Download Risk assessment - Office of the Gene Technology Regulator

Risk Assessment and
Risk Management Plan for
DIR 070/2006
Limited and controlled release of GM sugarcane
with altered plant architecture, enhanced water or
improved nitrogen use efficiency
Applicant: BSES Limited
February 2007
PAGE INTENTIONALLY LEFT BLANK
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Executive Summary
Introduction
The Gene Technology Regulator (the Regulator) has made a decision to issue a licence for
dealings involving the intentional release of genetically modified (GM) sugarcane lines which
are modified for altered plant architecture, enhanced water use efficiency or improved
nitrogen use efficiency into the environment, in respect of application (DIR 070/2006) from
the BSES Limited.
The DIR070/2006 licence permits the release of up to 2500 genetically modified (GM)
sugarcane lines on a limited scale and under controlled conditions.
The Gene Technology Act 2000 (the Act) and the Gene Technology Regulations 2001 (the
Regulations) govern the process undertaken by the Regulator before a decision is made on
whether or not to issue a licence. The decision is based upon a Risk Assessment and Risk
Management Plan (RARMP) prepared by the Regulator in accordance with the Risk Analysis
Framework and in consultation with a wide range of experts, agencies and authorities, and the
public.
More information on the comprehensive assessment required for licence applications to
release a genetically modified organism (GMO) into the environment is available from the
Office of the Gene Technology Regulator (OGTR) (Free call 1800 181 030) or at
<http://www.ogtr.gov.au/>.
The application
BSES applied for a licence to release up to 2500 GM sugarcane lines into the environment
under limited and controlled conditions. Up to 1900 of the GM sugarcane lines have been
modified to alter plant size and shape, enhance water use efficiency (WUE)1, or improve
nitrogen use efficiency (NUE)2. The remaining 600 GM sugarcane lines contain only
introduced selectable and/or visual marker genes. All of the introduced genes are derived
from plants (rice, sugarcane, barley, bean, thale cress, apple or maize) or the common gut
bacterium Escherichia coli.
The trial is to take place at up to 3 sites of no more than 2 ha during each of the 3 cropping
cycles between February 2007 to November 2010 (ie a total maximum area for the trial of
18 ha). The release may take place in the Queensland local government areas of Bundaberg,
Caboolture and/or Cairns.
The trial involves early stage ‘proof of concept’ research. The GM sugarcane lines containing
only marker genes would be used to compare different genetic modification methods. The
other introduced genes are intended to alter plant size and shape, enhance WUE or improve
NUE by regulating different biochemical pathways that may result in increased sugar yield.
The agronomic performance of GM sugarcane lines containing these genes will be evaluated
in the field; some under different irrigation and fertilizer treatments. No GM plant material
from the release will be used for human food, animal feed or for the production of other
sugarcane commodities.
BSES has proposed a number of measures to limit the spread and persistence of the GM
sugarcane lines and the introduced genetic materials that were considered during the
evaluation of the application.
1
2
WUE is defined as the measure of total yield (sugar) produced per unit of water supplied to the sugarcane crop.
NUE is a term used to describe how effectively plants acquire and utilise nitrogen.
Executive Summary (February 2007)
I
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Risk assessment
The hazard identification process considered the circumstances by which people or the
environment may be exposed to the GMOs, GM plant materials, GM plant by-products, the
introduced genes, or products of the introduced genes.
A hazard (source of potential harm) may be an event, substance or organism. A risk is
identified when a hazard is considered to have some chance of causing harm. Those events
that do not lead to an adverse outcome, or could not reasonably occur, do not advance in the
risk assessment process.
Sixteen events were identified and assessed whereby the release of the GM sugarcane lines
might give rise to harm to people or the environment.
These 16 events included consideration of whether, or not, expression of the introduced genes
could result in products that are toxic or allergenic to people or other organisms, alter
characteristics that may impact on the spread and persistence of the GM plants, or produce
unintended changes in their biochemistry or physiology. In addition, consideration was given
to the opportunity for gene flow to other organisms, and its effects if this occurred.
All events were characterised in relation to both the magnitude and probability of harm in the
context of the controls proposed by the applicant to limit the spread and persistence of the
GMOs in both time and space. This detailed consideration concluded that none of the sixteen
events gave rise to an identified risk that required further assessment. The principle reasons
comprise:
 small scale of the trial is limited in both area and duration
 suitability of containment and disposal measures to limit the spread and persistence of
the GM plants
 none of the GM plant materials will be used for any other purpose
 widespread presence of the same or similar proteins and enzymatic products in the
environment and lack of evidence of harm from these proteins and their products
 the lack of known toxicity or allergenicity of the proteins (and enzymatic products)
encoded by the introduced genes
 limited capacity of the GM sugarcane lines to spread and persist outside the areas of the
release
 limited ability and opportunity for the GM sugarcane lines to transfer the introduced
genes to commercial sugarcane crops or other sexually compatible species.
Therefore, any risks of harm to the health and safety of people, or the environment, from the
release of the GM sugarcane lines into the environment are considered to be negligible.
Risk management
The risk management process builds upon the risk assessment to determine whether measures
are required in order to protect people and/or the environment. As none of the 16 events
characterised in the risk assessment are considered to give rise to an identified risk that
requires further assessment, the level of risk is considered to be negligible.
The Regulator’s Risk Analysis Framework defines negligible risks as insubstantial, with no
present need to invoke actions for their mitigation. However, containment and disposal
measures have been imposed to restrict the release to the locations, size and duration
Executive Summary (February 2007)
II
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
requested by the applicant, as these were an important part of establishing the context for
assessing the risks.
The licence conditions require the applicant to limit the duration of the release to between
February 2007 and November 2010 on a maximum total area of up to 18 hectares; prevent the
use of the GMOs, or materials from the GMOs for any other purposes; maintain physical
isolation of the release sites; and conduct post-harvest monitoring to ensure all GM plants are
destroyed3.
Conclusions of the RARMP
The risk assessment concludes that this limited and controlled release of up to 2500 GM
sugarcane lines into the areas proposed in Queensland poses negligible risks to the health and
safety of people and the environment posed by, or as a result of, gene technology.
The risk management plan concludes that these negligible risks do not require specific risk
treatment measures. However, licence conditions have been imposed to contain the release to
the locations, size and duration requested by the applicant.
3
The licence for DIR 070/2006 is available on the OGTR website
(<http://www.ogtr.gov.au/gmorec/ir.htm#table> via the link to DIR 070/2006).
Executive Summary (February 2007)
III
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
PAGE INTENTIONALLY LEFT BLANK
Executive Summary (February 2007)
IV
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Table of Contents
EXECUTIVE SUMMARY .................................................................................................................................... I
INTRODUCTION .................................................................................................................................................... I
THE APPLICATION ................................................................................................................................................ I
RISK ASSESSMENT ............................................................................................................................................... II
RISK MANAGEMENT ............................................................................................................................................ II
CONCLUSIONS OF THE RARMP ........................................................................................................................ III
TABLE OF CONTENTS ......................................................................................................................................V
ABBREVIATIONS ........................................................................................................................................... VII
TECHNICAL SUMMARY .................................................................................................................................. 1
INTRODUCTION ................................................................................................................................................... 1
APPLICATION ...................................................................................................................................................... 1
RISK ASSESSMENT ............................................................................................................................................... 3
RISK MANAGEMENT ............................................................................................................................................ 4
LICENCE CONDITIONS TO MANAGE THIS LIMITED AND CONTROLLED RELEASE .................................................... 4
OTHER REGULATORY CONSIDERATIONS .............................................................................................................. 4
IDENTIFICATION OF ISSUES TO BE ADDRESSED FOR FUTURE RELEASES ................................................................ 5
CONCLUSIONS OF THE RARMP .......................................................................................................................... 5
CHAPTER 1
RISK ASSESSMENT CONTEXT .......................................................................................... 7
SECTION 1
SECTION 2
2.1
2.2
2.3
SECTION 3
SECTION 4
4.1
4.2
4.3
4.4
4.5
SECTION 5
5.1
5.2
5.3
5.4
SECTION 6
6.1
6.2
BACKGROUND ............................................................................................................................. 7
THE APPLICATION........................................................................................................................ 8
The proposed locations, size and duration of the trial ..................................................................... 8
The proposed dealings ..................................................................................................................... 8
The proposed measures to limit the spread and persistence of the GMOs ...................................... 9
THE PARENT ORGANISM .............................................................................................................. 9
THE GMOS, NATURE AND EFFECT OF THE GENETIC MODIFICATION ............................................ 9
Introduction to the GMOs ............................................................................................................... 9
Introduction to plant architecture, drought stress and nitrogen use efficiency .............................. 11
The introduced genes and regulatory sequences, and the gene products....................................... 13
Method of genetic modification .................................................................................................... 21
Characterisation of the GMOs ....................................................................................................... 22
THE RECEIVING ENVIRONMENT ................................................................................................. 23
Relevant abiotic factors ................................................................................................................. 23
Relevant agricultural practices ...................................................................................................... 23
Presence of related plants in the receiving environment ............................................................... 24
Presence of the introduced genes or similar genes in the environment ......................................... 25
