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Notification report


General information

Notification Number
B/SE/11/11399

Member State to which the notification was sent
Sweden

Date of acknowledgement from the Member State Competent Authority
21/10/2011

Title of the Project
Potato with increased amylose starch content, Potato with increased oil content

Proposed period of release:
15/04/2012 to 15/10/2016

Name of the Institute(s) or Company(ies)
Sveriges lantbruksuniversitet, Alnarp, Institutionen för växtförädling och bioteknik, ;


3. Is the same GMPt release planned elsewhere in the Community?
No

Has the same GMPt been notified elsewhere by the same notifier?
No

Genetically modified plant

Complete name of the recipient or parental plant(s)
Common NameFamily NameGenusSpeciesSubspeciesCultivar/breeding line
potatosolanaceaesolanumsolanum tuberosumtuberosumDinamo, Kuras, Verba

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
Potato starch with increased amylose content
The aim with the modification is to increase the amylose starch content in potato by inhibition of two starch branching enzyme genes, starch branching enzyme 1 (SBE1) and starch branching enzyme 2 (SBE2) and by that down regulate the synthesis of the second starch component amylopectin. An increase of amylose starch has been confirmed in both tissue culture and greenhouse grown tuber material using iodine staining and microscopic investigation of starch granules. The genes have been inhibited by introducing a RNAi fragment containing small parts of sbe1 and sbe2 from Solanum tuberosum synthetically produced in tandem and through recomblination inserted into a binary vector in duplicated inverted repeats. The repeats are seperated by an intron from Arabidopsis thaliana and a chloramphenicol (CmR) marker for bacteriel selection of the vector T-DNA is located inside the intron in the original vector. The resulting RNAi fragment is expressed using a tuber specific granule bound starch synthase (StGBSS) promoter from Solanum tuberosum. As a selection marker a neomycin phophotranferase (nptII) gene, yielding resistance towards kanamycin, has been inserted for selection of transgenic shoots. The aim with the field trial is to analyse if the observed high amyose starch effect is stable under normal growth conditions, to meassure the amylose- and starch content and to extract starch for further application studies.

Potato with increased oil content
The AtWRI1 gene from Arabidopsis thaliana has been inserted. This gene is coding for a transcription factor crucial for synthesis of seed oil. AtWRI1 is in Arabidopsis thaliana regulating the expression of genes involved in the glycolysis and the fatty acid biosynthesis. In the transformed potato this gene is expressed using a potato granule bound starch synthase (StGBSS) promoter. This promoter is expressing the gene mainly in tubers. In tissue culture microtubers generated from transgenic shoots the inserted gene stimulates triacylglycerol (TAG) biosynthesis. Microtubers are induced on tissue culture media containing a high concentration of sucrose which stimulates tuberisation, the potato StGBSS promoter and storage compound deposition. As a selection marker a neomycin phosphotransferase (nptII) gene, yielding kanamycin resistance, has been inserted for selection of transgenic shoots.The aim with the field trial is to investigate if the observed effect is maintained under normal growth conditions for potato in field.
AtWRI1 is expressed and is translated to protein in tuber tissue, pollen and root tip.The NptII gene is translated to protein and is considered to be expressed in the whole plant. The RNAi fragment containing sbe2, sbe1, intron and CmR is expressed mainly in tuber tissue where the transcript is being degraded into small peaces by the plants own RNAi system and no protein will be formed.


