Deliberate release into the
environment of GMOs for any other purposes than placing on the
market
Promoting food safety through a new
integrated risk analysis approach for foods - Plant Breeding and
Acclimatization Institute Radzików |
Date of publication: October 7, 2004
Source:
http://gmoinfo.jrc.it/gmp_browse_geninf.asp
Notification
report
General information
Notification Number:
B/PL/04/02-01
Member State: Poland
Date of Acknowledgement:07/04/2004
Title of the Project: Promoting Food Safety through a
New Integrated Risk Analysis Approach for Foods
Proposed period of release From: 20/04/2005
To: 31/10/2008
Name of the Institute(s) or Company(ies):
Plant Breeding and
Acclimatization Institute Radzików;
3. Is the same GMPt release planned elsewhere in the
Community?
No
4 - Has the same GMPt been notified elsewhere by the same
notifier?
No
Genetically
modified plant
1. Complete name of the
recipient or parental plant(s)
|
Common Name
|
Family Name
|
Genus |
Species
|
Subspecies
|
Cultivar/breeding line
|
| potato
|
solanaceae |
solanum |
solanum tuberosum |
tuberosum |
Irga
|
2. Description of the traits and characteristics which have
been introduced or modified, including marker genes and previous
modifications:
Two types of insterts were obtained, which contained: a)
truncated gene coding Potato virus Y (PVY) polymerase and b)
ligated non-translated sequences (5’ and 3’ NTR) of PVY genome.
This was achieved by constructing binary vectors of following
types:
1) plasmids pROKY1 and pROKY2, in which truncated (without 1.2
kb fragment without 400 closing nucleotides) gene coding PVY
polymerase (NIb) was cloned under control of promoter 35S CaMV.
The viral sequence originated from isolate PVYN–Fr (GenBank Acc.
No. D00441). Plasmid pROKY1 contain the insert in sense
orientation, while plasmid pROKY2 – in antisense orientation;
2) plasmids pRNTR1 i pRNTR2 carrying expression cassette, in
which ligated sequences from 5’ and 3’ ends of viral genome was
cloned under control of promoter 35S CaMV. These sequences
derived from isolate PVYN Wi and consisted of 184 nucleotides
from 5’ end (GenBank Acc. No. Z70238) and 331 nucleotides from
3’ end of viral genome. Plasmid pRNTR1 contain the insert in
sense orientation, while plasmid pRNTR2 – in antisense
orientation.
Vectors contained gene nptII, which enables selection of
transformed bacterial cells and plants growing on selective
media supplemented with antibiotics kanamycin and cefotaxime.
Genetically modified potato lines expressed resistance to
necrotic isolate of potato virus Y. The mechanical inoculation
of plants with isolate PVYN from cultivar Wilga (PVYN Wi) was
performed for assaying resistance. For virus detection DAS-ELISA
test was applied 4 and 6 weeks after inoculation to assay the
primary infection, and 4 and 6 weeks after planting tubers of
inoculated plants to test the secondary infection. In plants of
transgenic lines, which are to be tested in proposed field
experiment, virus was detected neither after inoculation nor in
their progeny plants.
It was observed, that plants of some transgenic lines differed
from plants of cultivar Irga (lowered vigour, increased tendency
toward secondary growth of tubers, decreased number of flowers,
increased or decreased tuber yield).
Genetic
modification
3. Type of genetic
modification:
Insertion;
4. In case of insertion of genetic material, give the source
and intended function of each constituent fragment of the region
to be inserted:
1) Fragments of cDNA corresponding with different regions of
PVY genome, which are expected to confer resistance to isolate
of PVY from necrotic strains group.
2) Sequence 35S from Cauliflower mosaic virus, which is promoter
of transgenes coding resistance.
3) Gene nptII from E. coli transpozone Tn5, which is a marker
enabling selection of transformed bacterial cells or transformed
plants growing on media supplemented with antibiotic kanamycine.
6. Brief description of the method used for the genetic
modification:
The vectors containing expression cassette with cloned
different fragments of the PVY genomic cDNA were constructed by
using binary plasmid pROK2. The PVY fragments originated from
isolates PVYN Fr and PVYN Wi. Fragments of cDNA were cloned
under control of promoter gene 35S CaMV. The vector contained
selective gene nptII, which enables growth of transformed
bacterial or plant cells in the presence of kanamycine in growth
medium.
The agroinfection method was applied for genetic transformation
of potato plants. The obtained binary vectors were used for
transformation of cells of Agrobacterium tumefaciens strain C58
pmp90 or LBA 4404. The plant transformation was done by
incubating cells of A. tumefaciens with fragments of fresh
potato leaves and internodes. Regenerating shoots were moved to
selective medium with kanamycin and cefotaxime. The presence of
inserts in bacterial cells was confirmed by growth of bacteria
on medium supplemented with kanamycine and submitting them to
the alkaline lysis and digestion with specific restriction
enzymes. Plant material and regenerating shoots were kept on
medium containing both antibiotics.
Experimental
Release
1. Purpose of the release:
Field trial with six transgenic potatoes is to be carried out
within the frame of the project „Promoting Food Safety through a
New Integrated Risk Analysis Approach for Foods” (SAFEFOODS).
This is integrated research project implemented within VI Frame
Program.. The general aim of the project is to work out new
methods of risk assessment related to human and animal health.
The risk factors depend on cultivation, storage technologies and
on methods applied for breeding new cultivars (conventional or
those using genetic engineering. To develop new methods of risk
assessment analytical methods will be applied such as proteomics
and metabolomics. Proteomic and metabolomic analysis methods
allow the abundance and distribution of many proteins and
metabolites to be determined simultaneously and globally in
potato tubers of different cultivars and transgenic lines, which
were grown under different cultivation regimes.
2. Geographical location of the site:
Mazowieckie Province, Pruszków District, Nadarzyn Commune,
experimental field of Plant Breeding and Acclimatization
Institute, Mlochów Research Department. In 2005 the field will
be located at Plot no. 197 according to Land Register of
Nadarzyn Commune.
3. Size of the site (m2):
Area occupied by transgenic potato lines – 90 m2. Area of
protective zone – 500 m2
4. Relevant data regarding previous releases carried out with
the same GM-plant, if any, specifically related to the potential
environmental and human health impacts from the release:
Area occupied by transgenic potato lines – 90 m2. Area of
protective zone – 500 m2
Environmental
Impact and Risk Management
Summary of the potential
environmental impact from the release of the GMPts:
Potato is vegetatively propagated by tubers, which are not
able to remain viable in soil after winter, hence the volunteer
potato plants are not observed. Potato is not able to cross with
other members of family Solanaceae in Poland. In breeding
practice potato cultivars and breeding lines are crossed, but
seeds are not able to survive in soil. The pollen spreading is
very limited.
The only factor influencing survivability of tubers in soil is
the low temperature. At IHAR Młochów experimental field the
volunteer plants are not observed, even after moderate
temperatures during winter.
There is possibility of transfer of genetic material from
transgenic potato resistant to necrotic isolate of PVY to the
same virus existing in ecosystem. The recombination between
transgene, which originate from the virus and viral genetic
material, is possible. However, in the case of transgenic lines
from cultivar Irga, the transgenes originate from conservative
regions of PVY genome. In laboratory condition recombination
between mRNA expressed by the trasnsgene and viral RNA was not
detected.
Moreover, transgenes introduced into the genome of cultivar Irga
derived from the most widespread isolate of PVY, i.e. PVYN Wi.
Hence, the genetic material of the virus existing in ecosystem
and that of transgene are identical. In central regions of
Poland in 1996 and 1997, more than 90% of PVY population
consisted of PVYN Wi.
The spreading of potato in natural ecosystems is not possible at
all, due to limited winter survivability of tubers. Moreover,
potato has no traits, which would increase its adaptation and
subsequent spreading in natural ecosystems. The only possible
ecosystems for potato are artificial agricultural biocenosis.
Plants of transgenic lines expressed higher level of resistance
to PVYN, as compared to non-transformed plants of cultivar Irga.
But many potato cultivars, which were obtained by methods of
traditional breeding, express similarly high or even higher
level of resistance to this virus.
Brief description of any measures taken for the management of
risks:
The design of planned filed trial is as follow: 1) randomized
complete block design with 3 blocks; 2) the treatments are
potato cultivars (about 15 cultivars) and 6 transgenic lines.
Each treatment in a bock consists of 15 plants growing in a row
(plot). The whole trial will be separated from other potatoes by
paths, which enable spraying potatoes without entering into
plants. To mark the trial, tables with trial symbol will be
placed in the field. The field trial will be surrounded by
additional protective belts of conventional potato cultivar. The
area of this zone will be 500 m2.. Potatoes from the protective
zone will be harvested together with experimental potatoes and
will not be used for further propagation.
The plots with transgenic lines will be placed at random and
will be able to identify on the trial map, which will be
accessible from person responsible for the trial.
All tubers from cultivars and transgenic lines will be harvested
and removed from the field. The environmental disturbances are
not expected. The field trial will be cultivated in the same way
as other breeding materials growing in the experimental field.
Pesticides and fungicides will be applied according to needs.
The harvest and post-harvest procedure:
1) before harvesting tubers the haulm will be cut by hand
2) tubers will be harvested by hand
3) standard description of harvested tubers (weighing tubers,
estimating of starch content, assessment of tubers morphology
from each plot)
4) monitoring of tubers and other remainders left in the field
and removing them.
5) the field will be monitored in the spring. All volunteers
will be removed..
The post harvest remainders will be burnt. Tubers, which will
not be used in further experiments in laboratory, will be
destroyed by steaming and subsequently composted.
Potato is vegetatively propagated, hence after genetic
transformation the inserted trait is established in the genome
and its transmission on progeny by sexual propagation is not
required. Field testing of generative progenies of transgenic
potato lines is not planned.
Summary of foreseen field trial studies focused to gain new
data on environmental and human health impact from the release:
Overall aim of the project is to develop new methods of
assessment risk related to food for human and animal, which is
connected with cultivation and storage technologies and to
cultivar. Cultivars are differentiated genetically and in
consequence phenotypically. The genetic differentiation might be
achieved by traditional breeding and also by methods of genetic
engineering. The new ways of risk assessment will be based on
methods, which simultaneously and globally determine quality and
quantity of proteins and metabolites in different cultivars or
transgenic lines. |
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