HORTSCIENCE 46(4):616–621. 2011.
In Vitro Regeneration and Genetic
Transformation of Cucumis metuliferus
through Cotyledon Organogenesis
Yu-Tsung Lin, Chia-Wei Lin, Chien-Hung Chung, and Mei-Hsiu Su
Department ofAgronomy, NationalChung Hsing University,Taichung 40227,
Hsiu-Yin Ho, Shi-Dong Yeh, and Fuh-Jyh Jan1
Department of Plant Pathology, National Chung Hsing University, Taichung
Department of Agronomy, National Chung Hsing University, Taichung
Additional index words. Agrobacterium transformation, b-glucuronidase, horned melon
Abstract. This study was undertaken to establish for the first time an efficient re-
generation and transformation system for Cucumis metuliferus line PI292190, which is
the source of a well-defined resistant gene, Wmv, that provides resistance against Papaya
ringspot virus type P (PRSV-P) and PRSV-W (formerly known as Watermelon mosaic
virus 1, WMV-1). Different combinations of growth regulators were evaluated for the
regeneration of cotyledon explants. Adventitious buds or shoot primordia were obtained
within 3 to 4 weeks on regeneration medium. After shoot development, adventitious buds
or shoot primordia were transferred to elongation medium for 3 to 4 weeks and these
shoots were subcultured onto rooting medium for another 1 to 2 weeks. Under optimal
from cotyledons. Furthermore, transgenic plants were successfully obtained using an
Agrobacterium tumefaciens-mediated transformation method as shown by polymerase
chain reaction analysis and histochemical b-glucuronidase (GUS) assay. A total of nine
transgenic plants were developed from 360 cotyledon explants, giving a transformation
frequency of 2.5%.
Cucumis metuliferus (Naud.) Mey, one of
Africa, provides high-quality nutrition sources
and is consumed daily as a vegetable by many
rural communities in Africa (Kristkova et al.,
2003). Its other names include jelly melon,
horned melon, or hedgehog gourd, and it is
generally marketed as Kiwano. It is an impor-
namely potassium, calcium and magnesium
(Odhav et al., 2007; Romero-Rodriguez et al.,
1992). The novel flavor of C. metuliferus fruit
may be useful in an innovative tea or bever-
age. The fruits contain high antioxidant com-
properties when applied to laboratory rats
(Motlhanka, 2008; Wannang et al., 2007).
Moreover, C. metuliferus was reportedly able
to provide competence for heat and salt tol-
erance (Benzioni et al., 1991), prolong fruit
shelf life (Mendlinger et al., 1992), and in-
crease resistance against Cucurbit yellowing
stunting disorder virus (Lopez-Sese and
Gomez-Guillamon, 2000), PRSV-P, PRSV-W
(formerly known as WMV-1) and Squash
mosaic virus (Provvidenti and Gonsalves,
1982; Provvidenti and Robinson, 1974,
1977), whitefly (Lopez-Sese and Gomez-
Guillamon, 2000), fusarium wilt caused by
Fusarium oxysporum f. sp. melonis (Nisini
et al., 2002), and root-knot nematode (Chen
and Adelberg, 2000; Nugent and Dukes,
1997; Siguenza et al., 2005; Wehner et al.,
The previously described regeneration
respect to the genotype of plants, the explant
1998; Beharav and Cohen, 1994; McCarthy
et al., 2001; Punja et al., 1990; Raharjo and
Punja, 1993; Tang and Punja, 1989). The fre-
quency of shoot formation in C. metuliferus
PI 292190 from petiole explants was 14.6%
(Raharjo and Punja, 1993); however, 0% to
30% from leaf explants and 0% to 10% from
In addition, no information on transfor-
mation of C. metuliferus is available so far,
although ample transformation systems for
melon, watermelon, squash, muskmelon, and
cucumber have been developed by Agro-
bacterium tumefaciens-mediated transforma-
tion (Choi et al., 1994; Fang and Grumet,
1990; Gonsalves et al., 1994; Pang et al.,
2000; Vasudevan et al., 2007) or micropro-
jectiles protocols (Chee and Slightom, 1992).
It was reported that genotype and growth
regulators are determining factors influencing
regeneration efficiency in the Cucurbitaceae
family (Oridate et al., 1992). 2, 4-dichlorophe-
noxy-acetic acid (2,4-D) is more frequently
used in genotype of Cucurbita species for
somatic embryogenesis, yet naphthaleneace-
tic acid (NAA) is used in Cucumis species. In
addition, benzyladenine (BA) was shown to
give the best response on shoot production
derived from cotyledon explants in Cucumis
species (Abrie and van Staden, 2001; Chee,
1991; Niedz et al., 1989) and shoot primordia
or NAA/zeatin combinations (Punja et al.,
In this study, a successful organogenic
regeneration procedure (with greater than
58% efficiency) for C. metuliferus is de-
scribed. Meanwhile, two binary vectors,
pBI121 (Chen et al., 2003) and TWBI, were
used in A. tumefaciens-mediated transforma-
tion. A. tumefaciens-mediated delivery of the
b-glucuronidase gene (gus) in C. metuliferus
was obtained at a frequency of 2.5%. This is
the first report on an efficient regeneration
and A. tumefaciens-mediated transformation
in C. metuliferus.
Materials and Methods
Plant material and regeneration system.
were removed and the naked seeds were
surface-disinfected by dipping in 0.5% so-
dium hypochlorite for 10 min followed by
three rinses in sterile distilled water. The
sterilized seeds were placed on Murashige
and Skoog (MS) medium (pH 5.7) including
Gamborg B5vitamins(Duchefa,The Nether-
lands) for 1 to 2 d. Embryos were removed
from sterilized seed and the residual cotyle-
dons were used as explants. Each enlarged
cotyledon was longitudinally and transver-
sally dissected to eight equal parts for re-
BA, and 2,4-D as shown in Table 1), callus,
adventitious buds, or shoot primordia derived
from cotyledon explants were excised and
transferred to elongation medium (0.1 mg?L–1
BA, 0.02 mg?L–1NAA). After further incuba-
tion for 3 to 4 weeks, the elongated shoots
were transferred to rooting medium (MS
medium containing 0.5 mg?L–1indole-3-bu-
tyric acid) and maintained in a tissue culture
room under 16-h light/8-h dark cycles with a
photon flux rate of 55 to 65 mEm–2?s–1from
Received for publication 10 Nov. 2010. Accepted
for publication 11 Feb. 2011.
This work was supported by a grant from the
‘‘Program for promoting academic excellence of
universities’’ from the National Science Council in
Taiwan (No. NSC 95-2752-B-005-004-PAE).
We are grateful to Dr. Wen-Hsiung Ko, Professor
Chung-Jan Chang, Professor of Plant Pathology at
the University of Georgia, Griffin Campus; and Dr.
Anne Frary, Professor of Izmir Institute of Tech-
suggestions of the manuscript.
1To whom reprint requests should be addressed;
e-mail email@example.com, firstname.lastname@example.org.
HORTSCIENCE VOL. 46(4) APRIL 2011
were acclimatized in a greenhouse under con-
trolled conditions of 26 ± 2 ?C. All plant
growth regulators were filter-sterilized and
added into the medium, which had been
autoclaved (15 min at 121 ?C and ?1 kPa)
after pH adjustment (pH 5.7). Approximately
15 mL of medium was dispensed into dispos-
able petri dishes (90 · 20 mm) for regener-
ation and elongation and 30 mL of rooting
medium was dispensed into Magenta GA7
vessels for rooting.
phosphinothricin. To determine the ap-
propriate concentration of kanamycin and
DL-phosphinothricin (PPT) for selection of
transgenic explants, Agrobacterium-infected
(MS medium supplemented with 1.0 mg?L–1
BA and 0.02 mg?L–1NAA) containing differ-
entconcentration ofkanamycinorPPT (Table
2). Surviving explants with adventitious buds
or shoot primordia were excised and trans-
ferred to S2 medium (MS medium supple-
mented with 0.1 mg?L–1BA, 0.02 mg?L–1
NAA) under the same selection conditions.
The number of surviving shoots was re-
corded after 2 weeks. The concentration of
kanamycin or PPT that gave the highest
frequency of shoot development was chosen
as the criterion for the transformation of C.
Transformation of Cucumis metuliferus.
A. tumefaciens strain LBA4404 harboring
the binary vector pBI121 (Clontech Labora-
tories, Palo Alto, CA) carrying the neomycin
phosphotransferase II (nptII) and gus genes
was used for plant transformation under
of binary vector pBI121 were driven by the
nopaline synthase (nos) promoter and Cauli-
flower mosaic virus (CaMV) 35S promoter,
respectively (Fig. 1A). Another binary vector,
TWBI, containing the chimeric bar (phosphi-
35S promoter (Fig. 1B) was also transformed
into Agrobacterium strain LBA4404 for C.
metuliferus transformation under PPT selec-
tion. Cotyledon explants were inoculated with
freshly grown-overnight bacterial suspension
at 100-fold dilution in MS liquid medium
supplemented with 20 mM acetosyringone.
After 10 min, the cotyledons were directly
transferred into TR medium (the same as S1
were then transferred to TS1 (S1 medium
supplemented with 150 mg?L–1kanamycin
and 250 mg?L–1carbenicillin) and subcul-
tured weekly for 3 to 4 weeks. The newly
generated buds or shoot primordia were
with 150 mg?L–1kanamycin and 250 mg?L–1
carbenicillin) media and subcultured weekly
for another 3 to 4 weeks. The number of
adventitious buds and shoot primordia formed
at the apical site of cotyledon explants were
recorded in TS2 medium for a period of 3 to
4 weeks. The rooting procedure (on rooting
medium supplemented with 250 mg?L–1car-
benicillin) and acclimation of transformants
were the same as described for regeneration
experiments. Acclimatized plants were ana-
lyzed for transgene by polymerase chain re-
action (PCR) and GUS assays.
Detection of transgenes. Plant genomic
DNA wasextracted fromleaves oftransgenic
plants by a modified CTAB method (Fulton
et al., 1995). Forty nanograms of plant geno-
mic DNA was used for PCR analysis per-
formed with denaturing at 94 ?C for 1 min,
annealing at 55 ?C for 1 min, and extension
at 72 ?C for 2 min for 30 cycles and a final
extension at 72 ?C for 5 min. PCRanalysis for
the nptII gene was performed using primers
GGGCGAAGAACTCCAG-3#) (Fig. 1A).
PCR analysis of the gus gene was performed
using primers FJJ1999-12 (5#-ATATGGA
located in the CaMV 35S promoter region
and FJJ2001-15 (5#-TGATAATCATCGC
AAGAC-3#) covering the nos terminator
region (Fig. 1A). Southern blot analysis
was performed to confirm the integration of
the gus gene in the transformants that gave
positive PCR results for both transgenes
(nptII and gus genes). Vector DNA (as posi-
and non-transformed plants were digested by
gels, and blotted onto nylon membranes. The
membranes were hybridized with a P32-labeled
probe of a 2.0 kb gus gene fragment, which
was PCR amplified from plasmid template
with primers FJJ1999-12 and FJJ2001-15.
In addition, a histochemical assay to detect
GUS activity was performed as described by
Jefferson (1987). Leaf tissues wereimmersed
in the reaction buffer (100 mM Tris, 50 mM
NaCl, and 0.1% Triton-X 100, pH 7.0) con-
taining 2 mM potassium ferricyanide and 2
mM 5-bromo-4-chloro-3-indolyl glucuronide
(X-gluc) as substrates. The reaction was per-
formed under a mild vacuum for 1 min after
incubation at 37 ?C for 1 to 2 weeks. The
tissues were washed with 70% ethanol before
observation. The efficiency of transformation
was calculated by dividing the number of
PCR-positive plants for both nptII and gus
genes by the total number of explants used in
three independent experiments.
Regeneration of cotyledon explants. Some
friable, white or pale yellow calli were ob-
served on the medium containing auxin only
(NAA and 2,4-D), whereas buds or shoot
primordia were observed after adding BA
at a concentration of 0.5 or 1.0 mg?L–1(Table
1). Generally, cotyledon explants enlarged
Table 1. The effect of auxins (NAA, 2,4-D) and cytokinin (BA) concentration on regeneration of buds or
shoot primordia derived from Cucumis metuliferus cotyledon explants.
Concn of growth regulator (mg?L–1)
zThe frequency was calculated by using the number of calli (-C), buds (-B), or shoot primordia (-SP)
divided by the number of cotyledon explants in each experiment.
NAA = naphthaleneacetic acid; 2,4-D = 2, 4-dichlorophenoxy-acetic acid; BA = benzyladenine.
Number of calli (-C),
buds (-B), or shoot
primordia (-SP) (frequency)z
27-C (27%), 20-B (20%), 15-SP (31.3%)
40-C (40%), 29-B (29%), 20-SP (41.7%)
23-C (23%), 19-B (39.6%), 8-SP (16.7%)
160-C (100%), 7-B (4.4%)
Table 2. Effect of kanamycin and DL-phosphinothricin (PPT) concentration on regeneration of buds or
shoot primordia from Agrobacterium-inoculated Cucumis metuliferus cotyledon explants.
Concn of selective agent
of surviving cotyledon
Number of calli (-C), buds
(-B), or shoot primordia
1-C (0.6%), 40-B (22.6%),
52-B (15.2%), 52-SP
30-B (10%), 6-SP (2%)
44-B (10.7%), 12-SP
21-B (22.6%), 21-SP
31-B (24%), 24-SP
150 379341 (89.9%)
zCotyledon explants surviving on regeneration medium containing kanamycin or PPT at different
concentrations after 2 weeks.
the number of surviving cotyledon explants, respectively.
HORTSCIENCE VOL. 46(4) APRIL 2011
PROPAGATION AND TISSUE CULTURE
considerably and developed into mature cal-
lus, buds, or shoot primordia on the margins
within 3 weeks after being transferred into
regeneration medium (Fig. 2A). When the
mature globular calli were transferred to
elongation medium, the regenerated calli
failed to form shoots. However, most adven-
titious buds or shoot primordia were observed
preferentially at the proximal cotyledons (Fig.
2B). When transferred to elongation medium,
these buds and shoot primordia continued to
grow and formed mature shoots after 3 to 4
weeks. In some cases, cotyledon explants
proliferated profusely and formed clusters of
shoot apices, which did not develop into
normal shoots on elongation medium (Fig.
2C). Some explants directly developed into
leaf-like structures without forming buds or
shoot primordia (Fig. 2D). In terms of bud
or shoot formation, the medium containing
0.02 mg?L–1NAA and 1.0 mg?L–1BA pro-
vided a suitable development. In most cases,
buds orshootprimordiadevelopedinto normal
shoots and leaves (Fig. 2E–F). All elongated
shoots were transferred to rooting medium for
1 to 2 weeks (Fig. 2G–H). The plantlets were
acclimatized in the greenhouse (Fig. 2I) with
the survival rate greater than 95%.
Sensitivity of cotyledons to kanamycin
and DL-phosphinothricin. Totestthe optimal
concentrations of kanamycin and PPT for
transformation, Agrobacterium-infected cot-
yledon explants were placed on the selection
medium supplemented with various concen-
3 weeks on selection medium, the survival
and 78.1% when kanamycin was supple-
mented at 50, 150, and 200 mg?L–1, respec-
tively. Fifty-two buds and 52 shoot primordia
were obtained from 341 surviving explants
when kanamycin was supplemented at 150
mg?L–1, whereas fewer buds or shoot primor-
dia were obtained at 50 or 200 mg?L–1(Table
became pale yellowish or developed partial
necrosis 3 weeks after being transferred to
media supplemented with various concentra-
tions of PPT from 1.0 to 33.8 mg?L–1. The
survival rates varied from 31.5% at 33.8
mg?L–1of PPT to 83.5% at 16.9 mg?L–1of
PPT (Table 2). Although it is cheaper and
PPTthankanamycin,morerestriction on buds
and shoot primordia development was noticed
on PPT selective medium. Therefore, kana-
mycin at a concentration of 150 mg?L–1was
used for further establishment of C. metulife-
Transformation of Cucumis metuliferus.
The frequency of buds or shoot differentia-
tion of inoculated explants varied from
43.3% to 68.3% in three independent exper-
iments (Table 3). Explants with buds or shoot
primordia were dissected and subcultured
weekly in TS2 medium for 3 to 4 weeks.
Most buds or shoots elongated but some did
not develop into normal shoots. These abnor-
mal shoots were hyperhydric and formed
unexpanded leaf structures. In contrast, the
elongated shoots showed various develop-
ments, including no root formation, short
root formation, and formation of a strong
and branched root system in rooting me-
dium. From these experiments, a total of 53
putative transgenic plantlets was success-
fully developed with healthy root systems.
All of them were acclimatized in the green-
house (Fig. 2I).
Ti plasmid contains neomycin phosphotransferase II (nptII) and b-gluduronidase (GUS) reporter
genes. These two genes were separately fused under the control of the nopaline synthase (NOS-pro)
herbicide Basta is driven by the CaMV 35S promoter and the NOS terminator (NOS-ter). RB
represents the right border and LB is the left border of the T-DNA.
Fig. 2. Plant regeneration and transformation of Cucumis metuliferus line PI 292190. (A) Formation of
callus at the cut surface of the cotyledon explants on medium containing 0.02 mg?L–1NAA and 0.2
mg?L–12,4-D after 3 weeks of culture (C = callus). (B) Adventitious shoots formation at the proximal
end of the cotyledon in regeneration medium (0.02 mg?L–1NAA and 1.0 mg?L–1BA) after 3 weeks of
culture (P = protuberance). (C) Adventitious buds showing shoot clustering and severe hyperhydricity
after several subcultures. (D–E) Explants showing differentiation into a leaf without the formation of
buds or shoot primordia. Formation of abnormal leaves that did not elongate (L = leaf-like structure).
(F) Healthy plantlets growing in elongation medium. (G–H) Healthy roots generated in transgenic C.
metuliferus plantlet obtained after 1 to 2 weeks culturing in the rooting medium. (I) Acclimatized
putative transgenic plants. NAA = naphthaleneacetic acid; 2,4-D = 2, 4-dichlorophenoxy-acetic acid;
BA = benzyladenine.
HORTSCIENCE VOL. 46(4) APRIL 2011
Detection of the presence of transgenes.
DNA isolated from the upper leaves of trans-
formants, untransformed C. metuliferus or
the pBI121 plasmid was used as template
DNA for PCR analysis of transgenes. The
presence of a 1.2-kb fragment corresponding
to the expected size of the nptII gene was
detected in transformants and pBI121 plas-
mid but not in the untransformed C. metuli-
ferus (Fig. 3A). A fragment of 2.0 kb for the
gus gene was found in transfomants and
pBI121 but not in untransformed C. metuli-
ferus (Fig. 3B). A total of 21 plants were
positive in PCR analysis of the nptII gene but
only nine of those plants were also positive
for the gus gene. Furthermore, the results of
Southern blot analysis showed the presence
of the gus gene in the transformants and
positive control but not in the untransformed
control (Fig. 4). Finally, a GUS expression
assay was conducted in transformants show-
ing positive PCR reaction for the gus gene
and non-transgenic C. metuliferus as a nega-
tive control. GUS activity was detected in
transformed shoots (Fig. 5A) along the mar-
gins of leaves in transgenic plant (Fig. 5B)
but not in the non-transgenic control (Fig.
5C) after incubation at 37 ?C for 2 d. Based
on the results of PCR analysis and GUS
assay, the transformation frequency of C.
metuliferus was measured to 2.5% (Table 3).
An efficient regeneration system (greater
than 58%) was obtained for C. metuliferus
after A. tumefaciens infection and the adven-
titious buds and shoot primordia developed
within 3 to 4 weeks. The buds and shoot
primordia were cultured on elongation me-
dium for 3 to 4 weeks and finally on rooting
medium for another 1 to 2 weeks. A total of
7 to 10 weeks were needed to obtain trans-
formants. An average transformation rate of
2.5% was achieved; however, a rate as high
as 24.2% was obtained in one of the exper-
iments (Table 3).
In this study, cotyledons were chosen as
the explant source to establish an efficient
regeneration and transformation system for
C. metuliferus line PI 292190. Direct organ-
ogenesis from cotyledons offers the follow-
ing advantages: the easier availability of
cotyledon, the avoidance of somatic muta-
tions that may be associated with callus, and
it is technically easy and rapid (Vasudevan
et al., 2007). It has been widely applied for in
vitro regeneration of different genotypes of
C. melo (Adelberg et al., 1994; Dong et al.,
1991; Ezura et al., 2000; Ficcadenti and
Rotino, 1995; Gaba et al., 1999; Galperin
et al., 2003; Gonsalves et al., 1994; Molina
and Nuez, 1995), C. sativus (Vasudevan et al.,
2007; Vengadesan et al., 2005), Cucurbita
pepo (Kathiravan et al., 2006), Citrullus lana-
tus (Chaturvedi and Bhatnagar, 2001), and C.
metuliferus line PI 482439 (Adelberg, 1998).
It was reported that the proximal edges of
cotyledon explants conferred the highest
regenerability in C. melo (Gaba et al., 1999;
Gonsalves et al., 1994). In this study, it was
also observed that proximal edges of cotyle-
don explants produced more buds and shoot
primordia (Fig. 2B). Normal adventitious
buds or shoot primordia were successfully
regenerated at a frequency of 58% from
cotyledon explants and from which the fre-
quency of shoot formation reached 37.2%
(Table 3). It was suggested that a higher
concentration of NAA (0.37 mg?L–1) or a
combination with 0.22 mg?L–1BA can hinder
the formation of callus from explants in C.
metuliferus linePI 292190 (Raharjo andPunja,
1993). In this study, we observed callus re-
generation in medium containing 0.02 mg?L–1
NAA and 0.2 to 0.5 mg?L–12,4-D but this
callus failed to regenerate shoots (Fig. 2A).
The enhancement of shoot development us-
ing higher concentrations of BA in regener-
ation medium has been reported for five
different selected cucurbit cultivars (Abrie
and van Staden, 2001). In a study conducted
by Gaba et al. (1999), the regeneration of
Cucumis species from epidermis and the
subepidermal layer was principally derived
from the adaxial surface of cotyledon ex-
plants when BA concentrations ranging from
0.22 to 2.2 mg?L–1were used in the culture
medium. In this study, 0.02 mg?L–1NAA
Table 3. Transformation frequency of Cucumis metuliferus.z
of adventitious buds
or shoot primordiay
(frequency) of shoot
zPrecultured cotyledon explants were inoculated with Agrobacterium tumefaciens strain LBA4404
harboring the binary vector pBI121.
yBuds or shoot primordia differentiation was recorded after 2 to 3 weeks of culture on TS1 medium. The
frequency was calculated by using the number of adventitious buds or shoot primordia divided by the
number of cotyledon explants inoculated in each experiment.
xOnly elongated adventitious buds or shoot primordia were counted. The frequency was calculated by
using the number of shoot differentiation divided by the number of cotyledon explants inoculated in each
wTP = transgenic plants that were polymerase chain reaction (PCR)-positive in the detection of nptII and
gus gene. Frequency was calculated by using total number of PCR-positive plantlets divided by the total
number of cotyledon explants.
vThe putative transgenic plants died after acclimatizing in the greenhouse and were not subject to PCR
Fig. 3. Polymerase chain reaction (PCR) analysis of nptII and b-glucuronidase genes. PCR amplified
fragments of (A) nptII gene and (B) b-glucuronidase (gus) gene from putative transgenic Cucumis
metuliferus (Lanes 1 to 18), non-transgenic Cucumis metuliferus PI 292190 (Lane 19), and plasmid
pBI121 (Lane 20). The expected fragment sizes of 1.2 kb for the nptII gene and 2.0 kb for the gus gene
and FJJ2002-15 were designed for nptII detection and a 1.2-kb fragment was amplified. A 2.0-kb
fragment was amplified for gus detection using primers FJJ1999-12 and FJJ2001-15.
Fig. 4. Southern blot analysis using a-32P labeled
probe derived from a fragment of the gus gene.
Lane 1, positive control (vector pBI121
digested by ScaI); Lanes 2 and 5, transformants
960822-5; Lanes 3 and 6, transformants
960822-17; Lanes 4 and 7, untransformed
negative control (Cucumis metuliferus line PI
292190). Genomic DNA of Lanes 2 to 4 and
5 to 7 plants was digested by ScaI and XhoI,
HORTSCIENCE VOL. 46(4) APRIL 2011
combined with 1.0 mg?L–1BA were used in
regeneration medium for cotyledon explants
and from which adequate buds or shoot pri-
mordia were regenerated and later developed
into normal shoots. However, some explants
in this study were found to produce many
buds but only few shoots (Fig. 2C). A similar
phenomenon has also been reported in re-
generation studies of other cucurbits (Colijn-
Hooymans et al., 1994; Compton and Gray,
1993). Gonsalves et al. (1994) reported that
buds developing from the epidermis or sub-
epidermal layer, especially those located at
the cut part of the cotyledon proximal to the
seed apex, and the first organogenesic meri-
stems formed in vitro inhibited the develop-
ment of further buds. It was suggested that
excising the larger shoots permits more buds
to develop further (Gaba et al., 1999).
The phenomenon of ‘‘escape’’ from anti-
biotic selection in the transformation of C.
melo was reported by Akasaka-Kennedy et al.
(2004). In previous work, cotyledon explants
under 75 to 100 mg?L–1kanamycin selection
regenerated at frequencies of 15% for C.
sativus (Vengadesan et al., 2005), 30% for
muskmelon (Fang and Grumet, 1990), and
75% to 90% for melon (Dong et al., 1991). A
high proportion of escapes was encountered
in cucumber when embryo suspension culture
was used under kanamycin selection (Schulze
et al., 1995). The occurrence of the escapes
may be the result of inadequate selective
ucts of contaminating microbial cells (Dong
et al., 1991). Therefore, it is important to
establish an efficient selection for the trans-
cotyledon explants (Gaba et al., 1999). In this
study, although a high concentration of kana-
mycin (150 mg?L–1) was used, the occurrence
of escape was difficult to avoid completely.
As shown in Table 3, an average of 58% of
the Agrobacterium-infected cotyledon ex-
plants formed multiple shoots on selective
media. However, only nine of 53 putative
nptII and gus genes in PCR analysis, in-
dicating a certain degree of escape or chime-
ric plants regenerated from C. metuliferus
PI 292190. It has been postulated that the
mycin occurred in melon or other Cucumis
species and that transformation rate of in-
oculated melon on selection media is higher
than on non-selective media (Dong et al.,
sure enables the transformed cells to compete
with the non-transformed cells more effec-
tively resulting in a slight decrease in the
level of escapes and chimeric plants. Other
selection agents such as methotrexate or PPT
were also used for efficient selection of trans-
genic plants in melon and cucumber trans-
formation (Dong et al., 1991; Vengadesan
et al., 2005). However, we observed a lower
surviving frequency of cotyledon explants on
PPT than on kanamycin. Very few shoots
regenerated under the various concentrations
of PPT, although buds or shoot primordia
developed at a low concentration. We spec-
ulated that C. metuliferus is very sensitive to
at concentrations of 50, 150, and 200 mg?L–1,
the survival rates reached 62.1%, 89.9%, and
78.1%, respectively (Table 2). Based on the
high survival rate of 89.9%, kanamycin at
150 mg?L–1was subsequently used for C.
metuliferus PI 292190 transformation. In the
case of the GUS histochemical assay, the
expression of the gus gene was detected only
along the margins of leaves and leaf petioles
in transformants, indicating that the CaMV
35S promoter was preferentially expressed
only in certain tissues (Fig. 5). GUS activity
driven by the CaMV 35S promoter was
reported to be very active in vascular bundles
and epidermal and parenchyma cells of
young leaf petioles and petal and gradually
decreases as the tissue matured (Dong et al.,
In conclusion, an efficient (greater than
58%) regeneration procedure that only re-
quired 3 to 4 weeks to complete was de-
veloped and with that transgenic plants of C.
metuliferus PI 292190 could be obtained in
less than 2 months. The transformation rate
of C. metuliferus line PI 292190 was reached
at 2.5%. This is the first successful attempt
for the transformation of any wild Cucumis
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