Genetic modification

3. 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:
pMAamy2
The T-DNA from pMAamy2 has a size of approximately 5.400 bp and contains from left to right border; left border (LB) from pTiT37 fragment originating from Agrobacterium tumefaciens, nopalin synthase polyadenylation sequence (nospA) from Agrobacterium tumefaciens, Tn5 fragment with marker gene neomycin phosphotransferase II (nptII) which yield kanamycin resistance and which can be isolated from different bacteria like for example Escherichia coli, nopaline synthase promoter (Pnos) from Agrobacterium tumefaciens which yield expression of nptII in the entire plant, polyadenylation sequence from cauliflower mosaic virus (CaMV) 35S transcript, GATEWAYTM recombination sequence from ”reading frame A” (rfA) from GATEWAYTM vector converting system (Invitrogen), RNAi fragment for inhibition of amylopectin sythesis with synthetically produced fragments of starch branching enzyme 2 (sbe2) and starch branching enzyme 1 (sbe1) in tandem and inserted as an inverted and duplicated fragment where both enzymes has its origin in Solanum tuberosum, between the duplications an intron from Arabidopsis thaliana ac007123.em_pl, inside the intron a chloramphenicol resistance marker gene (CmR) which is similar to a gene of Escherichia coli origin, GATEWAYTM recombination sequence from ”reading frame A” (rfA) from GATEWAYTM vector converting system (Invitrogen), promoter from granule bound starch synthase (StGBSS) from Solanum tuberosum which yields an expression of the RNAi fragment mainly in tuber tissue, right border (RB) from pTiT37 from Agrobacterium tumefaciens.

pHoWRI1
The T-DNA from pHoWRI1 has a size of approximately 6.000 bp and contains from the left to the right border sequence; pTiT37 fragment including ”left border sequence” (LB) from Agrobacterium tumefaciens, M13mp19 fragment with residues from a cloning cassette with an origin from phage M13 modified for laboratory use, pTiT37 fragment including 3´-sequence from nopaline synthase (nospA) functioning as a polyadenylation sequence with origin from Agrobacterium tumefaciens, synthetically synthesised gene coding for a protein, equivalent to AtWRI1 from Arabidopsis thaliana for stimulation of the oil biosynthesis, genomic StGBSS fragment functioning as a mainly tuber specific promoter in Solanum tuberosum, M13mp19 fragment with parts of a cloning cassette with origin from phage M13 modified for laboratory use, pTiT37 including 3´-sequence from a nopaline synthase (nospA) functioning as a polyadenylation sequence with origin from Agrobacterium tumefaciens, Ti-plasmid fragment with origin from Agrobacterium tumefaciens, Tn5 fragment with neomycin phosphotransferase II coding sequence (nptII gene) yielding the plant resistance towards kanamycin which can be isolated from several origins like for example Escherichia coli, pTiT37 fragment with ”right border sequence” (RB) including the 5´-untranslated part of a nopaline synthase gene (Pnos), functional as a promoter in plants from Agrobacterium tumefaciens.


6. Brief description of the method used for the genetic modification:
Potato starch with increased amylose content
Two starch branching enzymes (SBE) have been inhibited by using RNA interference (RNAi), which in cells are a naturally occurring system for regulation of genes. Furthermore, cells are using RNAi as a defence against pathogens like for example virus. Today RNAi is a common system for targeted down regulation of genes in plants. This is achieved by designing a small fragment of a target gene/genes to transcribe into a double stranded RNA, which trigger the plants own RNAi system to degrade the corresponding endogenous genes RNA transcript. For transformation of potato, Agrobacterium tumefaciens together with a binary vector system has been used where the genes to be transformed is located inside border sequences and is forming a transfer DNA (T-DNA). The DNA mobilising functions is located in a separate modified Ti plasmid which is not transferred to the plant. Between the T-DNA borders in the binary vector pK7GWIWG2(II), an RNAi fragment of 200 bp of each branching enzyme has been inserted in tandem as an inverted repeat. Furthermore a neomycin phosphotransferase II gene (nptII), coding for a protein yielding kanamycin resistance, is present in the T-DNA and used as a tool for selection of transgenic shoots. For expression of the RNAi fragment a tuber specific granular bound starch synthase (gbss) promoter from Solanum tuberosum has been used and for the expression of the nptII gene the consitutive nopalinsyntaspromoter (Pnos) from Agrobacterium tumefaciens has been used. The resulting vector is called pMAamy2.

