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Notification report


General information

Notification Number
B/NL/03/07

Member State to which the notification was sent
Netherlands

Date of acknowledgement from the Member State Competent Authority
29/09/2003

Title of the Project
Field trial with chicory plants expressing additional fructosyltransferases

Proposed period of release:
01/01/2004 to 31/12/2008

Name of the Institute(s) or Company(ies)
Plant Research International - Dept. Genetics and Breeding, NL-6700 AA Wageningen;


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
chicoryasteraceaecichoriumchicorium intybus95/9 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;

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

Final report
-

European Commission administrative information

Consent given by the Member State Competent Authority:
Not known