American Journal of Plant Sciences, 2012, 3, 1336-1340
http://dx.doi.org/10.4236/ajps.2012.39161 Published Online September 2012 (http://www.SciRP.org/journal/ajps)
Genetic Transformation of Citrus sinensis L. with an
antisense ACC oxidase Gene
Sumontip Bunnag*, Duangkamol Tangpong
Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand.
Received June 19th, 2012; revised July 13th, 2012; accepted July 24th, 2012
ABSTRACT
This work was carried out to optimize the conditions for highly effective embryogenic callus induction from mature
seeds, plantlet regeneration and genetic transformation of Citrus sinensis L. by Agrobacterium tumefaciens strain
EHA105 (pCAMBIA 1305.1). Embryogenic calli could be successfully induced from mature seeds employing the MT
medium supplemented with 500 mg/l malt extract. The percentage of embryogenic callus induction was 85. With the
same medium, the high proliferation rate of embryogenic callus was achieved. The liquid MT medium containing 500
mg/l malt extract in combination with 50 mg/l lactose could be used as the embryoid development medium. Somatic
embryos, however, could be regenerated with normal shoots and roots in the MS medium, with the regeneration per-
centage of 60 . The delivery of an antisense ACC oxida se gene into the species C. sinensis mediated by Agrobacterium
tumefaciens strain EHA 105 was successful by co-cultivating explants with the strain EHA 105 for 10 min, following
that by eliminating the bacterium with 200 mg/l cefotaxime, an d subsequently selecting transformed embryoid with 20
mg/l hygromycin. Verified histochemically by GUS assay, putative transformants showed the percentage of gus gene
expression of 100. Molecular analysis using PCR confirmed the integration of the antisense ACC oxidase gene into
plant genome.
Keywords: Citrus sinensis; antisense ACC oxidase; Agrobacterium tumefaciens; Cefotaxime
1. Introduction
Citrus is the most widely grown fruit crop throughout the
world [1]. It is the number one fruit of the world on ac-
count of its high n utritional value. A large n umber of the
production of fruits and fruit products, and the citrus in-
dustry is considered to be a major fruit industry [2]. Har-
vested fruits which are usually stored before they reach
the market for fresh consumption [3] are subjected to
biotic and abiotic stress conditions, especially stress from
excess ethylene production during the degreening pro-
cess of citrus fruits [4-8]. Even though citrus fruit is non-
climacteric and produces very low amounts of ethylene
at mature green state [9], a diurnal low temperature treat-
ment (5˚C) of attached and mature-green grapefruit (Cit-
rus paradisa Macf.) as well as harvested tangerine (C.
reticulata Blanco.) fruits were found to enhance ethylene
production and the yellowing of the citrus peel [10]. Fur-
thermore, ethylene treatment during the degreening pro-
cess of citrus fruits is involved in ethylene production as
well as enhances chlorophyllase activity [11-13] and the
synthesis of carotenoids [13]. Ethylene applied also en-
hances heat damage to flavedo tissue of cured citrus
fruits [6], increases the appearance of chilling injury
symptoms, stem-end rot decay and the co ntent of volatile
off-flavors in the juice head space and fruit internal at-
mosphere during postharvest storage [14]. Attempts to
produce transgenic citrus with ethylene-resistant traits by
introducing an antisense ACC oxidase gene into plant
genome to block ethylene biosynthesis has been widely
made in order to overcome the stress caused by ethylene
[15,16] as this method is powerful and feasible.
This study was therefore carried out to optimize the
conditions for highly effective embryogenic callu s induc-
tion from mature seeds, plantlet regeneration and genetic
transformation of Citrus sinensis L. by Agrobacterium
tumefaciens strain EH A105 (pCAMBIA 1305.1).
2. Materials and Methods
2.1. Embryogenic Callus Induction and Plantlet
Regeneration
Mature seeds of C. sinensis were initially washed with
mild detergent and rinsed with tap water for 3 times.
They were subsequently surface-sterilized in 70% (v/v)
*Corresponding author.

Genetic Transformation of Citrus sinensis L. with an antisense ACC oxidase Gene 1337
ethyl alcohol for 5 minutes and 20% (v/v) sodium hy-
pochlorite with 2 drops of Tween-20 for 10 minutes. Af-
ter being rinsed 3 times with sterile distilled water, seeds
were cultured on Murashige and Tucker (MT) medium
[17] supplemented with varied concentrations of malt
extract (0, 200, 300, 400 and 500 mg/l), 50 g/l sucrose
and 8 g/l agar, pH 5.8, kept at 25 ˚C ± 2˚C in dark condi-
tion for 8 weeks to determine the optimal concentration
of malt extract for embryogenic callus induction. For
embryogenic callus p roliferation, embryogenic calli were
monthly subcultured using the same medium and condi-
tions for 3 mon t hs.
For plantlet regeneration, embryogenic calli (1.0 g
fresh weight) were transferred to a 250 ml flask contain-
ing 50 ml liquid MT medium amended with 500 mg/l
malt extract and 5% (w/v) lactose, pH 5.8. The cultures
were maintained on a rotary shaker with a continuous
shaking (100 rpm) at 25˚C ± 2˚C under a long photope-
riod (16 h light: 8 h dark) with light intensity of 40
µmol·m–2·s–1 for 12 weeks for embryoid development.
After that, embryoids in the cotyledonary stage were se-
lected and rinsed with sterile distilled water for 3 minutes,
dried with sterile tissue papers. Then they were cultured
on solid MT medium supplemented with 500 mg/l malt
ex- tract and kept at 25˚C ± 2˚C under a long photoperiod
(16 h light: 8 h dark) with light intensity of 40
µmol·m–2·s–1 for 12 weeks.
2.2. Effect of Antibiotics on Embryoid
Regeneration
To determine the effect of antibiotics on embryoid re-
generation, embryoids developing in the torpedo shape,
sized 0.5 cm, were cultured on the MT medium supple-
mented with cefotaxime concentrations of 0, 100, 200,
300, 400, 500 and 600 mg/l. The effective concentrations
of hygromycin were also determined. The concentrations
tested were 0, 10, 15, 20 and 25 mg/l. All kinds of anti-
biotics were added to the medium after autoclaving. The
cultures were maintained at 25˚C ± 2˚C under a long
photoperiod (16 h light: 8 h dark) with light intensity of
40 µmol·m–2·s–1 for 5 weeks.
2.3. Agrobacterium-Mediated Transformation
Agrobacterium tumefaciens strain EHA105 (pCAMBIA-
1305.1) were used for the establishment of the transfor-
mation. The plasmid pCAMBIA1305.1 carried GUS
gene and hygromycin-resistant (hptII) gene, each ex-
pressed under the CaMV35S promoter. The bacterial
strain was cultured in Luria Broth (LB) liquid medium
supplemented with 100 mg/l kanamycin and maintained
on a reciprocal shaker at 28˚C for 48 hours until OD 600 =
1.5 - 1.8.
Embryoids in the torpedo stage were used as explants
for transformation in this experiment. The explants were
soaked in Agrobacterium suspension for 0, 5, 10, 15, 20
and 25 minutes. Then they were cocultivated on the MT
medium for 3 days. After cocultivation, the explants
were washed thoroughly in sterile distilled water con-
taining 300 mg/l cefotaxime for 15 minutes. Explants
were subsequently transferred to the MT medium sup-
plemented with 500 mg/l malt extract, 200 mg/l cefo-
taxime and 15 mg/l hygromycin for 6 weeks.
2.4. Histochemical GUS Assay
The histochemical assay for GUS gene expression was
performed using 5-bromo-4-chloro-3-indolyl glucuronide
(X-gluc) as a substrate [18]. Briefly, putative transfor-
mants were transferred to a 1.5 ml microtube containing
X-gluc and subsequently incubated overnight at a tem-
perature of 37˚C.
2.5. PCR Analysis
Total genomic DNA was extracted from embryoids of
transformed plantlets and nontransformed control plant-
lets by the CTAB method [19]. The primer sequences for
PCR were as follows: NOS forward sequence (F)5’-
GAATCCTGTTGCCGGTCTTG-3’, reverse sequence
(R)5’-TTATCCTAGTTTGCGCGCTA-3’ to yield a 180
bp fragment. The DNA was denatured at 94˚C for 4 min,
followed by 35 cycles of amplification (1 min at 92˚C; 1
min at 55˚C; 2 min at 72˚C). The final incubation at 72˚C
was extended to 4 min, and the reaction material was
cooled and kept at 4˚C. The PCR products were visua-
lized by running the completed reaction on a 2% agarose
gel containing ethidium bromide.
2.6. Statistical Analysis
Statistical significance was accepted at p < 0.05. All re-
sults were analyzed by One-way ANOVA using the Sta-
tistical Package for Social Sciences v17.0 software
(SPSS Inc. IL, USA).
3. Results
3.1. Embryogenic Callus Induction and Plantlet
Regeneration
The development of seeds into friable calli was distinct 4
weeks after being cultured on the MT medium supple-
mented with malt extract. Differences in callus induction
percentage were recorded (Figure 1). The maximum
callus induction percentage at 85 was obtained in the me-
dium containing 500 mg/l malt extract. Moreover, the
development of calli into emb ryogenic calli showing em-
bryoid s in the globular stage was evident in week 8 only
in the presence of 500 mg/l malt extract. It was found

Genetic Transformation of Citrus sinensis L. with an antisense ACC oxidase Gene 1338
120
100
80
60
40
20
0
Callus induction capacity (%)
1 2 3 4 5
Treatments
Figure 1. Callus induction percentage under different con-
ditions: 1) 100 mg/l; 2) 200 mg/l; 3) 300 mg/l; 4) 400 mg/l
and 5) 500 mg/l.
that embryogenic callus induction under high concentra-
tions of malt extract was more effective than that under
low concentrations.
The development of embryogenic calli into plantlets
was obtained in the MT medium supplemented with 500
mg/l malt extract and 5% lactose. After 6 weeks of cul-
ture, embryoids in the heart stage were seen and finally
developed into the torpedo stage in week 8. After 12
weeks of culture, embryoids in the cotyledonary stage
were obtained (Figure 2).
3.2. Effect of Antibiotics on Embryoid
Regeneration
Antibiotics used in the study strongly reduced regenera-
tion capacities of C. sinensis embryoids. In the presence
of 100 - 600 mg/l cefotaxime and 10 - 25 mg/l hygromy-
cin, a slight inhibitory effect was observed. The highest
dose of cefotaxime that yielded surviving embryoids was
200 mg/l (Figures 3(a) and 4(a)). The lowest dose of
hygromycin that completely inhibited embryoid growth
was 20 mg/l (Figures 3(b) and 4(b)). All of the embry-
oids turned brown and finally died in five weeks after
they were transferred to the selective medium.
3.3. Agrobacterium-Mediated Transformation
Differences in levels of GUS activities in embryoids after
being cocultivated for 0 - 25 minutes were detected (Fig-
ure 5(a)). The optimal cocultivation time for the maxi-
mum GUS activities was 10 min. Agrobacterium-medi-
ated transformation of C. sinensis yielded a maximum
percent expression (100%) (Figure 5(b)). To determine
the integration of T-DNA fragments in hygromycin-
resistant plantlets, polymerase chain reaction (PCR)
analysis was carried out. It was found that the size of
amplified fragment was 180 bp for NOS, whereas non-
transformed control plantlets did not show any expected
band size (Figure 6).
