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Notification report


General information

Notification Number
B/DE/05/165

Member State to which the notification was sent
Germany

Date of acknowledgement from the Member State Competent Authority
21/02/2005

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?
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 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 S. nigrum plants produce proteinase-inhibitors after herbivore attack.
These inhibitors affect the performance of herbivores by interfering with the insects' digestive
enzymes. The production of proteinase-inhibitors is very likely coupled to a plant encoded signal
polypeptide (SYSTEMIN) triggered by herbivory.
The aim of our field experiments is to analyze the ecological relevance of SYSTEMIN for S. nigrum.
For this purpose we transferred DNA fragments of a S. nigrum gene (nigpro) encoding an immature
form (precursor protein) of SYSTEMIN (prosystemin) back into S. nigrum plants. These fragments
interfere with the production of nigpro mRNA via RNA silencing reducing nigpro mRNA steady-state
levels. RNA silencing is triggered and maintained by constitutive transcription (enabled by the CaMV
35S promoter) of an antisense-intron-sense nigpro 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 nigpro 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 (SOL3-SYS lines) include one copy of the TDNA
per haploid genome. There are no further previous genetic 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 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
Synthetic
Function: linker DNA
d) internal fragment of the nigpro gene
Source: Solanum nigrum
Function: expression of sense RNA of the target nigpro gene to be silenced; forming
together with RNA from fragment f) an inverted repeat structure, triggering posttranscriptional
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. Following transcription, the intron is spliced and the remaining RNA forms an
inverted repeat dsRNA
f) internal fragment of the nigpro gene
Source: Solanum nigrum
Function: expression of antisense RNA of the target nigpro gene to be silenced; forming
together with RNA from fragment d) an inverted repeat structure, triggering posttranscriptional
gene silencing
g) 35S promoter of Cauliflower Mosaic Virus
Source: Cauliflower Mosaic Virus
Function: constitutive expression of the antisense-intron-sense constructs within the TDNA
of SOL3-SYS 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 nigpro gene from Solanum nigrum encoding prosystemin is presumed to be involved into
molecular defense mechanisms against herbivory, however, it is not known whether nigpro
plays an important role in the defense against caterpillars, other herbivorous insects or
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 nigpro gene by determining “darwinian fitness” parameters (e.g.
by measurements of biomass production), examining transcription profiles of selected and
specified genes and measuring the amounts of secondary metabolites and phytohormones 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):
about 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 they will be removed from the field,
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. 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
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 in nature.
For a short time there might be a slight increase in S. nigrum specific target organisms, caused
by reduced levels of SYSTEMIN in the transgenic plants. However, such an increase would
only occur temporarily because
1) only few transgenic plants are released for a short period 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 putative 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 (about 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.
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 from 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:
Yes
08/07/2005 00:00:00
Remarks: