General informationNotification NumberB/ES/03/34Member State to which the notification was sentSpainDate of acknowledgement from the Member State Competent Authority12/03/2003Title of the ProjectStudy on the ecological impact and the efficacy against the bacterial pathogens Ralstonia solanacearum and Erwinia spp. of transgenic lines of potato released in field under controlled conditions.Proposed period of release:30/04/2003 to 30/09/2003Name of the Institute(s) or Company(ies)Instituto Valenciano de Investigaciones Agrarias, Apartado oficial
46113 Moncada (Valencia)
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:The biological trait introduced in the transgenic potatoes is the increased production of lysozyme, due to the insertion in the plant genome of the expression cassette of the lysozyme from the bacteriophage T4. The foreign DNA, integrated in the plant genome through transformation with the soil bacteria Agrobacterium tumefaciens, also contains two attachment regions from tobacco plant and the resistance gene nptII-kanamycinGenetic 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:The three components of the inserted foreign DNA and their functions are the following:
|Common Name||Family Name||Genus||Species||Subspecies||Cultivar/breeding line|
a) the lysozyme gene from the bacteriophage T4, inserted by a fusion in the 5’ end, with a coding region for the barley -amylase signal-peptide, under the control of 35S promoter and terminator of cauliflower mosaic virus. This part of the integrated DNA contains the biological function of interest, which is to get resistance against bacterial invasions, as the lysozyme is a substance with antimicrobial properties. The signal-peptide is necessary to allow the correct secretion of the lysozyme into the extracellular space.
b) the resistance gene nptII-kanamycin from the Tn5 transposon of E. coli (Beck et al., 1982), under the control of the promoter of the nopaline-sinthetase (NOS) and the termination 4 gene from A. tumefaciens (Herrera-Estrella et al., 1983; Konzc and Schell, 1986; Konzc et al., 1987). The expression of this gene confers to transgenic cells tolerance to aminoglucosid antibiotics such as the kanamycin, so it is included as a selection marker in the transformation process.
c) two attachment regions from tobacco plant, flanking the expression region of the lysozyme.
The vector pSR 8-30, which is that containing the T-DNA with the interest genes to be transferred (Düring, 1994a), is arised from the vector pPCV 701 (Konzc et al., 1987). The T-DNA from the the base vector pSR 8-30 contains the marker gene nptII and a sinthetic polilinker for the insertion of the interest genes (Düring, 1994a). The T-DNA is limited by the two ends of the pMP90RK from the agrobacteria group GV3101 (Herrera-Estrella et al., 1983; Gielen et al., 1984; De Vos et al., 1981 ). The transformation vector pSR 8-40, used for the creation of DL12 line, is based in pSR 8-30 but containing the cassette with the T4 lysozyme gene. This is composed of the 35S promoter from the cauliflower mosaic virus, the -amylase signal-peptide gene from barley, the T4 lysozyme gene, and the termination gene 35S from the cauliflower mosaic virus.6. Brief description of the method used for the genetic modification:A method of genetic transformation by means of A. tumefaciens was used. The genes of interest, those from the bacteriophage T4 coding for lysozyme, were inserted in the segment of plasmidic DNA which is transferred and integrated in the genome of the plant cell. A binary vector, with two plasmids, was employed for the transfer (Konzc y Schell, 1986). One of the plasmids (the first vector) contained the T-DNA with the lysozyme genes, a marker gene of resistance against an antibiotic used in plants, an origin of replication to replicate itself both in A. tumefaciens and E. coli, and another marker gene of resistance against an antibiotic expressed in the bacterium. This vector was introduced in E. coli, and then transferred to A. tumefaciens containing the genes needed to move and integrate the T-DNA construction of the first vector in the plant genome.
The transformation system employed is based in the coculture of potato explants with the modified agrobacteria. Leaf discs from potato plants cultivated in a greenhouse were used as original plant material. The explants were inoculated in a liquid medium with the disarmed agrobacteria containing the construction with the genes of interest, and they were cocultivated in a suitable medium without antibiotics and with an hormone extract. After removing the agrobacteria, the leaves were cultivated in a medium with growth hormones, where the antibiotic kanamycin was added to select the transgenic shoots. These shoots were taken root in an hormone free medium. Afterwards, the reproduction was through cuttings.Experimental Release1. Purpose of the release:In the frame of the European Project “Impact of three selected biotechnological strategies for potato pathogen control on the indigenous soil microbiota” (QLRT-1999-31598) in which the Instituto Valenciano de Investigaciones Agrarias is a partner, a fundamental part of the planned trials request the culture and analysis of transgenic potatoes expressing the enzyme lysozyme. This study has a double purpose:
1. To assess the ecological impact of the use of the transgenic potatoes on the soil microbiota under Spanish conditions. This part will be carried out under field conditions.
2. To evaluate the resistance of these plants to two important bacterial pathogens that can cause serious economic losses to crops, Ralstonia solanacearum biovar 2 y Erwinia spp. This part will be carried out in a biosecurity laboratory type P3.
The study has to be made parallely in The Netherlands and Spain, with the objective of comparing the influence of the soil and the weather on the use of the transgenic potatoes, as both factors affect the composition of the microbial soil community.
It is not expected any change in the way and reproduction rate of the plant due to the genetic modification, as the inserted genes do not affect to the reproduction system. Neither is expected a change regarding to the survival ability, other than a greater resistance against phytopathogenic microorganisms. A smaller susceptibility to certain bacterial diseases does not offer a priori any potential to a more extended expansion ability in Spanish climatic conditions. The potato, S. tuberosum, is only able to cross with the same species, and there is no sign of change in the transgenic lines. Further, a transfer of foreign genetic material to non vegetal organisms is highly unlikely. In fact, in recent studies about culture of bacteria on genetically modified plants it has been shown the no transfer to bacteria of the antibiotic resistance genes introduced in transgenic plants as selection markers (Syvanen, 2000).
Moreover, by comparing the amount of endogen defense proteins (and other molecules) synthesized by the plant with the foreign protein produced by itself, it is not expected any significant change in the interaction with soil non-pathogen organisms; in fact, this has been shown in previous studies on field release in Germany of the same transgenic lines (Heuer et al., 2002; Lottmann et al., 1999). In addition, the lysozymes are a kind of enzymes widely distributed in nature, even in animals, human beings and plants. Thus, it can be considered that no strange substance is introduced in the transgenic potatoes.2. Geographical location of the site:A plot located in the municipal term of Torremocha, Teruel (Aragón, Spain) has been chosen. Teruel is an inner province situated in the Northestern of the Iberian Peninsule, characterized by a continental weather.3. Size of the site (m2):The selected plot is about 200 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:Field release of these transgenic potatoes under controlled conditions has been carried out in Germany since 1999 (Heuer et al., 2002; Lottmann et al., 1999, 2000) and in The Netherlands since 2001 (European Project QLRT-1999-31598). After 3 years of assays, it has been shown that in soil and climatic conditions from Germany the transgenic lines employed, which are the same whose release is requested in this present form, have no effect on the normal soil microbiota (Heuer et al., 2002) nor on that associated to the plant (Lottmann et al., 1999), nor even on the colonizer potential of the potato rhizosphere by bacteria with antagonistic activity against phytopathogenic microorganisms (Lottmann et al., 2000). The effect of the release of these transgenic potatoes on the bacterial communities of the rhizosphere is negligible compared to the influence of natural factors (Heuer et al., 2002), without differences in the abundance and the diversity of the bacteria associated with the plant between transgenic and non transgenic lines (Lottmann et al., 1999).Environmental Impact and Risk ManagementSummary of the potential environmental impact from the release of the GMPts:The potato for cultivation has no potential to expand in agricultural or natural environments. Therefore, the insertion of the bacteriophage T4 lysozyme and nptII genes does not involve any significant change in the general traits of the organism. So, it is not expected a greater persistence in cultivated field or a greater competitor ability in nature. Further, as the cultivated potato in Europe is not compatible sexually with other cultivars, it no vertical genetic transference is possible and, for this reason, advantages or disadvantages of the modification in other kind of potatoes can not be studied.
The induction of a greater resistance against pathogenic bacteria (due to the genetic modification 'expression of lysozyme'), does not imply necessarily changes in resistance of the pathogenic microorganism. The same conclusions are reached in the investigations with plants cultivated traditionally or with mutations, fusion of protoplasts, etc. On the other hand, the nptII gene, inserted as a marker, is considered in the specialized literature as biologically safe in transgenic plants (see for example Nap et al., 1992).
As explained in above sections C.1. and C.4., the comparison of the amount of endogen defense proteins (and other molecules), synthesized by the plant, with the foreign protein produced by itself allows to expect no significant change in the interaction with soil non-pathogen organisms, as shown in previous studies on field release in Germany of the same transgenic lines (Heuer et al., 2002; Lottmann et al., 1999). In addition, the lysozymes are a kind of enzyme widely distributed in nature, even in animals, human beings and plants. Thus, it can be considered that no strange substance is introduced in the transgenic potatoes. In soil and climatic conditions from Germany the transgenic lines employed, which are the same whose release is requested in this present form, have no effect on the normal soil microbiota (Heuer et al., 2002) nor on that associated to the plant (Lottmann et al., 1999), nor even on the colonizer potential of the potato rhizosphere by bacteria with antagonistic activity against phytopathogenic microorganisms (Lottmann et al., 2000). The effect of the release of these transgenic potatoes on the bacterial communities of the rhizosphere is negligible compared to the influence of natural factors (Heuer et al., 2002), without differences in the abundance and the diversity of the bacteria associated with the plant between transgenic and non transgenic lines (Lottmann et al., 1999).Brief description of any measures taken for the management of risks:The potatoes will be planted inside an area specially designed for the assay, separated by a row of plants different to potato and isolated by a non cultivated area of 5 m width. The flower buds will be removed periodically to prevent polinization, and all the tubers will be collected when assay finishes. The control tubers will be treated as normal tubers. After the crop, the rests of material corresponding to transgenic tubers will be removed by sterilization in autoclave, and the plot will be treated with the chemical disinfectant Vapam.Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:One of the objectives of the study (see section C.1) is to assess the ecological impact of the use of the transgenic potatoes on the soil microbiota under Spanish conditions. Potatoes will be collected at three growth stages, and the microbial community associated to rhizosphere will be analyzed and compared with that characterized in non transgenic potatoes.Final report-European Commission administrative informationConsent given by the Member State Competent Authority:Yes07/05/2003 00:00:00Remarks:The competent authority for the consent of field trials is Aragón.