Date of publication: June 21, 2004
Source:
http://gmoinfo.jrc.it/gmp_browse_geninf.asp
Notification
report
General information
Notification Number:
B/DE/04/156
Member State: Germany
Date of Acknowledgement: 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 From:01/07/2004
To:30/09/2006
Name of the Institute(s) or Company(ies): Max Planck
Institute for Chemical Ecology;
3. Is the same GMPt release planned elsewhere in the
Community?
No
4 - Has the same GMPt been notified elsewhere by the same
notifier?
No
Genetically
modified plant
1. Complete name of the
recipient or parental plant(s)
|
Common Name |
Family Name |
Genus |
Species |
Subspecies |
Cultivar/breeding line |
| black
nightshade |
solanaceae
|
solanum
|
solanum
nigrum |
|
sn30
|
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;
4. 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.
- 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 |
