European Commission
DG Joint Research Centre
Institute for Health and Consumer Protection
Biotechnology and GMOs Unit
http://gmoinfo.jrc.it/
January 12, 2004
Notification
report
General information
Notification Number:
B/NL/03/07
Member State:Netherlands
Date of Acknowledgement:29/09/2003
Title of the Project:
Field trial with chicory plants expressing additional
fructosyltransferases
Proposed period of release From:01/01/2004
To:31/12/2008
Name of the Institute(s) or Company(ies): Plant
Research International - Dept. Genetics and Breeding;
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 |
| chicory
|
asteraceae
|
cichorium
|
chicorium
intybus |
|
95/9 and
145/1 |
2. Description of the traits and characteristics which have
been introduced or modified, including marker genes and previous
modifications:
The genetic modification was performed to ensure that the
amount and the quality of the inulin (a carbohydrate belonging
to the fructan group), that is naturally accumulated in chicory
roots during the growing season, will remain stable during the
autumn or after frost.
Chicory naturally accumulates inulin which biosynthesis is
catalysed by the concerted action of the chicory’s native
sucrose: sucrose fructosyltransferase (SST) and fructan: fructan
fructosyltransferase (FFT) (Van Laere and Van den Ende, 2002).
In chicory, SST activity is maximum in June and decreases
gradually during the growing period of the root. This lead to an
accumulation of sucrose in the root, which in turn, enable a FFT
mediated- back transfer reaction of fructose moieties from
fructan to sucrose. This results in a lowering of the mean
degree polymerization (DP) of the inulin at harvest.
A high mean DP is preferable for industrial application (Frank
and De Leenheer, 2002). Fructan are used as gelling agent or as
soluble fibre, for these applications the longer the chain of
fructose the better the properties.
The GM chicory are expressing, constitutively, an extra SST (A33
or SST103 both isolated from Helianthus tuberosus, Koops and
Jonker, 1996, Van der Meer et al, 1998, Koops 1999). It is
expected that this extra SST will compensate for the decrease of
activity of the native SST. This compensation should result in
the maintenance, or even increase, of the mean degree of
polymerisation of the inulin in the transgenic chicory roots.
After a frost period fructan exohydrolase (FEH) come to
expression in chicory’s roots. FEH catalyses the
depolymerization of fructan, decreasing the value of the
extracted fructan at harvest. It is expected that the extra SST
introduced in the GM chicory will contribute to the re-synthesis
of fructan after a frost period.
Beside this main trait the transformed chicory’s also expressed
the nptII gene resulting in resistance to the antibiotic
kanamycin.
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 aim of the genetic modification is to reinforce the SST
activity naturally present in the chicory root. As described in
the literature, the native SST activity of chicory decrease
rapidly during the culture of the plant (Van Laere and Van den
Ende, 2002) the introduced genes are intended to compensate for
this decrease.
The a33 and sst103 gene cassettes (Enh35S CaMV-AMV-a33-Tnos,
Enh35S CaMV-AMV-sst103-Tnos, respectively) result in the
production in the enzymes A33 and SST103 that both are sucrose:
sucrose fructosyltransferases. They catalyse the first reaction
of fructan biosynthesis:
GF + GF = GFF + F
Where GF, GFF, G and F are sucrose, 1-kestose, glucose and
fructose, respectively. A33 and SST103 occur naturally in the
root of Helianthus tuberosus, they posses a signal peptide that
direct their accumulation into the vacuole of the plant cell
(Van der Meer 1998, Koops 1999).
The neomycin phosphotransferase gene cassette (Pnos-nptII-Tnos)
was used to select the transformed plant tissue during the
genetic transformation protocol by conferring kanamycin
resistance to the transformed cell.
Source of the different constituents:
T-DNA from pSST331 :
1)
Element: T-DNA borders: LB and RB
Origin: Agrobacterium tumefaciens
Function: Insertion in the chicory genome
2)
Element: Enh35S CaMV
Origin: Cauliflower Mosaic Virus
Function: 35S promoter from cauliflower mosaic virus with a
duplicated enhanced region (Kay et al 1987)
3)
Element: AMV
Origin: Alfafa Mosaic Virus
Function: Translational enhancer (Jobling and Gehrke, 1987)
4)
Element: sst103
Origin: Helianthus tuberosus
Function: Sucrose, sucrose fructosyltransferase (Van der Meer,
1998) EMBL accession number AJ009757
5)
Element:Tnos
Origin: A. tumefaciens
Function: 3’end of the nopalin synthase (Fraley et al 1983)
6)
Element: Pnos
Origin: A. tumefaciens
Function: Nopalin synthase promoter
7)
Element: nptII
Origin: Tn 5 transposon of E. coli
Function: Neomycin phosphostransferase II confer resistance to
kanamycin
8)
Element: LacZ
Origin: Escherichia coli
Function: Structural gene encoding beta galactosidase. Used to
select visually transformed bacteria.
T-DNA from pA33236:
1)
Element: T-DNA borders: LB and RB
Origin: Agrobacterium tumefaciens
Function: Insertion in the chicory genome
2)
Element: Enh35S CaMV
Origin: Cauliflower Mosaic Virus
Function: 35S promoter from cauliflower mosaic virus with a
duplicated enhanced region (Kay et al 1987)
3)
Element: AMV
Origin: Alfafa Mosaic Virus
Function: Translational enhancer (Jobling and Gehrke, 1987)
4)
Element: a33
Orign: Helianthus tuberosus
Function: Sucrose, sucrose fructosyltransferase (Koops, 1999)
5)
Element: Tnos
Origin: A. tumefaciens
Function: 3’end of the nopalin synthase (Fraley et al 1983)
6)
Element: Pnos
Origin: A. tumefaciens
Function: Nopalin synthase promoter
7)
Element: nptII
Origin: Tn 5 transposon of E. coli
Function: Neomycin phosphostransferase II confers resistance to
kanamycin
8)
Element: LacZ
Origin: Escherichia coli
Function: Structural gene encoding beta galactosidase. Used to
select visually transformed bacteria.
6. Brief description of the method used for the genetic
modification:
Genetic transformation mediated with Agrobacterium
tumefaciens (Horsch et al 1985).
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:
Inulin, a fructan (carbohydrate) is produced naturally by
about 15% of the flowering plants (Hendry and Wallace, 1993).
Chicory is one of them and is used for the industrial production
of inulin. Chicory was genetically transformed with a gene
encoding for the enzyme responsible for the first step of the
inulin biosynthesis. The introduced enzyme is intended to
sustain the endogenous chicory enzyme.
This complementation is expected to result in an improved inulin
quality. The purpose of the application is to assess under
agronomic conditions the fructan production capacity of the
transformed chicory and the effects of autumnal cold and frost
on the quality of the inulin produced.
2. Geographical location of the site:
Wageningen (sites Grebbedijk and De Haaff), province
Gelderland, The Netherlands.
3. Size of the site (m2):
1000 m2 (in the broad sense)
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 GM chicory possesses an extra SST enzyme that is expected
to compensate for the decrease of activity of the native chicory
SST. The result of this modification will be the accumulation in
the GM chicory taproot of inulin with a higher mean DP. This
alteration of the soluble carbohydrate content of the chicory
root could have effect on different aspects of the life of the
plants.
The modification of the soluble carbohydrates content observed
in the GM chicory could affect its ability to flower and to set
seeds.
Chicory has a biennial growth habit. During the first year the
plant develops a rosette of leaves and a tuberous root, the
taproot, where inulin (a fructan) is stored. The introduction of
an extra SST in the GM chicory was intended to render the inulin
less susceptible to degradation. One consequence could be that
the GM chicory would not be as efficient as the wild type
chicory to remobilize the inulin during the second year of
growth to sustain flowering. The consequence could be that the
GM chicory would be less competitive during the flowering
period.
We already performed crossing between the GM chicory and wild
type chicory, we observed that the development of the flower and
the seed setting was similar for both plants. There could be two
explanations for this absence of difference. Cultivated chicory
is bred to have a high content of inulin, wild chicory root
accumulate much less fructan, but are still able to flower. It
seems that chicory does not need to flower as much fructan as
the cultivated chicory is able to accumulate. Beside this
quantitative aspect it could also be that during the flowering
phase FEH, the enzyme responsible for the degradation of
fructans, is still able to degrade the inulin and counteract the
effect of the extra SST.
To avoid any effects, the GM chicory will be grown only during
the vegetative part of their reproductive cycle and will not be
allowed to flower.
The modification of the soluble carbohydrates content observed
in the GM chicory could affect its resistance to frost.
Two hypotheses can be developed:
- The presence of the extra SST would limit the degradation of
inulin, the leaves of the GM plant would then receive less
energy to overcome the frost and resume growth. The chicory with
an extra SST would then be more sensitive to frost.
- The presence of the extra SST would allow the plant to
synthesise more and longer inulin during the year of growth.
This extra pool of carbohydrate, compare to the wild type
chicory, could be used by FEH (fructan exohydrolase). FEH
degrades fructan, it is induced after frost or cold period, it
produces fructose from inulin. This fructose can be transported
to the leaves to help resist frost and favour re-growth.
To date there are no data which would allow to chose between
these two hypotheses. The experiment described in the present
application will help to give some answers about this aspect
(growth parameters and sugar content are going to be measured
during the experiment and especially after cold and frost). To
avoid any effect on the environment, the effect of the
modification will be confined to the plants used for the field
trial. These plants will be grown only during the vegetative
part of their reproductive cycle to minimise the risk of
spreading by pollen and seeds.
