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Notification report


General information

Notification Number
B/CZ/14/01

Member State to which the notification was sent
Czech Republic

Date of acknowledgement from the Member State Competent Authority
05/12/2014

Title of the Project
Deliberate release of genetically modified CKX barley (pEXP:CKX1, pEXP:CKX2, bGLU:CKX1, CKX1:RNAi) producing ectopically additional cytokinin dehydrogenase in roots

Proposed period of release:
01/04/2015 to 30/09/2017

Name of the Institute(s) or Company(ies)
Palacky University in Olomouc, ;


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
barleypoaceaehordeumhordeum vulgarevulgare

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
pEXP:CKX1
Spring barley was genetically modified by the CKX1 gene (At2g41510) encoding an Arabidopsis cytokinin dehydrogenase under the control of a root-specific promoter derived from an expressed protein gene (LOC_Os04g11040.1) of rice (Oryza sativa L. cv. Nipponbare). The Arabidopsis cytokinin oxidase / dehydrogenase 1 enzyme (CKX1) catalyzes the irreversible degradation of the hormone cytokinin in a single enzymatic step by oxidative side chain cleavage causing ectopic depletion of cytokinins in roots. In addition, the transgenic line has also integrated the hpt selection gene (YP_006952299) coding for hygromycin phosphotransferase that confers resistance to the antibiotic Hygromycin B. This antibiotic resistance has been used in in vitro culture to screen for genetically modified cells and plants.
pEXP:CKX2
Spring barley has been genetically modified by the CKX2 gene (At2g41500), encoding an Arabidopsis cytokinin dehydrogenase under the control of a root-specific promoter derived from an expressed protein gene (LOC_Os04g11040.1) of rice (Oryza sativa L. cv. Nipponbare). The Arabidopsis cytokinin oxidase / dehydrogenase 2 enzyme (CKX2) catalyzes the irreversible degradation of the hormone cytokinin in a single enzymatic step by oxidative side chain cleavage causing ectopic depletion of cytokinins in roots. In addition, the transgenic line has also integrated hpt selection gene coding for hygromycin phosphotransferase that confers resistance to antibiotic Hygromycin B. This antibiotic resistance has been used during in vitro culture to screen for genetically modified cells and plants.
bGLU:CKX1
Spring barley has been genetically modified by the CKX1 gene encoding the A. thaliana cytokinin dehydrogenase 1 under the control of the root-specific maize β-glucosidase promoter, which was synthesized according to the sequence deposited in the Genbank database under the number DQ333310. The Arabidopsis cytokinin oxidase / dehydrogenase 1 enzyme (CKX1) catalyzes the irreversible degradation of the cytokinin phytohormones in a single enzymatic step by oxidative side chain cleavage causing ectopic depletion of cytokinins in roots. In addition, the transgenic line has also integrated the hpt selection gene coding for the hygromycin phosphotransferase that confers resistance to the antibiotic Hygromycin B. This antibiotic resistance has been used during in vitro culture to screen for genetically modified cells and plants.
CKX1:RNAi
Spring barley has been genetically modified by construct CKX1:RNAi, harboring silencing cassette of barley cytokinin dehydrogenase gene 1 under the control of maize (Zea mays L.) constitutive ubiquitin promoter. HvCKX1 gene naturally occurs in the barley genome and its product catalyzes irreversible degradation of the cytokinin phyhormones in a single enzymatic step by oxidative side chain cleavage causing ectopic depletion of cytokinins in young roots and grains. In addition, the transgenic line has also integrated the hpt selection gene coding for the hygromycin phosphotransferase that confers resistance to the antibiotic Hygromycin B. This antibiotic resistance has been used during in vitro culture to screen for the genetically modified cells.


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:
pEXP:CKX1 barley was genetically modified by insertion of the Arabidopsis thaliana ecotype Columbia gene AT2G41510 coding for cytokinin dehydrogenase 1. The CKX1 gene was positioned under the control of a root-specific promoter. The root-specific promoter was derived from an expressed protein gene (LOC_Os04g11040.1) of rice (Oryza sativa L. cv. Nipponbare). Cytokinin dehydrogenases catalyze the irreversible degradation of cytokinins in a single enzymatic step by oxidative side chain cleavage. Cytokinins, which are chemically N6-substituted purine derivatives, are a class of plant hormones that regulate cell division as well as a large number of developmental events in plants. An important trait regulated by cytokinin is the size of the root system. A reduced cytokinin status in plants (including barley) causes an enhanced root system which might render plants more tolerant to drought and nutrient deficiencies in the soil. Crop yield is often limited by the availability of water and soil-derived mineral nutrients. A larger root system may enable plants to gain access to more water and nutrients and in this way to cope with adverse environmental conditions. The root-specific promoter from rice will restrict the expression of CKX gene to the roots and thus not affect shoot growth.
The transgenic line shows an up to 8-fold higher CKX enzyme activity in roots than the wild type. In contrast, CKX enzyme activity is similar to the control plants in leaves.
Genetic elements contained in the vectors:
pEXP:CKX unit
Root specific promoter: promoter of expressed protein gene (LOC_Os04g11040.1) from rice (Oryza sativa L. cv. Nipponbare)
CKX1 gene (AT2G41510) from Arabidopsis thaliana
Selection unit
ubi promoter: promoter region from Zea mays polyubiquitin gene (NCBI S94464.1)
hpt gene: Hygromycin phosphotransferase gene (hpt) from E. coli, which confers resistance to the antibiotic Hygromycin B
pEXP:CKX2 barley was genetically modified by insertion of the Arabidopsis thaliana ecotype Columbia gene AT2G41500 coding for cytokinin dehydrogenase 1. The CKX1 gene was positioned under the control of a root-specific promoter. The root-specific promoter was derived from an expressed protein gene (LOC_Os04g11040.1) of rice (Oryza sativa L. cv. Nipponbare). Cytokinin dehydrogenases catalyze the irreversible degradation of cytokinins in a single enzymatic step by oxidative side chain cleavage. Cytokinins, which are chemically N6-substituted purine derivatives, are a class of plant hormones that regulate cell division as well as a large number of developmental events in plants. An important trait regulated by cytokinin is the size of the root system. A reduced cytokinin status in plants (including barley) causes an enhanced root system which might render plants more tolerant to drought and nutrient deficiencies in the soil. Crop yield is often limited by the availability of water and soil-derived mineral nutrients. A larger root system may enable plants to gain access to more water and nutrients and in this way to cope with adverse environmental conditions. The root-specific promoter from rice will restrict the expression of CKX gene to the roots and thus not affect shoot growth.
The transgenic line shows an up to 8-fold higher CKX enzyme activity in roots than the wild type. In contrast, CKX enzyme activity is similar to the control plants in leaves.
Genetic elements contained in the vectors:
pEXP:CKX unit
Root specific promoter: promoter of expressed protein gene (LOC_Os04g11040.1) from rice (Oryza sativa L. cv. Nipponbare)
CKX2 gene (AT2G19500) from Arabidopsis thaliana
Selection unit
ubi promoter: promoter region from Zea mays polyubiquitin gene (NCBI S94464.1)
hpt gene: Hygromycin phosphotransferase gene (hpt) from E. coli, which confers resistance to the antibiotic hygromycin B
bGLU:CKX1 barley has been genetically modified by insertion of the Arabidopsis thaliana ecotype Columbia gene At2g41510 coding for cytokinin dehydrogenase 1 (NM_129714.3). CKX1 gene was positioned under the control of a root-specific promoter. The root-specific β-glucosidase promoter from maize (Zea mays) was synthesized according to the sequence deposited in Genbank database (DQ333310). Cytokinin dehydrogenases catalyze the irreversible degradation of cytokinins in a single enzymatic step by oxidative side chain cleavage. Cytokinins, which are chemically N6-substituted purine derivatives, are a class of plant hormones that regulate cell division as well as a large number of developmental events in plants. An important trait regulated by cytokinin is the size of the root system. A reduced cytokinin status in plants (including barley) causes an enhanced root system which might render plants more tolerant to drought and nutrient deficiencies in the soil. Crop yield is often limited by the availability of water and soil-derived mineral nutrients. A larger root system may enable plants to get better access to water and nutrient resources, therefore to better cope with adverse environmental conditions. The root-specific promoter from maize will restrict the expression of CKX1 gene to the roots and thus not affect shoot growth.
The transgenic line shows an up to 8-fold higher CKX enzyme activity in roots as compared to the wild type. In contrast, CKX enzymatic activity in the leaves of transgenic plants is similar to the control plants.
Genetic elements contained in the vectors:
bGLU:CKX1 unit
Root specific promoter: promoter of expressed protein gene (DQ333310) from maize (Zea mays) was synthesized according to the sequence deposited in Genbank database.
CKX1 gene (At2g41510; NM_129714.3) from Arabidopsis thaliana
Selection unit
35S promoter: 35S tobacco mosaic virus TMV promoter
hpt gene: Hygromycin phosphotransferase gene (hpt) from E. coli, which confers resistance to the antibiotic hygromycin B.
CKX1:RNAi
Spring barley (Hordeum vulgare L.) has been genetically modified by insertion of the silencing cassette CKX1:RNAi. Hordeum vulgare gene HvCKX1 coding for cytokinin dehydrogenase 1. Silencing cassette for HvCKX1 gene was positioned under the control of a constitutive promoter derived from maize ubiquitin gene. Cytokinin dehydrogenases catalyze the irreversible degradation of cytokinins in a single enzymatic step by oxidative side chain cleavage. Cytokinins, chemically N6-substituted purine derivatives, are a class of plant hormones that regulate cell division as well as a large number of developmental events in plants. Beside the others, cytokinins influence grain filling by regulation of the cell-wall invertase, the important enzyme in a polysacharides metabolism. Increased cytokinin levels in the grains might enhance the process of grain filling and lead to the production of bigger grains. As the HvCKX1 gene is naturally expressed in the young barley roots and grains, the constitutive expression should not influence the other parts of the plant.
The transgenic line showed an up 4-fold lower CKX activity in the barley grains compared to the wild type plants. The total yield from CKX1:RNAi barley was 20 % higher than the yield of wild type barley.
Genetic elements contained in the vectors:
CKX1:RNAi unit.
silencing cassette, Hordeum vulgare gene HvCKX1 coding for cytokinin dehydrogenase 1.
Selection unit
ubi promoter: promoter region from Zea mays polyubiquitin gene (NCBI S94464.1)
hpt gene: Hygromycin phosphotransferase gene (hpt) from E. coli, which confers resistance to the antibiotic Hygromycin B


6. Brief description of the method used for the genetic modification:
Zygotic immature embryos of spring barley cv. Golden Promise were transformed by Agrobacterium-mediated transformation. Infected embryos were cultivated on a callus inducing media under the selection pressure of Hygromycin B. Proliferated calli were transferred onto a shoot and later a root inducing media. Intact regenerated plants were transferred into the soil and grown till maturity to obtain T1 seeds.

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:
The purpose of the trialing program is to check the growth of the CKX modifications (pEXP:CKX1, pEXP:CKX2, bGLU:CKX1, CKX1:RNAi) under field conditions, compare yield parameters and tolerance to water deficit with non-modified barley.

2. Geographical location of the site:
Campus of the Faculty of Science, Palacky University, Olomouc – Holice, region Olomouc

3. Size of the site (m2):
The trial site does not exceed 1 500 m2, including the safety borders of non-modified barley.

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:
When grown in a GMO greenhouse, the plants showed typical cytokinin deficient phenotype with an increase in size of root system.

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
No risks to human or animal health or the environment from the deliberate release of genetically modified CKX barley: pEXP:CKX1, pEXP:CKX2, bGLU:CKX1, CKX1:RNAi, with higher activity of cytokinin dehydrogenase in roots, are expected as per information contained in the environmental risk assessment included in the notification.

Brief description of any measures taken for the management of risks:
Barley is a self-pollinated plant with cleistogamic flowers. The shedding of pollen from the genetically modified plants will be controlled by maintaining a 100-meter isolation distance from any other non-experimental barley crop. In addition, the trial site will be surrounded by a 2-meter isolation distance of conventional barley of a similar maturity that will be also destroyed at the end of the release.
Grains are fixed on a cob and enclosed in many husks that protect the seeds from outside contact. Thus, seed dispersal of individual kernels is not likely to occur.
No plant or plant product coming from the trials will enter the food or feed chain.
After the release, the plot will be regularly visited during the following year in order to ensure removal of barley volunteers, if any. Although volunteer barley cannot generally survive a hard winter, barley volunteers, if any, will be monitored in order to ensure their destruction.
There will be no commercial barley grown on the field the following year.


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
01/04/2015 00:00:00
Remarks: