General informationNotification NumberB/CZ/07/03Member State to which the notification was sentCzech RepublicDate of acknowledgement from the Member State Competent Authority19/03/2007Title of the ProjectEvaluation of genetic modifications for use in flax breedingProposed period of release:30/04/2007 to 30/09/2016Name of the Institute(s) or Company(ies)Agritec, Research, Breeding & Services, Ltd., ;
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:Flax and linseed plants were genetically modified using one of four different types of Agrobacterium-based vectors to: a) induce mutations of existing plant genes (a vector for insertion mutagenesis), b) introduce bacterial gene bar coding for tolerance to herbicide phosphinotricine (syn. gluphosinate ammonium) to obtain flax plants more tolerant to the herbicide application, c) introduce genes coding for inhibitors of serine-type proteases (SPI-2) with the aim to enhance tolerance of flax plants to fungal diseases and/or insect pests, 4) enhance the ability of flax to accumulate heavy metal polutants from the soil in plants and their parts by introducing one of folowing gene sequences either for human metalothioneine (αHMT1A) or short synthetic peptide (CP).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:a) In case of the vector for insertion mutagenesis the transferred DNA (T-DNA) does not contain any target gene(s) showing a direct effect on acceptory plant. Transformation of plants results in the integration of T-DNA into plant genome. Cassette contains a tetramer of 35S promoter enhancer (4x35Sen) derived from Cauliflower mosaic virus (CaMV), which integration close to a plant gene regulatory region could potentially cause an activation of the proper gene (has not been found yet in case of flax). This could be recognized as a dominant mutation. If the T-DNA insertion takes place at functional gene sequence it can cause, in opposite, a plant gene inactivation. Impairment of the gene function is expressed as a recessive mutation. Such mutations may lead to metabolic changes and in specific cases to changes of morphological or biological traits some of which could be of breeder’s interest. Next to 4x35Sen cassette component is the hpt gene coding for enzyme hygromycin phosphotransferase (which detoxifies the aminocyclitol antibiotic hygromycin B) a plant selectable marker derived from common bacteria Escherichia coli. The selectable gene hpt is driven by nopaline synthase promoter (pnos) of Agrobacterium tumefaciens and terminates with pAg4 a polyadenylation sequence of gene 4 from A. tumefaciens, which ends transcription and directs polyadenylation of the mRNA.
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b) Binary vector plasmid (pMG8)(pBin19) was used to deliver an insert bearing the bar gene of Streptomyces sp. coding for tolerance to herbicide phoshinotricine and driven by 35S CaMV promoter to plant genome. Another gene nptII (selectable marker) from E. coli which is under the control of Nos promoter of A. tumefaciens and confers the resistance of transgenic plants to antibiotic kanamycin is also present in a gene cassette. Both bar and nptII genes are linked to nos 3' Agrobacterium tumefaciens non coding regulatory sequence for termination of transcription.
c) Plasmid pUC19 (Nirmala et al., 2001) bearing a sequence of the SPI-2 gene encoding inhibitor of serine proteases of the Greater Wax Moth (Galleria mellonella) driven by 35S CaMV promoter was used for flax transformation. To terminate the gene transcription the OCTSt sequence of octopine synthase terminator from A. tumefaciens was linked to the gene. Presence of the SPI-2 inhibitor in flax plants could enhance their resistance to microbial pathogenes (as indicated by in vitro experiments) and using the next gene variants also to insect pests. The SPI-2 gene is fused to a selectable marker gene nptII with pNOS and NOSt (promoter and terminator sequences of A. tumefaciens).