AUSTRALIAN AND INTERNATIONAL APPROVALS ....................................................................... 25
Australian approvals of the GM sugarcane lines ........................................................................... 25
International approvals .................................................................................................................. 26
CHAPTER 2
RISK ASSESSMENT ............................................................................................................. 27
SECTION 1
SECTION 2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
SECTION 3
INTRODUCTION.......................................................................................................................... 27
HAZARD CHARACTERISATION ................................................................................................... 28
Production of a substance toxic to people ..................................................................................... 32
Production of a substance allergenic to people ............................................................................. 34
Production of a substance toxic to organisms other than people ................................................... 36
Spread and persistence of the GM sugarcane in the environment ................................................. 37
Vertical transfer of genes or genetic elements to sexually compatible plants ............................... 44
Horizontal transfer of genes or genetic elements to sexually incompatible organisms. ................ 47
Unintended changes in biochemistry, physiology or ecology ....................................................... 48
Unauthorised activities .................................................................................................................. 50
RISK ESTIMATE PROCESS FOR IDENTIFIED RISKS ........................................................................ 50
CHAPTER 3
RISK MANAGEMENT ......................................................................................................... 51
Table of Contents (February 2007)
V
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
SECTION 1
BACKGROUND ........................................................................................................................... 51
SECTION 2
OTHER AUSTRALIAN REGULATORS ........................................................................................... 51
SECTION 3
RISK TREATMENT MEASURES FOR IDENTIFIED RISKS ................................................................. 52
SECTION 4
GENERAL RISK MANAGEMENT ................................................................................................... 52
4.1
Summary of proposed licence conditions ...................................................................................... 52
4.2
Other risk management considerations ......................................................................................... 53
SECTION 5
ISSUES TO BE ADDRESSED FOR FUTURE RELEASES ..................................................................... 54
SECTION 6
CONCLUSIONS OF THE RARMP................................................................................................. 55
SECTION 7
DIR 070/2006 LICENCE............................................................................................................. 55
REFERENCES .................................................................................................................................................... 56
APPENDIX A
DEFINITIONS OF TERMS IN THE RISK ANALYSIS FRAMEWORK USED BY
THE REGULATOR ........................................................................................................................................... 69
APPENDIX B
SUMMARY OF ISSUES RAISED IN SUBMISSIONS RECEIVED FROM
PRESCRIBED EXPERTS, AGENCIES AND AUTHORITIES ON APPLICATION DIR 070/2006 ........ 71
APPENDIX C
SUMMARY OF ISSUES RAISED IN SUBMISSIONS RECEIVED FROM THE
PUBLIC ON APPLICATION DIR 070/2006 ................................................................................................... 72
Table of Contents (February 2007)
VI
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Abbreviations
ai
APVMA
ADP
ATP
AQIS
C-terminal
DIR
DNA
dsRNA
EMBL
FAO
FARRP
FSANZ
g
GA
GM
GMAC
GMO
GTTAC
ha
kDa
kg
km
LD50
m
mg
mRNA
μg
N/A
ng
NHMRC
NICNAS
NUE
OECD
OGTR
PC2
PCR
RARMP
RNA
SDAP
T-DNA
TGA
US FDA
USDA
WUE
Active ingredient
Australian Pesticides and Veterinary Medicines Authority
Adenosine diphosphate
Adenosine triphosphate
Australian Quarantine and Inspection Service
Carboxy-terminal end of a protein
Dealing involving Intentional Release
Deoxyribonucleic acid
Double stranded RNA
European Molecular Biology Laboratory
Food and Agricultural Organization of the United Nations
Food Allergy Research and Resource Program
Food Standards Australia New Zealand (formerly ANZFA)
Gram
Gibberellic acid
Genetically Modified
Genetic Manipulation Advisory Committee
Genetically Modified Organism
Gene Technology Technical Advisory Committee
Hectare
Kilodalton
Kilogram
Kilometre
Amount of a substance given in a single dose that causes death in
50% of a test population of an organism
Metre
milligram
Messenger ribonucleic acid
Microgram
Not applicable
Nanogram
National Health and Medical Research Council
National Industrial Chemicals Notification and Assessment Scheme
Nitrogen use efficiency
Organisation for Economic Co-Operation and Development
Office of the Gene Technology Regulator
Physical containment level 2
Polymerase chain reaction
Risk Assessment and Management Plan
Ribonucleic acid
Structural database of allergenic proteins
Transfer deoxyribonucleic acid
Therapeutic Goods Administration
United States Food and Drug Administration
United States Department of Agriculture
Water use efficiency
Abbreviations (February 2007)
VII
DIR 70/2006 – Risk Assessment and Risk Management Plan
WHO
Office of the Gene Technology Regulator
World Health Organisation
Abbreviations (February 2007)
VIII
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Technical Summary
Introduction
The Gene Technology Regulator (the Regulator) has decided to issue a licence
(DIR 070/2006) to BSES Limited for dealings involving the intentional release of genetically
modified (GM) sugarcane lines into the environment, on a limited scale and under controlled
conditions.
The DIR 070/2006 licence permits the limited and controlled release of up to 2500 GM
sugarcane lines. The release will occur at up to 3 sites per cropping cycle between February
2007 to November 2010 in the local government areas of Bundaberg, Caboolture and/or
Cairns, Queensland. Each site would be a maximum 2 ha in area, with a total maximum area
for the trial of 18 ha.
The Gene Technology Act 2000 (the Act), the Gene Technology Regulations 2001 (the
Regulations) and corresponding State and Territory law govern the comprehensive and highly
consultative process undertaken by the Regulator before making a decision whether to issue a
licence to deal with a GMO.
The Regulator’s Risk Analysis Framework explains the approach used to evaluate licence
applications and to develop the Risk Assessment and Risk Management Plans (RARMPs) that
form the basis of her decisions4.
This RARMP for DIR 070/2006 has been finalised in accordance with the gene technology
legislation. Matters raised in the consultation process regarding risks to the health and safety
of people and the environment from the proposed dealings were taken into account by the
Regulator in deciding to issue a licence and the licence conditions that have been imposed.
Application
Title:
Applicant:
Common name of the parent organism:
Scientific name of the parent organism:
Modified traits:
Identity of the genes responsible for the
modified traits:
Proposed locations:
Proposed release size:
Proposed time of release:
Limited and controlled release of GM sugarcane with altered plant architecture,
enhanced water or improved nitrogen use efficiency*
BSES Limited
Sugarcane
Saccharum spp. hybrid
Plant architecture (shoot number, stalk size and height), water use efficiency,
nitrogen use efficiency, and marker gene expression (antibiotic resistance and
reporter genes)
 14 genes for altered plant architecture from the plants Hordeum vulgare
subsp. vulgare, Oryza sativa, Phaseolus coccineus and Saccharum spp.
 3 genes for enhanced water use efficiency from the bacterium Escherichia
coli, and plants Arabidopsis thaliana, Malus x domestica.
 1 gene for improved nitrogen use efficiency from Zea mays
 uidA gene (-glucuronidase reporter gene) from the bacterium E. coli
 nptII gene (antibiotic resistance selectable marker) from the bacterium E. coli
 bla gene (antibiotic resistance selectable marker) from the bacterium E. coli.
Local government areas of Bundaberg, Caboolture and/or Cairns (Qld)
Maximum total area 18 ha, comprising up to 3 sites of no more than 2 ha per
cropping cycle
February 2007 to November 2010
* The original title of licence application submitted by BSES was GM sugarcane field trial – testing the effect on
sugar yield of transformation methods.
4
More information on the assessment of licence applications and copies of the Risk Analysis Framework are
available from the Office of the Gene Technology Regulator (OGTR). Free call 1800 181 030 or at
<http://www.ogtr.gov.au/ir/process.htm> and <http://www.ogtr.gov.au/pdf/public/raffinal2.2.pdf>, respectively.
Technical Summary (February 2007)
1
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
BSES applied for a licence to release up to 2500 GM sugarcane lines into the environment
under limited and controlled conditions. Up to 1900 of the GM sugarcane lines have been
modified to alter plant architecture (shoot number, stalk size and height), enhance water use
efficiency (WUE)5, or improve nitrogen use efficiency (NUE)6. Up to 600 of the GM
sugarcane lines contain only introduced selectable antibiotic resistance marker and/or visual
marker genes. The trial is intended to take place at up to 3 sites per cropping cycle between
February 2007 to November 2010 in the local government areas of Bundaberg, Caboolture
and/or Cairns, Queensland. Each site would be a maximum 2 ha in area, with a total
maximum area for the trial of 18 ha.
The GM sugarcane lines for release were derived by transforming plants of the commercially
grown Saccharum spp. hybrid Q117. Up to 1900 of the GM sugarcane lines contain either
individual or combinations of 18 different introduced genes intended to improve agronomic
characteristics and sucrose yields by either altering plant architecture, or enhancing WUE or
improving NUE.
The 14 genes for altered plant architecture are derived from the plants Oryza sativa (rice),
Saccharum spp. (sugarcane), Hordeum vulgare subsp. vulgare (barley) and Phaseolus
coccineus (bean). The 3 genes for enhanced WUE are derived from the plants Arabidopsis
thaliana (thale cress), Malus x domestica (apple) and the gut bacterium Escherichia coli. The
one gene for enhanced NUE is derived from Zea mays (maize). The introduced genes encode
proteins or double-stranded RNAs (dsRNAs) that are intended to alter plant architecture,
enhance WUE or improve NUE, by modulating biochemical pathways, either through direct
participation (proteins) or regulation of endogenous gene expression (dsRNA) in the
sugarcane plants, which in turn may result in increased sugar yield.
The GM sugarcane lines modified to alter plant architecture, enhance WUE or improve NUE
also contain the E. coli derived nptII marker gene, which confers resistance to some
aminoglycoside antibiotics (eg neomycin, paromomycin, geneticin). The nptII gene enabled
the identification and selection of GM plant tissues during the initial development of the GM
sugarcane lines in the laboratory.
All except 100 of the 1900 lines contain the E.coli bla gene (conferring ampicillin resistance).
However, this gene is not expressed in the GM sugarcane lines as it is linked to a bacterial
promoter that does not function in plants. The bla gene was used to select bacteria carrying
the gene construct of interest that were used in the initial plant transformations.
In addition to these 1900 GM sugarcane lines, up to 600 more lines contain only the nptII
gene (100 lines), or the nptII gene with the bla bacterial marker gene (100 lines), or the nptII
gene with the visual reporter gene uidA (400 lines). These lines will be compared during the
trial to determine the best transformation technique for generating GM sugarcane lines.
The purpose of the trial is to conduct early stage (‘proof of concept’) research to assess the
agronomic performance of GM sugarcane lines in the field, including some under different
irrigation and fertilizer treatments, and to collect data to assist in optimising the
transformation process.
The applicant proposed measures to limit the spread and persistence of the GM sugarcane
lines in the environment. These were taken into account in establishing the risk assessment
context for the release, and their suitability for limiting the release to the locations, size and
duration proposed by the applicant was considered as part of the risk assessment process. No
5
6
WUE is defined as the measure of total yield (sugar) produced per unit of water supplied to the sugarcane crop.
