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APPLICATION FOR LICENCE FOR INTENTIONAL RELEASE OF GMOs INTO THE
ENVIRONMENT: Application No. DIR 052/2004
SUMMARY INFORMATION
Project Title:
Field trial of genetically modified rice (Oryza sativa L.)
– functional characterisation of the rice genome
Applicant:
CSIRO
PO BOX 255
DICKSON ACT 2602
Common name of the parent organism:
Cultivated rice
Scientific name of the parent organism:
Oryza sativa L. cv Nipponbare
Modified traits:
A number of genes have been introduced into the rice
plants as selectable markers (ie. herbicide tolerance and
antibiotic resistance genes) and as visual markers (ie.
reporter genes enabling detection of gene expression via
staining or fluorescence).
In addition, other plant traits may be modified due to
insertion of the new genes disrupting the expression of
endogenous rice genes (such altered traits will be studied
to help identify rice gene function).
Identities of the genetic elements
responsible for the modified traits:
Selectable markers:
- bar gene (glufosinate ammonium herbicide
tolerance) from Streptomyces hygroscopicus
- hph gene (Hygromycin antibiotic resistance) from
Escherichia coli
- nptII gene (Kanamycin antibiotic resistance) from
bacterial Tn5 transposon
Visual markers:
- uidA gene (encoding GUS protein) from E. coli
- gfp gene (encoding Green Fluorescent Protein)
from a jellyfish, Aequorea victoria
- eyfp gene (encoding Enhanced Yellow Fluorescent
Protein) modified from the A. victoria gfp gene
Genetic elements used for inserting genes into the rice
genome:
- Transfer DNA (T-DNA) border regions from
Agrobacterium tumefaciens
- Ds transposable element border regions from Maize
(Zea mays L.)
Proposed Location
Local Government Area of Wagga Wagga, NSW
Proposed Release Size:
0.03 ha in each of three growing seasons within a four
year period.
October 2004 to May 2008
Proposed Time of Release
Address: MDP 54 PO Box 100 Woden ACT 2606 Website: www.ogtr.gov.au
Telephone: 1800 181 030 Facsimile: 02 6271 4202
Introduction
The Gene Technology Act 2000 (the Act) took effect on 21 June 2001. The Act, supported by
the Gene Technology Regulations 2001, an inter-governmental agreement and corresponding
legislation that is being enacted in each State and Territory, underpins Australia’s nationally
consistent regulatory system for gene technology. Its objective is to protect the health and
safety of people, and the environment, by identifying risks posed by or as a result of gene
technology, and managing those risks by regulating certain dealings with genetically modified
organisms (GMOs).
The Act establishes a statutory officer, the Gene Technology Regulator (the Regulator), to
administer the legislation and make decisions under the legislation. The Regulator is
supported by the Office of the Gene Technology Regulator (OGTR), an Australian
Government regulatory agency located within the Health and Ageing portfolio.
The legislation sets out the requirements for considering applications for licences for dealings
with GMOs and the matters that the Regulator must take into account before deciding
whether, or not, to issue a licence.
The application and the proposed dealings
The OGTR has received an application from CSIRO for a licence for the intentional release of
genetically modified (GM) rice (Oryza sativa L. cv Nipponbare) into the environment, on a
limited scale and under controlled conditions.
CSIRO proposes to carry out the release at one site in the local government area of Wagga
Wagga City Council, NSW over three growing seasons between October 2004 and May 2008,
including provision for one fallow season if required. However, the statutory timeframe for
consideration of the application extends until February 2005. Therefore if a licence were to be
issued it would be likely to cover the growing seasons between 2005 and 2009.
The aims of the proposed release are:
 to identify rice genes influencing traits of biological or agronomic interest by observing
alterations in the visible characteristics (phenotypes) of GM rice lines which were
generated under contained (laboratory and glasshouse) conditions; and
 to characterise gene flow in rice under Australian field conditions.
The proposed trial involves the planting of approximately 1500 different GM rice lines
(usually 30 plants of each line) containing randomly located insertions of T-DNA or Ds
elements carrying a combination of the selectable and visual marker genes listed in the
summary information above.
Herbicide tolerance and antibiotic resistance selectable marker genes enable the selection of
plants that have been successfully modified, because applying the herbicide or antibiotic
would kill any cell or plant without the gene. Visual marker genes enable the detection of
gene expression because they produce proteins that can be detected by staining or UV light.
The insertion of the T-DNA or Ds genetic elements into rice genes is likely to disrupt the
function of those genes. Disrupting a gene can alter the phenotype of a plant. Hence,
observing changes in the appearance of the plant can give an indication of the gene’s function
and whether it may be of biological or agronomic interest. As the rice genes into which the
new genetic elements have been inserted are ‘tagged’ both by these elements and their
associated marker genes, this facilitates the later identification and isolation of the altered
genes in the laboratory for further study.
In addition, some of the inserted gene constructs are designed to only express a visual marker
protein if they are inserted near an endogenous rice gene promoter or within a rice gene. The
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location of expression of the marker protein within these GM rice plants will reflect the
normal expression pattern of the tagged rice gene.
Field observations of traits such as flowering time and growth rate will be made throughout
the growing seasons. Seeds will be harvested at the end of each season to allow future
laboratory experiments on each line as required. None of the GM rice plants or their products
would be used for human or animal food.
For characterisation of gene flow, in the first season, the applicant proposes that
approximately 500 plants of one GM rice line will be surrounded by approximately 5000
non-GM rice plants in one plot within the trial. This will allow the applicant to generate data
on gene flow under Australian field conditions in order to establish the effective pollen trap
size, for application to potential future field trials (which would be subject to separate
evaluation and approval processes).
To minimise dissemination of the GM rice, CSIRO proposes to cover the entire site with
bird-proof netting, locate the site within a locked, double-fenced area and surround the site
with a 150 m isolation zone, in which no rice plants will be grown. In addition, the proposed
trial site is at least 85 km from the nearest commercial rice growing areas.
Previous releases of the GMO
There have been no previous releases of genetically modified rice in Australia. However other
crops expressing some of these genes have been released in Australia (for example: bar in
DIR 010/2001, DIR 015/2002, DIR 016/2002, DIR 021/2002, and DIR 038/2003; hph in
DIR 034/2003; nptII plus uidA in DIR 005/2001, DIR 006/2001, DIR 009/2001,
DIR 012/2002 and DIR 028/2002; gfp in DIR 019/2002 and DIR 031/2002). There have been
no reports of adverse effects on human health and safety or the environment resulting from
these releases. In addition, field trials of other genetically modified rice plants have been
approved in China, the Philippines, India, Mexico and the USA in recent years.
Parent organism
The parent organism is cultivated rice (Oryza sativa L.), which is exotic to Australia and is
grown as an agricultural crop in New South Wales and Victoria. The cultivar used in this
project, Nipponbare, originated in Japan where it is grown commercially. It is not a
commercial cultivar in Australia.
Genetic modifications and their effects
The GM rice lines proposed for release contain various combinations of selectable (bar, nptII
and/or hph) and visual (uidA, gfp and/or eyfp) marker genes. These have been inserted to
enable detection of the transformed plants and identification of the disrupted genes. Two
types of genetic elements were used to insert the marker genes into the GM rice lines:
 transfer DNA (T-DNA) border sequences from Agrobacterium tumefaciens (a common
soil bacterium), required for the generation of GM plants by Agrobacterium-mediated
transformation (see Method of genetic modification below); and
 Ds transposable element border sequences from maize, which, once present in the rice
genome, can be induced to move to new genomic locations. In the GM plants proposed
for release, the Ds transposable element is immobile (see Method of genetic
modification below);
A number of other genetic elements are also present in a variety of combinations in the
inserted gene constructs:
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 gene promoters controlling the expression of selectable or visual marker genes:
Cauliflower mosaic virus (CaMV) 35S promoter; manopine synthase 2’ (mas2’) promoter
from A. tumefaciens; and ubiquitin (Ubi1) promoter from maize. A promoter is a genetic
regulatory element controlling the level and location of the expression of an adjacent
gene;
 ‘transcriptional activator’ or ‘minimal promoter’ (TA) sequence derived from the CaMV
35S promoter, which interacts with adjacent rice gene promoters to drive expression of an
introduced visual marker gene;
 synthetic splice acceptor (SA) sequence, which interacts with other messenger RNA
(mRNA) processing signals of rice genes into which it is inserted, leading to expression of
an introduced visual marker gene;
 G protein gene intron from Arabidopsis thaliana, involved in mRNA processing, which
improves expression of the associated introduced gene; and
 transcription terminator sequences (including polyadenylation signals), involved in
mRNA processing and required for the correct expression of the inserted genes in rice
plants: nopaline synthase (nos) gene terminator and octopine synthase (ocs) gene
terminator from A. tumefaciens;
Two other genetic elements are present that will not be active in the GM plants but will
facilitate the subsequent laboratory isolation of the disrupted rice genes from the GM plants:
 ColE1 plasmid origin of replication from E. coli, allows plasmid DNA replication in
bacterial cell; and
 the bla (ampicillin resistance) gene from E. coli.
