Ministry of Science and Technology-MCT
National Technical Biosafety Commission-CTNBio

Process No.:01200.005154/1998-36
Petitioner: Bayer S.A.
CNPJ: 18.459.628/0043-74
Address: Rua Verbo Divino, 1207 – Bloco B – 2 andar –
Chácara Santo Antônio; São Paulo – SP; CEP: 04719-002
Subject: Commercial Release of Genetically Modified Corn
Previous Extract: Communication No. 070/1999 published on
DOU of January 06th, 1999.
Meeting: 102 Ordinary Meeting of CTNBio that took place on
May 16th, 2007.
Decision: GRANTED

After appreciation of Technical Opinion for commercial
release of corn tolerant to glufosinate (phosphinotricin)
of ammonium herbicide, genetically modified, as well, as of
all the progenies coming from the transformation event T25
and its derivatives of the crossing of lines of nontransgenic
populations of corn with lines bearing the event
T25, CTNBio decided to GRANT it, on the terms of this
technical opinion.
Bayer S.A. requested from CTNBio a Technical Opinion for
the free registration, use, essays, tests, seeding,
transportation, storage, commercialization, consume,
importation, release and discard of corn tolerant to
glufosinate of ammonium herbicide – Liberty Link Corn –
event T25. The plant received gene pat, which is
responsible for the syntheses of enzyme phosphinotricin –
N- acetyltransferase (PAT), that catalyzes the conversion
of L-phosphinotricin (glufosinate of ammonium) to non-toxic
products, inactivating the active ingredient, conferring
then, on the plant the characteristic of tolerance to the
herbicide. Gene pat is a modified version of the isolated
gene of the soil natural bacteria, Streptomycin
viridochromogenes, Tü 494 race, and was inserted into the
vegetable cells through direct incorporation of DNA in corn
protoplasts (electroporation), through vector plasmid
pUC/Ac. The initiating sequences drawn aiming at
identifying the event, that are represented as confidential
information in the document entitled “Previous
communications to the presentation of the biosafety report
of event T25” shall be made available to the public. Due to
the fact that it is a commercial release, there is no need
of keeping confidentiality. Protein PAT was detected in low
levels on the vegetable tissues analyzed, and is fast
degraded in gastric and intestinal fluids, presenting great
susceptibility to digestion and thermal desnaturation,
being highly improbable that it may have any toxic or
allergenic effect. Genetic modification introduced in event
T25 did not result in important differences of chemical
composition regarding nutrients, being within the normal
variation scope among the conventional varieties. The
sylvan species closer to corn is the teosinte found in
Mexico and in some Central America places. Therefore, there
is no sylvan species in Brazil with which corn can be
crossed. The coexistence between conventional corn
cultivations (improved or creoles) and transgenic
cultivations of corns is possible from the agronomic point
of view. Thus, the probability of fixation of allele
containing the gene sequence that confers tolerance to
glufosinate of ammonium on the population is much reduced
on the absence of selection pressure. Corn is a plant that
is incapable of surviving in natural conditions, when not
technically assisted. Therefore, there is no possibility of
corn being transformed into an invasive plant or weed.
Genetically modified cultures behave like correspondent
conventional cultures, and up to now there is no
registration of great alterations on the structures of
microbial communities of soils. Additionally, gene pat
already exists in the soil, once it comes from the soil
natural bacteria, S. viridochromogenes. Glufosinate of
ammonium is registered in Brazil, at the Ministry of
Agriculture, Cattle Breeding and Supply (MAPA), at IBAMA,
and its monograph is approved by the Ministry of Health,
being commercialized in Brazil and in many other countries.
Thus, other norms should be observed when corn T25 is
registered, such as Law 7.802, of July 11th, 1989 (Agro
toxic Law), especially regarding the limits acceptable for
herbicides residues to be established by the registration
and inspection organs and entities. The restrictions to the
use of the GMO in analysis, and its derivatives are
conditioned to the coexistence norms, and to the postcommercialization
monitoring plan, to be eventually
published by CTNBio. Thus, CTNBio considers that this
activity is not potentially causing meaningful degradation
of the environment, or aggravations to human and animal’s
health.

CTNBio TECHNICAL OPINION

I. GMO Identification
Designation of GMO: Liberty Link Corn, Event T25
Petitioner: Bayer S.A.
Species: Zea mays – Corn
Inserted Characteristics: Tolerance to Glufosinate of
Ammonium Herbicide
Method of characteristics introduction: direct
transformation of protoplasts through electroporation
process.
Proposed Use: Silage and grains production for human and
animal consume of GMO and its derivatives.
II. General Information
Zea gender belongs to Gramineae family and has four kinds,
being corn Zea mays ssp.mays L., the species that has the
biggest economical importance. The number of chromosomes in
Z. mays is 2n = 20, 21, 22, 24(5). It has been widely known
that the center of origin of Zea mays ssp.mays includes
Mexico and Central America (12). Corn is an allogamous and
annual plant. Genes dissemination may occur via crossed
pollination with a sexually compatible plant or sylvan
parental plants in the surroundings. Corn is pollinated by
the wind, and the dissemination of the pollen is determined
by the speed and direction of the winds. However, corn
pollen feasibility, in extremely favorable conditions, is
maximum 24 hours. The sylvan species closer to corn is
teosinte, found in Mexico and in some places in Central
America, where it can be crossed with corn cultivated in
production fields. The cultivated corn can also be crossed
with the most distant genre Tripsacum. However, this
crossing occurs with great difficulty, and results on
sterile-male progeny.
Corn history is over eight thousand years old in the
Americas, and nowadays it is the cultivated species that
reached the highest degree of domestication, and only
survives in nature when it is cultivated by men (1).
Of all the cultivated plants, it is probably the one that
has the biggest genetic variability. Today, there are
around 300 identified corn races, and within each race,
thousands of cultivations. The maintenance of this genetic
variability has been usually made through individualized
storage, in germoplasm banks, with controlled conditions of
humidity and temperature. There are many corn germoplasm
banks in Brazil and in the world. Embrapa has two
germoplasm banks, one at Embrapa Genetic Resources and
Biotechnology, in Brasília-DF, and another one at Embrapa
Corn and Sorghum, in Sete Lagoas-MG.
Corn is commercially cultivated in more than 100 countries,
with a total production estimated in 705 million tons/year.
The biggest corn world producers are: The United States,
China, Brazil, Mexico, France and India. Corn is used
mainly for the production of animal food and processed
food, and recently, it has been used on the production of
fuel alcohol. In the last harvest Brazil cultivated 12
million hectares of corn. While the average productivity in
the USA is 9.0, and in Argentina it is 7.0 tons/ha, the
average productivity in Brazil was 3.5 tons/ha. Such low
productivity of corn culture in Brazil is not because of
lack of technology, but for the fact that a meaningful part
of Brazilian agriculturists who plant corn do not use
improved seeds, or do not have access to modern
technologies of cultivation. Agriculturists of the
Brazilian Center-West that use modern technology and
tropical simple hybrid seeds manage to produce an average
similar to the one obtained by its peers in the USA, that
is, 9.0 tons/ha.
Scientific work of corn improvement (“hybrid corn era”)
started in Brazil around 1930, at the Agronomic Institute
of Campinas – IAC, and at the Federal University of Viçosa
– UFV. Today, we have in Brazil many national and foreign
companies that at the 2006/2007 harvest made available
around 275 different kinds of corn cultivation, improved
and adapted to the tropical conditions of the country. This
is the result of more than 50 years of genetically improved
tropical corn that started with the so-called races of
Creole corn. It is also important to highlight that, in
this universe of 275 commercial genotypes, we have creoles
varieties (corn improved by small agriculturists, whose
seeds may be reused), and also state of the art simple
hybrids (for high technology plantation, with production
potential above 12 tons/ha). Today, Brazil develops the
biggest, the most efficient, and the most traditional
program of tropical corn improvement in the world.
The commercial event Liberty Link Corn was obtained by the
direct transformation of protoplasts through
electroporation process. The tolerance to glufosinate of
ammonium herbicide was obtained through the introduction of
the gene that expresses the protein PAT (Phosphinotricin Nacetyltransferase)
isolated from Streptomycin
viridochromogenes that after catalyzing the acetilation of
L-phosphinotricin (glufosinate of ammonium), promotes the
inactivation of the active component. As a consequence,
plants of the referred event of transformation are
resistant to the herbicide, allowing its use in the control
of invasive plants.
In Brazil, many planned releases of corn T25 in the
environment, in experimental character, were conducted
after approval by CTNBio in regions that represent the corn
culture, including the states of São Paulo, Minas Gerais,
Mato Grosso do Sul, Paraná, Goiás, Rio Grande do Sul and
Bahia.
III. GMO description and expressed proteins
The commercial event Liberty Link was obtained through the
direct transformation of protoplasts of corn lineage He/89
through the use of polyethylene glycol (PEG) with the
plasmodium pUC/Ac as a whole containing genic elements of
interest. The transformed protoplasm were cultivated under
selection conditions in the presence of glufosinate of
ammonium herbicide, also known as L-Phosphinotricin (PPT –
Phosphinotricin) until they originate cellular agglomerate
that were later regenerated to normal plants according to
protocol established by Mórocz and collaborators (14).
The main elements that compose the cassette of expression
containing pat gene, as well as the main elements present
on the plasmid pUC/Ac are:
a. plasmid pUC18: plasmid of Escherichia coli with
high number of copies, used for cloning fragments of
DNA;
b. ampR – gene that confers resistance to ampicillin
obtained from E. coli, and that codified b – Lactamase
(bla), being express4ed only in bacteria, once it is
under control of prokaryotic promoter;
c. Ori-pUC – replication origin (ColE1) of plasmid
pUC18;
d. P-35S – promoter of transcribed 35S of virus of
cauliflower mosaic;
e. pat – codifying sequence of pat gene of S.
viridochromogenes modified with codons preferential
for plants, once the original sequence presents high
content of G:C, atypical for plants;
f. T-35S – terminator region not translated into
proteins, obtained from the transcribed 35S of virus
of cauliflower mosaic.
Molecular studies of event T25 presented in the process
allow to visualize how a copy of the insert was introduced
into the genome of LL corn. Bla gene that confers
resistance to ampicillin is present, but it was fragmented,
and its portion of nucleotide 6 to 195 was eliminated from
the event. The studies also show that a sequence similar to
the promoter 35s is at the end of the insert flanking the
other portion of bla gene present in the insert,
corresponding to nucleotides 196 to 861. The replication
region (ColE1) of the plasmid pUC18 is also present in the
insert, as well as the cassette of expression with the
codifying sequence of pat gene with the promoters and
terminators regions of transcribed 35S in the correct
conformation for the expression of pat gene, that was
introduced into the corn genome in a sole copy through
direct incorporation by the electroporation method.
In the event T25 characterization, regions of genomic DNA
of corn, which flank the place of the insert were also
sequenced. That allowed identifying that the region where
it was inserted presented high similarity with one of the
gene alleles of alcoholic desidrogenase of corn. Such
allele was probably inactivated, but as this gene presents
high number of copies on the genome of the species (17),
the insertion of the elements described above have not
apparently harmed the development and the agronomic
characteristics of the plants. No molecular data was
presented confirming or not the inactivation of the
alcohol-desidrogenase (gene bank access No. AF1223535).
However, the event T25 was tested on the field, and in
contention in the United States and in Canada, and the
comparison between the event T25, and not genetically
modified hybrid corn plants did not identify alterations on
agronomic characteristics that are out of the normal range
of variability for characteristics such as productivity,
plants height, cycle, susceptibility to diseases and
plagues, profitability components, and others. On planned
releases in the environment conducted in Brazil, no
alterations were observed in the agronomic characteristics
of corn T25, which would be different from the patterns
found in hybrids, and in not genetically modified corn
lineages. Thus, it is possible to assume that the insertion
of the fragment described above in the corn genome did not
alter its normal fenotypical characteristics.
The sequencing of the genomic regions of corn that flank
the insert are also important, for they allow identifying
this event as unique. The flanks were sequenced on region
5’, 151pb, and on the region 3’, 121pb from the insert. The
starting sequences drawn to identify the event, and that
are presented as confidential information on the document
“Previous Communications to the submission of the biosafety
report of event T25” are available to the public.
The sequencing of the entire insert present in the event
T25 also allowed to identify, on the junction between the
major fragment of bla gene, and the region with elements
similar to promoter 35S, two open reading frames (ORF –
Open Reading Frame): ORF-1, codifying 253 amino acids and
ORF-2, codifying 109 amino acids. The petitioner does not
describe if these ORF codify some protein of known
function. No data confirming or not the expression of these
hypothetical proteins in event T25 have not been presented
either. On both ORF, the initiation codon is found inside
the fragment similar to promoter 35S, and not inside the
fragment of bla gene. This situation, as well as the
incomplete presence of the entire sequence of the promoter
35S in this region, would probably(16) make impractical the
expression of ORF 1 and 2.
The construction used on the transformation puts bla gene
under control of a prokaryotic promoter, making this gene
to be expressed only in bacteria(10). Even being the bla
gene incomplete in event T25, tests were conducted to
identify the presence of enzyme b-lactamase and of
transcribed ones. Enzymatic or transcribed activity of bla
gene was not detected in none of the vegetative and
reproductive parts of Liberty Link corn.
The analysis of PAT protein expression was made on leaves,
roots, stems, grains and pollen through TLC, HPLC and
ELISA. It was not possible to detect the presence of
protein activity in pollen grains. In seeds, roots, leaves
and stems the detected activity was of 0.68, 5.36, 41.32
and 50.95 mU/mg, respectively. Considering that the
promoter used is constructive CaMV 35S, one could expect
PAT expression in every tissue on similar levels. However,
it is already known that promoter 35S, depending on the
tissue and place of insertion in the genetically modified
plant genome, may present variations in its gene activation
capacity (9,10).
The number of inserts was estimated through Southern Blots
made with five enzymes of restriction, and confirmed as a
copy through tests of segregation in the progeny of
crossings made between hemizygote plants and nongenetically
modified lineages. These results indicated that
pat gene is transmitted in a stable way between generations
and behaves as a normal and dominant gene.
IV. Aspects related to Human and Animal’s Health
The evaluation of food safety derived from genetically
modified raw material is based on risk analysis, scientific
methodology that encompass evaluation, management, and risk
communication phases. On the risk evaluation phase one
looks for the qualitative and quantitative characterization
of potential adverse effects, having as base the concept of
substantial equivalence, for the identification of eventual
differences between the new food and its conventional
correspondent.
To evaluate safety of genetically modified food raw
material, or its equivalence to conventional food, it is
recommended that four main elements are analyzed, more
notably: (1) parental variety, that is, the plant that
originated the new genetically modified raw material; (2)
the transformation process, including the characterization
of the construction used, and of the resulting event; (3)
the gene product inserted and the potential of toxicity and
allergenicity, and finally; (4) the composition of the new
variety deriving from the genetic transformation. The group
of data of these analysis should allow for the
identification and characterization of the potential
different effects associated to the consume of the new raw
material, subsidizing the management and risk communication
phases.
According to the petitioner, event T25 derives from the
transformation of cells of lineage He/89 of common corn Zea
mays, a species deeply characterized, and about which there
is solid safety background for human and animal consume.
Information about identity, origin and chemical composition
are reported, and a copy of the publication was attached to
the process, which provides abundant data regarding its
composition, highlighting the variations naturally observed
in the presence of nutrients (21).
The analysis of chemical composition of the variety
obtained through gene manipulation, mainly of the levels of
its nutrients and eventual toxic compositions naturally
present, aims at guaranteeing that this new variety is as
nutritive and safe as its conventional equivalent. Thus,
it serves to confirm that intentional effects of
modification do not compromise its safety, or results in
unintended effects. The introduction of cassette of
expression containing pat gene, as well as other gene
elements described before, do not alter the substantial
equivalence of Liberty Link corn in relation to the quality
and quantity of metabolite normally found in corn. The data
presented by the petitioner are related to the centesimal
composition, to the profiles of amino acids, fatty acids,
mineral and vitamins, besides the content of phitate, both
for the genetically modified variety, with and without the
use of glufosinate, and for the conventional variety,
cultivated under the same conditions and on the same region
during the same period. At the beginning, results from
analysis conducted with plants cultivated abroad in two
regions were presented. Late on, due to questionings made
by CTNBio’s members, data regarding plants cultivated in
the country in different environments were presented, in
the state of Goiás and Paraná. These compositions analysis
were made in the country at the Institute Adolfo Lutz, of
São Paulo.
In general, for all the parameters analyzed, there was a
meaningful difference between the genetically modified
variety and the conventional one, or the differences noted
were within the variability normally observed among
conventional corn varieties. Anyway, the small differences
found in relation to event T25 do not affect the
nutritional value, or safety, for they were similar to the
ones usually found in other varieties, or under different
conditions of cultivation. In this regard, it is important
to highlight that there were differences among the results
obtained for the cultivations executed in Goiás and in
Paraná, even for the conventional variety, without, thou,
resulting in meaningful difference of the latter for the
genetically modified variety. Thus, it is clear that the
environmental conditions were more determining for the
differences in the chemical composition, than the presence
of pat gene in the genome of the transformation event T25.
In relation to the levels of residues of glufosinate of
ammonium left in the plant, due to its use during the
cultivation of transgenic variety, studies executed in
Brazil showed that there were no differences between those
levels found in the parental variety when compared to the
transgenic variety (event T25), when the herbicide is
applied in accordance with the patterns of the Brazilian
legislation for the evaluation of the maximum limits of
residues.
PAT protein is degraded by the gastric juice of animals and
by similar artificial gastric juice of human beings, losing
its physical-chemical characteristics after oral
exposition. Thus, the protein is not expected to be fully
absorbed, and, therefore, it is highly improbable that the
protein may reduce different or toxic effects. Besides, PAT
protein activity in the different parts of corn is low
(around mU/mg of protein).
References about acute toxicity were described in documents
of the Environmental Protection Agency of the United States
and of DG Health and Consumer Protection of the European
Commission indicating lack of toxicity. “The oral acute
toxicity test of PAT protein produced in bacteria showed
lack of effects in a dose of 2,500 mg/kg”(19). “The enzyme
phosphinotricin acetyltransferase (PAT) should not present
biosafety problems. The quantitative level of PAT in grains
is very low. Its enzymatic function is specific for a
substrate that is naturally absent in human beings, called
phosphinotricin. Besides, it is degraded and inactivated in
human artificial gastric fluid containing pepsin in pH 1.0-
1.2. Therefore, it is improbable that it maintains any
enzymatic activity in vivo. What’s more, no homology
between PAT protein and toxins known was found. Native PAT
protein (51% of purity) was evaluated for acute toxicity in
rats, and no effect in 5.0 g doses per kg of corporal
weight was reported” (4).
There are no reports saying that PAT protein has allergenic
activity. Analysis of sequences of PAT protein show that it
does not present places capable of glicolisation what could
theoretically, confer allergenicity to protein. However, a
possible source of PAT protein allergenicity would be the
expression of protein in pollen, what does not seem to
occur. Gene pat sequence and PAT protein were compared to
data banks, and it was observed that they do not present
meaningful homology with other sequences of nucleotides and
proteins, except for others genes of resistance to
phosphinotricin including gene pat of S. viridochromogenes
and bar gene of S. hygroscopicus.
V. Environmental Aspects
Corn is a monoic plant: a sole individual contains male and
female flowers located separately. Corn plants are plants
of crossed fecundation and widely pollinated with the wind
help, insects, gravity and others. The introduction of gene
elements previously described did not alter the
reproductive characteristics of the plant. Therefore, the
probability of crossed fecundation between plants of the
event T25 and other corn plants is the same as the one that
occurs between hybrids and corn lineages not genetically
modified.
Corn is the species that reached the highest level of
domestication among cultivated plants, losing its
characteristics of survival in nature as, for example,
degrana elimination. Thus, corn is a plant that is
incapable of surviving in natural conditions, when not
technically assisted. Therefore, there is no possibility of
corn being transformed into an invasive plant or weed.
Gene flow in corn may occur through pollen transfer and
seeds dispersion. Seeds dispersion is easily controlled,
once the domestication of corn eliminated the ancient
mechanisms of seeds dispersion, and, therefore, the pollen
movement is the only effective escape mean of genes of corn
plants.
Studies about corn pollen dispersion have been conducted,
and some of them show that corn pollen may travel long
distances. However, most pollen that is released is
deposited near the culture, with very low translocation
rate outside the source culture. The predominant
pollination agent for corn is the wind and the distance
that viable pollen may travel depends on wind patterns,
humidity and temperature. Luna et al. (11) evaluate the
distance of isolation and control of pollen and demonstrate
that crossed pollination occurs in a maximum distance of
200m and no crossed pollination happened in the same
distances or over 300 meters in relation to pollen sources,
in non detasseling condition. The results indicate that
pollen viability is kept for 2h, and that crossed
pollination was not observed in 300 meters distances from
the pollen source.
Under low to moderate winds, it was estimated that
comparing concentrations to 1 of source culture,
approximately 2% of pollen are annotated at 60m, 1.1% at
200m and 0.75-0.5% at 500m of distance. At 10m from a
field, in average, the number of pollen grains per area
unit is ten times smaller than the one observed at 1m from
the border. Therefore, if the established distances of
separation developed for corn seed production are observed,
it is expected that the pollen transfer to the adjacent
varieties are minimized, and it shall not have any genetic
material with tolerance to herbicide. Even in case of
having a gene escape, the probability of allele fixation
containing the gene sequence that confers tolerance to
glufosinate of ammonium in the population is much reduced
in the absence of selection pressure.
The secondary ecological impacts, that refer to effect in
the environment, especially in the ecosystem of the soil,
are very discussed nowadays due to the complexity of the
soil. The organisms of the soil are usually very exposed to
the contact with genetically modified plants, both with
direct contact, and through the fall of leaves, reticular
exudates or even decomposition of roots. Through the
reports in literature, genetically modified cultures behave
similarly to conventional correspondent cultures, and
meaningful alterations have not been detected until now on
the structures of microbial communities of the soils.
Studies of plants intact genes transfer to microorganisms
show extremely low possibilities of transfer, suggesting
that the probability of occurrence of this event is, in
practice, extremely low. There is no evidence that plants
genes have even been transferred to bacteria in natural
conditions. Moreover, gene pat already exists in the soil,
once it comes from a natural soil bacteria, S.
viridochromogenes.
Glufosinate of ammonium is a non systemic and non selective
herbicide used, mainly, in the control of post-emergency
invasive plants of wide leaves, and of thin ones. Such
herbicide is registered in Brazil, at the Ministry of
Agriculture, Cattle Breeding, and Supply (MAPA), at the
Brazilian Institute of Environment and Renewable Natural
Resources (IBAMA), and with monograph approved by the
Ministry of Health, being commercialized in Brazil and in
other countries. Its wide use in the world is due to the
fact that it is biodegradable, presents low residual
activity, low toxicity to men, animals, and other organism
of the food chain. It is considered persistent and movable
in the soil, and in sandy soils up to 80% may be lixiviate.
Depending on handling conditions, edafoclimatic conditions,
microbial activity, and other factors, glufosinate of
ammonium has half-life in the soil that varies from 12 to
70 days. However, residues have been found in the soil
after 100 days (16). Therefore, if it is used outside its
recommendations, glufosinate of ammonium herbicide has
potential for contaminating water courses and groundwater
(18) as any other herbicide used in genetically modified
cultures or not. However, it is highlighted that other
norms should be observed when T25 corn is registered, such
as Law 7.802, of July 11th, 1989 (Agro Toxic Law)
especially regarding the acceptable limits for residues of
herbicides to be established by the registration and
inspection organs and entities mentioned above.
VI. Restriction to the use of GMO and its derivatives:
Technical opinions regarding the agronomic performance came
to the conclusion that there is equivalence between
transgenic and conventional plants. Thus, information
indicate that transgenic plants do not fundamentally differ
from non transformed corn genotypes, except for the
tolerance to glufosinate of ammonium herbicide.
Additionally, there is no evidence of different reactions
to the use of Liberty Link corn. So, there is no
restriction to the use of this corn, or of its derivatives,
be it for human or for animal food.
The vertical genic flow for local varieties (called creoles
corns) of open pollination is possible and presents the
same risk caused by commercial genotypes available in the
market (80% of planted conventional corn in Brazil comes
from commercial seeds that went through a process of
genetic improvement). The coexistence between conventional
corns cultivation (improved or creoles) and transgenic
cultivations of corns is possible from the agronomic point
of view (3,13). Therefore, CTNBio will eventually publish
norms about coexistence of genetically modified corn with
non modified varieties.
VII. Considerations about the particularities of different
regions of the country (subsidies to inspection organs):
Small variations of centesimal composition, not meaningful
ones, were found between the corn planted in the South
region (Paraná), and the one planted in the Center West
Region (Goiás) that were imputed, not to the presence of
the transformation event, but to environment conditions.
Therefore, there are no restrictions to the use of this
corn, at least in relation to the South, Southeast and
Center West of the country.
The registration and inspection organs, in the ambit of
their competences, will establish acceptable limits of
residues to herbicide glufosinate of ammonium used in
plantations cultivated with corn T25.
According to what is established on art. 1 of Law 11.460,
of March 21st, 2007, “it is vetoed the research and
cultivation of genetically modified organisms in indigenous
lands and areas of conservation units”. CTNBio will
eventually publish norms about the coexistence of
genetically modified corn with non modified varieties.
VII. Conclusion
Considering that event T25 derives from the lineage He/89
of common corn (Zea mays), species with solid safety
background for human and animal consume, and that the
introduced gene, pat, does not codified proteins known as
toxic or allergenic, and results on the enzyme Lphosphinotricin-
N-acetyltransferase, with high specificity
for the herbicide glufosinate of ammonium.
Considering that the gene construction used on the
transformation resulted on the instable insertion of a copy
of pat gene, and regulating regions of the mosaic virus of
cauliflower on the genome of corn, besides a non-functional
incomplete sequence of the gene of resistance to
ampicillin, resulting only on PAT expression, without
apparent harm to the plant or to the environment.
Considering also that:
1. Corn is the species that reached the highest degree of
domestication among plants cultivated, being able to
survive in nature without human intervention.
2. The sylvan species closer to corn is teosinte, found in
Mexico and in some places in Central America, where it can
be crossed with corn cultivated in production fields.
Therefore, there are no sylvan species in Brazil with which
corn can be crossed.
3. Protein PAT has rapid degradation in gastric and
intestinal fluids (7,20).
4. Recombinant enzyme PAT was detected in low levels of
analyzed vegetable tissues, and presented great
susceptibility to digestion and thermal denaturation by the
processing, being highly improbable that it may have any
toxic or allergenic effect (7,20).
5. There are many proteins similar to PAT in nature without
any evidence of adverse effect to men, animals, or plants
have been described, and that its subtract is highly
specific, not having any sequence of amino acids with
homology to toxins or allergens.
6. Genetic modification introduced in event T25 did not
result in important differences of chemical composition
regarding nutrients, being within the normal variation
scope among the conventional varieties.
7. Microorganisms S. hygroscopicus and S. viridochromogenes
are saprophyte bacteria of the soil, and there are many
species of Streptomycin genre similar to S. hygroscopicus
and S. viridochromogenes, and among them, many containing
pat and bar genes(2).
8. DNA molecule is a natural food component, not presenting
any evidence that such molecule may have adverse molecule
effect to men when ingested in food in acceptable
quantities (no direct toxic effect).
9. There is no evidence that intact genes of plants may be
transferred and functionally integrated in human genome, or
of other mammals exposed to this DNA or foods manufactured
with these elements(6).
10. Plants tolerant to glufosinate of ammonium containing
PAT protein have been cultivated in the United States and
in Canada for approximately a decade without any
registration of adverse effect to human and animal food.
Besides, many regulating agencies of varied countries have
approved these plants for these plants for human and animal
use including Australia, Japan and European Union.
11. The petitioner answered to all questionings postulated
on the Normative Instruction No. 20 at CTNBio.
12. None of the questions indicate that this corn may
present adverse effects on human and animal food.
13. After ten years of use in different countries, no
problem was detected for human, animal’s health, or to the
environment that may be attributed to transgenic corns. It
is necessary to emphasize that the lack of negative effects
resulting of corn transgenic plants does not mean that they
may not happen. Zero risk and absolute safety does not
exist in biologic world, although an accumulation of
trustworthy scientific information already exists, and a
safe background of ten years use allows us to state that
corn T25 is as safe as conventional versions. Thus, the
petitioner is conditioned to conduct monitoring of postcommercial
release on the terms to be eventually
established by CTNBio.
14. The coexistence among cultivations of conventional
corns (improved or creoles) and transgenic cultivations is
possible from the agronomic point of view.
15. The probability of allele fixation, containing the gene
sequence that confers tolerance to glufosinate of ammonium
on the population is much reduced in the absence of
selection pressure.
16. There is no need of maintenance of confidentiality in
relation to the commercial release.
CTNBio thinks that this activity is not potentially causing
meaningful degradation of the environment, or aggravations
to human and animal’s health. The use restrictions of GMO
in analysis, and its derivatives are conditioned to
coexistence norms, and to the after commercialization
monitoring plan to be eventually published by CTNBio.
VIII - Bibliography
1. BAHIA FILHO, A.F.C.; GARCIA, J.C. 2000. Analysis and
evaluation of Brazilian market of corn seeds. In: UDRY,
C.V.; DUARTE, W.F. (Org.) Uma história brasileira do milho:
o valor de recursos genéticos. Brasília: Paralelo 15,167-
172.
2. BARTSCH K; TEBBE C.C. 1989. Initial steps in degradation
of Phosphinothricin (glufosinate) by soil bacteria. Applied
and Environmental Microbiology 55: 711-716.
3. BROOKES, G.; BARFOOT, P.; MELÉ, E.; MESSEGUER, J.;
BÉNÉTRIX, F. BLOC, D.; FOUEILLASSAR, X, FABIÉ,A.;
POEYDOMENGE, C.2004. Genetically modified maize; pollen
movement and crop co-existence. Dorchester, UK: PG
economics, 20pp.
(www.pgeconomics.co.uk/pdf/Maizepollennov2004final.pdf)
4. EUROPEAN COMMISSION – Food safety the Farm to the Fork.
1996. Opinion On the Potential For Adverse Health Effects
From the Consumption of Genetically Modified Maize (Zea
mays L).
(http://ec.europa.eu/food/fs/sc/oldcomm7/out02_en.html)
5. FAO/WHO – Food and Agriculture Organization of the
United Nations/World Health Organization. 2000 Grassland
index. Zea mays L.
(http://www.fao.org/WAICENT/faoinfo/agricult/agp/doc/gbase/
data/pf000342.htm)
6. FAO/WHO – Food and Agriculture Organization of the
United Nations. 2000. Safety Aspects of Genetically
Modified Foods of Plant Origin. Report of a Joint FAO/WHO
Expert June 2000. World Health Organization, WHO
Headquarters, Geneva, Switzerland. 35pp.
(http://www.who.int/foodsafety/publications/biotech/en/ec_j
une2000_en.pdf)
7. HEALTH CANADA. 1997 Glufosinate Ammonium Tolerant Corn
(T14 and T25). Novel Food Information – Food Biotechnology.
4pp. (http://www.hc-sc.gc.ca/fn-an/alt_formats/hpfbdgpsa/
pdf/gmf-agm/32bg_agrevo-ct_agrevo_e.pdf).
8. HULL, R.; COVEY, S.N.; DALE, P. 2000. Genetically
modified plants and the 35S promoter: assessing the risks
and enhancing the debate. Microb. Ecol. In Health and Dis.
12:1-5.
9. KOOTER, J.M.; MATZKE, M.A.; MEYER, P. 1999. Listening to
the silent genes: transgenic silencing. Gene regulation and
pathogen control. Trends in Plant Sci. 4: 340-347.
10. LEWIN, B.2004. Genes VII. New York: Oxford University
Press, 1ed. 1056pp.
11. LUNA, S.V.; FIGUEROA, J.M.; BALTAZAR, M.B.; GOMEZ ,
L.R.; TOWNSEND , R.E SCHOPER, J.B. 2001. Maize pollen
longevity and distance isolation requirements for effective
pollen control. Crop Sci. 41:1551-1557.
12. MANGELSDORF.P.C.; REEVES, R.G. 1938. The origin of
maize. Proc. Natl. Acad. Sci. USA 24: 303-312.
13. MESSEGUER, J.; PEÑAS,G.; BALLESTER, J.; BAS, MAS.;
SERRA, H.; SALVIA, J.; PALAUDELMÀS, M.; MÉLE, E. 2006.