d) Agrobacterial vectors used to enhace the accumulation of heavy metals in flax plants contain either αHMT1A (a short nucleotide sequence of the gene coding for human metallothionenine 1A protein – GenBank accesion #:T27991) or a short nucleotide sequence for synthetic peptide CP (Kotrba et al. 1996) under control of 35S CaMV promoter and flanked by selectable marker gene nptII with pNOS promoter and NOSt terminator of A. tumefaciens.6. Brief description of the method used for the genetic modification:Transformation was carried out by A. tumefaciens-mediated transfer of foreign genes into hypocotyl explants. Hypocotyl explants of in vitro grown flax plantlets were incubated and co-cultured with Agrobacterium. The bacteria were washed off after 3 days, and the hypocotyl explants were cultured on a selection medium containing antibiotics to kill the bacteria and hygromycin or kanamycin, depending on a vector used for transformation, for selection of transgenic shoots. The hygromycin selection was based on the presence the selectable marker hpt gene and kanamycin selection was based on the selectable marker gene nptII of the T-DNA. Regenerated transgenic shoots were transferred on a proliferation- and later on a rooting medium for further transfer to the greenhouse.7. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination:Not applicableExperimental Release1. Purpose of the release:Field experiments aimed at the verification of biological characteristics and breeding parameters of selected (glasshouse tested) transgenic lines of flax and linseed obtained by:
a) insertion mutagenesis
b) introducing of bar gene to enhance plant tolerance to the herbicide application
c) introducing of genes for inhibitors of serine proteases to enhance the ability of flax to resist microbial pathogenes and/or insect pests
d) introducing of genes for metallothionenine αHMT1A or short peptide CP to enhance the accumulation of heavy metals in flax plants and their parts (phytoremediation)2. Geographical location of the site:Vikyrovice, region Olomouc3. Size of the site (m2):max. 300 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:Numerous transgenic lines obtained by method a) – insertion mutagenesis, were field-tested since 1999 based on approvals of the Ministry of Environment CZ. No adverse effects were found both to the environment and human health.Environmental Impact and Risk ManagementSummary of the potential environmental impact from the release of the GMPts:Analysis of the characteristics of transgenic flax lines has shown that the risk for potential adverse effects on human and animal health and the receiving environment, resulting from the planned field trials with GM flax and linseed lines, is consistently negligible:
Cultivation of flax depends to a great deal on human care because its competetivness is very low. Current flax and linseed cultivars are not able to withstand winter under condition of mid Europe both on field areas and in natural environments either as seeds of plants. Under cold and wet conditions flax plants are immediately attacked by numerous fungal and bacterial pathogenes and crop has to be chemically protected against harm. Flax propagates using the seed exclusively.
The risk of the introduced traits in flax to be the cause of any competitive advantage or disadvantage in natural environments is also negligible. Like for any other conventional flax, the likelihood of these GM flax and linseed lines to spread into non-agronomic environments is negligible. Moreover, there is no potential for gene transfer from GM flax to any wild plant species. There is a limited number of wild flax species common in Europe, which are sexually compatible to cultivated flax but their natural habitats in CZ do not correspond to areas of flax cultivation. Even our attempts to obtain hybrids through artificial crossings with L. flavum were unsuccessful (Tejklová 2005).
GM flax lines pose negligible risk for adverse environmental effects through their interactions with target organisms. The ecological interactions of GM lines derived from insertion mutagenesis were not different from traditional flax. Based on target genes characteristics the same is expected for other modified lines.Brief description of any measures taken for the management of risks:The distance from the nearest cultivation of unmodified flax will be at least 300 m. At least 3 m-wide separating distance of other crops will be kept to decrease dissemination of seeds and to monitor effects of GM organism on field flora and fauna closed to experimental plots too. Sowing and harvesting machinery will be cleaned on site to prevent the dispersal of GM seed. Harvested material and seed will be transported from the site in closed and labelled containers to the laboratories for analyses. Remaining seed which will not be used in following seasons will be inactivated as well as vegetative plant material. The area will be controlled for volunteers for a period of 1 consecutive year. During the next 6 years time there will be no conventional flax cultivation, which equals conventional crop rotation practice. Volunteers will be treated using conventional agricultural practices. During the release the project leader and trained personnel will monitor the trial site at defined intervals.Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:Not applicable. No such trials are planned.Final report-European Commission administrative informationConsent given by the Member State Competent Authority:Yes04/05/2007 00:00:00Remarks: