In Vitro Cell.Dev.Biol.
—Plant (2010) 46:1–7
DOI 10.1007/s11627-009-9245-3
BIOTECHNOLOGY/GENETIC TRANSFORMATION/FUNCTIONAL GENOMICS
Characterization of a field-grown transgenic pineapple clone
containing the genes chitinase, AP24, and bar
Lourdes Yabor & Bárbara Valle & Carol Carvajal & Carlos Aragón &
Martha Hernández & Justo González & Marcos Daquinta & Ariel Arencibia &
José Carlos Lorenzo
Received: 18 March 2009 / Accepted: 20 August 2009 / Published online: 17 September 2009 / Editor: W. C. Otoni
# The Society for In Vitro Biology 2009
Abstract We previously introduced the bar gene, along
with chitinase and AP24 genes, into the pineapple genome.
The present report focuses on the evaluation of the first
vegetative generation of a transgenic clone containing these
genes. Three materials were compared: macropropagated
controls (non-transformed), micropropagated controls (non-
transformed), and micropropagated transformed plants.
From each group, 50% of the plants were sprayed with
FINALE® 3 mo after the experiment initiation. The
characterization was performed after 1 yr of field growth.
FINALE® killed all non-transgenic plants. Plants that
survived the herbicide application showed 2n=50 chromo-
somes in their roots after 1 yr in the field. Micropropagated
transformed plants sprayed with FINALE® did not show
phenotype differences from micropropagated transformed
plants not sprayed with the herbicide. Between the micro-
propagated transformed plants sprayed with FINALE®
and the micropropagated control plants, the following
differences were observed: modifications in levels of cell
wall-linked, free and total phenolics, and total proteins.
Moreover, changes of the fruit mass without crown were
also recorded. Between the micropropagated transformed
plants sprayed with FINALE® and the macropropagated
control plants, levels of chlorophyll b, total chlorophyll
pigments, and proteins were different. Furthermore,
activities of phenylalanine ammonia-lyase, superoxide
dismutase, and glutamine synthetase were dissimilar. The
L. Yabor (*) : B. Valle : C. Carvajal : C. Aragón :
M. Hernández : J. González : M. Daquinta : A. Arencibia :
J. C. Lorenzo
Laboratory for Plant Breeding, Bioplant Center,
University of Ciego de Avila,
Ciego de Ávila 69450, Cuba
e-mail: lyabor@bioplantas.cu
plant height and diameter, and the crown height were also
different. Until now, we have evaluated transformed
pineapple plants during hardening and field growth.
Although some unexpected variations were recorded, we
believe they are not relevant enough to justify rejection of
transgenesis as an important tool for pineapple genetic
improvement.
Keywords Ananas comosus (L.) Merr .
Plant transformation . Field performance
Introduction
World pineapple production reached 18.2 million tons in
2006 (FAOSTAT 2008). Therefore, several research groups
are developing basic and applied studies to create new
varieties with better agronomic performance. We previously
developed a protocol for pineapple genetic transformation,
introducing the chitinase, AP24, and bar genes into the
pineapple genome under the control of the following
promoters: OCS-35S CaMV-rice actin I, 35S CaMV, and
maize Ubi1, respectively (Espinosa et al. 2002). These
promoters have been described as constitutive transcription
promoters (Franck et al. 1980; Christensen et al. 1992).
Chitinase and AP24 have been depicted as antifungal
genes. The chitinase gene (from Phaseolus vulgaris)
product degrades chitin, an essential compound of most of
the fungal cell walls (Broglie et al. 1986; Schlumbaum et
al. 1986). The AP24 gene (from Nicotiana tabacum) codes
for a protein that destabilizes the fungal membrane (Singh
et al. 1989; Woloshuk et al. 1991). We introduced these two
antifungal genes into the pineapple genome in an attempt to
reduce the great losses caused by Phytophthora nicotianae
var. parasitica (Kamoun 2001). These phytopathological
2 YABOR ET AL.
studies are in progress in our experimental field station.
Regarding the safety profile studies of these two genes, we
have not found reports about AP24. However, class-I
chitinases have been identified as the major panallergens in
fruits associated with the latex-fruit syndrome, such as the
rawly consumed avocado, banana, and chestnut (Blanco et
al. 1994; Brehler et al. 1997; Sanchez-Monge et al. 2000).
We used the bar gene as a selectable marker. It was cloned
from Streptomyces hygroscopicus and encodes for phosphi-
notricin acetyltransferase (Thompson et al. 1987). This
enzyme is capable of inactivating phosphinotricin that is
the active compound of the non-selective herbicide
FINALE® (Torres et al. 1999). After application of FINALE®
onto non-transformed plants, phosphinotricin inhibits the
enzyme glutamine synthetase (Mohapatra et al. 1999).
Such an inhibition causes plant toxicity to ammonium,
provoking death (D´Halluin et al. 1992). Phosphinotricin
acetyltransferase proteins have been widely studied
(Wehrmann et al. 1996) and have an excellent safety
profile (Herouet et al. 2005).
The aforementioned references indicate that genes and
promoters used for pineapple genetic transformation are
well known, at least in their primary effect. However, the
biochemical side effects of the herbicide FINALE® on bar
gene-containing transgenic pineapple plants have not been
explored to date. Although several metabolic studies of
transgenic plants have shown the effects of transformation
(Momma et al. 1999; Wilson and Latham 2006), investiga-
tion into the unexpected biochemical side effects could help
to positively impact the public perception of genetically
modified plant food (Kuiper et al. 2001).
Based on these prospects, we previously studied some of
the biochemical side effects of pineapple genetic transfor-
mation in interaction with the herbicide FINALE® during
hardening (Yabor et al. 2006; Yabor et al. 2008). Levels of
aldehydes and chlorophylls, and peroxidase activity were
recorded in middle-aged leaves. The transformed clone not
sprayed with FINALE® showed the following side effects
because of transgenesis only: levels of malondialdehyde,
other aldehydes, chlorophyll b, and total chlorophyll pig-
ments decreased. The most remarkable biochemical differ-
ences between transgenic and non-transgenic plants after
application of FINALE® follow. Levels of malondialdehyde
and other aldehydes in transgenic material were not
decreased by FINALE®, perhaps because these levels were
already low as a result of transformation. FINALE®
increased peroxidase activity in transgenic plant. but this
increase was higher in non-transgenic material. The
herbicide increased contents of chlorophyll pigments (a, b,
total) in transformed plants. However, as expected, non-
transgenic plants decreased levels of chlorophylls (a, b,
total) after application of FINALE®. The genetic transfor-
mation of pineapple with the bar gene not only conferred
resistance to the herbicide FINALE® but also promoted
other biochemical changes during in vitro plantlet harden-
ing (Yabor et al. 2006; Yabor et al. 2008).
Several phenotype characterizations of transgenic plants
have been performed in a field environment: tobacco,
potato (De Greef et al. 1989), maize (Wehrmann et al.
1996; Betz et al. 2000; Hagerty et al. 2005; Meihls et al.
2008), sugarcane (Enriquez-Obregón et al. 1998; Carmona
et al. 2005), Pupulus alba L. (Confalonieri et al. 2000),
cabbage (Al-Kaff et al. 2000), grapevines (Vigne et al.
2004), pineapple (Botella and Fairbairn 2005), tomato
(Accotto et al. 2005), Cucurbita pepo (Quemada et al.
2008), and cotton (Gore et al. 2008).
As far as we know, Sripaoraya et al. (2006) pioneered
the field evaluation of bar gene-containing transgenic
pineapple plants (Thailand, cv. Phuket) after 14 mo of
growth. They published relevant data comparing transgenic
and non-transgenic plants. However, two important control
treatments were not included: (1) transgenic plants not
sprayed with FINALE® and (2) macropropagated plants.
On the other hand, only a limited number of phenotype
traits were considered. In brief, Sripaoraya et al. (2006)
recorded more shoots and leaves, and bigger fruits in
transgenic (sprayed with FINALE®) than in non-transgenic
(not sprayed with FINALE®) pineapple plants.
The research shown here provides a more detailed
evaluation of a transgenic pineapple clone under field
environment, including relevant control plant materials. We
evaluated the first vegetative generation after 1 yr of field
growth because this is a common practice among pineapple
growers.
Materials and Methods
Pineapple c.v. Serrana Smooth Cayenne was used. Trans-
genic plants were obtained according to Espinosa et al.
(2002). Embryogenic calluses were gently passed through
polypropylene meshes (2,000 μm pore diameter, Spectrum,
Belmont, CA) and dried for 15 min in the laminar flow
cabinet. Agrobacterium tumefaciens suspension was then
added (strain AT2260, pHCA58, containing a bar gene
controlled by maize Ubi 1 promoter, a class-I bean
chitinase gene controlled by a hybrid OCS-35S CaMV-
rice actin I promoter and a tobacco AP24 gene controlled
by 35S CaMV promoter).
After 10 min, infected calluses were washed with distilled
water and dried with filter paper. Co-culture was allowed for
24 h (darkness, 25°C). Calluses were then transferred to the
callus proliferation medium supplemented with 0.2 gl−1
cefotaxime (4 wk, 25°C, 16-h light photoperiod, 2,000 lx).
Calluses were transferred to temporary immersion bioreac-
tors for plant regeneration in a selective medium (2.5 mg l−1
 CHARACTERIZATION OF A FIELD-GROWN TRANSGENIC PINEAPPLE CLONE
phosphinotricin). After 45 d, phosphinotricin-resistant plants
were recovered.
Non-transformed plants (micropropagated control treat-
ment) were obtained following the protocol described
above but avoiding contact with Agrobacterium tumefaciens
and phosphinotricin. In a previous experiment (data not
shown), we compared the resistance to herbicide FINALE® of
100 transformed clones during hardening. In the present
study, we used the transgenic clone that previously
showed the lowest foliar damage after application of
FINALE®.
Non-transformed and transformed (one clone) plants
were transferred to a greenhouse for hardening according to
Yanes et al. (2000). Plants were placed in plastic trays
containing 82 cm3 of a mixture zeolite + filter cake (1:1).
Microject automated irrigations for 25 s every 30 min were
applied. Plants were kept under a photosynthetic photon
flux density of 458µmol m−2 s−1. Standard phytosanitary
controls were applied. Plants were transferred to the field
environment after 6 mo of hardening.
Three plant materials were compared in the Experimen-
tal Field Station at the Bioplant Center: macropropagated
control plants (non-transformed), micropropagated control
plants (non-transformed), and micropropagated transformed
plants. From each group, 50% of the plants were sprayed
with FINALE® at 12 lha−1 as recommended by Bayer
CropScience (2005) after 3 mo of field growth. Therefore,
six treatments were compared in a random block design: 60
plants/treatment, four replications/treatment and 15 plants/
replication. Plant survival was recorded at 3 mo after
application of FINALE®.
The field management was performed according to
instructions recommended by the Cuban Ministry of
Agriculture. The most important pineapple agricultural
phenotypic traits were recorded after 1 yr of field growth.
The following biochemical determinations were also carried
out.
Pineapple D leaf (middle-aged) samples were stored in
liquid nitrogen. Each biochemical determination started
from three independently pooled samples (100 mg each).
They were finely ground in liquid nitrogen. Contents of
malondialdehyde and other aldehydes (Heath and Packer
1968); chlorophyll [a, b, total (Porras 1991)]; and phenolics
[cell wall-linked, free, total (Gurr et al. 1992)] were
determined. Peroxidase activity and specific peroxidase
activity (Hammerschmidt et al. 1982), phenylalanine
ammonia-lyase activity (Jorrin and Dixon 1990), superoxide
dismutase activity (McCord and Fridovich 1969), and
glutamine synthetase activity (Habash et al. 2001) were also
measured. Moreover, the chromosome numbers of each plant
material were determined according to Bor et al. (1987). The
Statistical Package for Social Sciences (Version 8.0 for
Windows, SPSS Inc., New York, NY) was used to perform
3
one-way analysis of variance (ANOVA), ANOVA, and
Tukey tests (p≤0.05).
Results and Discussion
FINALE® had killed all non-transgenic plants when plant
survival was recorded 30 d after herbicide application
(Table 1). Plant materials that survived the herbicide
application showed 2n=50 chromosomes in their roots
after 1 yr in the field.
Many lines of evidence have suggested that T-DNA
integration causes significant chromosomal rearrangements
in tobacco (Ohba et al. 1995). In addition, Castle et al.
(1993), studying 36 Arabidopsis embryo-defective mutants
produced following seed T-DNA transformation, found
indications for chromosomal translocations in nine of them.
The authors concluded that chromosomal rearrangements
could be a common feature of T-DNA-transformed plants.
Moreover, Takano et al. (1997) described massive rear-
rangements (inversions and duplications) of genomic DNA
at integration sites.
We did not perform a detailed study of pineapple
chromosomal rearrangements, but observed that the number
and shape of chromosomes appear to be similar. However,
we do not ignore that the modifications mentioned above
could have happened.
Micropropagated transformed plants sprayed with
FINALE® did not show phenotype differences from micro-
propagated transformed plants not sprayed with the
herbicide after 1 yr of field growth (Tables 2 and 3).
In Tables 2 and 3, between the micropropagated trans-
formed plants sprayed with FINALE® and the micro-
propagated control plants, the following differences were
observed: modifications in levels of cell wall-linked, free
and total phenolics, as well as, total proteins (Table 2).
Moreover, changes of the fruit mass without crown were
also recorded (Table 3).
As shown in Tables 2 and 3, between the micro-
propagated transformed plants sprayed with FINALE® and
the macropropagated control plants, levels of chlorophyll b,
total chlorophyll pigments, and proteins were different.
Furthermore, activities of phenylalanine ammonia-lyase,
superoxide dismutase, and glutamine synthetase were
dissimilar (Table 2). The plant height and diameter, and
the crown height were also different (Table 3).
As mentioned above, during hardening, leaf levels of
malondialdehyde, other aldehydes, chlorophyll b, and total
chlorophyll pigments decreased because of genetic trans-
formation only (Yabor et al. 2006). Such differences
between transgenic and non-transgenic materials were not
observed after 1 yr of field growth (Table 2). However, levels
of phenolics (free, cell wall-linked, total) and proteins, and
4 YABOR ET AL.

Table 1. Pineapple (C.V. serrana smooth cayenne) plant survival after 3 mo of finale® application (%, average±SE)

Macropropagated control Micropropagated Micropropagated

plants (non-transformed) control plants transformed plants
(non-transformed)

Not sprayed Sprayed Not sprayed Sprayed Not sprayed Sprayed

with FINALE® with FINALE® with FINALE® with FINALE® with FINALE® with FINALE®
100.0±0.0 a 0.0±0.0 b 93.3±2.7 a 0.0±0.0 b 91.7±3.2 a 93.3±2.7 a

Results with the same letter are not statistically different (ANOVA, Tukey, p>0.05). FINALE® was sprayed after plants had grown for 3 mo in the
field.

the fruit mass without crown were statistically different
between these two groups of plants (Tables 2 and 3).
On the other hand, FINALE® caused some biochemical
side effects when sprayed over young transgenic plants
during hardening (Yabor et al. 2008). These effects were
not observed in adult plants (Table 2). Perhaps the interval
between the herbicide application and the evaluations
performed was relevant. When FINALE® was sprayed
during hardening, biochemical indicators were recorded
15 d later. In contrast, during field growth, biochemical
determinations were performed 9 mo after the herbicide
application.
The effect of in vitro culture was remarkable in the
experiment shown here. Comparing the macropropagated
control plants with the other materials, statistical differences
were found in levels of chlorophyll b and phenolics (free,
cell wall-linked, total). Moreover, activities of phenylala-
nine ammonia-lyase, superoxide dismutase, and glutamine

Table 2. Biochemical evaluations performed in pineapple (C.V. serrana smooth cayenne) D leaves after 1 yr of field growth (averages±SE)

Macropropagated control Micropropagated control Micropropagated transformed plants

plants (non-transformed) plants (non-transformed)
Not sprayed with FINALE® Not sprayed with FINALE® Not sprayed with Sprayed with
FINALE® FINALE®

Malondialdehyde content 09.23±0.08 a 06.86±0.57 a 11.30±1.60 a 09.74±0.48 a

(μmol g−1 fresh leaf mass)
Other aldehyde content 172.01±14.78 a 163.09±21.16 a 178.04±20.14 a 169.09±18.78 a
(μmol g−1 fresh leaf mass)
Chlorophyll a content 61.99±17.83 a 109.56±26.82 a 144.34±14.59 a 142.78±14.48 a
(μg g−1 fresh leaf mass)
Chlorophyll b content 77.59±11.02 b 125.66±32.14 a 129.93±32.48 a 134.72±41.21 a
(μg g−1 fresh leaf mass)
Total content of chlorophyll 139.58±26.13 b 239.22±58.23 ab 340.27±43.79 a 368.36±45.34 a
(μg g−1 fresh leaf mass)
Content of cell wall-linked 1,890.54±98.63 a 1,081.26±47.49 b 1,728.46±231.81 a 1,598.89±211.45 a
phenolics (mg g−1 fresh
leaf mass)
Content of free phenolics 843.27±33.94 b 303.62±45.32 c 1,103.89±66.79 a 1,098.67±51.45 a
(mg g−1 fresh leaf mass)
Total content of phenolics 2,733.82±113.67 a 1,384.88±90.14 b 2,832. 35±272.24 a 2,798.25±264.72 a
(mg g−1 fresh leaf mass)
Protein content 0.0159±0.0006 b 0.0172±0.0002 b 0.0187±0.0004 a 0.0198±0.0005 a
(mg g−1 fresh leaf mass)
Peroxidase activity 70.51±2.72 a 59.46±5.70 a 65.07±8.59 a 62.09±7.42 a
(U mg−1 fresh leaf mass)
Peroxidase specific activity 04.49±0.22 a 03.45±0.32 a 03.46±0.43 a 03.42±0.40 a
(U mg−1 of protein)
Phenylalanine ammonia-lyase 0.0142±0.0018 a 0.0105±0.0017 b 0.0116±0.0016 b 0.0989±0.0098 b
activity (U g−1 of protein)
Superoxide dismutase activity 138.20±9.60 a 82.97±6.58 b 93.03±13.33 b 87.50±11.47 b
(U mg−1 of protein)
Glutamine synthetase activity 0.0145±0.0035 b 0.0251±0.0022 a 0.0247±0.0029 a 0.0282±0.0034 a
(U μg−1 of protein)

Results with the same letter are not statistically different (one-way ANOVA, Tukey, p>0.05). FINALE® was sprayed after plants had grown for
3 mo in the field
 CHARACTERIZATION OF A FIELD-GROWN TRANSGENIC PINEAPPLE CLONE 5

Table 3. Other phenotypic traits of pineapple (C.V. serrana smooth cayenne) after 1 yr of field growth (averages ± SE)

Macropropagated control plants Micropropagated control plants Micropropagated transformed plants

(non-transformed) (non-transformed)
Not sprayed with FINALE® Not sprayed with FINALE® Not sprayed with Sprayed with
FINALE® FINALE®

Plant height (cm) 63.70±6.37 b 71.11±7.02 a 79.42±7.94 a 77.34±7.73 a

Plant diameter (cm) 73.04±7.30 b 93.57±9.86 a 97.63±9.76 a 91.76±9.17 a
Peduncle diameter (cm) 1.95±0.14 a 1.81±0.08 a 1.74±0.05 a 1.87±0.07 a
Peduncle length (cm) 18.31±1.42 a 17.01±1.22 a 20.8±1.79 a 22.43±1.62 a
Fruit mass with crown 1.80±0.18 a 1.40±0.14 b 1.81±0.18 ab 1.71±0.17 ab
(kg)
Fruit mass without 0.62±68.85 a 0.30±35.49 b 0.62±112.60 a 0.52±110.16 a
crown (kg)
Fruit crown mass (kg) 0.48±0.05 a 0.40±0.06 b 0.37±0.03 ab 0.32±0.01 ab
Height of crown (cm) 22.75±2.02 a 18.20±135 ab 14.80±2.21 b 12.34±2.10 b
Number of eyes in the 99.83±9.80 a 50.83±5.33 b 72.67±6.61 ab 74.68±7.43 ab
fruit
Number of spirals in the 2.50±0.25 a 2.00±0.21 a 2.33±0.23 a 3.00±0.47 a
fruit
Depth of fruit eyes (cm) 0.20±0.04 a 0.17±0.02 a 0.28±0.06 a 0.24±0.04 a
Diameter of fruit heart 1.87±0.20 a 1.75±0.15 a 2.08±0.23 a 1.98±0.12 a
(cm)
Fruit height (cm) 12.53±0.61 a 8.28±0.43 b 10.52±0.87 ab 9.99±0.72 ab
Fruit superior diameter 9.22±0.25 a 8.90±0.39 a 9.18±0.53 a 8.56±0.32 a
(cm)
Fruit inferior diameter 9.62±0.31 a 8.97±0.29 a 8.73±0.24 a 8.56±0.19 a
(cm)
Fruit vitamin C (mg/ 15.19±1.40 a 16.87±1.51 a 14.25±1.37 a 15.32±1.44 a
100 mL juice)
Fruit acidity (%) 1.53±0.07 a 1.36±0.01 a 1.71±0.25 a 1.67±0.15 a
°Brix 17.63±1.72 a 15.04±1.57 a 16.87±1.67 a 18.5±1.79 a
Maturity index 12.22±1.22 a 11.10 ± M1.11 a 10.58±1.05 a 12.67±1.47 a
Number of shoots 0 0 0 0
Number of slips 0 0 0 0
Number of suckers 0 0 0 0
Number of crowns per 1 1 1 1
fruit
Plant generation cycle 18 18 18 18
(mo)
Presence of thorns in Few (all over the leaf edge) Few (all over the leaf edge) Few (all over the Few (all over the
leaves leaf edge) leaf edge)
Leaf color Greenish with red zones Greenish with red zones Greenish with red Greenish with red
zones zones
Plant architecture Slightly wide Slightly wide Slightly wide Slightly wide
Shape of fruit eyes Rectangular Rectangular Rectangular Rectangular
Fruit color Reddish orange Reddish orange Reddish orange Reddish orange
Fruit shape Cylindrical Cylindrical Cylindrical Cylindrical
Presence of thorns in Few (only on leaflet extreme) Few (only on leaflet extreme) Few (only on Few (only on
fruit crowns leaflet extreme) leaflet extreme)

Results with the same letter are not statistically different (one-way ANOVA, Tukey, p>0.05). FINALE® was sprayed after plants had grown for
3 mo in the field

synthetase activities were also modified (Table 2). The In vitro culture has frequently produced rejuvenation and
plant height and diameter, the fruit mass (with or without epigenetic changes in many plants. However, these effects
crown), the fruit crown mass, the number of eyes in the generally decrease during the course of field growth and the
fruit, and the fruit height were affected too (Table 3). first clonal multiplication (Ventura et al. 1996; Lorenzo et
6 YABOR ET AL.
al. 2001; Sripaoraya et al. 2001; Martínez et al. 2002). Our
institute has been cultivating pineapple in vitro (via
different techniques) for more than 20 yr, and only two
confirmed variants have been found. Therefore, we regard
somaclonal variation as a rare event in pineapple (Pérez et
al. 2009). Tables 2 and 3 show the first vegetative
generation. Probably the effect of in vitro culture will
decrease in the next ones.
Until now, we have evaluated transformed pineapple
plants during hardening and field growth. Although some
unexpected variations were recorded, we believe that they
are not relevant enough to justify rejection of transgenesis
as an important tool for pineapple genetic improvement.
The second vegetative generation of pineapple transgenic
plants is being studied at the Bioplant Center’s Experimental
Field Station.
Acknowledgement This research was supported by the Cuban
Ministry of Science, Technology, and the Environment through a grant
to Mrs. Lourdes Yabor Cabrera. The authors are grateful to Dr. Lazaro
Hernandez (CIGB, Havana, Cuba) for providing gene constructs; to Mrs.
Glyn Jabbour for her critical reading of the manuscript; and to Mrs.
Mayda Arzola, Mrs. Julia Martínez, Mrs. Alitza Iglesias, and Mr. Abel
González for their excellent technical assistance.
References
Accotto G. P.; Nervo G.; Acciarri N.; Tavella L.; Vecchiati M.; Schiavi
M.; Mason G.; Vaira A. Field evaluation of tomato hybrids
engineered with tomato spotted wilt virus sequences for virus
resistance, agronomic performance, and pollen-mediated trans-
gene flow. Phytopathology 95: 800–807; 2005.
Al-Kaff N. S.; Kreike M. M.; Covey S. N.; Pitcher R.; Page A. M.;
Dale P. J. Plants rendered herbicide-susceptible by cauliflower
mosaic virus-elicited suppression of a 35S promoter-regulated
transgene. Nat Biotech. 18: 995–999; 2000.
Bayer. Technical information. In: Bayer (ed) Glufosinate-Ammonium.
Bayer CropScience. Monheim, Germany, 38; 2005.
Betz F. S.; Hammond B. G.; Fuchs R. L. Safety and advantages of
Bacillus thuringiensis-protected plants to control insect pests.
Reg Toxicol Pharmacol. 32: 156–173; 2000.
Blanco C.; Carrillo T.; Castillo R.; Quiralte J.; Cuevas M. Latex
allergy: clinical features and cross-reactivity with fruits. Ann
Allergy 73: 309–314; 1994.
Bor Y.; Silva P.; Francisco R. Chromosomal number of Bromeliaceae
plants. Rev. Bras. Frut. 9: 49–55; 1987.
Botella J. R.; Fairbairn D. J. Present and future potential of pineapple
biotechnology. Acta Hort. 622: 23–28; 2005.
Brehler R.; Thiessen U.; Mohr C.; Luger T. "Latex-fruit syndrome":
frequency of cross-reacting IgE antibodies. Allergy 52: 404–410;
1997.
Broglie K. E.; Gaynor J. J.; Broglie R. M. Ethylene-regulated gene
expression: molecular cloning of the genes encoding an endochi-
tinase from Phaseolus vulgaris. Proc Natl Acad Sci U S A 83:
6820–6824; 1986.
Carmona E. R.; Arencibia A. D.; López J.; Simpson J.; Vargas D.;
Sala F. Analysis of genomic variability in transgenic sugarcane
plants produced by Agrobacterium tumefaciens infection. Plant
Breed 124: 33–38; 2005.
Castle L.; Errampalli D.; Atherton L.; Franzmann E. Genetic and
molecular characterization of embryonic mutants identified
following seed transformation in Arabidopsis. Mol Gen Genet
241: 504–514; 1993.
Confalonieri M.; Belenghi B.; Balestrazzi A. Transformation of elite
white poplar (Populus alba L.) cv. “Villafranca” and evaluation
of herbicide resistance. Plant Cell Rep. 19: 978–982; 2000.
Christensen A. H.; Sharrock R. A.; Quail P. H. Maize polyubiquitin
genes: structure, thermal perturbation of expression and transcript
splicing, and promoter activity following transfer to protoplasts
by electroporation. Plant Mol Biol. 18: 675–689; 1992.
D´Halluin K.; De Block M.; Denecke J.; Janssens J.; Leemans J.;
Reynaerts A.; Botterman J. The bar gene as selectable and
screenable marker in plant genetic engineering. Meth Enzymol
216: 415–426; 1992.
De Greef W.; Delon R.; De Block M.; Leemans J.; Botterman J.
Evaluation of herbicide resistance in transgenic crops under field
conditions. BioTechnology 7: 61–64; 1989.
Enriquez-Obregón G. A.; Vázquez-Padrón R. I.; Prieto-Samsonov D.
L.; De la Riva G. A.; Selman-Housein G. Herbicide-resistance
sugarcane (Saccharum officinarum L.) plants by Agrobacterium-
mediated transformation. Planta 206: 20–27; 1998.
Espinosa P.; Lorenzo J. C.; Iglesias A.; Yabor L.; Menéndez E.;
Borroto Y.; Hernández L.; Arencibia A. Production of pineapple
transgenic plants assisted by temporary immersion bioreactors.
Plant Cell Rep. 21: 136–140; 2002.
FAOSTAT (2008) FAO STATISTIC DIVISION. Available http://
faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567.
Accessed 23 May 2008.
Franck A.; Guilley H.; Jonard G.; Richards K.; Hirth L. Nucleotide
sequence of cauliflower mosaic virus DNA. Cell 21: 285–294; 1980.
Gore J.; Adamczyk J. J.; Catchot A.; Jackson R. Yield response of
dual-toxin Bt cotton to Helicoverpa zea infestations. J Econ
Entomol. 101: 1594–1599; 2008.
Gurr, S. I.; McPherson, J.; Bowles, D. J. Lignin and associated
phenolic acids in cell walls. In: Wilkinson D. L. (ed) Molecular
Plant Pathology. Oxford, pp. 51–56; 1992
Habash D.; Massiah J.; Rong H.; Wallsgrove R.; Leigh R. The role of
cytosolic glutamine synthetase in wheat. Ann Appl Biol. 138: 83–
89; 2001.
Hagerty A. M.; Kilpatrick A. L.; Turnipseed S. G.; Sullivan M. J.;
Bridges W. C. Predaceous arthropods and lepidopteran pests on
conventional, bollgard, and bollgard II cotton under untreated
and disrupted conditions. Environ Entomol. 34: 105–114; 2005.
Hammerschmidt R.; Nuckleus E. M.; Kuc J. Association of enhanced
peroxidase activity with induced systemic resistance of cucumber
to Colletotrichum lagenarium. Physiol Plant Pathol. 20: 61–71;
1982.
Heath R. L.; Packer J. Photoperoxidation in isolated chloroplast: I.
Kinetics and stoichiometry of fatty acid peroxidation. Arch
Biochem Biophys. 125: 189–198; 1968.
Herouet C.; Esdaile D. J.; Mallyon B. A.; Debruyne E.; Schulz A.;
Curtier T.; Hendricx K.; Klis Var der R. J.; Rovan D. Safety
evaluation of the phosphinothricin acetyltransferase proteins
encoded by the pat and bar sequences that confer tolerance to
glufosinate-ammonium herbicide in transgenic plants. Reg
Toxicol Pharmacol. 41: 134–149; 2005.
Jorrin J.; Dixon R. A. Stress responses in alfalfa (Medicago sativa L.).
II. Purification, characterization, and induction of phenylalanine
ammonia-lyase isoforms from elicitor-treated cell suspension
cultures. Plant Physiol. 92: 447–455; 1990.
Kamoun S. Non-host resistance to Phytophthora: novel prospects for a
classical problem. Plant Biol. 4: 295–300; 2001.
Kuiper H. A.; Kleter G. A.; Noteborn H. P.; Kok J. Assessment of the
food safety issues related to genetically modified foods. Plant J.
27: 503–528; 2001.
 CHARACTERIZATION OF A FIELD-GROWN TRANSGENIC PINEAPPLE CLONE
Lorenzo J. C.; Ojeda E.; Espinosa A.; Borroto C. Field performance of
temporary immersion bioreactor-derived sugarcane plants. In
Vitro Cell Dev Biol-Plant 37: 803–806; 2001.
Martínez M.; Ojeda E.; Espinosa A.; Sánchez M.; Castillo R.;
González M. T.; Engelmann F.; Lorenzo J. C. Field performance
of sugarcane plants derived from cryopreserved calluses. Cryoletters
23: 21–26; 2002.
McCord J.; Fridovich I. Superoxide dismutase: an enzymic
function for erythrocuprein. J Inorg Biochem. 244: 6049–
6055; 1969.
Meihls L. N.; Higdon M. L.; Siegfried B. D.; Miller N. J.; Sappington
T. W.; Ellersieck M. R.; Spencer T. A.; Hibbard B. E. Increased
survival of western corn rootworm on transgenic corn within
three generations of on-plant greenhouse selection. Proc Natl
Acad Sci U S A 49: 19177–19182; 2008.
Mohapatra U.; McCab M.; Power J.; Schepers F.; Van den Arend A.;
Davey M. R. Expression of the bar gene confers herbicide
resistance in transgenic lettuce. Transg Res. 8: 33–44; 1999.
Momma K.; Hashimoto W.; Ozawa S.; Kawai S.; Katsube T.;
Takaiwa F.; Kito M.; Utsumi S.; Murata K. Quality and safety
evaluation of genetically engineered rice with soybean
glycinin: analyses of the grain composition and digestibility
of glycinin in transgenic rice. Biosci Biotechnol Biochem. 63:
314–318; 1999.
Ohba T. Y.; Machida C.; Machida Y. DNA rearragements associated
with the integration of T-DNA in tobacco: an example for
multiple duplications of DNA around the integration target. Plant
J. 7: 157–164; 1995.
Pérez G.; Yanes E.; Isidrón M.; Orenzo J. C. Phenotypic and AFLP
characterization of two new pineapple somaclones derived from
in vitro culture. Plant Cell Tiss Org Cult. 96: 113–116; 2009.
Porras, R. J. Recent advances and re-assessments in chlorophyll
extraction and assay procedures for terrestrial, aquatic and marine
organisms including recalcitrant algae. In: Scheer H (ed)
Chemistry of Chorophyll, Boca Raton: 320; 1991
Quemada H.; Strehlow L.; Decker-Walters D.; Staub J. Population
size and incidence of virus infection in free-living popula-
tions of Cucurbita pepo. Environ Biosafety Res. 7: 185–196;
2008.
Sanchez-Monge R.; Blanco C.; Díaz-Perales A.; Collada C. Carrillo
T.; Aragoncillo C.; Salcedo G. Class I chitinases, the panaller-
gens responsible for the latex-fruit syndrome, are induced by
ethylene treatment and inactivated by heating. J Allergy Clin
Immunol. 106: 190–195; 2000.
Schlumbaum A.; Match F.; Vogeli U.; Boller T. Plant chitinases differ
in antifungal activity. Nature 324: 325–367; 1986.
7
Singh N. K.; Nelson D. E.; Kuhn D.; Hasegawa P. M.; Bressant R. A.
Molecular cloning of osmotin and regulation of its expression by
ABA and adaptation to low water potential. Plant Physiol. 90:
1096–1101; 1989.
Sripaoraya S.; Blackhall N.; Marchant R.; Power J.; Lowe K.
Relationships in pineapple by random amplified polymorphic
DNA (RAPD) analysis. Plant Breed 120: 265–267; 2001.
Sripaoraya S.; Keawsompong S.; Insupa P.; Power J. B.; Davey M. R.;
Srinives P. Genetically manipulated pineapple: transgene stabil-
ity, gene expression and herbicide tolerance under field con-
ditions. Plant Breed 125: 411–413; 2006.
Takano M.; Egawa H.; Ikeda E.; Wakasa K. The structures of
integration sites in transgenic rice. Plant J. 11: 353–361; 1997.
Thompson C. J.; Movva N. R.; Tizard R.; Crameri R.; Davies J. E.;
Lauwereys M.; Botterman J. Characterization of the herbicide-
resistance gene bar from Streptomyces higroscopicus. EMBO J 6:
2523–2527; 1987.
Torres A. C.; Nagata R. T.; Ferl R. J.; Bewick T. A.; Cantliffe D. J. In
vitro assay selection of glyphosate resistance in lettuce. J Am Soc
Hort Sci. 1: 86–89; 1999.
Ventura J.; Zambolim L.; Chaves G. Tissue culture technique for rapid
clonal propagation of pineapple cultivars. Acta Hort. 425: 161–
166; 1996.
Vigne E.; Komar V.; Fuchs M. Field safety assessment of recombi-
nation in transgenic grapevines expressing the coat protein gene
of grapevine fanleaf virus. Transg Res. 13: 165–179; 2004.
Wehrmann A.; Van Vliet A.; Opsomer C.; Bottermann J.; Schulz A.
The similarities of bar and pat gene products make them equally
applicable for plant engineers. Nat Biotechnol 14: 1274–1278;
1996.
Wilson A. K.; Latham J. R. Transformation-induced mutations in
transgenic plants: analysis and biosafety implications. Biotechnol
Genet Eng Rev. 23: 209–234; 2006.
Woloshuk C. P.; Meulenhoff J. S.; Sela-Buurlage M.; Van den Elzen P.
J. M.; Cornelissen B. J. C. Pathogen-induced proteins with
inhibitory activity toward Phytophthora infestans. The Plant Cell
3: 619–628; 1991.
Yabor L.; Aragón C.; Hernández M.; Arencibia A. D.; Lorenzo J. C.
Biochemical side effects of the herbicide FINALE on bar gene-
containing transgenic pineapple plantlets. Euphytica 164: 515–
520; 2008.
Yabor L.; Arzola M.; Aragón C.; Hernández M.; Arencibia A.;
Lorenzo J. C. Biochemical side effects of genetic transformation
of pineapple. Plant Cell Tiss Org Cult. 86: 63–67; 2006.
Yanes P. E.; González O. J.; Sánchez R. R. A technology of
acclimatization of pineapple vitroplants. Pineap News 7: 24; 2000.