Pollen-mediated gene flow in maize in real situations of
coexistence. Plant Biotechnology Journal. 4:633-645.
14. MÓROCZ, S.; DONN, G.; NÉMETH, J.; DUDITS, D. 1990. An
improved system to obtain fertile regenerants via maize
protoplasts isolated from highly embryogenic suspension
culture. Theor Appl. Genet.80: 721-726.
15. PAULI, S.; ROTHNIE, H.M.; CHEN, G.; HE, X.; HOHN, T.
2004. The cauliflower mosaic virus 35S promoter extends
into the transcribed region. Journal of Virology 78: 12120-
12128.
16. SMITH, A.E.; BELIK, M.B. 1989. Field persistence
studies with the herbicide glufosinate-ammonium in
Saskatchewan soils. J. Environ. Qual. 18: 475-479.
17. TIKHONOV, A.; SANMIGUEL, P.; NAKAJIMA, Y.; GORESTEIN,
N.; BENNETZENAND , J.; AVRAMOVA,Z. 1999. Colinearity and
its exceptions in orthologous and regions of maize and
sorghum. Proc. Natl. Acad. Sci. USA 96: 7409-7414.
18. US ENVIRONMENTAL PROTECT AGENCY- EPA. 1993. Office of
pesticides and toxicity substances. Pesticide fact sheet.
(http://www.epa.gov/fedrgstr/EPA-PEST/1997/September/Day-
10/bac.pdf)
19. US ENVIRONMENTAL PROTECTION AGENCY – EPA. 1997.
Phosphinothricin Acetyltransferase and the Genetic Material
Necessary for its Production in all Plants; Exemption From
the Requirement of a tolerance on all Agricultural
Commodities. (ww.epa.gov/fedrgstr/EPA-PEST/1997/April/Day-
11/p9373.htm)
20. US FOOD AND DRUG ADMINISTRATION – FDA. 1995.
Biotechnology Consultation – note to file BNF No. 000029.
(http://www.cfsan.fda.gov/~rdb/bnfm029.html)
21. WATSON, S.A.; RAMSTAD, P.E. 1987. Corn: chemistry and
technology. St. Paul: American Association of Cereal
Chemists, 1 ed. 604pp.
Walter Colli
President of CTNBio
Divergent Vote
The reporter Dr. Lia Giraldo da Silva Augusto (Permanent
Environmental Sector Sub-commission) issued contrary
opinion to this product approval based on:
1. Inexistence of normative instruction for commercial
release;
2. Un-fulfillment of precaution principle;
3. Lack of monitoring credits of environmental impact of
short, medium and long term for transgenic cultivations.

I. GMO IdentificationDesignation of GMO: Liberty Link Corn, Event T25Petitioner: Bayer S.A.Species: Zea mays – CornInserted Characteristics: Tolerance to Glufosinate ofAmmonium HerbicideMethod of characteristics introduction: directtransformation of protoplasts through electroporationprocess.Proposed Use: Silage and grains production for human andanimal consume of GMO and its derivatives.II. General InformationZea gender belongs to Gramineae family and has four kinds,being corn Zea mays ssp.mays L., the species that has thebiggest economical importance. The number of chromosomes inZ. mays is 2n = 20, 21, 22, 24(5). It has been widely knownthat the center of origin of Zea mays ssp.mays includesMexico and Central America (12). Corn is an allogamous andannual plant. Genes dissemination may occur via crossedpollination with a sexually compatible plant or sylvanparental plants in the surroundings. Corn is pollinated bythe wind, and the dissemination of the pollen is determinedby the speed and direction of the winds. However, cornpollen feasibility, in extremely favorable conditions, ismaximum 24 hours. The sylvan species closer to corn isteosinte, found in Mexico and in some places in CentralAmerica, where it can be crossed with corn cultivated inproduction fields. The cultivated corn can also be crossedwith the most distant genre Tripsacum. However, thiscrossing occurs with great difficulty, and results onsterile-male progeny.Corn history is over eight thousand years old in theAmericas, and nowadays it is the cultivated species thatreached the highest degree of domestication, and onlysurvives in nature when it is cultivated by men (1).Of all the cultivated plants, it is probably the one thathas the biggest genetic variability. Today, there arearound 300 identified corn races, and within each race,thousands of cultivations. The maintenance of this geneticvariability has been usually made through individualizedstorage, in germoplasm banks, with controlled conditions ofhumidity and temperature. There are many corn germoplasmbanks in Brazil and in the world. Embrapa has twogermoplasm banks, one at Embrapa Genetic Resources andBiotechnology, in Brasília-DF, and another one at EmbrapaCorn and Sorghum, in Sete Lagoas-MG.Corn is commercially cultivated in more than 100 countries,with a total production estimated in 705 million tons/year.The biggest corn world producers are: The United States,China, Brazil, Mexico, France and India. Corn is usedmainly for the production of animal food and processedfood, and recently, it has been used on the production offuel alcohol. In the last harvest Brazil cultivated 12million hectares of corn. While the average productivity inthe USA is 9.0, and in Argentina it is 7.0 tons/ha, theaverage productivity in Brazil was 3.5 tons/ha. Such lowproductivity of corn culture in Brazil is not because oflack of technology, but for the fact that a meaningful partof Brazilian agriculturists who plant corn do not useimproved seeds, or do not have access to moderntechnologies of cultivation. Agriculturists of theBrazilian Center-West that use modern technology andtropical simple hybrid seeds manage to produce an averagesimilar to the one obtained by its peers in the USA, thatis, 9.0 tons/ha.Scientific work of corn improvement (“hybrid corn era”)started in Brazil around 1930, at the Agronomic Instituteof Campinas – IAC, and at the Federal University of Viçosa– UFV. Today, we have in Brazil many national and foreigncompanies that at the 2006/2007 harvest made availablearound 275 different kinds of corn cultivation, improvedand adapted to the tropical conditions of the country. Thisis the result of more than 50 years of genetically improvedtropical corn that started with the so-called races ofCreole corn. It is also important to highlight that, inthis universe of 275 commercial genotypes, we have creolesvarieties (corn improved by small agriculturists, whoseseeds may be reused), and also state of the art simplehybrids (for high technology plantation, with productionpotential above 12 tons/ha). Today, Brazil develops thebiggest, the most efficient, and the most traditionalprogram of tropical corn improvement in the world.The commercial event Liberty Link Corn was obtained by thedirect transformation of protoplasts throughelectroporation process. The tolerance to glufosinate ofammonium herbicide was obtained through the introduction ofthe gene that expresses the protein PAT (Phosphinotricin Nacetyltransferase)isolated from Streptomycinviridochromogenes that after catalyzing the acetilation ofL-phosphinotricin (glufosinate of ammonium), promotes theinactivation of the active component. As a consequence,plants of the referred event of transformation areresistant to the herbicide, allowing its use in the controlof invasive plants.In Brazil, many planned releases of corn T25 in theenvironment, in experimental character, were conductedafter approval by CTNBio in regions that represent the cornculture, including the states of São Paulo, Minas Gerais,Mato Grosso do Sul, Paraná, Goiás, Rio Grande do Sul andBahia.III. GMO description and expressed proteinsThe commercial event Liberty Link was obtained through thedirect transformation of protoplasts of corn lineage He/89through the use of polyethylene glycol (PEG) with theplasmodium pUC/Ac as a whole containing genic elements ofinterest. The transformed protoplasm were cultivated underselection conditions in the presence of glufosinate ofammonium herbicide, also known as L-Phosphinotricin (PPT –Phosphinotricin) until they originate cellular agglomeratethat were later regenerated to normal plants according toprotocol established by Mórocz and collaborators (14).The main elements that compose the cassette of expressioncontaining pat gene, as well as the main elements presenton the plasmid pUC/Ac are:a. plasmid pUC18: plasmid of Escherichia coli withhigh number of copies, used for cloning fragments ofDNA;b. ampR – gene that confers resistance to ampicillinobtained from E. coli, and that codified b – Lactamase(bla), being express4ed only in bacteria, once it isunder control of prokaryotic promoter;c. Ori-pUC – replication origin (ColE1) of plasmidpUC18;d. P-35S – promoter of transcribed 35S of virus ofcauliflower mosaic;e. pat – codifying sequence of pat gene of S.viridochromogenes modified with codons preferentialfor plants, once the original sequence presents highcontent of G:C, atypical for plants;f. T-35S – terminator region not translated intoproteins, obtained from the transcribed 35S of virusof cauliflower mosaic.Molecular studies of event T25 presented in the processallow to visualize how a copy of the insert was introducedinto the genome of LL corn. Bla gene that confersresistance to ampicillin is present, but it was fragmented,and its portion of nucleotide 6 to 195 was eliminated fromthe event. The studies also show that a sequence similar tothe promoter 35s is at the end of the insert flanking theother portion of bla gene present in the insert,corresponding to nucleotides 196 to 861. The replicationregion (ColE1) of the plasmid pUC18 is also present in theinsert, as well as the cassette of expression with thecodifying sequence of pat gene with the promoters andterminators regions of transcribed 35S in the correctconformation for the expression of pat gene, that wasintroduced into the corn genome in a sole copy throughdirect incorporation by the electroporation method.In the event T25 characterization, regions of genomic DNAof corn, which flank the place of the insert were alsosequenced. That allowed identifying that the region whereit was inserted presented high similarity with one of thegene alleles of alcoholic desidrogenase of corn. Suchallele was probably inactivated, but as this gene presentshigh number of copies on the genome of the species (17),the insertion of the elements described above have notapparently harmed the development and the agronomiccharacteristics of the plants. No molecular data waspresented confirming or not the inactivation of thealcohol-desidrogenase (gene bank access No. AF1223535).However, the event T25 was tested on the field, and incontention in the United States and in Canada, and thecomparison between the event T25, and not geneticallymodified hybrid corn plants did not identify alterations onagronomic characteristics that are out of the normal rangeof variability for characteristics such as productivity,plants height, cycle, susceptibility to diseases andplagues, profitability components, and others. On plannedreleases in the environment conducted in Brazil, noalterations were observed in the agronomic characteristicsof corn T25, which would be different from the patternsfound in hybrids, and in not genetically modified cornlineages. Thus, it is possible to assume that the insertionof the fragment described above in the corn genome did notalter its normal fenotypical characteristics.The sequencing of the genomic regions of corn that flankthe insert are also important, for they allow identifyingthis event as unique. The flanks were sequenced on region5’, 151pb, and on the region 3’, 121pb from the insert. Thestarting sequences drawn to identify the event, and thatare presented as confidential information on the document“Previous Communications to the submission of the biosafetyreport of event T25” are available to the public.The sequencing of the entire insert present in the eventT25 also allowed to identify, on the junction between themajor fragment of bla gene, and the region with elementssimilar to promoter 35S, two open reading frames (ORF –Open Reading Frame): ORF-1, codifying 253 amino acids andORF-2, codifying 109 amino acids. The petitioner does notdescribe if these ORF codify some protein of knownfunction. No data confirming or not the expression of thesehypothetical proteins in event T25 have not been presentedeither. On both ORF, the initiation codon is found insidethe fragment similar to promoter 35S, and not inside thefragment of bla gene. This situation, as well as theincomplete presence of the entire sequence of the promoter35S in this region, would probably(16) make impractical theexpression of ORF 1 and 2.The construction used on the transformation puts bla geneunder control of a prokaryotic promoter, making this geneto be expressed only in bacteria(10). Even being the blagene incomplete in event T25, tests were conducted toidentify the presence of enzyme b-lactamase and oftranscribed ones. Enzymatic or transcribed activity of blagene was not detected in none of the vegetative andreproductive parts of Liberty Link corn.The analysis of PAT protein expression was made on leaves,roots, stems, grains and pollen through TLC, HPLC andELISA. It was not possible to detect the presence ofprotein activity in pollen grains. In seeds, roots, leavesand stems the detected activity was of 0.68, 5.36, 41.32and 50.95 mU/mg, respectively. Considering that thepromoter used is constructive CaMV 35S, one could expectPAT expression in every tissue on similar levels. However,it is already known that promoter 35S, depending on thetissue and place of insertion in the genetically modifiedplant genome, may present variations in its gene activationcapacity (9,10).The number of inserts was estimated through Southern Blotsmade with five enzymes of restriction, and confirmed as acopy through tests of segregation in the progeny ofcrossings made between hemizygote plants and nongeneticallymodified lineages. These results indicated thatpat gene is transmitted in a stable way between generationsand behaves as a normal and dominant gene.IV. Aspects related to Human and Animal’s HealthThe evaluation of food safety derived from geneticallymodified raw material is based on risk analysis, scientificmethodology that encompass evaluation, management, and riskcommunication phases. On the risk evaluation phase onelooks for the qualitative and quantitative characterizationof potential adverse effects, having as base the concept ofsubstantial equivalence, for the identification of eventualdifferences between the new food and its conventionalcorrespondent.To evaluate safety of genetically modified food rawmaterial, or its equivalence to conventional food, it isrecommended that four main elements are analyzed, morenotably: (1) parental variety, that is, the plant thatoriginated the new genetically modified raw material; (2)the transformation process, including the characterizationof the construction used, and of the resulting event; (3)the gene product inserted and the potential of toxicity andallergenicity, and finally; (4) the composition of the newvariety deriving from the genetic transformation. The groupof data of these analysis should allow for theidentification and characterization of the potentialdifferent effects associated to the consume of the new rawmaterial, subsidizing the management and risk communicationphases.According to the petitioner, event T25 derives from thetransformation of cells of lineage He/89 of common corn Zeamays, a species deeply characterized, and about which thereis solid safety background for human and animal consume.Information about identity, origin and chemical compositionare reported, and a copy of the publication was attached tothe process, which provides abundant data regarding itscomposition, highlighting the variations naturally observedin the presence of nutrients (21).The analysis of chemical composition of the varietyobtained through gene manipulation, mainly of the levels ofits nutrients and eventual toxic compositions naturallypresent, aims at guaranteeing that this new variety is asnutritive and safe as its conventional equivalent. Thus,it serves to confirm that intentional effects ofmodification do not compromise its safety, or results inunintended effects. The introduction of cassette ofexpression containing pat gene, as well as other geneelements described before, do not alter the substantialequivalence of Liberty Link corn in relation to the qualityand quantity of metabolite normally found in corn. The datapresented by the petitioner are related to the centesimalcomposition, to the profiles of amino acids, fatty acids,mineral and vitamins, besides the content of phitate, bothfor the genetically modified variety, with and without theuse of glufosinate, and for the conventional variety,cultivated under the same conditions and on the same regionduring the same period. At the beginning, results fromanalysis conducted with plants cultivated abroad in tworegions were presented. Late on, due to questionings madeby CTNBio’s members, data regarding plants cultivated inthe country in different environments were presented, inthe state of Goiás and Paraná. These compositions analysiswere made in the country at the Institute Adolfo Lutz, ofSão Paulo.In general, for all the parameters analyzed, there was ameaningful difference between the genetically modifiedvariety and the conventional one, or the differences notedwere within the variability normally observed amongconventional corn varieties. Anyway, the small differencesfound in relation to event T25 do not affect thenutritional value, or safety, for they were similar to theones usually found in other varieties, or under differentconditions of cultivation. In this regard, it is importantto highlight that there were differences among the resultsobtained for the cultivations executed in Goiás and inParaná, even for the conventional variety, without, thou,resulting in meaningful difference of the latter for thegenetically modified variety. Thus, it is clear that theenvironmental conditions were more determining for thedifferences in the chemical composition, than the presenceof pat gene in the genome of the transformation event T25.In relation to the levels of residues of glufosinate ofammonium left in the plant, due to its use during thecultivation of transgenic variety, studies executed inBrazil showed that there were no differences between thoselevels found in the parental variety when compared to thetransgenic variety (event T25), when the herbicide isapplied in accordance with the patterns of the Brazilianlegislation for the evaluation of the maximum limits ofresidues.PAT protein is degraded by the gastric juice of animals andby similar artificial gastric juice