The net effect of the genetic modifications on rice gene disruption is analogous to that
achieved in conventional breeding programs utilising chemical mutation or radiation to
disrupt gene function. However, in the latter case the extent of the genetic changes, other than
expression of the trait of interest, is unknown. A key difference with this technique is that the
location and number of genetic changes can be precisely determined.
Field observations of the growth of GM plants containing these various genetic elements will
enable the function and expression patterns of previously uncharacterised rice genes to be
investigated. The introduced ‘tagging’ mechanism will then permit these genes to be isolated
for further study.
Method of genetic modification
The gene constructs (eight in total) located between T-DNA borders were initially introduced
into rice from standard plasmid vectors carried by Agrobacterium tumefaciens, using rice
callus tissue. Genetically modified rice cells were selected in the laboratory by using the
appropriate antibiotic or herbicide to which the inserted selectable marker gene conferred
resistance, and regenerated into whole plants. Polymerase chain reaction (PCR) assays were
used to confirm the presence of each of the introduced genes in the GM rice plants.
Some of the GM plants have undergone a further genetic modification step, utilising Ds
transposable element border sequences within the original T-DNA insertions. These Ds
border sequences can be induced to move from their original location to a new location within
the rice genome, carrying any inserted selectable or visual marker genes with them. This
movement is dependent upon the expression of a second maize transposable element
sequence, the Ac transposase gene. The Ac transposase gene was introduced into a separate
group of GM rice plants, which are not proposed for release. In contained laboratory or plant
house facilities, these Ac transposase GM rice plants were conventionally bred with some of
the T-DNA-containing GM rice lines described in the preceding sections. In the presence of
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the Ac transposase, the Ds elements (along with any intervening genes) become mobile.
Subsequent generations of plants were grown to achieve genetic separation (segregation) of
the Ac and Ds elements. It is important to note that only plant lines in which the Ac
transposase gene is absent are proposed for release. This ensures that the Ds element is
immobile in the GM plants that would be grown in the trial.
Consultation on preparation of the Risk Assessment and Risk Management
Plan
The Regulator has made an initial assessment as to whether the proposed release may pose
significant risks to human health and safety or the environment, in accordance with section 49
of the Act. Due to the low risk potential of the GMOs, the control measures that will be
imposed, and the limited scale of the dealings, the Regulator has decided that the proposed
release does not pose a significant risk to human health and safety or the environment.
This means that the Regulator is not required to seek public comment on the assessment of
this proposal until after a risk assessment and risk management plan (RARMP) has been
prepared. In the interim, copies of the application are available on request from the OGTR.
Please quote application number DIR 052/2004.
In preparing the RARMP, the Regulator will seek input from a wide range of key stakeholders
and expert groups comprising State and Territory Governments, relevant Australian
Government agencies, the Minister for the Environment and Heritage, the Gene Technology
Technical Advisory Committee and appropriate local councils, as required by section 50 of
the Act. In accordance with section 52 of the Act, the Regulator will again consult with these
prescribed agencies and authorities as well as the public in finalising the RARMP.
At this stage, the consultation version of the RARMP is expected to be issued for a six week
consultation period in October 2004. The public will be invited to provide submissions on the
RARMP via advertisements in the media and direct mail to anyone registered on the OGTR
mailing list. Summaries and copies of the RARMP will be available from the OGTR, or on
the OGTR website.
If you have any questions about the application or the assessment process, please contact the
OGTR at:
The Office of the Gene Technology Regulator
PO Box 100
WODEN ACT 2606
Tel: 1800 181 030
Fax: 02 6271 4202
Email: [email protected]
Website www.ogtr.gov.au
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