Back

Notification report


General information

Notification Number
B/DE/04/156

Member State to which the notification was sent
Germany

Date of acknowledgement from the Member State Competent Authority
15/01/2004

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:
01/07/2004 to 30/09/2006

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?
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
black nightshadesolanaceaesolanumsolanum nigrumsn30

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
It was demonstrated that S. nigrum plants produce proteinase-inhibitors after herbivore attack. These inhibitors affects the herbivore performance by inhibiting the insects' digestive enzymes. The production of the inhibitors is very likely induced by a signal molecule (systemin) after wounding and herbivory.
The aim of our field experiment is to analyze the ecological relevance of proteinase inhibitors and systemin for S. nigrum. For this purpose we transferred DNA fragments of S. nigrum genes encoding a proteinase inhibitor (pin2b) and an immature form of systemin (prosystemin; nigpro), respectively, into S. nigrum plants. These fragments interfere with the production of the two respective proteins by induction of RNA-silencing, hence reducing the steady-state levels of the respective gene products. RNA silencing has been engineered and is effected during constitutive expression (enabled by the 35S promoter) of an antisense-intron-sense gene cassette. Silencing of the respective mRNA is triggered and maintained after splicing of the intron 3 from the pyruvate-orthophosphate-dikinase gene (pdk i3) from Flaveria trinervia, and subsequent mRNA interference is provoked by the respective double-stranded RNAs.
Plants bearing the antisense-intron-sense construct of the nigpro gene showed a reduction of 67 % compared to wildtype plants of trypsine proteinase inhibitor activity in wounded as well as non-treated leaves. In plants bearing the antisense-intron-sense construct of the pin2b gene a reduction of trypsine proteinase inhibitor activity also has been determined, measurements are in progress.
Agrobacterium tumefaciens was used to transfer T-DNA into plant nuclei. 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 (pSOL3PIS lines: S03/111, S03/114; pSOL3SYS lines : S03/71, S03/73) are bearing one copy of the respective T-DNA and do not contain sequences of the npt III gene. There are no previous modifications of the plants.


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:
The following DNA fragments, present in the respective T-DNAs, were introduced into Solanum nigrum:


a) 3ยด T-DNA Right Border
- Source: Agrobacterium tumefaciens.
- Function: border between transferred and non-transferred DNA of the vector.
- Transgenic plant lines: pSOL3PIS, pSOL3SYS.

b) terminator of Cauliflower Mosaic Virus
- Source: Cauliflower Mosaic Virus.
- Function: termination of mRNA transcription.
- Transgenic plant lines: pSOL3PIS, pSOL3SYS.

c) linker
- Synthetic.
- Function: linker-DNA.
- Transgenic plant lines: pSOL3PIS, pSOL3SYS.

d1) internal fragment of the proteinase inhibitor encoding pin 2b gene
- Source: Solanum nigrum.
- Function: expression of sense RNA of the target gene pin2b to be silenced; forming together with RNA from fragment f1) an inverted repeat structure, triggering posttranscriptional gene silencing of pin2b.
- Transgenic plant lines: pSOL3PIS.

d2) internal fragment of the prosystemin encoding nigpro gene
- Source: Solanum nigrum.
- Function: expression of sense RNA of the target gene nigpro to be silenced; forming together with RNA from fragment f2) an inverted repeat structure, triggering posttranscriptional gene silencing of nigpro.
- Transgene plant lines: pSOL3SYS.

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 double stranded structure triggering RNA silencing of the respective target gene.
- Transgenic plant lines: pSOL3PIS, pSOL3SYS.

F1) internal fragment of the proteinase inhibitor encoding pin2b gene
- Source: Solanum nigrum.
- Function: expression of antisense RNA of the target gene pin2b to be silenced; forming together with RNA from fragment d1) an inverted repeat structure, triggering posttranscriptional gene silencing of pin2b.
- Transgenic plant lines: pSOL3PIS.

f2) internal fragment of the prosystemin encoding nigpro gene
- Source: Solanum nigrum.
- Function: expression of antisense RNA of the target gene nigpro to be silenced; forming together with RNA from fragment d2) an inverted repeat structure, triggering posttranscriptional gene silencing of nigpro.
- transgenic plant lines: pSOL3SYS.

g) 35S promoter of Cauliflower Mosaic Virus
- Source: Cauliflower Mosaic Virus.
- Function: constitutive expression of the antisense-intron-sense constructs within the T-DNAs of pSOL3PIS and pSOL3SYS.
- Transgenic plant lines: pSOL3PIS, pSOL3SYS.

h) promoter of the nopaline synthase encoding nos gene
- Source: Agrobacterium tumefaciens.
- Function: constitutive promoter to transcribe hptII mRNA.
- Transgenic plant lines: pSOL3PIS, pSOL3SYS.

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.
- Transgenic plant lines: pSOL3PIS, pSOL3SYS.

j) terminator of the nopaline synthase encoding nos gene
- Source: Agrobacterium tumefaciens.
- Function: termination of mRNA transcription.
- Transgenic plant lines: pSOL3PIS, pSOL3SYS.

k) T-DNA left border
- Source: Agrobacterium tumefaciens.
- Function: border between transferred and non-transferred DNA of the vector.
- Transgenic plant lines: pSOL3PIS, pSOL3SYS.


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:
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 is important for a better understanding of plant 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 (near the city of Jena).
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):
1479

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 genetically modified plants will not be left on the field until flowering. Already before the appearance of the first flower buds the plants will be removed from the field (including root material), autoclaved at the MPI for Chemical Ecology and professionally disposed of so that any transfer of the genes to surrounding S. nigrum plants or to other sexually compatible species can be excluded. Furthermore, S. nigrum is 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 an increase in S. nigrum specific target organisms, in particular of herbivores, but such an increase would only occur temporarily because
1) only a 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, the population of other pests which might get temporarily in contact with the transgenic plans will not be influenced.
The transgenic plants do not have any environmental benefit. In contrast, as the expression of each one of the two defense genes 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 crop plant it is not hazardous to humans e.g. because of accidental consumption. However because, to our knowledge, this is the first field trial using transgenic S. nigrum plants and hence no experiences do exist for such experiments, the following measures are taken to control putative risks:
1) Only a small number of plants (400 - 600) is bedded out on a small area (1479 m2).
2) The plants remain for a maximum of three weeks on the field 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 plants the field is scrutinized every two days by at least two scientists who document any feature going on during the experiment, i.e. check for the integrity of the transgenic plants, phenotypic changes, microbial pathogen attacks, herbivory, or irruptions by mammals. These observations continue until no transgenic plants are on the field and for additional four weeks. All observations are documented on paper and if needfully by photographs and archived by the project leader of the field trial.
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:
Yes
30/08/2004 00:00:00
Remarks: