earlier. The inoculated

plants were incubated in a growth chamber maintained at

25�C and 100% relative humidity under a 16/8-h (light/dark)

photoregime. After 1 week, leaves and stem sections from

the inoculated plants were cultured on PDA in petri plates

and incubated again under the same conditions. The cultures

were photographed (Fig. 7e, f) 1 week after inoculation.

Results and discussion

Production of marker-free normal plants

Leaf explants of P. hybrida were infected with the A.

tumefaciens harboring pMAT21-WD, an ipt-type MAT

vector containing the ipt, GUS, and R (recombinase) genes

in the removable cassette flanked by directly oriented

recombination sites (RS) and cultured on hormone- and

antibiotic-free MS medium. Uninfected control explants

were also cultured under the same conditions. Approxi-

mately 2 weeks after infection, nodular compact calli

appeared on the infected leaf explants (Fig. 2a); in contrast,

the uninfected control leaf explants were unable to produce

calli and ultimately died (Fig. 2c). Adventitious shoot lines

(ASLs) were separated from the explants, approximately

1 month after infection, and transferred to PGR- and

selective antibiotic-free 0.25% gelrite-solidified MS med-

ium supplemented with 3% sucrose and 20 mg l-1 me-

ropenem (Fig. 2b). These ASLs differentiated into normal

looking shoots and ipt shoots. The ipt shoots were identi-

fied and selected from each subculture based on distinctive

morphological characteristics, including abnormal mor-

phology, short internodes, and a lack of apical dominance

and rooting ability (Fig. 2d, e). The selected ipt shoots

were subcultured at 2-week intervals on the same MS

medium.

Approximately 6 months after infection, 23 normal-

looking shoots were obtained from the separation of ipt

shoots of different ASLs. The recombinase of the R/RS

system mediated excision of the ipt gene (selectable mar-

ker) and, consequently, the reduction in cytokinin pro-

duction caused the emergence of normal-looking shoots.

Morphologically normal-looking shoots and ipt shoots

were subjected to the histochemical GUS assay (Fig. 3). As

expected, all of the ipt shoots showed GUS expression and

all of the normal shoots did not. The ipt GUS? and normal

GUS- shoots were analyzed by PCR to confirm that the

gene of interest, WD, was intact and the selection marker

had been excised from the normal-looking plants.

Molecular analyses

Three oligonucleotide primers that were able to amplify the

ipt, GUS, and WD genes were used in the PCR analyses.

The predicted 1.2-kb GUS, 0.5-kb WD, and 0.8-kb ipt

fragments were amplified in all ipt shoots (Fig. 4, lanes

3–5), and the ipt and GUS genes, as expected, were not

detected in normal shoots (Fig. 4, lanes 6–8). WD was

detected in four of the 23 normal shoots, verifying that the

selection marker was excised and that these normal shoots

were marker-free and contained only the gene of interest,

WD. No transgene was detected in genomic DNA of the

remaining normal-looking shoots based on the results of

the PCR analysis (data not shown). The cytokinin produced

in the transgenic cells may diffuse to adjacent cells, leading

to the production of escape plants. Importantly, all of the

transgenic normal-looking shoots were marker-free, in

contrast to our findings in an earlier study (Khan et al.

2006a, 2010a) in which two of the transgenic normal-

looking shoots in Nierembergia caerulea and one of the

eight well-rooted transgenic plants in P. hybrida contained

the intact ipt gene. It can be concluded that the R/RS site-

specific recombination caused excision of the ipt gene from

all cells of the normal shoots and the latter were free from

chimerism. However, autoexcision of the selection marker

gene by recombinase driven by the constitutively expressed

promoter, CaMV35S, took a long time (6–8 months). The

induction of site-specific recombination and the excision of

the ipt gene by inducible promoters directing recombinase

expression is an alternative approach to overcome the

problems of chimerism and inefficient excision (Endo et al.

2002; Matsunaga et al. 2002).

Southern blot analysis of genomic DNA from the ipt and

morphologically normal marker-free transgenic plants was


123

performed to determine the copy number of the WD gene in

transgenic plants. Genomic DNA from ipt and morpho-

logically normal transgenic and non-transformed control

plants was digested with HindIII and hybridized with the

DIG-labeled probe of the WD gene. Southern blot hybrid-

ization indicated that the transgenic plants had one to three

T-DNA insertions (Fig. 5). Hybridization was not detected

with the non-transformed plant DNA (Fig. 5, lane 1).

Fig. 2 Regeneration of ipt and normal shoots from leaf explants of

Petunia hybrida through ipt shoot formation after infection with

Agrobacterium tumefaciens harboring the ipt-type MAT vector,

pMAT21-WD. a Calli and shoot formation by infected leaf explants

of Petunia on hormone- and selective antibiotic-free MS medium

3 weeks after infection. b Adventitious shoot lines (ASLs) isolated

from the regenerated explants and cultured on MS medium for ipt or

normal shoot formation. c Control uninfected explants showing no

calli or shoot formation 5 weeks after co-cultivation. d Differentiation

of ASLs into ipt-like shoots (arrows) 5 weeks after infection. e iptshoots (arrows) that could be visually distinguished from other shoots

3 months after infection. f Morphologically normal shoot free from

selection marker, produced from ipt shoots approximately 6 months

after infection

Fig. 3 Histochemical GUS assay of ipt and morphologically normal

shoots of P. hybrida ‘Dainty Lady’ obtained through ipt shoot

formation after infection with A. tumefaciens harboring pMAT21-WD.

Ipt shoots (a) and marker-free transgenic shoots (b) were tested for

histochemical GUS expression by soaking in X-Gluc solution. After

overnight (15–16 h) staining, chlorophyll was removed by soaking

the tissues for several hours in 70% ethanol


123

RT–PCR was carried out to confirm the expression of

the WD gene at the mRNA level (Fig. 6a). Expression was

confirmed in all of the transgenic marker-free lines

(Fig. 6a, lanes 3–6); no PCR product was detected in the

control plant (lane 2). When DNase-treated RNA samples

(without reverse transcriptase) from control and transgenic

plants were used as a PCR template, the absence of the

product confirmed that the RNA samples were not con-

taminated by genomic DNA (data not shown).

Northern blot analysis of total RNA from marker-free

and non-transformed control plants was performed to

further confirm the expression of WD at the mRNA level.

The findings revealed that all of the transgenic plants had

detectable levels of WD gene transcripts (Fig. 6b, lanes

2–5). mRNA transcripts were not detected in the control

plant (Fig. 6b, lane 1).

To confirm the expression of WD gene at protein level,

we carried out western blot analysis of protein extracts

from marker-free and non-transformed petunia plants. In

the western blot analysis, the primary antibody immuno-

reacted with the antigen (WD) and the secondary antibody

(goat-anti-rabbit IgG). The secondary antibody, conjugated

with a HRP enzyme, converted the luminol substrate (ECL)

to a light-releasing substance, which was detected as a spot

on film and a 5-kDa fragment was detected in the total

Fig. 4 PCR analysis of ipt and morphologically normal shoots of P.hybrida ‘Dainty Lady’ obtained through ipt shoot formation after

infection with A. tumefaciens harboring the ipt-type MAT vector,

pMAT21-WD. Lanes M Size marker (øX174/HaeIII digests), 1negative control (DNA from non-transformed plant), 2 positive

control (plasmid DNA) for the GUS (1.2 kb, upper band), ipt (0.8 kb,

middle band), and WD (0.5 kb, lower band) genes, 3–5 amplification

of GUS (1.2 kb, upper band), ipt (0.8 kb, middle band), and WD(0.5 kb, lower band) genes in ipt shoots, 6–8 amplification of WDgene in marker-free transgenic shoots. Ipt and GUS genes were not

detected in normal-looking transgenic shoots

Fig. 5 Southern blot analysis of the WD gene in ipt and morpholog-

ically normal shoots of P. hybrida ‘Dainty Lady’ obtained through iptshoot formation after infection with A. tumefaciens harboring

pMAT21-WD. Genomic DNA from ipt shoots and morphologically

normal transgenic plants was digested with HindIII and hybridized

with the DIG-labeled probe of the WD gene. Lanes M DIG-labeled

DNA molecular weight marker III, 1 nontransformed control, 2–4 iptshoots, 5–7 marker-free transgenic normal shoots produced from iptshoots

Fig. 6 Reverse transcription (RT)–PCR, northern blot, and western

blot analyses of marker-free transgenic and control P. hybrida plants

‘Dainty Lady’ obtained through ipt shoot formation after infection

with A. tumefaciens harboring the ipt-type MAT vector pMAT21-WD.

a RT–PCR was performed to confirm the expression of the WD gene

at the mRNA level. Lanes M Size marker (øX174/HaeIII digests), 1positive control (plasmid cDNA) for WD gene, 2 negative control

(cDNA from non-transformed plant), 3–6 amplification of WDtranscripts in marker-free transgenic plants. b Northern blot analysis

of marker-free petunia plants. Northern hybridization was carried out

using the digoxigenin (DIG)-labeled DNA probe of the WD gene.

Lanes 1 Non-transformed control, 2–5 mRNA transcription from the

WD gene in marker-free transgenic petunia plants. c Western analysis

for the expression of WD protein in marker-free transgenic plants of

petunia. Protein extracts of leaves from in vitro-grown transgenic

independent clones and non-transformed control plants were frac-

tionated on a 15% polyacrylamide gel and subjected to immunoblot

analysis using a rabbit polyclonal antiserum for WD. Lanes: 1 Non-

transformed control, 2–5 marker-free transgenic plants. Arrowindicates the 5-kDa band of the WD protein


123

protein extracts of only transgenic plants (Fig. 6c, lanes

2–5). No reaction was detected in the non-transformed

control plants (Fig. 6c, lane 1).

Disease resistance assay of marker-free transgenic

plants

Defensin, a protein belonging to the defensin family, is one

of the antimicrobial proteins expressed in plants against

stresses such as infection by a foreign pathogen. Low

concentrations of WD was found to inhibit the growth of

the rice blast fungus in vitro (Saito et al. 2001). Earlier

studies on the expression of WD in transgenic Nicotiana

benthamiana (Saito et al. 2001; Kiba et al. 2003), rice

(Kanzaki et al. 2002), potato (Khan et al. 2006b), Pha-

laenopsis (Sjahril et al. 2006), and ‘‘Egusi’’ melon (Colo-

cynthis citrullus) (Ntui et al. 2010) all showed the

enhanced antimicrobial activities of this protein.

Fungal resistance of transgenic plants expressing the

WD gene was evaluated against B. cinerea, one of the main

pathogenic fungus that causes severe damage to the leaves

and flowers of P. hybrida. In vitro transgenic and non-

transformed control plants were challenged with B. cinerea

strain 40 grown on PDA to evaluate the resistance imparted

by the expression of WD protein in transgenic petunia

plants. Following the placement of an agar block (1 cm2)

with grown mycelia at the base of 2- to 3-week-old in vitro

control and transgenic plants, the fungal mycelia readily

penetrated the stem of the control plants, resulting in

dehydration and stem softening. Approximately 10 days

after inoculation, the control plant could not stand upright

and fell over (Fig. 7b). In contrast transgenic plants

expressing the WD gene remained green and continued to

grow through the fungal hyphae (Fig. 7a).

In another set of experiments, detached leaves from

control and marker-free transgenic petunia plants were

placed in petri dishes with wet filter paper, wounded, and

inoculated with the B. cinerea strain 40 spore suspension

(two inoculation spots per leaf). Water-soaked spots on the

leaf disks could be observed after 24 h. One day later,

necrotic lesions appeared which expanded over time as

increasingly more tissue was damaged by the pathogen.

After 5 days of incubation at room temperature, most of the

leaf area from the control plant turned brown and decom-

posed (Fig. 7d). In contrast, the leaf from the transgenic

plants expressing WD protein was still green, with small

necrotic spots only at the sites of inoculation (Fig. 7c). The

Fig. 7 Disease resistance assay of marker-free transgenic plants of P.hybrida ‘Dainty Lady’ expressing the WD gene against B. cinerea. a,

b Transgenic and non-transformed control in vitro plants were

challenged with B. cinerea grown on potato dextrose agar (PDA).

Two weeks after inoculation, the control plant could not stand upright

and fell over (b), whereas the transgenic plants expressing the WDgene remained green and continued to grow through the fungal

hyphae (a). c, d Detached leaf assay of control and marker-free

transgenic petunia plants. Leaf from the control plant (d) developed

expanded lesions around the inoculation spot, whereas the leaf from

the transgenic plant had only a small necrotic region at the site of

inoculation (c). e, f Leaves and stem sections from B. cinerea-

inoculated control and transgenic petunia plants were cultured on

PDA. Explants from the control favored the growth of the fungus (f),whereas growth of the fungus was restricted by the WD-expressing

plant parts from the transgenic petunia (e). g Area (mm2) of necrotic

lesion developed on detached leaves of control and transgenic petunia

plants after inoculation with spores of B. cinerea. WT Wild type

b


123

necrotic area (mm2) affected by B. cinerea around the

inoculation spot was threefold larger in non-transformed

control leaf (Fig. 7g). In our previous study, WD protein in

transgenic potato imparted partial resistance to B. cinerea,

as evidence in the detached leaf assay (Khan et al. 2006b).

The results of the present study further confirm the resis-

tance potential of WD protein against B. cinerea.

The antimicrobial activity of WD protein in restricting

the multiplication of B. cinerea in plants was further con-

firmed by challenging in vitro transgenic and control

Petunia plants with the fungal spores in glass bottles and

incubating the challenged plants at room temperature and

100% relative humidity. After 1 week, leaf and stem sec-

tions from the inoculated plants were cultured on PDA in

petri plates and incubated again under the same conditions.

Following the second incubation period, fungal growth

appeared to be unrestricted on all plant parts from the

control plants (Fig. 7f). In comparison, the multiplication

of B. cinerea in the leaves and stem sections from the

transgenic petunia expressing WD was restricted (Fig. 7e).

Based on the results reported here, we suggest that the

WD gene was successfully integrated into the genome of

transgenic petunia plants, producing the defensin protein.

Expression of the antimicrobial peptide, WD, provided

enhanced resistance to infections of B. cinerea in marker-

free transgenic petunia. However, further research is nee-

ded to test the resistance provided by WD in transgenic

petunia to other pathogenic fungi and bacteria.

Acknowledgments We would like to thank the Japan Society for

Promotions of Sciences (JSPS) for their financial support for this

research project. Our thanks are also extended to Pulp and Paper

Research group, Nippon Paper Industries, Tokyo who kindly provided

the MAT vector constructs.

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Botrytis cinerea-resistant marker-free Petunia hybrida produced using the MAT vector system