Date of publication: May 24,
2005
Source:
http://gmoinfo.jrc.it/gmp_browse_geninf.asp
Notification number:
B/DE/05/164
Member State:Germany
Date of Acknowledgement:05/01/2005
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 From:01/05/2005
To:30/10/2007
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 |
|
|
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:
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 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 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 |