Figure 2. Embryoids in different stages: 1) globular stage, 2)
heart stage; 3) torpedo stage; and 4) cotyledonary stage.
120
100
80
60
40
20
0
Viability (%)
Cefotaxime co ncentrat ions
0100 200 300 400 500 60
0
(a)
120
100
80
60
40
20
0
010 15 20 25
Hygromycin conc entr ations
Vi abil ity (%)
(b)
Figure 3. Effect of different concentrations of cefotaxime (a)
and hygromycin; (b) on C. sinensis embryoi d gr owth.
4. Discussion
Addition of malt extract in the medium is essential to
embryoid induction in citrus. In this study, the optimal
concentration of malt extract for embryogenic callus in-
duction was 500 mg/l. The results were in agreement
with the finding reporting that inducing ovules derived

Genetic Transformation of Citrus sinensis L. with an antisense ACC oxidase Gene 1339
(a)
(b)
Figure 4. Embryoids on the MT medium containing diffe-
rent concentrations of cefotaxime (a) and hygromycin (b).
4
3
2
1
0
Levels of GUS activity (+)
0 5 10 15 20 25
Cocul tivatio n time (m in)
(a)
120
100
80
60
40
20
0
Percentage of GUS activity
0 5 10 15 20 25
Cocul tiva t io n time (min)
(b)
Figure 5. Levels (a) and percentages (b) of GUS expression
in embryoids after being cocultivated for 0 - 25 min.
from 8 week seeds of C. sinensis into somatic embryos
was obtained in the MT medium supplemented with 500
mg/l malt extract [20]. Another report also claimed that
addition of 500 mg/l malt extract in the Murashige and
Skoog (MS) medium could induce the development of C.
sinensis style tissues into somatic embryos [21].
A selective agent is crucial for selection of the trans-
formants and for avoiding development of undesirable
numbers of the escapes. It was suggested that hygromy-
cin is an excellent selective agent and needs to be opti-
1 2 3 4 M
180 b p
Figure 6. PCR analysis in transformed embryoids in C.
sinensis using primers to detect NOS; lane M: 100 bp lad-
der, lane 1: transformed plantlets; lane 2: nontransformed
control plantlets; lane 3: pCAMBIA1305.1 (positive control),
and lane 4: negative control.
mized for each plant species [22]. In this report, hptII
encoding resistance to hygromycin was used in the pro-
duction of transgenic citrus. Hygromycin are aminogly-
coside antibiotics which cause harmful death to plant
cells by inhibiting transcription and translation.
In order to eliminate A. tumefaciens after cocultivation,
addition of antibiotics in the medium is required. In this
study, cefotaxime showed strong inhibition of the regen-
eration potential in C. sinensis. However, it was found
that the concentration of 200 mg/l did not inhibit the re-
generation of explants. Our findings are in agreement
with the finding reporting that cefotaxime concentration
of 200 mg/l did not inhibit the regeneration of explants in
Malus sylvestris var. “Delicious” [23].
In this study, a number of transformed C. sinensis
were produced using Agrobacterium-mediated transfor-
mation system. The results confirmed that embryoids can
be used as explants for this transformation system. More-
over, our findings showed that the CaMV35S promoter
was useful for C. sinensis transformation.
5. Conclusions
In summary, this report described the use of A. tumefa-
ciens strain EHA105 (pCAMBIA 1305.1) to transfer
screenable and selectable marker genes into C. sinensis
and showed molecular evidence of primary transgenic
plants which demonstrated stable integration of trans-
genes. We confirm that embryoids of C. sinensis are the
suitable target tissues for Agrobacterium-mediated trans-
formation.

Genetic Transformation of Citrus sinensis L. with an antisense ACC oxidase Gene 1340
6. Acknowledgements
The authors would like to thank the Graduate School,
Khon Kaen University for financial support, and the De-
partment of Biology, Faculty of Science, Khon Kaen
University for granting us permission to use the devices
necessary for the study.
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