of human beings, losingits physical-chemical characteristics after oralexposition. Thus, the protein is not expected to be fullyabsorbed, and, therefore, it is highly improbable that theprotein may reduce different or toxic effects. Besides, PATprotein activity in the different parts of corn is low(around mU/mg of protein).References about acute toxicity were described in documentsof the Environmental Protection Agency of the United Statesand of DG Health and Consumer Protection of the EuropeanCommission indicating lack of toxicity. “The oral acutetoxicity test of PAT protein produced in bacteria showedlack of effects in a dose of 2,500 mg/kg”(19). “The enzymephosphinotricin acetyltransferase (PAT) should not presentbiosafety problems. The quantitative level of PAT in grainsis very low. Its enzymatic function is specific for asubstrate that is naturally absent in human beings, calledphosphinotricin. Besides, it is degraded and inactivated inhuman artificial gastric fluid containing pepsin in pH 1.0-1.2. Therefore, it is improbable that it maintains anyenzymatic activity in vivo. What’s more, no homologybetween PAT protein and toxins known was found. Native PATprotein (51% of purity) was evaluated for acute toxicity inrats, and no effect in 5.0 g doses per kg of corporalweight was reported” (4).There are no reports saying that PAT protein has allergenicactivity. Analysis of sequences of PAT protein show that itdoes not present places capable of glicolisation what couldtheoretically, confer allergenicity to protein. However, apossible source of PAT protein allergenicity would be theexpression of protein in pollen, what does not seem tooccur. Gene pat sequence and PAT protein were compared todata banks, and it was observed that they do not presentmeaningful homology with other sequences of nucleotides andproteins, except for others genes of resistance tophosphinotricin including gene pat of S. viridochromogenesand bar gene of S. hygroscopicus.V. Environmental AspectsCorn is a monoic plant: a sole individual contains male andfemale flowers located separately. Corn plants are plantsof crossed fecundation and widely pollinated with the windhelp, insects, gravity and others. The introduction of geneelements previously described did not alter thereproductive characteristics of the plant. Therefore, theprobability of crossed fecundation between plants of theevent T25 and other corn plants is the same as the one thatoccurs between hybrids and corn lineages not geneticallymodified.Corn is the species that reached the highest level ofdomestication among cultivated plants, losing itscharacteristics of survival in nature as, for example,degrana elimination. Thus, corn is a plant that isincapable of surviving in natural conditions, when nottechnically assisted. Therefore, there is no possibility ofcorn being transformed into an invasive plant or weed.Gene flow in corn may occur through pollen transfer andseeds dispersion. Seeds dispersion is easily controlled,once the domestication of corn eliminated the ancientmechanisms of seeds dispersion, and, therefore, the pollenmovement is the only effective escape mean of genes of cornplants.Studies about corn pollen dispersion have been conducted,and some of them show that corn pollen may travel longdistances. However, most pollen that is released isdeposited near the culture, with very low translocationrate outside the source culture. The predominantpollination agent for corn is the wind and the distancethat viable pollen may travel depends on wind patterns,humidity and temperature. Luna et al. (11) evaluate thedistance of isolation and control of pollen and demonstratethat crossed pollination occurs in a maximum distance of200m and no crossed pollination happened in the samedistances or over 300 meters in relation to pollen sources,in non detasseling condition. The results indicate thatpollen viability is kept for 2h, and that crossedpollination was not observed in 300 meters distances fromthe pollen source.Under low to moderate winds, it was estimated thatcomparing concentrations to 1 of source culture,approximately 2% of pollen are annotated at 60m, 1.1% at200m and 0.75-0.5% at 500m of distance. At 10m from afield, in average, the number of pollen grains per areaunit is ten times smaller than the one observed at 1m fromthe border. Therefore, if the established distances ofseparation developed for corn seed production are observed,it is expected that the pollen transfer to the adjacentvarieties are minimized, and it shall not have any geneticmaterial with tolerance to herbicide. Even in case ofhaving a gene escape, the probability of allele fixationcontaining the gene sequence that confers tolerance toglufosinate of ammonium in the population is much reducedin the absence of selection pressure.The secondary ecological impacts, that refer to effect inthe environment, especially in the ecosystem of the soil,are very discussed nowadays due to the complexity of thesoil. The organisms of the soil are usually very exposed tothe contact with genetically modified plants, both withdirect contact, and through the fall of leaves, reticularexudates or even decomposition of roots. Through thereports in literature, genetically modified cultures behavesimilarly to conventional correspondent cultures, andmeaningful alterations have not been detected until now onthe structures of microbial communities of the soils.Studies of plants intact genes transfer to microorganismsshow extremely low possibilities of transfer, suggestingthat the probability of occurrence of this event is, inpractice, extremely low. There is no evidence that plantsgenes have even been transferred to bacteria in naturalconditions. Moreover, gene pat already exists in the soil,once it comes from a natural soil bacteria, S.viridochromogenes.Glufosinate of ammonium is a non systemic and non selectiveherbicide used, mainly, in the control of post-emergencyinvasive plants of wide leaves, and of thin ones. Suchherbicide is registered in Brazil, at the Ministry ofAgriculture, Cattle Breeding, and Supply (MAPA), at theBrazilian Institute of Environment and Renewable NaturalResources (IBAMA), and with monograph approved by theMinistry of Health, being commercialized in Brazil and inother countries. Its wide use in the world is due to thefact that it is biodegradable, presents low residualactivity, low toxicity to men, animals, and other organismof the food chain. It is considered persistent and movablein the soil, and in sandy soils up to 80% may be lixiviate.Depending on handling conditions, edafoclimatic conditions,microbial activity, and other factors, glufosinate ofammonium has half-life in the soil that varies from 12 to70 days. However, residues have been found in the soilafter 100 days (16). Therefore, if it is used outside itsrecommendations, glufosinate of ammonium herbicide haspotential for contaminating water courses and groundwater(18) as any other herbicide used in genetically modifiedcultures or not. However, it is highlighted that othernorms should be observed when T25 corn is registered, suchas Law 7.802, of July 11th, 1989 (Agro Toxic Law)especially regarding the acceptable limits for residues ofherbicides to be established by the registration andinspection organs and entities mentioned above.VI. Restriction to the use of GMO and its derivatives:Technical opinions regarding the agronomic performance cameto the conclusion that there is equivalence betweentransgenic and conventional plants. Thus, informationindicate that transgenic plants do not fundamentally differfrom non transformed corn genotypes, except for thetolerance to glufosinate of ammonium herbicide.Additionally, there is no evidence of different reactionsto the use of Liberty Link corn. So, there is norestriction to the use of this corn, or of its derivatives,be it for human or for animal food.The vertical genic flow for local varieties (called creolescorns) of open pollination is possible and presents thesame risk caused by commercial genotypes available in themarket (80% of planted conventional corn in Brazil comesfrom commercial seeds that went through a process ofgenetic improvement). The coexistence between conventionalcorns cultivation (improved or creoles) and transgeniccultivations of corns is possible from the agronomic pointof view (3,13). Therefore, CTNBio will eventually publishnorms about coexistence of genetically modified corn withnon modified varieties.VII. Considerations about the particularities of differentregions of the country (subsidies to inspection organs):Small variations of centesimal composition, not meaningfulones, were found between the corn planted in the Southregion (Paraná), and the one planted in the Center WestRegion (Goiás) that were imputed, not to the presence ofthe transformation event, but to environment conditions.Therefore, there are no restrictions to the use of thiscorn, at least in relation to the South, Southeast andCenter West of the country.The registration and inspection organs, in the ambit oftheir competences, will establish acceptable limits ofresidues to herbicide glufosinate of ammonium used inplantations cultivated with corn T25.According to what is established on art. 1 of Law 11.460,of March 21st, 2007, “it is vetoed the research andcultivation of genetically modified organisms in indigenouslands and areas of conservation units”. CTNBio willeventually publish norms about the coexistence ofgenetically modified corn with non modified varieties.VII. ConclusionConsidering that event T25 derives from the lineage He/89of common corn (Zea mays), species with solid safetybackground for human and animal consume, and that theintroduced gene, pat, does not codified proteins known astoxic or allergenic, and results on the enzyme Lphosphinotricin-N-acetyltransferase, with high specificityfor the herbicide glufosinate of ammonium.Considering that the gene construction used on thetransformation resulted on the instable insertion of a copyof pat gene, and regulating regions of the mosaic virus ofcauliflower on the genome of corn, besides a non-functionalincomplete sequence of the gene of resistance toampicillin, resulting only on PAT expression, withoutapparent harm to the plant or to the environment.Considering also that:1. Corn is the species that reached the highest degree ofdomestication among plants cultivated, being able tosurvive in nature without human intervention.2. The sylvan species closer to corn is teosinte, found inMexico and in some places in Central America, where it canbe crossed with corn cultivated in production fields.Therefore, there are no sylvan species in Brazil with whichcorn can be crossed.3. Protein PAT has rapid degradation in gastric andintestinal fluids (7,20).4. Recombinant enzyme PAT was detected in low levels ofanalyzed vegetable tissues, and presented greatsusceptibility to digestion and thermal denaturation by theprocessing, being highly improbable that it may have anytoxic or allergenic effect (7,20).5. There are many proteins similar to PAT in nature withoutany evidence of adverse effect to men, animals, or plantshave been described, and that its subtract is highlyspecific, not having any sequence of amino acids withhomology to toxins or allergens.6. Genetic modification introduced in event T25 did notresult in important differences of chemical compositionregarding nutrients, being within the normal variationscope among the conventional varieties.7. Microorganisms S. hygroscopicus and S. viridochromogenesare saprophyte bacteria of the soil, and there are manyspecies of Streptomycin genre similar to S. hygroscopicusand S. viridochromogenes, and among them, many containingpat and bar genes(2).8. DNA molecule is a natural food component, not presentingany evidence that such molecule may have adverse moleculeeffect to men when ingested in food in acceptablequantities (no direct toxic effect).9. There is no evidence that intact genes of plants may betransferred and functionally integrated in human genome, orof other mammals exposed to this DNA or foods manufacturedwith these elements(6).10. Plants tolerant to glufosinate of ammonium containingPAT protein have been cultivated in the United States andin Canada for approximately a decade without anyregistration of adverse effect to human and animal food.Besides, many regulating agencies of varied countries haveapproved these plants for these plants for human and animaluse including Australia, Japan and European Union.11. The petitioner answered to all questionings postulatedon the Normative Instruction No. 20 at CTNBio.12. None of the questions indicate that this corn maypresent adverse effects on human and animal food.13. After ten years of use in different countries, noproblem was detected for human, animal’s health, or to theenvironment that may be attributed to transgenic corns. Itis necessary to emphasize that the lack of negative effectsresulting of corn transgenic plants does not mean that theymay not happen. Zero risk and absolute safety does notexist in biologic world, although an accumulation oftrustworthy scientific information already exists, and asafe background of ten years use allows us to state thatcorn T25 is as safe as conventional versions. Thus, thepetitioner is conditioned to conduct monitoring of postcommercialrelease on the terms to be eventuallyestablished by CTNBio.14. The coexistence among cultivations of conventionalcorns (improved or creoles) and transgenic cultivations ispossible from the agronomic point of view.15. The probability of allele fixation, containing the genesequence that confers tolerance to glufosinate of ammoniumon the population is much reduced in the absence ofselection pressure.16. There is no need of maintenance of confidentiality inrelation to the commercial release.CTNBio thinks that this activity is not potentially causingmeaningful degradation of the environment, or aggravationsto human and animal’s health. The use restrictions of GMOin analysis, and its derivatives are conditioned tocoexistence norms, and to the after commercializationmonitoring plan to be eventually published by CTNBio.VIII - Bibliography1. BAHIA FILHO, A.F.C.; GARCIA, J.C. 2000. Analysis andevaluation of Brazilian market of corn seeds. In: UDRY,C.V.; DUARTE, W.F. (Org.) Uma história brasileira do milho:o valor de recursos genéticos. Brasília: Paralelo 15,167-172.2. BARTSCH K; TEBBE C.C. 1989. Initial steps in degradationof Phosphinothricin (glufosinate) by soil bacteria. Appliedand Environmental Microbiology 55: 711-716.3. BROOKES, G.; BARFOOT, P.; MELÉ, E.; MESSEGUER, J.;BÉNÉTRIX, F. BLOC, D.; FOUEILLASSAR, X, FABIÉ,A.;POEYDOMENGE, C.2004. Genetically modified maize; pollenmovement and crop co-existence. Dorchester, UK: PGeconomics, 20pp.()4. EUROPEAN COMMISSION – Food safety the Farm to the Fork.1996. Opinion On the Potential For Adverse Health EffectsFrom the Consumption of Genetically Modified Maize (Zeamays L).()5. FAO/WHO – Food and Agriculture Organization of theUnited Nations/World Health Organization. 2000 Grasslandindex. Zea mays L.(data/pf000342.htm)6. FAO/WHO – Food and Agriculture Organization of theUnited Nations. 2000. Safety Aspects of GeneticallyModified Foods of Plant Origin. Report of a Joint FAO/WHOExpert June 2000. World Health Organization, WHOHeadquarters, Geneva, Switzerland. 35pp.(une2000_en.pdf)7. HEALTH CANADA. 1997 Glufosinate Ammonium Tolerant Corn(T14 and T25). Novel Food Information – Food Biotechnology.4pp. (pdf/gmf-agm/32bg_agrevo-ct_agrevo_e.pdf).8. HULL, R.; COVEY, S.N.; DALE, P. 2000. Geneticallymodified plants and the 35S promoter: assessing the risksand enhancing the debate. Microb. Ecol. In Health and Dis.12:1-5.9. KOOTER, J.M.; MATZKE, M.A.; MEYER, P. 1999. Listening tothe silent genes: transgenic silencing. Gene regulation andpathogen control. Trends in Plant Sci. 4: 340-347.10. LEWIN, B.2004. Genes VII. New York: Oxford UniversityPress, 1ed. 1056pp.11. LUNA, S.V.; FIGUEROA, J.M.; BALTAZAR, M.B.; GOMEZ ,L.R.; TOWNSEND , R.E SCHOPER, J.B. 2001. Maize pollenlongevity and distance isolation requirements for effectivepollen control. Crop Sci. 41:1551-1557.12. MANGELSDORF.P.C.; REEVES, R.G. 1938. The origin ofmaize. Proc. Natl. Acad. Sci. USA 24: 303-312.13. MESSEGUER, J.; PEÑAS,G.; BALLESTER, J.; BAS, MAS.;SERRA, H.; SALVIA, J.; PALAUDELMÀS, M.; MÉLE, E. 2006.Pollen-mediated gene flow in maize in real situations ofcoexistence. Plant Biotechnology Journal. 4:633-645.14. MÓROCZ, S.; DONN, G.; NÉMETH, J.; DUDITS, D. 1990. Animproved system to obtain fertile regenerants via maizeprotoplasts isolated from highly embryogenic suspensionculture. Theor Appl. Genet.80: 721-726.15. PAULI, S.; ROTHNIE, H.M.; CHEN, G.; HE, X.; HOHN, T.2004. The cauliflower mosaic virus 35S promoter extendsinto the transcribed region. Journal of Virology 78: 12120-12128.16. SMITH, A.E.; BELIK, M.B. 1989. Field persistencestudies with the herbicide glufosinate-ammonium inSaskatchewan soils. J. Environ. Qual. 18: 475-479.17. TIKHONOV, A.; SANMIGUEL, P.; NAKAJIMA, Y.; GORESTEIN,N.; BENNETZENAND , J.; AVRAMOVA,Z. 1999. Colinearity andits exceptions in orthologous and regions of maize andsorghum. Proc. Natl. Acad. Sci. USA 96: 7409-7414.18. US ENVIRONMENTAL PROTECT AGENCY- EPA. 1993. Office ofpesticides and toxicity substances. Pesticide fact sheet.(-10/bac.pdf)19. US ENVIRONMENTAL PROTECTION AGENCY – EPA. 1997.Phosphinothricin Acetyltransferase and the Genetic MaterialNecessary for its Production in all Plants; Exemption Fromthe Requirement of a tolerance on all AgriculturalCommodities. (ww.epa.gov/fedrgstr/EPA-PEST/1997/April/Day-11/p9373.htm)20. US FOOD AND DRUG ADMINISTRATION – FDA. 1995.Biotechnology Consultation – note to file BNF No. 000029.()21. WATSON, S.A.; RAMSTAD, P.E. 1987. Corn: chemistry andtechnology. St. Paul: American Association of CerealChemists, 1 ed. 604pp.Walter ColliPresident of CTNBioDivergent VoteThe reporter Dr. Lia Giraldo da Silva Augusto (PermanentEnvironmental Sector Sub-commission) issued contraryopinion to this product approval based on:1. Inexistence of normative instruction for commercialrelease;2. Un-fulfillment of precaution principle;3. Lack of monitoring credits of environmental impact ofshort, medium and long term for transgenic cultivations.

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