Pak. J. Bot., 45(5): 1761-1766, 2013.

TRANSFER OF THE GAFP AND NPI, TWO DISEASE-RESISTANT GENES, INTO A

PHALAENOPSIS BY AGROBACTERIUM TUMEFACIENS

JIE LI
1*
, PING KUANG
1
, REN-DAO LIU
1
, DAN WANG
1
, ZHI-NA WANG
1
AND MIN-REN HUANG
2

1
College of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China
2
Laboratory of Forest Genetics and Gene Engineering, Nanjing Forestry University, Nanjing 210037, China
*
Corresponding authors: e-mail: jay0224@sina.com

Abstract

Gastrodia Antifungal Protein (GAFP) and Neutrophils Peptide-I (NPI), two disease-resistant genes were successfully
transferred into the Dtps.Tailin Angel Phalaenopsis by using Agrobacterium strain off gradual methods. Two hundred and
eighty kanamycin-resistant plants were regenerated by this method. PCR, Southern blot and RT-PCR confirmed that GAFP
and NPI genes have been integrated into the genome of Phalaenopsis. In vitro antibiosis assay of the Colletotrichum
gloeosporioides Sacc suggested that the transgenic plants were disease-resistant. Disease-resistance experiments proved that
both GAFP and NPI genes were expressed efficiently in Phalaenopsis.

Introduction

As an expensive pot-flower, Phalaenopsis plays an
important role in the global flower trading market.
However, Phalaenopsis is susceptible to diseases caused by
fungi and bacteria, causing great loss to orchid production.
Conventional breeding methods to improve the disease
resistant of Phalaenopsis are limited mainly due to the long
reproduction cycles. Genetic modification of Phalaenopsis
is one of the newly emerged promising methods to enhance
the disease resistant of Phalaenopsis.
Gastrodia antifungal protein (GAFP) was isolated from
the top corm of Gastrodia elata. GAFP can inhibit more
than 20 fungi strains, including Armillaria mellea. GAFP
was cloned and the prokaryotic expression of this protein
showed bacteriostatic activity (Wang et al., 2003).
Neutrophils Peptide-I (NPI) increases permeability of
microbeless lipid membrane in the electromagnetic field,
which directly or indirectly affect energy metabolism and
reduce the Electric Potential on both sides of the
membrane. NPI has the widest antibiotic spectrum to
bacteria, fungi, viruses, and certain malignant tumor cells
(Hammond et al., 1996). Chen et al., (2005) transferred
pBin35SGAFP-NPI, a binary resistance gene expression
plasmid, into Populus deltoidsP.simonii and the genes
were expressed well in the transgenic plants.
Transformation of plants mediated by Agrobacterium
tumefaciens has gradually increased its contribution to the
production of transgenic plants( Bhatti et al., 2013; Shen et
al., 2012).Successful transformation of some mark genes like
GUS/ HPT and BP/KNAT1 into Phalaenopsis by using
Agrobacterium have been previously reported (Belarmino et
al., 2000; Endang et al., 2007; Sreeramanan et al., 2010).
However, there is no report on the successful transferring
some economically important traits genes into orchids, such
as genes associated with flower color and size, and genes
resistance to insects and diseases. In order to improve the
Phalaenopsis disease resistance, we transferred GAFP and
NPI genes into the Dtps.Tailin Angel Phalaenopsis to
investigate the diseases resistance of these transplants.

Materials and Methods

Gene transformation acceptor system: Young leaves of
the tissue-cultured plantlets of Phalaenopsis Dtps.Tailin
Angel (a red-flowered cultivar) were first cut
perpendicularly to their rachis into 5~10 mm slices. The
slices were put on the inducing medium with their rachis
facing the medium. The medium used in this experiment
was half-strength MS ( MS) medium (Chen et al., 2000)
with sucrose at 30 mg/L supplemented with N
6
-
benzyladenin (6-BA) at 0, 5, 10, 15 and 20 mg/L, and -
naphthaleneacetic acid (NAA) at 0, 1.0 and 2.0 mg/L,
respectively. After optimization of the medium for
stimulating Protocorm-like bodies (PLBs) formation,
kanamycin (Km) at 0, 5, 10, 15 and 20mg/L and
cefotaxime sodium (Cef) at 0, 50,100,200 and 300mg/L,
were added respectively to the medium to test the
sensitivity of explants to antibiotics.
Eight weeks later, the PLBs were subcultured for 12
h on MS medium (pH 5.7) with 7.0 g/L agar, peptone at
2g/L, banana juice at 3%, 2 g/L active carbon, and
sucrose at 10, 20, 30, 40 mg/L, respectively. The cultures
were incubated for 12 h at 262
o
C under cool-white
fluorescent light of 1500 lx and relative humidity at
60%~80%. Km was added at 0, 5, 10, 15 and 20 mg/L
respectively to test the effect of antibiotics to the growth
of the roots.

Bacterial strain and plasmid vector: A. tumefaciens
strains LBA4404 was used for the transformation. It
harbors a binary vector pBin35SGAFP-NPI (Chen et al.,
2005). The T-DNA region of pBin35SGAFP-NPI
contains a GAFP gene (380bp) and a NPI gene (170bp)
under the control of cauliflower mosaic virus (CaMV)
35S promoter (Fig. 1).

Transformation: As genetic transformation is influenced
by acetosyringone in monocotyledons (Rashid et al.,
2012), A.tumefaciens strains LBA4404 was grown in YEB
medium containing 50mg/L kanamycin and also 200M
acetosyringone (AS) overnight at 28
o
C to OD
600
0.5. Prior
to transformation, explants were cut into 11 cm sections
and pre-cultured on MS medium for 0, 1, 2, 3, 4, 5, 6 and
7 days, respectively, and then immersed the explants in the
above mentioned cultures of LBA4404 for 5, 15, 25, 35, 45
and 55 min, respectively. Co-cultivation of these explants
with 200M AS-treated A.tumefaciens was carried out
under a 12-h (light of 400lx) /12-h (dark) photoperiod at
25
o
C for 1, 2, 3,4,5,10,15 and 20 d respectively.
JIE LI ET AL.,
1762


Fig. 1. Construct of pBin35S GAFP-NPI. RB: right border;
LB: left border; Kan+: kanamycin resistance gene; NOS:
nopaline synthase promoter; 35S-promoter: cauliflower mosaic
virus (CaMV) 35S promoter; GAFP: Gastrodia Antifungal
Protein gene (380bp); NPI: Neutrophils Peptide-I genes (170bp);
multiple cloning sites, including HindIII, XbaI, ScaI, and EcoRI.

Regeneration of transformants: The explants were
washed thoroughly with sucrose-free MS liquid medium
containing 500mg/L cefotaxime. The cultures were
transferred to a selective medium containing kanamycin
and cefotaxime in darkness. When the light yellow
granular embryogenic callus was visible on the leaf edge,
the cultures were transferred on medium to induce
plantlets formation.

Nucleic acid isolation: Genomic DNAs and total RNA of
the plantlets were isolated from fresh leaf tissue as
described previously (Endang et al., 2007).

Molecular detect of the genes in the transformants: PCR
amplification was performed to investigate where the
plantlets contains the transferred genes. The forward primer
for GAFP gene is 5-ACG TCT AGA AGG GAT CGG
TTG AAT-3and the reverse primer is 5-GAT CTC GAG
GCC AGA AGC CGC CGC TGT-3,while the forward
primer for NPI genes is 5-GTG TAG GAT CCA TGG
TGG TCT GTG GGT GCA GAG-3and the reverse primer
is 5-GGG AGA GCT CAG TAG TCC AAA CAT GT-3.
Southern hybridization and detection were performed
by using the Dig-dUTP DNA probe, which was prepared
from the GAFP cDNA and NPI cDNA by using the Dig
DNA Labeling Kit (Boehringer Mannheim).
RT-PCR analysis was carried out by one-step RT-
PCR which was used Access RT-PCR kit (Promega) as
described previously (Awan et al., 2010).

Expression of GAFP-NPI genes: Colletotrichum
gloeosporioides Sacc isolated from pathologic leaves of
Phalaenopsis which was incubated on PDA at 30
o
C by
using agarose medium hole diffusion method. When
mycelium grew up to 3 cm in diameter, two holes per
plate were punched symmetrically to remove the agar
located 1 cm away from the mycelium. 200l of total
protein extract from the transgenic plants and non-
transgenic plants were added to the holes separately. The
plates were incubated at 25~28
o
C for 24~36 h in
darkness.

Inoculation of Colletotrichum gloeosporioides Sacc to
Phalaenopsis transplants: Suspension of Colletotrichum
gloeosporioides Sacc was prepared as described previously
(Zhang et al., 2005). 2l suspension of spores was added to
the five needle-wounded holes on the second leaf counted
from top of the transformated and non-transformated
Phalaenopsis. The plants were grown at room temperature.
The disease was sored 10 days later as the spot in diameter
on the leaves infected by Colletotrichum gloeosporioides
Sacc as 0:no symptom, 1:less than 5 mm, 2:5~10mm,
3:10mm~15mm and 4: >15mm.

Results and Discussion

Establishment of the leaf dish acceptor system: 6-BA
and NAA can stimulate PLBs formation. The best results
were those grown on MS with 6-BA at 10.0mg/L and
NAA at 1.0mg/L (Table 1).

Table 1. Frequency of Protocorm Like Bodies
(PLBs) induction in Phalaenopsis.
Treatments
BANAA (mg/L)
Mean PLBs
per explant
PLBs-forming
explants (%)
0.0 0.0 0.0
e
0.0%
h

5.0 0.0 0.0
e
0.0%
h

10.0 0.0 1.0
d
26.0%
f

15.0 0.0 0.5
de
20.0%
g

20.0 0.0 0.0
e
0.0%
h

0.0 1.0 0.0
e
0.0%
h

5.0 1.0 2.0
cd
35.0%
de

10.0 1.0 13.0
a
85.0%
a

15.0 1.0 8.0
b
80.0%
a

20.0 1.0 3.0
c
45.0%
c

0.0 2.0 0.0
e
0.0%
h

5.0 2.0 1.0
d
30.0%
ef

10.0 2.0 1.5
d
40.0%
cd

15.0 2.0 3.8
c
55.0%
b

20.0 2.0 3.3
c
50.0%
bc

BA ** **
NAA ** **
BANAA ** **
Means with the same letter are not significantly different.

The correlations between the concentration of
sucrose and plantlets regenerations from PLBs was
y=0.01992x
2
+0.07751x+0.17000. Plantlets developed
well on MS containing 20g/L sucrose without any
growth regulators.
Antibiotic sensitivity tests showed no significant
difference of Cef concentration on the mean protocorm
induction from Phalaenopsis between 0 to 300 mg/L
(Table 2).
TRANSFER OF THE GAFP AND NPI, TWO DISEASE-RESISTANT GENES, BY AGROBACTERIUM TUMEFACIENS
1763
Table 2. Effect of cefotaxime sodium concentration on
induction of protocorm from Phalaenopsis leaves.
Cefotaxime
sodium
(mg/L)
PLBs
forming
explant (%)
Number of
PLBs per
explant
PLBs-
forming
capacity
0 85.0 14 11.9
50 83.0 13 10.8
100 83.0 12 10.2
200 87.0 10 8.7
300 84.0 10 8.4

Protocorms (Table 3) and roots (Table 4) were not
produced when Km was at 10mg/L. Based on the results,
Cef at 50mg/L may be used to eliminate the
Agrobacterium in selective medium, and Km at 10mg/L
may be added to discriminate between transformants and
non- transformants.

Tranformation and regeneration of transformants:
Using leaf dish of Phalaenopsis as initial explants, we
tested several factors affecting transformation in
Agrobacterium tumefaciens mediated system. The
optimized system for genetic transformation in
Phalaenopsis was established. The best condition is as
following: pre-culture time to induce kanamycin-resistant
(Km
r
) PLBs 4~5 days, LBA4404 OD
600
at 0.05, infection
time 10 min and pH 5.4. The co-culture time is the most
critical on the induction Km
r
PLBs. We divided this time
as two stages, the first stage is 5 days, when the explants
were co-cultivated with Agrobacterium tumefaciens
without adding any antibiotics; the second stage is
2~3weeks, with Cef concentration at 20 mg/L. The Km
r

PLBs was as high as 16.2%, five times as the first stage of
co-cultivation (Table 5).

Table 3. Effect of kanamycin concentration on
induction of protocorm from Phalaenopsis leaves.
Kanamycin
(mg/L)
PLBs
forming
explant (%)
Number of
PLBs per
explant
PLBs-
forming
capacity
0 86.0 13 11.2
5 8.0 4 0.3
10 0.0 0 0.0
15 0.0 0 0.0
Table 4. Effect of kanamycin concentration on
induction rooting from protocorm.
Kanamycin
(mg/L)
Roots-
forming
PLBs (%)
Number of
roots per
PLBs
Roots-
forming
capacity
0 100.0 4.1 4.1
5 48.0 1.8 0.9
10 0.0 0.0 0.0
15 0.0 0.0 0.0
20 0.0 0.0 0.0

Table 5. Induction of Km
r
protocorm from
Phalaenopsis leaves in optimum conditions (%).
Treats
batch
Frequency of
Km
r
in CK+
Frequency of
Km
r
in CK-
Frequency of
Km
r
in
inoculated
1 84 1.0 12.0
2 77 1.5 13.0
3 80 0.5 12.5
4 80 1.0 12.0
5 79 0.5 16.2
6 80 1.0 15.0
CK+: Frequency of protocorm formation without Km and
Agrobacterium tumefaciens immersed; CK-: Frequency of
protocorm formation only with Km.

Km-resistant buds induced from leaf explants were
cut for proliferation on selective medium supplemented
with Km at 10mg/L with monthly subculture. After three
rounds of selection, the putative transformants were
transferred to MS medium containing Km10mg /L for
root development (Fig. 2).The plantlets regenerated from
Km-resistant buds were used for further analyses.

Molecular analyses of transformants: The presence of
GAFP-NPI genes in eleven randomly chosen Km-
resistant plantlets was firstly examined by PCR. As
shown in Fig. 3, the fragments showed the expected size
of 380bp for GAFP and 170bp for NPI in putatively
transformed plantlets. As expected, there were no PCR
bands shown with the DNA from non-transgenic plantlets.



Fig. 2. The kanamycin-resistant buds and regeneration of transgenic plants. (A) protocorm formation from leaves without Km and
Agrobacterium tumefaciens immersed; (B) Km-resistant buds; (C)Km-resistant plantlets.
JIE LI ET AL.,
1764


Fig. 3. PCR detection on NPI-GAFP genes in transgenic plantlets. M: 200bp ladder; CK+: pBin35SGAFP-NPI control; CK-: non-
transgenic plantlets; 1-11: transgenic plantlets.



Fig. 4. Southern blot screening for foreign NPI-GAFP genes in transformed plantlets. CK+:pBin35SGAFP-NPI control; CK-: non-
transgenic plantlets; 1-8: transformed plantlets.

Southern blot analysis confirmed the presence and
integrity of the GAFP gene and NPI gene in transformed
plantlets (Fig. 4). To further assess the expression of the
GAFP gene and NPI gene driven by the CaMV 35S
promoter, we also analyzed the amounts of GAFP mRNA
and NPI mRNA in twelve transformed plantlets by RT-PCR.
As shown in Fig. 5, the GAFP gene and NPI gene were
expressed in ten transgenic lines analyzed. Only two
transformed plantlets presented no corresponding fragments.

Pathogen-resistance of the transformants: GAFP and
NPI protein were constitutively expressed in plants. The
expressed product is fungi-resistant. Colletotrichum
gloeosporioides Sacc isolated from pathologic leaves of
Phalaenopsis was incubated at agarose medium by hole
diffusion method. The results showed the clear inhibition
zones around crude extracts protein of twenty four Km
r

plantlets; however, there was no inhibition zone from
non-transgenic plantlets (Fig. 6). In the vitro antibiotics
assay suggested that the transgenic plantlets were disease-
resistant and the two genes can be expressed efficiently in
transgenic Phalaenopsis.

In vivo experiment of disease resistant of transgenic
Phalaenopsis: After 10 ds incubation, the disease spots
of non-transgenic Phalaenopsis reached scale 3-4 while
the transgenic Phalaenopsis reached only scale 1, which
indicated transgenic Phalaenopsis was resistant to
C.gloeosporioides Sacc, even they are not immune to the
pathogen (Fig. 7).
TRANSFER OF THE GAFP AND NPI, TWO DISEASE-RESISTANT GENES, BY AGROBACTERIUM TUMEFACIENS
1765


Fig. 5. RT-PCR for foreign NPI-GAFP genes in transformed plantlets. CK-: non-transgenic plants; 1-12: transgenic plants.



Fig. 6. In vitro antifungal assay of transgenic plantlets. CK: no inhibition zone around crude extracts protein of non-transgenic plants;
2, 4, 6,8: inhibition zones around crude extract protein of transgenic plants.



Fig. 7. Antifungal assay on transgenic Phalaenopsis. 1~4: transgenic plants; 5~8: non-transgenic plants.
JIE LI ET AL.,
1766
In this study, we showed several factors, e.g., pre-
culture time, co-culture time, bacteria concentration
infection time, medium pH, which contributed to success
of the transformation process. Optimized conditions for
co-cultivation were also shown in this study. The Leaf
dish acceptor system of gene transformation has been
established. In this experiment, leaf dish of Phalaenopsis
as initial explants can not only regenerate efficiently, but
also avoid producing transgenic chimera. Time of co-
cultivation is one of the main factors among different
factors affecting Agrobacterium mediated gene
transformation (Raja et al., 2010; Narusaka et al.,
2003).The target explants produced more infecting cells
by Agrobacterium strain off gradual methods, which
improved the frequency of transformation greatly by
adjusting the concentration of Agrobacterium to balance
the maximal frequency of transformation and the
minimum frequency of Agrobacterium contamination.
This method increased the percent of putatively
transformed plantlets to 16.2%. These results also
indicated that the long co-culture period was essential for
successful transformation of plants (Nakashima et al.,
2000; Yu et al., 2001; Liau et al., 2003; Zia et al., 2010).
GAFP-NPI genes were integrated into the genome of
Phalaenopsis confirmed by PCR, Southern blot and RT-
PCR analysis. However, mRNA transcription was not
detected in a few of transgenic plants, which suggested
that some unknown reasons silenced the transformed
foreign genes. In vitro antibiotics assay and antifungal
assay in greenhouse showed that there was a discrepancy
in antibacterial activity in different transgenic plants. The
difference between lab results and field application still
exists, and much remains to be done.

Acknowledgements

Authors sincerely thank Dr. Chen Ying for providing
the plasmid pBin35SGAFP-NPI.Grants from Education
Department of Sichuan Province (Grant No.10zd1129),
the Doctoral Program of Southwest University of Science
and Technology (Grant No. 09zx7107) and Sichuan
academic and technical leader training projects (Grant No.
11sd3105).

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(Received for publication 18 Marcy 2012)