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:
01/05/2007 to 31/10/2009

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 nigrumSn 30

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 affect herbivore performance by inhibiting the insects' digestive enzymes.
The aim of our field experiments is to analyze the ecological relevance of proteinase inhibitors for S. nigrum. For this purpose we transferred DNA fragments of two S. nigrum proteinase inhibitor genes (pin1 and pin2b) back into S. nigrum plants. These fragments interfere with the production of the respective proteins by inducing RNA-silencing which reduces the steady-state levels of pin1 and pin2b mRNA in the transgenic plants. Silencing has been engineered and is effected by constitutive expression (enabled by the CaMV 35S promoter) of an antisense-intron-sense gene cassette. Silencing of respective mRNAs is triggered and maintained after splicing of the intron 3 from the pyruvate-orthophosphate-dikinase gene (pdk i3) from Flaveria trinervia, and subsequent RNA-silencing is provoked by double-stranded RNAs.
Agrobacterium tumefaciens was used to insert T-DNA into plant nuclei. A hygromycin resistance gene from Escherichia coli (hptII under the control of the Pnos promoter) was utilized to select for transgenic plants.
The transgenic genotypes selected for the field trials (S06/336, S06/353, S06/354, S06/356) are bearing one copy of the respective T-DNA and do not contain complete sequences of the nptIII gene. There are no previous modifications of the plants.

Genetic modification

3. Type of genetic modification:
Insertion; Other;

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) 3´ T-DNA Right Border
Source: Agrobacterium tumefaciens
Function: border between transferred and non-transferred DNA of the vector

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

c) Linker
Function: linker-DNA

d) Internal fragments of the proteinase inhibitor encoding pin1 and pin 2b genes
Source: Solanum nigrum
Function: Expression of sense RNA of the target genes pin1 and pin2b to be silenced; forming together with RNA from fragment f) an inverted repeat structure triggering posttranscriptional gene silencing of pin1 and pin2b

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
f) Internal fragments of the proteinase inhibitor encoding pin1 and pin2b genes
Source: Solanum nigrum
Function: Expression of antisense RNA of the target genes pin1 and pin2b to be silenced; forming together with RNA from fragment d) an inverted repeat structure triggering posttranscriptional gene silencing of pin1 and pin2b

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

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 between transferred and non-transferred DNA of the vector

6. Brief description of the method used for the genetic modification:
Agrobacterium mediated T-DNA transfer to tissues of Solanum nigrum. 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:
Brief introduction: Plants are not only exposed to adverse environmental conditions like drought, heat, cold and noxious gases (e.g. ozone) but they also have to cope with pathogens and herbivores which can strongly affect the life-span and fitness of a plant. In competition with herbivores plants have developed a range of mechanisms to protect themselves from infection and herbivory.
Project: Two genes from Solanum nigrum, pin1 and pin2b, encoding proteinase inhibitors, have been experimentally shown to be involved in the defense against herbivores. However, it is not clear whether the genes play important roles in the defense against herbivores when the 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 the ecological relevance of the pin1 and pin2b genes by determining for example “darwinian fitness” parameters (e.g. by measurements of biomass production) of the transgenic S. nigrum plants in comparison to isogenic wild type plants. The overarching question is: What are the ecological consequences of non-expressing proteinase-inhibitors?
S. nigrum is used as a model plant because the genome of this species – in contrast to cultivated or crop plants – is not influenced by, or under selection pressure of, human plant breeding processes. Our approach is based on fundamental ecological questions (e.g. defense vs. generative growth/darwinian fitness) and hence is needed 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 a quadrangle):
A: 51° 00,703’ N, 011° 38,999’ E
B: 51° 00,725’ N, 011° 38,982’ E
C: 51° 00,672’ N, 011° 38,833’ E
D: 51° 00,648’ N, 011° 38,857’ E

3. Size of the site (m2):
About 600 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 buds are to be removed, collected, autoclaved at the MPI for Chemical Ecology, and professionally disposed of. Hence, any transfer of transgenes to surrounding S. nigrum plants or to other potentially compatible plant species can be excluded. In addition, 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
i.) S. nigrum is an annual plant,
ii.) all tissues are frost sensitive and
iii.) so far it was not demonstrated that S. nigrum can 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, caused by reduced levels of proteinase inhibitors in the transgenic plants, but such an increase would only occur temporarily because
i.) only few transgenic plants are released for a short period (2 – 6 weeks) and
ii.) the field site will be surrounded by wild type plants of S.nigrum. 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 pests which might get temporarily in contact with the transgenic plans will not be influenced.
The transgenic plants do not have any environmental benefits. In contrast, as the expression of 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:
The following measures are taken to control putative risks:
i.) Only a small number of plants (maximum of 200) is bedded out on a small area in a given time period of 2 – 6 weeks about three times per year on a field of about 600 m2.
ii.) The plants remain for a maximum of six weeks on the field and will not grow larger than about 20 cm.
iii.) Plants at early developmental stages do not set flower buds and if, flower buds are immediately removed, collected, and autoclaved. At the end of each experiment, plants will be removed from the field and autoclaved at the Max Planck Institute.
iv.) After bedding out of transgenic plants, the field is scrutinized every three days by at least one person involved in the experiments who documents any feature going on during the experiment, i.e. check for the integrity of the transgenic plants, occurrence of flower buds, phenotypic changes, microbial attacks, herbivory, or irruptions by mammals. These observations continue until no transgenic plants are on the field and for additional eight weeks or until November 30 at the latest. All observations are documented in an official field journal and are archived by the project leader of the field trials.
v.) If naturally grown S. nigrum plants and related species occur to a distance of 35 m surrounding the field, these plants will be removed. In addition, the field is surrounded by a mixture of clover and grasses which potentially out-compete S. nigrum seedlings.
vi.) The field site is scrutinzed in the following year for potential occurrence of transgenic S. nigrum plants which immediately are to be removed and autoclaved.

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:
13/08/2007 00:00:00