General informationNotification NumberB/PL/04/02-01Member State to which the notification was sentPolandDate of acknowledgement from the Member State Competent Authority07/04/2004Title of the ProjectPromoting Food Safety through a New Integrated Risk Analysis Approach for FoodsProposed period of release:20/04/2005 to 31/10/2008Name of the Institute(s) or Company(ies)Plant Breeding and Acclimatzation Institute
Radzików, 05-870 Blonie, Poland
tel. +48 22 725 36 11
3. Is the same GMPt release planned elsewhere in the Community?NoHas the same GMPt been notified elsewhere by the same notifier?NoGenetically modified plantComplete name of the recipient or parental plant(s)
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:
|Common Name||Family Name||Genus||Species||Subspecies||Cultivar/breeding line|
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 modification3. Type of genetic modification:Insertion; 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 Release1. 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 m24. 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 m2Environmental Impact and Risk ManagementSummary 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.Final report-European Commission administrative informationConsent given by the Member State Competent Authority:Not known