Potato with increased oil content
A gene construct has been inserted in potato using Agrobacteium tumefaciens mediated transformation. Inserted genes are located between two T-DNA border sequences in the binary vector pBIN19 in Agrobacterium tumefaciens. The T-DNA borders are considered to enclose and define the transferred genetic material (T-DNA). The pBIN19 derivative used for transformation is called pHoWRI1. Between the T-DNA borders in the vector is a neomycin phosphotransferase II gene (nptII) present, coding for a protein yielding resitance towards kanamycin, which is used as a tool for selection of transgenic shoots. The nptII gene is regulated by a nopalin synthase promoter.
For transformation of T-DNA to potato, Agrobacterium tumefaciens containing pMAamy2 or pHoWRI1 has been used. Transformation has been performed on cut leaf tissue and transgenic shoots have been selected on 50 ug/ml kanamycin for Kuras and Dinamo and 75 ug/mg kanamycin for Verba. After transformation has Agrobacterium been killed with 400 mg/ml Cefotaxim.


7. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination:
Not applicable

Experimental Release

1. Purpose of the release:
The purpose of the release of potato with increased content of amylose starch is to;
• Study the modified property's stability.
• Study any morphological abnormalities that might arise through somatic mutations as a result of in vitro cultivation.
• Study the mature tubers amylose- and starch content.
• Study if modification of the parental variety Verba can give amylose potatoes with a higher starch content than in the other two parental varieties Dinamo and Kuras.
• Extract starch from modified tubers to be used in small application studies.

Long-term goal is to gain knowledge about the amylose starch potential to be used for different industrial applications as well as to investigate if Verba as parental variety can produce an amylose potato with slightly higher starch content than those previously obtained. The trial is for research purposes only.

The purpose of the release of potato with increased oil content is to:
• Study if oil formation in tubers can be observed under field conditions with quantification and determination of the composition.
• Study any morphological abnormalities that might arise through somatic mutations as a result of in vitro cultivation.
• Study the possible effects of oil biosynthesis on the plant morphology characters.
• Study the possible effects of oil biosynthesis on tuberisation and maturity.
• Study the possible effects of oil biosynthesis on starch formation and composition.

The objective of the field experiment is to answer basic research questions regarding the ability to produce oil in an underground storage organ.


2. Geographical location of the site:
The experiment will be performed by Hushållningssällskapet, which has extensive experience in trials with modified potatoes. Locations considered for field trials are sited in Kristianstad, Lomma, Kävlinge and Halmstad municipality.

3. Size of the site (m2):
Maximum 1000 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:
Not applicable

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
The modified potatoes are not assumed to have increased environmental impact compared to their parental varieties. No alteration in pollen production, seed size, germination capacity or any other characteristic that has an impact on the dispersal or reproduction can be assumed. No traits has been inserted in the modified potatoes are to increase the selective advantage of the potato. Generally, potato tubers are frost sensitive and are therefore not competitive outside the growing season. Potatoes are also very susceptible to a number of plant diseases, including Phytophthora infestans, which gives a lethal infection in potato and therefore has no increased competitiveness outside the field compared with other plants. Potatoes are not a plant that is exceptionally exposed to herbivores. Neither of these characters are expected to be influenced by the genetic modification.

Brief description of any measures taken for the management of risks:
Potato is only found in the agricultural landscape. No indications of invasiveness has been reported for potato. Potato has no compatible relatives in Europe and therefore can only cross with other cultivated potatoes. To minimise the dispersal of pollen to other cultivated potatoes, a safety distance of 20 m will be applied. Cleaning of machinery, tools and vehicles will be performed after contact with modified lines. In order to monitor any post-harvest surviving tubers, the site will remain in fallow for one year and no potatoes will be grown in the field for the following two years. The trial will be inspected at least once a week and observations as well as differences between the parental varieties and the transgenic lines will be documented.

Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
Not applicable

Final report


European Commission administrative information

Consent given by the Member State Competent Authority:
Yes
03/06/2012 00:00:00
Remarks: