Notification report

General information

Notification Number

Member State to which the notification was sent

Date of acknowledgement from the Member State Competent Authority

Title of the Project
Ecological relevance of potentially defensive genes during the interaction between Solanum nigrum (Black Nightshade) and environmental factors.

Proposed period of release:
01/05/2005 to 30/10/2007

Name of the Institute(s) or Company(ies)
Max Planck Institute for Chemical Ecology, Hans-Knoell-Str. 8, D-07745 Jena;

3. Is the same GMPt release planned elsewhere in the Community?

Has the same GMPt been notified elsewhere by the same notifier?

Genetically modified plant

Complete name of the recipient or parental plant(s)
Common NameFamily NameGenusSpeciesSubspeciesCultivar/breeding line
black nightshadesolanaceaesolanumsolanum nigrum

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
It has been demonstrated that plants produce a specific set of different proteins after pathogen attack. Consequently, these proteins were denominated “pathogenesis related proteins - PR-proteins”. For one protein, PR-1, its mode of action as well as its presumed function to prevent herbivore attack is an ongoing object of research.
The aim of our field experiments is to analyze the ecological relevance of PR-1 utilizing wildtype and transgenic S. nigrum plants. We transferred two short DNA fragments of a S. nigrum PR-1 gene (pr-1S) back into S. nigrum plants. These fragments interfere with the production of pr-1S mRNA via RNA silencing reducing pr-1S mRNA steady-state levels. RNA silencing is triggered and maintained by constitutive transcription (enabled by the CaMV 35S promoter) of an antisense-intron-sense pr-1S gene cassette, subsequent splicing of the intron (no. 3 from the pyruvate-orthophosphate-dikinase gene (pdk i3) from Flaveria trinervia), and subsequent production of siRNAs derived from pr-1S double-stranded RNA.
Agrobacterium tumefaciens was used to transfer T-DNA into plant chromosomal DNA. A hygromycin resistance gene from Escherichia coli (hpt II under the control of the Pnos promoter) was utilized to select for transgenic plants.
The transgenic genotypes selected for the field trials (SOL3PR-1S lines) include one copy of the T-DNA per haploid genome and do not contain the npt III gene. There are no further previous genetic modifications of the plants.

Genetic modification

3. Type of genetic modification:

In case of insertion of genetic material, give the source and intended function of each constituent fragment of the region to be inserted:
The following DNA fragments were introduced into Solanum nigrum:

a) 3´ T-DNA Right Border
Source: Agrobacterium tumefaciens
Function: border to transferred DNA

b) terminator of Cauliflower Mosaic Virus
Source: Cauliflower Mosaic Virus
Function: termination of mRNA transcription

c) linker
Function: linker DNA

d) internal fragment of the pr-1S gene
Source: Solanum nigrum
Function: expression of sense RNA of the target pr-1S gene to be silenced; forming together with RNA from fragment f) an inverted repeat structure, triggering post-transcriptional gene silencing

e) intron 3 (i3) of the pyruvate, orthophosphate dikinase gene pdk
Source: Flaveria trinervia
Function: spacer between antisense- and sense gene fragments enhancing vector stability. When expressed, the intron is spliced and the remaining RNA forms an inverted repeat dsRNA

f) internal fragment of the pr-1S gene
Source: Solanum nigrum
Function: expression of antisense RNA of the target pr-1S gene to be silenced; forming together with RNA from fragment d) an inverted repeat structure, triggering post-transcriptional gene silencing

g) 35S promoter of Cauliflower Mosaic Virus
Source: Cauliflower Mosaic Virus
Function: constitutive expression of the antisense-intron-sense constructs within the T-DNA of SOL3PR-1S plants

h) promoter of the nopaline synthase encoding nos gene
Source: Agrobacterium tumefaciens
Function: constitutive promoter to transcribe hptII mRNA

i) hygromycine phosphotransferase gene hptII, cloned from pCAMBIA-1301
Source: Escherichia coli
Function: selectable marker for the transformation of plant cells and seedling selection of respective progenies

j) terminator of the nopaline synthase encoding nos gene
Source: Agrobacterium tumefaciens
Function: termination of mRNA transcription

k) T-DNA left border
Source: Agrobacterium tumefaciens
Function: border to transferred DNA

6. Brief description of the method used for the genetic modification:
Agrobacterium mediated T-DNA transfer to tissues of Solanum nigrum. Subsequent regeneration of plants from calli using phytohormones for shoot induction

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:
Plants are not only exposed to adverse environmental conditions like drought, heat, cold and noxious gases (e.g. ozone). They also have to cope with pathogens and herbivores which can strongly affect their life span and fitness. In competition with herbivores plants have developed a range of mechanisms to protect themselves from infection and herbivory.
The pr-1S gene from Solanum nigrum encoding an archetypical pathogenesis related protein is presumed to be involved into the molecular defense mechanisms against herbivory, however, it is not known whether pr-1S plays an important role in the defense against herbivores and microbial pathogens when plants are challenged by a plethora of biotic and abiotic factors in a natural habitat, i.e. in the field. The aim of our work is to analyze and summarize the ecological relevance of the pr-1S gene by determining “darwinian fitness” parameters (e.g. by measurements of biomass production) of the transgenic S. nigrum plants compared with isogenic wild types.
S. nigrum is used as a model plant because this species – in contrast to cultivated plants – has not been modified by breeding. Our approach is based on fundamental ecological questions and important for a better understanding of plant-plant, plant-pathogen and plant-insect interactions in nature and the functioning of ecosystems.
There are neither agronomic purposes nor tests of hybridisation and disseminations.

2. Geographical location of the site:
Germany, federal state of Thuringia, county (Landkreis) Dornburg
GPS coordinates (corners of the quadrangle):
A: 5100,7029 N, 1138,9988 E
B: 5100,7253 N, 1138,9816 E
C: 5100,6717 N, 1138,8325 E
D: 5100,6477 N, 1138,8567 E

3. Size of the site (m2):
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:
not applicable

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
As soon as an appearance of flower buds can be detected the plants will be removed from the field (including root material), autoclaved at the MPI for Chemical Ecology and professionally disposed of. Hence, any transfer of the transgene to surrounding S. nigrum plants or to other potentially sexual compatible plant species can be excluded. S. nigrum has been determined to be a predominant self pollinating plant species.
Small mammals and birds could carry off vegetative plant parts. However, an unintended release is impossible as
1) S. nigrum is an annual plant,
2) all tissues are frost sensitive and
3) so far it could not be demonstrated that tissues of S. nigrum are able to regenerate new plants from vegetative plant parts.
For a short time there might be a slight increase in S. nigrum specific target organisms, caused by reduced levels of PR-1 protein. However, such an increase would only occur temporarily because
1) only few transgenic plants are released for a short period (14-21 days) and
2) the field site will be surrounded by wild type plants of S. nigrum. The number of wild type plants will exceed the number of transgenic plants. A putative positive effect on the number of herbivores due to the transgenic plants will be equalized by the surrounding wild type plants. Hence, populations of pests which might get temporarily in contact with the transgenic plants will not be influenced.
The transgenic plants do not have any environmental benefit. In contrast, as the expression of a pathogen defense gene is suppressed in the transformed plants we expect that they are less resistant to herbivore attack in the field.

Brief description of any measures taken for the management of risks:
Because S. nigrum is not a common crop plant it is not hazardous to humans following accidental consumption. However, the following measures are taken to control putative risks:
1) Only a small number of plants (a total of 600 at maximum) is bedded out consecutively on one small area (500 m2).
2) The plants remain on the field for a maximum of three weeks and will not grow larger than about 20 cm.
3) The plants will not set flower buds. The plants will be removed from the field and autoclaved at the Max Planck institute.
4) After bedding out of the first set of plants the field is scrutinized every day by scientists who will document any feature going on during the experiment, i.e. checking for flower buds, integrity of the transgenic plants, phenotypic changes, microbial pathogen attacks, herbivory, or irruption by any other animals. These observations continue until no transgenic plants are on the field. Within eight weeks after the end of the experiment, the field will be monitored every seven days. All observations are documented on paper (in german language) and if needed by photographs.
5) If there is an occurance of S. nigrum plants naturally growing up to a distance of 25 m surrounding the field, these plants will be removed. Additionally, the field is surrounded by a mixture of clover and grasses.

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:
02/06/2005 00:00:00