The modification of the soluble carbohydrates content observed
in the GM chicory could affect its attractiveness toward small
animal.
Because the sugar composition of the GM chicory has been altered
attractiveness toward small animal such as rodent could be
modify and result in a higher attraction of these animals.
Fructans naturally occur in chicory and it is not expected that
the GM chicory will be more attractive to small animal. In the
event that a small animal would eat a piece of the GM chicory it
is not expected to have detrimental effect on this animal.
Fructans are natural component of chicory, they also are one of
the main carbohydrates present in grass and other wild
Asteraceae. Fructan are not toxic or allergenic, they are
considered as a functional food for human: a food component that
also favours health. Fructans act as soluble fibre to facilitate
the intestinal tract, they are also known to favour the
bifidogenic bacterial population in the colon of mammals,
limiting in turn the development of other harmful bacterial. To
limit the risk of small animal eating the GM chicory a fence
will surround the field to limit the access to the plants.
The modification of the soluble carbohydrates content observed
in the GM chicory could affect its sensitivity to disease.
Because the soluble carbohydrate composition of the GM chicory
taproot will be altered, the development of disease could be
modified. The composition of the soluble carbohydrate of the
leaves of the GM chicory is expected to be similar to the one of
the wild type chicory. The GM chicory is not expected to be
modified for its sensitivity to leaf pathogens. There are no
data concerning the importance of fructan in relation with
attack by pathogens. During the monthly root sampling, roots
will be visually inspected to detect if any difference in
pathogen attack can be observed between the GM chicory and the
wild type control.
To limit the effect on the environment, the effect of the
modification will be confined to the plants used for the field
trial. These plants will be grown only during the vegetative
part of their reproductive cycle to minimise the risk of
spreading by pollen and seeds. At the end of the experiment any
plants left will be destroyed. During the weekly control for the
presence of plant initiating flowering, attention will be
devoted to the detection of disease.
The modification of the soluble carbohydrates content observed
in the GM chicory could affect its relation with the soil
microbial flora.
The development of fungi and bacteria on and in the close
neighbourhood of roots is partially controlled by substances
released by the roots. Although fructans are stored in the
vacuole of the plant cell, it is not excluded that if a damage
occurs on the root some fructans could be released into the
soil. The presence of this fructans could in turn influence the
composition of the soil microflora. As explain before fructans
are natural component of the chicory taproot and about 15% of
the flowering plants naturally accumulate fructans. It is not
expected that the GM chicory will have a different influence on
the soil microflora compared with other naturally fructan
accumulating plants.
To limit the potential influence of the GM chicory on the soil
microflora, only few plants will be grown during the experiment.
Taproots will be harvested by hand to limit the spreading of
root pieces, the soil adhering to the taproot (containing some
chicory’s hairy roots) will be collected and treated as waste.
To achieve the modification of the soluble carbohydrate content
in the root of the GM chicory, new DNA sequences have been
introduced. It is not impossible that these sequences could be
transferred to soil bacteria and modify their fitness.
Horizontal transfer of transgene to soil bacteria, although
never demonstrated, can not be excluded. Acquisition of the
nptII gene cassette by bacteria is not likely to increase their
fitness. During the field experiment kanamycin is not going to
be used, there will be thus no selection pressure that could
favour the development of bacteria that would acquire the nptII
sequence. Resistance to kanamycin is naturally largely present
in soil bacterial population. The unlikely horizontal transfer
of the nptII sequence would not result in a new situation.
The transfer of the fructosyl transferase gene cassettes to
bacteria is not expected to result in an increase
competitiveness of these ones. Fructan (levan) synthesis already
occurs in bacteria such as Bacillus, Streptococcus, Pseudomonas,
Xanthomonas, Azotobacter, Erwinia and Actinomyces (Hendry and
Wallace, 1993).
To limit the effect of potential transfer of DNA sequences to
soil bacteria, only few plants will be grown during the
experiment. Taproots will be harvested by hand to limit the
spreading of root pieces, the soil adhering to the taproot
(containing some chicory’s hairy roots) will be collected and
treated as waste.
Brief description of any measures taken for the management of
risks:
A fence, to limit the access for animals, will surround the
area receiving the plants.
The GM chicory will be grown only for the production of the
taproot to prevent the spreading via pollen or seeds.
Chicory plants (GM and wild type) initiating flowering will be
destroyed. In flowering chicory fructans are partially degraded
to sustain the growth of the stem and the flower development.
Flowering plant can not be used for the purpose of the
experiment. When plants will be present on the field (May to
December) the culture will be checked every week to detect and
destroy any plants initiating flowering.
Summary of foreseen field trial studies focused to gain new
data on environmental and human health impact from the release:
Not applicable
|