NUE is a term used to describe how effectively plants acquire and utilise nitrogen.
Technical Summary (February 2007)
2
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
material from the GM sugarcane plants will be used for human food, animal feed or other
sugarcane products.
Risk assessment
The risk assessment considered information contained in the application, current scientific
knowledge, and issues relating to risks to human health and safety and the environment raised
in submissions received from consultation with a wide range of prescribed experts, agencies
and authorities on the application (summarised in Appendix B of the RARMP). No new risks
to people or the environment were identified from the advice received on the consultation
RARMP. However, feedback on some previously raised issues has enabled their clarification
in the final RARMP.
Two submissions from the public on the application and how they were considered are
summarised in Appendix C of the RARMP. No submissions were received from the public on
the consultation RARMP.
A reference document, The Biology and Ecology of Sugarcane (Saccharum spp. hybrids) in
Australia, was produced to inform the risk assessment process for licence applications
involving GM sugarcane plants. The document is available from the OGTR or from the
website <http://www.ogtr.gov.au>.
The hazard identification process considered the circumstances or events by which people or
the environment may be adversely affected by exposure to the GMOs, GM plant materials,
GM plant by-products, the introduced genes, or products of the introduced genes.
A hazard (source of potential harm) may be an event, substance or organism. A risk is
identified when a hazard is considered to have some chance of causing harm. Those events
that do not lead to an adverse outcome, or could not reasonably occur, do not advance in the
risk assessment process.
Sixteen events were identified and assessed whereby the release of the GM sugarcane lines
might give rise to harm to people or the environment.
These 16 events included consideration of whether expression of the introduced genes could
result in products that are toxic or allergenic to people or other organisms, alter characteristics
that may impact on the spread and persistence of the GM plants, or produce unintended
changes in their biochemistry or physiology. In addition, consideration was given to the
opportunity for gene flow to other organisms, and its effects if this occurred.
All events were characterised in relation to both the magnitude and probability of harm in the
context of the controls proposed by the applicant to limit the spread and persistence of the
GMOs in both time and space. This detailed consideration concluded that none of the sixteen
events gave rise to an identified risk that required further assessment. The principle reasons
comprise:
 small scale of the trial is limited in both area and duration
 suitability of containment and disposal measures to limit the spread and persistence of
the GM plants
 none of the GM plant materials will be used for any other purpose
 widespread presence of the same or similar proteins and enzymatic products in the
environment and lack of evidence of harm from these proteins and their products
 the lack of known toxicity or allergenicity of the proteins (and enzymatic products)
encoded by the introduced genes
Technical Summary (February 2007)
3
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
 limited capacity of the GM sugarcane lines to spread and persist outside the areas for
release
 limited ability and opportunity for the GM sugarcane lines to transfer the introduced
genes to commercial sugarcane crops or other sexually compatible species.
Therefore, as no risks to the health and safety of people, or the environment were identified
from the limited and controlled release of the GM sugarcane lines into the environment, the
level of risk is considered to be negligible.
Risk management
A risk management plan builds upon the risk assessment to consider whether any action is
required to mitigate the identified risks, and what can be done to protect the health and safety
of people and the environment.
As none of the 16 events that were characterised in the risk assessment process are considered
to give rise to an identified risk that requires further assessment, the level of risk to human
health and safety and the environment from the release of GM sugarcane lines is considered
to be negligible.
The Regulator’s Risk Analysis Framework defines negligible risks as insubstantial with no
present need to invoke actions for their mitigation. However, containment measures have
been imposed to restrict the release to the locations, size and duration requested by the
applicant, as these were important parameters in establishing the context for assessing the
risks.
Licence conditions to manage this limited and controlled release
A number of licence conditions have been imposed to limit and control the release, including
requirements to:
 surround the trial sites by one guard row of non-GM sugarcane and an isolation zone of
at least 6 metres
 locate the trial sites at least 50 m away from natural waterways
 harvest and process sugarcane from the trial separately from any commercial sugarcane
 destroy all plant materials not required for experimentation or new plantings
 following cleaning of sites, monitor for and destroy any GM sugarcane that may grow
for at least 12 months until the site is clear of volunteers for a continuous 6 month
period.
Other regulatory considerations
Australia’s gene technology regulatory system operates as part of an integrated legislative
framework. The Regulator sought input on the preparation of the RARMP from other
agencies that also regulate GMOs or GM products including Food Standards Australia New
Zealand (FSANZ), Australian Pesticides and Veterinary Medicines Authority (APVMA),
Therapeutic Goods Administration (TGA), National Industrial Chemicals Notification and
Assessment Scheme (NICNAS), National Health and Medical Research Council (NHMRC)
Technical Summary (February 2007)
4
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
and Australian Quarantine Inspection Service (AQIS). Dealings conducted under a licence
issued by the Regulator may also be subject to regulation by one or more of these agencies7.
FSANZ is responsible for human food safety assessment, including GM food. As the trial
involves early stage research the applicant does not intend any material from the GM
sugarcane lines to be used in human food. Accordingly the applicant has not applied to
FSANZ to evaluate any of the GM sugarcane lines. FSANZ approval would need to be
obtained before they could be used in human food.
Identification of issues to be addressed for future releases
The risk assessment identified additional information that may be required to assess an
application for a larger scale trial, reduced containment conditions or commercial release of
any of these GM sugarcane lines. These include:
 molecular characterisation of GM sugarcane lines selected for possible future releases
 additional data on the potential toxicity or allergenicity of proteins encoded by the
introduced genes for altered plant architecture, enhanced WUE and improved NUE, and
of plant materials from the GM sugarcane lines selected for further research
 biochemical, physiological and agronomic characteristics indicative of weediness in the
selected GM sugarcane lines including measurement of tolerance to environmental
stresses and reproductive capacity.
Conclusions of the RARMP
The risk assessment concludes that this proposed limited and controlled release of up to 2500
GM sugarcane lines into the local government areas of Bundaberg, Caboolture and/or Cairns
in Queensland poses negligible risks to the health and safety of people and the environment
posed by, or as a result of, gene technology.
The risk management plan concludes that these negligible risks do not require specific risk
treatment measures. However, licence conditions have been imposed to limit the release to the
locations, size and duration requested by the applicant, as these were important parameters in
establishing the context for assessing the risks.
More information on Australia’s integrated regulatory framework for gene technology is contained in the Risk
Analysis Framework available from the Office of the Gene Technology Regulator (OGTR). Free call 1800 181
030 or at <http://www.ogtr.gov.au/pdf/public/raffinal2.2.pdf>.
7
Technical Summary (February 2007)
5
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
PAGE INTENTIONALLY LEFT BLANK
Technical Summary (February 2007)
6
DIR 70/2006 – Risk Assessment and Risk Management Plan
Chapter 1
Section 1
Office of the Gene Technology Regulator
Risk assessment context
Background
1.
This chapter describes the parameters within which risks that may be posed to the health
and safety of people and the environment by the proposed release are assessed. These include
the scope and boundaries for the evaluation process required by the gene technology
legislation8, details of the intended dealings, the GMO(s) and parent organism(s), previous
approvals and releases of the same or similar GMOs in Australia or overseas, environmental
considerations and relevant agricultural practices. The parameters for the risk assessment
context are summarised in Figure 1.
Figure 1
Components of the risk context considered during the preparation of the Risk Assessment
RISK ASSESSMENT CONTEXT
LEGISLATIVE REQUIREMENTS
Gene Technology Act and Regulations
DEALINGS
Activities involving the GMO
Location, size and duration of release
Proposed containment measures
GMO
Introduced genes (genotype)
Novel traits (phenotype)
PARENT ORGANISM
RECEIVING ENVIRONMENT
Environmental conditions
Agronomic practices
Sexually compatible relatives
Presence of similar genes
PREVIOUS RELEASES
2.
Sections 49 to 51 of the Gene Technology Act 2000 (the Act) outline the matters which
the Regulator must take into account, and who she must consult with, in preparing the
RARMPs that form the basis of her decision on licence applications.
3.
For this application, establishing the risk assessment context includes consideration of:
 the locations, size and duration of the trial requested by the applicant
 proposed dealings
 containment measures for the GMOs proposed by the applicant
 characteristics of the parent organism
 the nature and effect of the genetic modifications
 the environmental conditions in the location(s) where the release would occur
 relevant agricultural practices
 presence of related plants in the environment
 presence of the introduced or similar genes in the environment
8
The legislative requirements and the approach taken in assessing licence applications are outlined in more
detail at <http://www.ogtr.gov.au/ir/process.htm> and in the Risk Analysis Framework (OGTR 2005)
<http://www.gov.au/pdf/raffinal2.2pdf>.
Chapter 1 - Risk assessment context (February 2007)
7
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
 any previous releases of these or other GMOs relevant to this application.
4.
Initial consideration of the application under section 49 of the Act determined that
public consultation was not required for the preparation of the consultation version of the
RARMP. In accordance with section 50 of the Act, the Gene Technology Technical Advisory
Committee (GTTAC), State and Territory governments, prescribed Australian Government
agencies, the Minister for Environment and Heritage and the local councils where the release
may take place were consulted on matters relevant to the preparation of the RARMP. This
advice, and where it was taken into account in the RARMP, is summarised in Appendix B.
Two submissions from the public were also received. This advice and its consideration is
summarised in Appendix C.
5.
In accordance with section 52 of the Act, the Regulator notified the public when the
consultation version of the RARMP had been prepared and invited written submissions. No
submissions were received from the public on the consultation RARMP. Advice on the
RARMP was also sought from the same experts, agencies and authorities as before. None of
the latter raised any new issues relating to risks to human health and safety and the
environment that required further consideration. However, feedback on some previously
considered issues has enabled their clarification in the final RARMP.
Section 2
The application
6.
BSES proposes to release up to 2500 GM sugarcane lines into the environment under
limited and controlled conditions. The GM sugarcane lines have been modified to alter plant
architecture (shoot number, stalk size and height), enhance water use efficiency (WUE)9 or
improve nitrogen use efficiency (NUE)10.
2.1 The proposed locations, size and duration of the trial
7.
The trial is proposed to take place at up to 3 sites per cropping cycle from February
2007 to November 2010 in the local government areas of Bundaberg, Caboolture and/or
Cairns, Queensland. Each site would be a maximum of 2 ha, with a total maximum area for
the trial of 18 ha.
2.2 The proposed dealings
8.
The purpose of the trial is to conduct early stage (‘proof of concept’) research to assess
the agronomic characteristics of the GM sugarcane lines and to compare different plant
transformation procedures used to generate the GM sugarcane lines.
9.
The applicant proposes to assess the performance of the GM sugarcane lines in order to
determine whether any may warrant further development. Standard sugarcane industry
growing practices will be applied, including harvesting the GM sugarcane before flowering to
maximise sugar content (flowering reduces potential sugar yields significantly). The WUE
and NUE lines will be subject to different irrigation treatments and fertilizer applications
(nitrogen), respectively. Phenotypic measurements of the plants would be made periodically
during their growth (including stalk number and structure, sugar and fibre content, and leaf
morphology) and plant materials would be analysed after harvest (including stalk weight and
sugar yield). Some biochemical and molecular analyses of the plant materials would also be
made. No products from the release would be used for human food, animal feed or other
sugarcane products.
9
WUE can be defined as the measure of total yield (sugar) produced per unit of water supplied to the sugarcane
crop.
10
NUE is a term used to describe how effectively plants acquire and utilise nitrogen.
Chapter 1 - Risk assessment context (February 2007)
8
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
2.3 The proposed measures to limit the spread and persistence of the GMOs
10. The applicant proposes measures to limit the spread and persistence of the GM
sugarcane lines into the environment. These are taken into account in the risk assessment
context (this chapter) and their suitability is evaluated in the following chapter for limiting the
release to the locations, size and duration proposed by the applicant.
11.
The applicant proposes the following containment measures:
 surround the trial sites by one guard row of non-GM sugarcane and an isolation zone of
at least 6 metres
 locate the trial sites at least 50 m away from natural waterways
 harvest and process sugarcane from the trial separately from any other commercial
sugarcane
 analyse plant materials at the trial site or in a PC2 laboratory
 destroy all plant materials not required for experimentation or propagation
 following cleaning of sites, monitor for and destroy any GM sugarcane that may grow
for 12 months and thereafter until the site is free of volunteers for a continuous 6 month
period
 transport of GM plant materials in accordance with OGTR transportation guidelines.
Section 3
The parent organism
12. The parent organism is the sugarcane cultivar Q117, which resulted from a cross
between cultivars Q77 and Q58-829 (Saccharum spp. hybrids) from a BSES breeding
program. Because of domestic quarantine restrictions, this cultivar is only approved for
commercial planting and ratooning north of Rockhampton (information supplied by the
applicant). More detailed information on sugarcane can be found in the document, The
Biology and Ecology of Sugarcane (Saccharum spp. hybrids) in Australia, which was
produced to inform the risk assessment process for licence applications involving GM
sugarcane plants (OGTR 2002). This document is available at <http://www.ogtr.gov.au>.
Section 4
The GMOs, nature and effect of the genetic
modification
4.1 Introduction to the GMOs
13.
Up to 2500 GM sugarcane lines are proposed for the trial.
14. Up to 1900 GM sugarcane lines with altered plant architecture, enhanced WUE or
improved NUE are proposed for release, each line generated using 1 of 19 different
transformation vectors (see Table 1). Each transformation vector was used to make up to 100
GM sugarcane lines. Each line contains up to four introduced partial (in both sense and antisense orientations for silencing of endogenous genes) or complete gene sequences for the
above traits. Seventeen of the introduced genes are derived from the plants Oryza sativa
(rice), Saccharum spp. (sugarcane), Hordeum vulgare subsp. vulgare (barley), Phaseolus
coccineus (bean), Arabidopsis thaliana (thale cress), Malus x domestica (apple) and Zea mays
(maize). One gene is derived from the common gut bacterium Escherichia coli. Details of the
genes are provided in Section 4.3.
15. All of the 1900 GM sugarcane lines with altered plant architecture, enhanced WUE or
improved NUE also contain the selectable antibiotic resistance marker gene nptII, which
Chapter 1 - Risk assessment context (February 2007)
9
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
encodes neomycin transferase (NPTII), derived from E. coli. The nptII gene confers resistance
to some aminoglycoside antibiotics (eg neomycin, paromomycin, geneticin) and enabled the
identification and selection of GM plant tissue during the initial development of the GM
sugarcane lines in the laboratory.
16. All of the GM sugarcane lines with altered plant architecture, enhanced WUE or
improved NUE also contain the E. coli derived bla gene (except those made using the
transformation vector 15, see Table 1), which confers ampicillin resistance to bacteria. This
bacterial marker gene is not expressed in the GM sugarcane lines as it is linked to a bacterial
promoter that does not function in plants. The bla gene was used to select bacteria carrying
the gene construct of interest that were used in the initial plant transformation.
17. An additional 600 GM sugarcane lines, each made using 1 of 3 different gene
constructs, contain only the nptII gene (100 lines), or the nptII gene with the bla bacterial
marker gene (100 lines), or the nptII gene with the plant reporter gene uidA (400 lines). These
lines would be analysed during the trial to compare the effectiveness of different
transformation techniques used to generate the GM sugarcane lines.
18. Short regulatory sequences (promoters, and sequences for ending transcription) that
control expression of the introduced genes are present in all the gene constructs. These are
derived from the plants Flaveria trinervia (clustered yellowtops) and Z. mays, and the
bacteria Agrobacterium tumefaciens and E. coli. Although A. tumefaciens is a plant pathogen
and E. coli is an opportunistic human pathogen, the regulatory sequences comprise only a
small part of their respective total genomes, and are not capable of causing disease.
19. All of the introduced genes, except the bla gene, which is under the control of its own
bacterial promoter from E. coli, are under the control of the maize ubiquitin1 (ubi1) gene
promoter (Christensen et al. 1992). All of the genes, except the bla gene, which again has its
own bacterial terminator from E. coli, have the nopaline synthase (nos) gene terminator from
A. tumefaciens.
Table 1
Details of the GM sugarcane lines modified for altered plant architecture, enhanced WUE or
improved NUE
Transformation
vector*#
1
Trait(s)
Altered plant architecture
Partial gene sequence
(source)
None
2
Altered plant architecture
None
3
Altered plant architecture
None
4
Altered plant architecture
None
5
Altered plant architecture
None
6
Altered plant architecture
None
7
Altered plant architecture
None
8
Altered plant architecture
None
9
Altered plant architecture
None
Chapter 1 - Risk assessment context (February 2007)
Complete gene sequence
(source)
PcGA2ox-1
(P. coccineus)
HvGA3ox-2
(H. vulgare subsp. vulgare)
HvGA20ox-1
(H. vulgare subsp. vulgare)
HvGA20ox-2
(H. vulgare subsp. vulgare)
OsMAX3
(O. sativa)
OsMAX4-1
(O. sativa)
OsMAX4-2
(O. sativa)
OsTB1
(O. sativa)
SoTB1
(Saccharum spp.)
10
DIR 70/2006 – Risk Assessment and Risk Management Plan
Transformation
vector*#
10
Trait(s)
Altered plant architecture
11
Altered plant architecture
12
Altered plant architecture
Office of the Gene Technology Regulator
Partial gene sequence
(source)
SoMAX3
(Saccharum spp.)
SoTB1
(Saccharum spp.)
None
Complete gene sequence
(source)
None
None
HvGA20ox-2
(H. vulgare subsp. vulgare)
SoTB1
(Saccharum spp.)
OsMAX3
OsMAX4-1
OsMAX4-2
(O. sativa)
13
Altered plant architecture
SoTB1
(Saccharum spp.)
14
Altered plant architecture
OsMAX3
OsMAX4-1
OsMAX4-2
(O. sativa)
SoTB1
(Saccharum spp.)
15
Enhanced WUE
None
16
Enhanced WUE
None
17
Enhanced WUE
None
18
Enhanced WUE
None
19*
Improved NUE
None
MdS6PDH
(M x domestica)
MdS6PDH
(M x domestica)
EcTPSP
(E. coli)
AtMYB2
(A. thaliana)
ZmDOF1
(Z. mays)
*
All lines containing the introduced genes for altered plant architecture, enhanced WUE or improved NUE
contain the E. coli nptII and bla antibiotic resistance genes (except the lines made using vector 15).
#
Vector 15 does not contain the E. coli bla antibiotic resistance gene
4.2 Introduction to plant architecture, drought stress and nitrogen use
efficiency
Plant architecture and yield
20. Changes in plant architecture have been important in the domestication and improved
yields of many plant species such as the single stalk in maize compared to its multiple stalked
ancestor teosinte (Galinat 1998).
21. Sugarcane yields may also benefit from changes to plant architecture. This can be
achieved by increasing sugar concentration of the cane, increasing the cane yield or both.
Cane yield increases can be achieved by increasing the number of stalks produced and/or the
weight of the individual stalks through manipulating thickness and/or height. However, an
increase in stalk number generally leads to a reduction in stalk weight and diameter (Milligan
et al. 1990) leading to limited yield responses (Bell & Garside 2005).
22. Suckering, the late season outgrowth of lateral buds, is another factor affecting sugar
yield. Suckers are morphologically different to main stalks and tillers. These differences
include a greater stem base diameter and shorter, wider leaves than the primary stalk of a
similar age (Bonnett et al. 2001). Suckers can typically reduce sugar yield as their sugar
content is much lower than the primary mature cane (Bonnett et al. 2004) and can thus lead to
a dilution of yields (information supplied by applicant).
Chapter 1 - Risk assessment context (February 2007)
11
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
23. The falling of crops due to stem or root weakness, known as lodging, can decrease
yields and this has been investigated through selection of lodging-resistant varieties.
However, little effort has been undertaken towards the breeding of lodging-resistant varieties
(Singh et al. 2000).
24.
Lodging resistance could be achieved by either:
 increasing stalk number to reduce individual stalk weight and allow increased stalk
flexibility in windy conditions; or
 reducing the stalk number resulting in thicker stems, possibly leading to greater
resistance to lodging from high winds placing strains on the base of the plant.
25. A better understanding of the genes controlling plant architecture is critical for
developing a purposeful molecular or conventional breeding strategy. Although in sugarcane
this is poorly understood, much progress has recently been made in other species including
maize, rice and in model dicots, tobacco, garden pea and Arabidopsis. In these species several
transcription factors have been identified which regulate plant architecture, including the
maize TB1 (Doebley et al. 1997; Takeda et al. 2003), pea RMS (Schmitz & Theres 2005) and
Arabidopsis MAX genes (McSteen & Leyser 2005).
26. A transcription factor is any protein required for recognition, by RNA polymerases, of
specific regulatory sequences in genes (eg a promoter) (Lewin 1994a). A particular
transcription factor may affect the initiation of transcription of many genes, such as those
involved in abiotic stress reactions (Shinozaki et al. 2003; Chinnusamy et al. 2004;
Chinnusamy et al. 2006). The activity of a regulatory transcription factor may be controlled
by its rate of synthesis, covalent modification (eg phosphorylation), binding to a ligand (eg
hormone), or inhibitors that can sequestrate or affect the transcription factor’s ability to bind
to DNA (Lewin 1994b). Approximately 7.4% (~2000) of the expressed genes in the
experimental model plant Arabidopsis encode transcription factors (Iida et al. 2005).
27. Genes involved in the biosynthesis of the plant hormone gibberellin are also known to
play a role in plant architecture (Biemelt et al. 2004). Gibberellic acids (GAs) are a large
family of diterpenoid compounds of which some are bioactive growth regulators. GAs control
seed germination, stem elongation and leaf, trichome, flower and fruit development (reviewed
in Davies 2004).
Drought stress tolerance (enhanced WUE) and plant responses
28. Drought stress is an abiotic stress; a non-living factor that causes harmful effects to
plants. Other types of primary abiotic stresses include salinity, cold, heat and chemical
pollution. Plants respond to different abiotic stresses, often through an interconnecting series
of signalling and transcription controls that ultimately aim to increase the plant’s ability to
tolerate the initial stress through different biochemical and physiological mechanisms.
29. Enhanced WUE is a trait of increasing importance for sugarcane growers. Water
allocations in many sugarcane production regions are insufficient to make up the difference
between crop water requirement and effective rainfall (Holden 1998). Also, irrigation
requirements differ from year to year because of the high variability in rainfall across the
areas of sugarcane cultivation. Thus, competition for water, increasing costs, and
environmental concerns require that major users of water achieve best-practice in terms of
efficiency of water use (Inman-Bamber et al. 2000).
30. At the molecular level there are three broad categories of genes that play a role in a
plant’s response to drought stress (Wang et al. 2003; Vinocur & Altman 2005). These include:
Chapter 1 - Risk assessment context (February 2007)
12
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
 signal sensing, perception and transduction
 transcriptional control
 stress tolerance response (via proteins involved in detoxification, osmoprotection,
chaperone functions and the control of water and ion movement).
31. Specific receptors are thought to enable plants to directly sense stress (Xiong & Zhu
2001; Zhu 2002). Once sensed, a drought stress signal may be relayed to eventually regulate
the transcription of numerous genes involved in the stress response. The transduction of the
stress signal may be accomplished through various signalling molecules such as Ca2+,
phospholipids, reactive oxygen species, sugars and plant hormones, or via protein messengers
such as mitogen activated protein kinases (MAPKs) and serine/threonine phosphatases (Xiong
& Zhu 2001; Ramanjulu & Bartels 2002; Zhu 2002; Chaves & Oliveira 2004; Chinnusamy et
al. 2004).
32. Drought stress signals activate genes that control the transcription of genes involved in
the stress tolerance response mechanisms of cell protection. The majority of these can be
categorised as transcription factors (Wang et al. 2003; Shinozaki et al. 2003), but other genes
involved in transcription control include those encoding proteins involved in chromatin
remodelling (Singh 1998). Certain regulatory genes can be induced by more than one type of
abiotic stress (eg drought, cold and salinity) (Seki et al. 2002).
Nitrogen use efficiency (NUE) in plants
33. NUE is an important factor in crop plant productivity and as such nitrogen based
fertilizers are used extensively in modern agriculture, including sugarcane. Due to the rapid
growth and size of sugarcane, current agricultural practices are to apply the fertiliser in a large
single quantity early in the crops life cycle. This has implications in nitrogen run-off due to
heavy early seasonal rains (information supplied by applicant). The use of fertilizers has an
environmental impact through run-off into nearby streams (Addiscott et al. 1991).
34. There are several ways to improve nitrogen use in plants without adding extra nitrogen
based fertilizers. These can include: crop rotation practices with nitrogen fixing crops;
traditional plant breeding and marker assisted technologies to develop plants with increased
NUE; selection of soil microbes that can better fix nitrogen; and genetic modification of the
plants for nitrogen use efficiency or to create/enhance their relationship with symbiotic
nitrogen fixing bacteria.
35. Several studies have attempted to improve NUE in plants using genetic modification
with limited success (Good et al. 2004). These studies have focussed on introducing genes
encoding nitrogen transporters, nitrate and nitrite reductases, glutamine synthetase and
synthases, aminotransferases and dehydrogenases, and transcription factors.
4.3 The introduced genes and regulatory sequences, and the gene products
36. Genetic material from various sources was introduced into sugarcane. The genetic
elements, their sources and function in the source organisms are listed below (see Table 2).
37. The introduced genes that are intended to alter plant architecture, enhance WUE or
improve NUE, consist of partial gene sequences that encode dsRNAs (double-stranded
RNAs) or complete gene sequences that encode proteins. The introduced genes are intended
to modulate biochemical pathways in the GM sugarcane lines, either through encoding
enzymes or regulating endogenous gene expression.
Chapter 1 - Risk assessment context (February 2007)
13
DIR 70/2006 – Risk Assessment and Risk Management Plan
Table 2
Office of the Gene Technology Regulator
Source organisms of the introduced genes and regulatory sequences*
Genetic Element
Source Organism
Function in source organism
PcGA2ox-1 (complete gene sequence)
HvGA3ox-2 (complete gene sequence)
Phaseolus coccineus
Hordeum vulgare subsp.
vulgare
H. vulgare subsp. vulgare
H. vulgare subsp. vulgare
Oryza sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
Saccharum spp.
Saccharum spp.
Saccharum spp.
Malus x domestica
Escherichia coli
Arabidopsis thaliana
Zea mays
E. coli
E. coli
E. coli
Z. mays
Flaveria trinervia
Agrobacterium tumefaciens
E. coli
E. coli
Gibberellin biosynthesis
Gibberellin biosynthesis
HvGA20ox-1 (complete gene sequence)
HvGA20ox-2 (complete gene sequence)
OsMAX3 (complete gene sequence)
OsMAX3 (partial gene sequence)
OsMAX4-1 (complete gene sequence)
OsMAX4-1 (partial gene sequence)
OsMAX4-2 (complete gene sequence)
OsMAX4-2 (partial gene sequence)
OsTB1 (complete gene sequence)
SoTB1 (complete gene sequence)
SoTB1 (partial gene sequence)
SoMAX3 (partial gene sequence)
MdS6PDH (complete gene sequence)
EcTPSP (complete gene sequence)
AtMYB2 (complete gene sequence)
ZmDOF1 (complete gene sequence)
nptII
bla
uidA
ubi1 promoter*
pdk intron^
nos terminator*
bla promoter
bla terminator
Gibberellin biosynthesis
Gibberellin biosynthesis
Transcription factor
N/A#
Transcription factor
N/A#
Transcription factor
N/A#
Transcription factor
Transcription factor
N/A#
N/A#
Sorbitol biosynthesis
Trehalose biosynthesis
Transcription factor
Transcription factor
Antibiotic resistance
Antibiotic resistance
Visual marker
Transcription initiation of ubi1 gene
Intron
Transcription termination of nos gene
Transcription initiation of bla gene
Transcription termination of bla gene
*All of the introduced genes, except the bla gene, which is under the control of its own bacterial promoter and
terminator from E. coli, are under the control of the maize ubiquitin1 (ubi1) gene promoter from Z. mays and the
nopaline synthase (nos) gene terminator from A. tumefaciens (see Section 4.1).
^All of the introduced genes that consist of partial gene sequences encoding dsRNAs also contain the intron
sequence of the pyruvate orthophosphate dikinase (pdk) gene from Flaveria trinervia. The intron helps in the
formation of a dsRNA hairpin structure necessary for the efficient silencing of the targeted endogenous gene.
#
Not Applicable.
4.3.1
The introduced genes for altered plant architecture
38. In total, 14 genes or partial gene sequences for altered plant architecture (transformation
vectors 1-14, Table 1) have been introduced (up to 4 genes or partial gene sequences per line)
into the GM sugarcane lines.
39. Six of the transformation vectors (see Table 1) used to make the GM sugarcane lines
contain the complete gene sequences of OsTB1 (O. sativa Teosinte Branching 1) and/or
complete or partial gene sequences of SoTB1 (Saccharum spp. Teosinte Branching 1). Both of
these gene homologs encode transcription factors that may affect lateral branching and sugar
yield in the GM sugarcane lines. The OsTB1 gene reduces lateral branching in its parent
species (Takeda et al. 2003; McSteen & Leyser 2005). Both the OsTB1 and SoTB1 genes are
homologous to the maize TB1 gene responsible for control of branching in the maize plant
(Doebley et al. 1997; Hubbard et al. 2002).
40. Other regulatory genes introduced into the GM sugarcane lines that may alter the plant
architecture include genes that affect auxin transport in the plant. The genes, which include
the homologs OsMAX3 (O. sativa More Axillary Growth 3), SoMAX3 (Saccharum spp. More
Axillary Growth 3), OsMAX4-1 and OsMAX4-2, are present in six of the transformation
vectors (Table 1) used to make the GM sugarcane lines. These introduced genes consist of
Chapter 1 - Risk assessment context (February 2007)
14
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
complete or partial gene sequences. Auxin plays an important role in the regulation of bud
outgrowth and the More Axillary Growth (MAX) 1, 3 and 4 genes of Arabidopsis, and related
genes from other plants, have been shown to be responsible for the regulation of shoot
branching (Booker et al. 2004; Schwartz et al. 2004; Bainbridge et al. 2005; Bennett et al.
2006) and bud inhibition (Sorefan et al. 2003; Booker et al. 2004; Schwartz et al. 2004;
Bainbridge et al. 2005; Snowden et al. 2005; Bennett et al. 2006 and reviewed in McSteen
and Leyser, 2005).
41. Some of the GM sugarcane lines contain all three introduced O. sativa MAX genes as
complete sequences along with the introduced partial gene sequence of the SoTB1 gene
(transformation vector 13, Table 1). GM sugarcane lines containing all three introduced O.
sativa MAX as partial sequences along with the introduced complete gene sequence of the
SoTB1 gene (transformation vector 14, Table 1) are also proposed for release.
42. Five of the transformation vectors (see Table 1) used to make the GM sugarcane lines
contain the complete gene sequences of PcGA2ox-1 (P. coccineus gibberellin 2-oxidase-1),
HvGA3ox-2 (H. vulgare subsp. vulgare gibberellin 3-oxidase-2), HvGA20ox-1 and
HvGA20ox-2. These gene homologs encode enzymes involved in active gibberellin (GA)
(HvGA3ox-2, HvGA20ox-1 and HvGA20ox-2) and inactive GA biosynthesis (PcGA2ox-1)
(Hedden & Kamiya 1997) and may affect the height and yield of the GM sugarcane lines. The
biologically active GAs includes the compounds GA1 and GA4 and the inactive forms include
GA51, GA29, GA34 and GA8 (Hedden & Phillips 2000).
43. One of the transformation vectors used to make the GM sugarcane lines contains both
the introduced HvGA20ox-2 and SoTB1 complete gene sequences (transformation vector 12,
Table 1). These GM sugarcane lines may have decreased lateral branching, altered plant
height and sugar yield.
4.3.2
The introduced genes for enhanced WUE
44. The 3 introduced genes (MdS6PDH, EcTPSP and AtMYB2) intended to enhance WUE
(transformation vectors 15-18, Table 1) encode a D-sorbitol-6-phosphate dehydrogenase
(S6PDH), a fusion enzyme consisting of a trehalose-6-phosphate synthase and trehalose-6phosphate phosphatase (TPSP), and a myeloblastosis interacting protein 2 (MYB2). The
genes are derived from Malus x domestica, E. coli and A. thaliana, respectively.
45. The MdS6PDH and EcTPSP genes encode enzymes involved in sorbitol and trehalose
production, respectively. Increased trehalose production in GM rice (Jang et al. 2003) has
been shown to increase tolerance to different abiotic stresses, in particular drought stress
tolerance (Garg et al. 2002), also known as WUE. Similarly, sorbitol is expected to have an
osmoprotective function in the plant as has been seen in marsh plant (Ahmad et al. 1979).
Introduction of MdS6PDH into tobacco led to increased boron uptake and increased growth
and yield when grown under conditions of limited boron, often seen following drought
conditions (Brown et al. 1999; Bellaloui et al. 1999).
46. The EcMYB2 gene encodes the MYB2 transcription factor, which is known to be
expressed under salt stress and dehydration (Urao et al. 1993; Abe et al. 2003).
4.3.3
The introduced gene for improved nitrogen use efficiency
47. The introduced gene ZmDOF1 (Z. mays DNA binding with one finger 1) for improved
NUE (transformation vector 19, Table 1) encodes the transcription factor DOF1. DOF1 can
enhance the expression of many genes simultaneously and has been shown to enhance plant
growth when expressed in Arabidopsis plants under low-nitrogen conditions (Yanagisawa et
al. 2004).
Chapter 1 - Risk assessment context (February 2007)
15
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
48. The DOF protein family of transcription factors share a unique highly conserved DNAbinding domain, a novel zinc finger motif (Yanagisawa 1995; Umemura et al. 2004). DOF
proteins have been shown to have diverse functions and associated with a diverse range of
promoters (reviewed in (Yanagisawa & Schmidt 1999; Yanagisawa 2002; Yanagisawa 2004).
While the DOF binding domain has been shown to function in plant, animal and yeast cells
(Yanagisawa 2001), the proteins are plant specific.
49. A study in Arabidopsis (Yanagisawa et al. 2004) showed that expression of the
introduced ZmDOF1 gene can improve nitrogen assimilation and enhance growth on a fresh
weight basis under low nitrogen input. Previous studies have associated the ZmDOF1 gene
with the enhancement of expression of several genes involved in carbon metabolism
(Yanagisawa & Sheen 1998; Yanagisawa 2000) in higher plants and micro-organisms,
specifically:
 the C4-type phosphoenol-pyruvate carboxylase (C4PEPC), which catalyses the initial
fixation of CO2 in the C4 photosynthetic pathway (Yanagisawa & Sheen 1998)
 cytosolic orthophosphate dikinase (cyPPDK), which catalyses the production of
phosphoenol-pyruvate (PEP) (Edwards et al. 1985), the substrate in the reaction
catalysed by PEPC (Chollet et al. 1996)
 a non-photosynthetic gene encoding PEPC.
4.3.4
Regulatory sequences for expression of the introduced genes
50. Expression of the introduced genes for altered plant architecture, enhanced WUE and
improved NUE in GM sugarcane lines is controlled by the ubi1 promoter from maize
(Christensen et al. 1992). The ubi1 promoter is thought to lead to constitutive expression of
the inserted genes in plants (Christensen et al. 1992).
51. Also required for gene expression in plants is an mRNA termination region, including a
polyadenylation signal. The mRNA termination region for the introduced genes for altered
plant architecture, enhanced WUE and improved NUE in GM sugarcane lines is the 3’
untranslated region derived from the nopaline synthetase (nos) gene from Agrobacterium
tumefaciens (Depicker et al. 1982). Although A. tumefaciens is a plant pathogen, the
regulatory sequence comprises only a small part of its total genome and is not capable of
causing disease.
52. Five of the introduced gene constructs for altered plant architecture consist of partial
gene sequences encoding dsRNAs. These also contain the intron sequence of the pyruvate
orthophosphate dikinase (pdk) gene from the plant Flaveria trinervia. The intron helps in the
formation of a dsRNA hairpin structure necessary for the efficient silencing of the targeted
endogenous gene.
53. The bla gene is under the control of its own bacterial promoter and terminator, both
from E. coli. Since the bacterial promoter and terminator sequence do not operate in plants,
the bla gene is not expressed in the GM plants.
4.3.5
Toxicity and allergenicity of the proteins (and enzymatic products) encoded
by the introduced genes for altered plant architecture
54. All of the introduced genes intended to alter plant architecture were derived from
several common crop plants including barley, rice, sugarcane and bean. These source
organisms are not known to be toxic to humans or other organisms.
55. Given that expression of the endogenous genes SoTB1 and SoMAX3 is suppressed in
sugarcane lines 13 and 14, the amount of TB1 transcription factor or MAX3 enzyme present
Chapter 1 - Risk assessment context (February 2007)
16
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
in GM plant tissue is likely to be less than in parent sugarcane. On this basis, if the proteins
had any toxicity potential then it should be reduced in these sugarcane lines.
56. Known food allergens generally share a number of characteristics including amino acid
sequence homology, a molecular weight (Mr) of 15-70 kDa, are highly expressed,
glycosylated, stable [resistant to digestion in the gastrointestinal tract (GIT)] and derived from
a food or biological source known to be allergenic (Metcalfe et al. 1996; FAO 2001a).
57. FAO/WHO (FAO 2001a; Codex Alimentarius Commission 2003) guidelines propose
that cross-reactivity between an unknown protein and a known allergen has to be considered
when there is more than 35% identity in the amino acid sequence over a sliding window of 80
amino acids or a match of six contiguous amino acids. Matches of six amino acids with any
allergen have been shown to occur frequently by chance which limits the utility of this criteria
for predicting allergenicity (Hileman et al. 2002; Stadler & Stadler 2003; Thomas et al. 2005;
Silvanovich et al. 2006). The International Food Biotechnology Council/International Life
Sciences Institute have suggested that an optimal length of between eight and twelve amino
acids is required for binding to T-cells and that an immunological significant sequence
identity requires a match of at least eight contiguous amino acids (Metcalfe et al. 1996).
58. Many protein allergens are N-glycosylated (Herouet et al. 2005) leading to the concern
that glycosylation may contribute to the allergenicity (Buchanan 2001).
59. All the introduced genes for altered plant architecture are in the molecular weight range
identified as being typical of allergens (15-70 kDa). A motif search
(http://www.expasy.org/prosite/) with the amino acid sequences derived from the introduced
gene sequences identified that some of the proteins contained putative N-glycosylation sites
(Asp-X-Ser/Thr).
60. Amino acid sequence comparisons using SDAP11, Agmobiol12, Food Allergy Research
and Resource Program (FARRP) Protein Allergen13, Allergen14, and Allermatch15 databases
revealed that none of the sequences of the genes for altered plant architecture contained a
region of 80 amino acids with greater than 35% sequence identity to any known allergens.
The GA20 OXIDASE-1 sequence from barley showed a match of eight identical continuous
amino acids to a known pollen allergen, Amb a 1, from Ambrosia artemisiifolia (short
ragweed)(Rafnar et al. 1991). However, this may be a chance occurrence since the related
barley GA20 OXIDASE-2 protein did not contain the same match and there were no other
regions of similarity between GA20 OXIDASE-1 and Amb a 1.
61. Furthermore, an extensive search of the scientific literature yielded no data or
information to suggest that any of the proteins encoded by the introduced genes for altered
plant architecture have any toxicity or allergenicity potential to people or other organisms.
4.3.6
Toxicity and allergenicity of the proteins encoded by the introduced genes
(and enzymatic products) for enhanced WUE.
62. The MdS6PDH gene encoding for the S6PDH enzyme was isolated from Malus x
domestica (apple). Apple is not considered to be toxic or allergenic. The S6PDH enzyme
produces sorbitol in apple fruit (information supplied by applicant) and it is not expected that
11
<http://fermi.utmb.edu/SDAP/sdap_who.html
<http://ambl.lsc.pku.edu.cn/english/index.phpallergen>
13
<http://www.allergenonline.com>
14
<http://allergen.csl.gov.uk/>
15
<http://allermatch.org/>
12
Chapter 1 - Risk assessment context (February 2007)
17
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
sorbitol will have any allergenic or toxic effects. Sorbitol is approved by Food Standard
Australia New Zealand (FSANZ) as a food additive16.
63. The EcTPSP gene encoding for the TPSP protein was derived from E. coli, a bacterium
present in the gastrointestinal tract of humans and other animal species. The EcTPSP gene is a
fusion gene of two E. coli genes (trehalose-6-phosphate synthase and trehalose-6-phosphate
phosphatase) for the synthesis of trehalose (Seo et al. 2000), a sugar found naturally in honey,
mushrooms, lobster, shrimp and foods produced using baker's and brewer's yeast. Trehalose
has been gazetted into the FSANZ code 31 July 2003, A453 Trehalose as a Novel Food.
Trehalose has also been evaluated by the US Food and Drug Authority (US FDA) as
‘generally recognised as safe’ (GRAS Notice No. GRN 00004517).
64. The gene coding for the transcription factor, AtMYB2, is derived from A. thaliana, a
member of the Brassicaceae family which includes many edible members such as broccoli,
cabbage and radish. Many other edible plant species, and possibly all plants, contain MYB
genes, such as barley, maize, wheat, rye, rice, soybean, and tomato. Arabidopsis is native to
Europe and central Asia but is naturalised worldwide. It is widely used throughout the world
for scientific research and there have been no reports of allergenicity or toxicity. It is not
commonly eaten, but there are no reports of it causing disease or ill-health in humans. There
is no other evidence that this gene exists for any biological role apart from its role in the stress
response cycle.
65. MYB2, S6PDH and TPSP are in the molecular weight range identified as being typical
of allergens (15-70 kDa). A motif search (http://www.expasy.org/prosite/) with the amino
acid sequences identified that MYB2 and S6PDH contain putative N-glycosylation sites (AspX-Ser/Thr).
66. Amino acid sequence comparisons using SDAP9, Agmobiol10, FARRP Protein
Allergen11, Allergen12, and Allermatch13 databases revealed no indication that the proteins
encoded by the MdS6PDH, EcTPSP and AtMYB2 genes share significant sequence homology
with any known allergens. On this basis, the encoded proteins are unlikely to have any
allergenic potential in people
67. An extensive search of the scientific literature yielded no data or information to suggest
that the proteins encoded by the introduced genes for enhanced WUE have any toxicity or
allergenicity potential in people or other organisms.
4.3.7
Toxicity and allergenicity of the introduced gene (ZmDOF1) for improved
nitrogen use efficiency
68. The introduced ZmDOF1 gene is derived from the crop plant maize (Z. mays). Maize is
a common food and fodder plant and it is not expected that the DOF1 protein encoded by the
ZmDOF1 gene would be toxic or allergenic.
69. The DOF1 protein from maize is in the molecular weight range identified as being
typical of allergens (15-70 kDa). A motif search (http://www.expasy.org/prosite/) identified
one putative N-glycosylation site (Asp-X-Ser/Thr).
70. A comparison of the deduced amino acid sequence of the gene for nitrogen use
efficiency to amino acid sequences in the publicly available SDAP9, Agmobiol10, FARRP
Protein Allergen11, Allergen12, and Allermatch13 databases revealed no matches of at least
35% identity in a segment of 80 amino acids with any known allergens. One match of eight
contiguous identical amino acids to a known allergen was identified with the Gal d
16
17
<http://www.foodstandards.gov.au/thecode/foodstandardscode.cfm#_FSCchapter1> Part 1.3, Standard 1.3.1
<http://www.cfsan.fda.gov/~rdb/opa-gras.html>
Chapter 1 - Risk assessment context (February 2007)
18
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
vitellogenin epitope from Gallus domesticus (chicken)(Nardelli et al. 1987). However, this
may be a chance occurrence since there were no other regions of similarity between DOF1
and Gal d vitellogenin.
71. Furthermore, an extensive search of the scientific literature yielded no data or
information to suggest that DOF1 has any toxicity or allergenicity potential to people or other
organisms.
4.3.8
The antibiotic resistance marker gene (nptII) and its encoded protein
72. All of the GM sugarcane lines contain the nptII antibiotic resistance marker gene. The
nptII gene was isolated from the bacterial Tn5 transposon (from E. coli) (Beck et al. 1982). It
encodes an enzyme, neomycin phosphotransferase type II (NPTII), which confers resistance
to some aminoglycoside antibiotics, eg neomycin, paromomycin and geneticin. NPTII uses
ATP to phosphorylate those antibiotics, thereby inactivating and preventing them from killing
the NPTII-producing cells. The nptII gene functioned as a selectable marker during the
laboratory stages of sugarcane plant tissue selection following genetic modification, allowing
GM cells to grow in the presence of the antibiotic while inhibiting the growth of non-GM
cells. The nptII gene is in common use as a selectable marker in the production of GM plants
(Miki & McHugh 2004).
73. Other regulatory agencies, in Australia and in other countries, have previously assessed
the nptII gene as safe for use in human food (US FDA 1998; ANZFA 2001a; ANZFA 2001b;
ANZFA 2001c; ANZFA 2001d; FSANZ 2003). In addition, a number of genetically modified
food crops containing the nptII gene have been approved for commercial release both in
Australia (DIRs 012/2002, 021/2002, 022/2002 and 059/2005) and overseas. No adverse
effects on humans, animals or the environment have been reported from these releases (US
FDA 1998; Flavell et al. 1992; EFB 2001).
Regulatory sequences for the expression of the nptII gene
74. The bacterial nptII gene was modified by the addition of regulatory sequences including
the ubi1 gene promoter from maize (Christensen et al. 1992) and the nopaline synthase gene
(nos) transcription termination region from A. tumefaciens to allow efficient expression in
plant cells.
75. A. tumefaciens is a common gram-negative soil bacterium that causes crown gall
disease in a wide variety of plants (Van Larebeke et al. 1974). Although A. tumefaciens is a
plant pathogen, the regulatory sequence comprises only a small part of its total genome, and is
not capable of causing disease.
Toxicity of NPTII
76. Protein and DNA sequence comparisons using sequences from four separate databases
(Genbank, EMBL, PIR29, Swiss-Prot) indicated that NPTII does not have significant
homology to any proteins listed as food toxins in these databases (FDA 1994).
77. Humans (and, by implication, other animals) continually ingest kanamycin-resistant
microorganisms, some containing the NPTII enzyme. The diet, especially raw salad, is the
major source: estimated conservatively, each human ingests 1.2 x 106 kanamycin-resistant
microorganisms daily (Flavell et al. 1992). Large numbers of kanamycin- or neomycinresistant bacteria already inhabit the human digestive system (Levy et al. 1998) with Flavell et
al. (1992) estimating about 1012 per person. Kanamycin-resistant bacteria have been isolated
from soil, river water and sewage (Smalla et al. 1993).
Chapter 1 - Risk assessment context (February 2007)
19
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
78. The insertion of the nptII gene into a wide range of GMOs has not resulted in any
adverse effects (Flavell et al. 1992). The nptII gene was introduced into mammalian cell lines
with no effects on viability or growth. During gene therapy experiments, mammalian cells
expressing the NPTII protein have been infused into cancer patients. Again, no adverse
effects have been observed (Flavell et al. 1992).
79. The NPTII protein produced in GM tomatoes has been fed to rodents and reported to be
rapidly inactivated and degraded (Calgene Inc. 1990). An acute oral toxicity study in mice, in
which the purified NPTII protein was fed at doses of up to 5000 mg/kg of body weight
(2500 mg/kg administered twice, four hours apart), did not show any adverse effects
(Berberich et al. 1993). A similar study in mice also reported no adverse effects of NPTII at
5000 mg/kg of body weight (Fuchs et al. 1993b).
Allergenicity of NPTII
80. The NPTII protein is approximately 29 kDa in size, which is within the typical size
range of allergenic proteins. However, it does not possess glycosylation sites, is not stable in
the mammalian digestive system and is heat labile, decreasing the probability that it is
allergenic (US FDA 1998; Fuchs et al. 1993a; FDA 1994; ANZFA 2001e; ANZFA 2001f).
Fuchs et al. reported that no NPTII was detected 10 seconds after addition of simulated gastric
fluid as measured by both western blot and enzymatic activity (Fuchs et al. 1993b). Protein
sequence comparisons using sequences from four separate protein databases (EMBL,
GenBank, PIR29 and Swiss-Prot) indicated that NPTII does not have significant sequence
identity to any known protein food allergens (Fuchs & Astwood 1996).
81. The FDA has evaluated data submitted for deliberate releases of GMOs expressing the
NPTII protein and concluded that NPTII does not have any of the characteristics associated
with allergenic proteins (US FDA 1998). The UK Royal Society have concluded that there is
at present no evidence that available GM foods cause allergic reactions, and that the risks
posed by GM plants are in principle no greater than those posed by conventional breeding or
by plants introduced from other areas of the world (The Royal Society 2002).
4.3.9
β-lactamase gene or ampicillin antibiotic resistance gene (bla or amp)
82. Most of the GM sugarcane lines (up to 2400) contain the bacterial β-lactamase gene
(bla; also known as amp) derived from E. coli (Spanu et al. 2002). The bla gene encodes the
β-lactamase enzyme, which confers ampicillin resistance.
83. The β-lactamase enzyme is widespread in the environment and in food. Naturally
occurring ampicillin-resistant microorganisms have been found in mammalian digestive
systems (Spanu et al. 2002). The bla gene was originally isolated from antibiotic resistant
strains of E. coli found in hospital patients.
84. The bla gene in the GM sugarcane lines is controlled by bacterial regulatory sequences
and therefore will not be expressed. The gene was used in the laboratory prior to the
production of the genetically modified plants.
4.3.10
Reporter gene (uidA) and its encoded protein (GUS)
85. Up to 400 of the GM sugarcane lines contain the uidA reporter gene. The uidA gene
encodes the enzyme β-glucuronidase (GUS), which is derived from the common gut
bacterium E. coli. It is the most widely used reporter gene in GM plants (Miki & McHugh
2004) as it allows GM tissues to be identified using a simple assay. The uidA gene was used
as a ‘reporter’ in the laboratory for assessing GM sugarcane plant materials for levels of gene
Chapter 1 - Risk assessment context (February 2007)
20
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
expression and can also be used to assess gene expression levels in plants grown under field
conditions.
86. The GUS protein is a monomer with a molecular weight of 68 kDa, and the GUS
enzyme is active as a tetramer. GUS catalyses the hydrolysis of β-glucuronides and, less
efficiently, some β-galacturonides. E. coli lives in the digestive tract of vertebrates, including
humans (Jefferson et al. 1986), and the GUS enzyme enables it to metabolise β-glucuronides
as a main source of carbon and energy.
Regulatory sequences for the expression of the uid gene
87. Expression of the uidA gene in the GM sugarcane lines is controlled by the ubi1 gene
promoter from maize (Christensen et al. 1992) and the termination sequence of this gene is
the 3′ non-translated region of the nos gene from A. tumefaciens (Rogers et al. 1985).
Although A. tumefaciens is a plant pathogen, the regulatory sequence comprises only a small
part of its total genome and is not capable of causing disease.
Toxicity of GUS
88. Acute oral toxicity studies in mice with purified GUS protein at doses of up to
100 mg/kg did not show any adverse effects (Naylor 1992). Feeding in humans and animals
with 1010 GUS-containing E. coli bacteria per ingestion also did not show any toxic or
pathogenic reactions (Gilissen et al. 1998). Metabolites of the GUS enzyme are non-toxic
(Gilissen et al. 1998).
Allergenicity of GUS
89. The GUS protein is approximately 68 kDa in size, which is within the typical size range
of allergenic proteins. However, the widespread occurrence of GUS and the constant exposure
of humans to the protein without known ill effects indicates that the likelihood that GUS
being an allergen is extremely low (Gilissen et al. 1998). In addition, the GUS protein does
not possess glycosylation sites and is rapidly denatured in simulated mammalian digestive
system (Fuchs & Astwood 1996). The GUS protein from E. coli is rapidly (<15 seconds)
degraded in simulated gastric fluid and loses its activity by heating/cooking (Fuchs &
Astwood 1996). Protein sequence analysis indicates that GUS does not have significant
sequence homology to any known protein food allergens.
4.4 Method of genetic modification
4.4.1
Agrobacterium mediated transformation
90. Up to 400 GM sugarcane lines were generated by Agrobacterium-mediated
transformation using standard sugarcane transformation protocols (Arencibia et al. 1998).
These 400 GM sugarcane lines only contain the introduced genes nptII and uidA. They do not
contain the introduced genes for altered plant architecture, enhanced WUE or improved NUE.
Four different strains of A. tumefaciens (Ag10, Ag11, EHA105 and LBA4404) were used in
the plant transformation process for the generation of the 400 lines (up to 100 lines per strain).
91. A. tumefaciens is a common gram-negative soil bacterium that causes crown gall
disease in a wide variety of plants (Van Larebeke et al. 1974). Plants can be genetically
modified by the transfer of DNA (transfer-DNA or T-DNA, located between specific border
sequences on a resident plasmid) from A. tumefaciens through the mediation of genes from
the virulence region of Ti plasmids.
92. Disarmed Agrobacterium strains have been constructed specifically for plant
transformation. The disarmed strains do not contain the genes responsible for the
Chapter 1 - Risk assessment context (February 2007)
21
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
overproduction of auxin and cytokinin (iaaM, iaaH and ipt), which are required for tumour
induction and rapid callus growth (Klee & Rogers 1989). Agrobacterium plasmid vectors
used to transfer T-DNAs contain well characterised DNA segments required for their
replication and selection in bacteria, and for transfer of T-DNA from Agrobacterium and its
integration into the plant cell genome (Bevan 1984; Wang et al. 1984).
93. Agrobacterium-mediated transformation has been widely used in Australia and overseas
for introducing genes, and regulatory sequences for their expression, into plants. Unintended
changes in phenotype can occur as a result of transformation, or because of the tissue culture
process used after transformation, that are similar to those encountered in conventional
breeding and mutation breeding (Cellini et al. 2004; Bradford et al. 2005). Such ‘off types’
are visually identified and eliminated from the breeding program.
94. A disarmed binary plasmid vector was used to introduce the gene construct containing
the nptII and uidA genes into sugarcane cultivar (Saccharum hybrid), Q117, using standard
Agrobacterium transformation protocols. Following transformation, regeneration of plants
was performed in the presence of paromomycin.
95. The 400 GM sugarcane lines were generated from independent transformation events,
and therefore the introduced genes are expected to be located at different sites in the
sugarcane genome in each line.
4.4.2
Biolistic mediated transformation
96. The remaining 2100 GM sugarcane lines were generated using standard sugarcane
biolistic transformation protocols (Bower & Birch 1992; Bower et al. 1996; Birch et al. 2000).
This involved coating very small metal (tungsten) particles with the transformation vector
containing the introduced genes. The particles were then ‘shot’ into sugarcane embryonic
callus. Successfully transformed tissue was selected by its resistance to the antibiotic
geneticin, conferred by the introduced nptII gene and regenerated into GM sugarcane plants.
97. All of the GM sugarcane lines were generated from independent transformation events,
and therefore the introduced genes are expected to be located at different sites in the
sugarcane genome in each line.
4.5 Characterisation of the GMOs
4.5.1
Stability and molecular characterisation
98. The applicant stated that all 2500 sugarcane lines are at an early development stage and
have not been tested for genotypic stability. Such data would be required for GM sugarcane
lines selected for further development.
99. PCR analyses of the GM sugarcane lines generated by Agrobacterium-mediated and
biolistic transformation have confirmed the presence of the introduced genes for altered plant
architecture, enhanced WUE or improved NUE. Real Time PCR indicates that the genes are
being expressed (information supplied by applicant). Further detailed molecular
characterisation may be performed during the trial on selected GM sugarcane lines grown
under field conditions.
100. No assessment has been made of the number of copies of the introduced genes inserted
into the sugarcane genome. The number of gene copies integrated into a plant genome varies
depending on the method of introduction. Copy number of an introduced gene following
microprojectile bombardment usually varies from 1 to more than 20 (Pawlowski & Somers
1996), whereas 1-3 copies of introduced genes are commonly seen in transgenic lines
obtained through Agrobacterium-mediated transformation (Arencibia et al. 1998).
Chapter 1 - Risk assessment context (February 2007)
22
DIR 70/2006 – Risk Assessment and Risk Management Plan
4.5.2
Office of the Gene Technology Regulator
Characterisation of the phenotype of the GMOs
101. One of the aims of the proposed trial is to assess whether the GM sugarcane lines
demonstrate improved agronomic performance (eg sugar yield), as compared to non-GM
sugarcane, under field conditions.
102. The applicant reported that under glasshouse conditions, necrosis is observed on leaves
of the GM sugarcane expressing the introduced MdS6PDH gene. The necrosis is more
noticeable at the leaf apex where the sorbitol content is the highest. The condition is more
severe in the plants producing larger amounts of sorbitol. These GM sugarcane lines are up to
30% shorter compared to non-GM parent. All other aspects of growth seemed to be normal.
GM sugarcane lines containing the introduced genes HvGA20ox-1 or 2 appear to produce
stalks more rapidly (1-2 months earlier), and GM sugarcane lines containing the introduced
gene PcGA2ox-1 grow more slowly and are shorter, than their non-GM sugarcane parent. The
GM sugarcane lines containing the other introduced genes appear morphologically similar to
the non-GM sugarcane parent (information supplied by applicant).
Section 5
The receiving environment
103. The receiving environment forms part of the context in which the risks associated with
dealings involving the GMOs are assessed. This includes the size, duration and regions of the
dealings, any relevant biotic/abiotic properties of the regions where the release would occur;
intended agronomic practices, including those that may be altered in relation to normal
practices; other relevant GMOs already released; and any particularly vulnerable or
susceptible entities that may be specifically affected by the proposed release (OGTR 2005).
5.1 Relevant abiotic factors
104. The locations, size and duration of the proposed limited and controlled release of the
GM sugarcane lines are outlined in Section 2.1 of this Chapter. The release is proposed to
take place in the Queensland local government areas of Bundaberg, Caboolture and/or Cairns.
These geographically distinct commercial sugarcane growing regions have typical climates
for sugarcane growing in Australia (see Table 3) with hot and wet summers, and warm and
dry winters.
Table 3
Climatic data for sites representative of proposed GM sugarcane trial areas
Representative site
(within local
government area)
Average daily
max/min
temperature
(summer)
29.6/21.0ºC
Average daily
max/min
temperature
(winter)
22.5/10.7ºC
Average
monthly rainfall
(summer)
Average
monthly rainfall
(winter)
Bundaberg airport
151.8 mm
39.8 mm
(Bundaberg)
Somerset Dam#
30.2/19.0ºC
20.9/7.5ºC
136.1 mm
45.5 mm
(Caboolture)
Cairns airport*
31.3/23.5ºC
26.0/17.4ºC
340.0 mm
34.4 mm
(Cairns)
Source: <http://www.bom.gov.au>
#The Bureau of Meteorology does not have any averages for the shire of Caboolture. Data from the closely located weather
station of Somerset Dam was used instead.
* The Summer averages for Cairns were based on January to March and the winter averages were based on July to
September.
5.2 Relevant agricultural practices
105. The applicant intends to follow standard agricultural protocols used at BSES research
stations to grow the GM sugarcane lines. The sugarcane plants at the trial sites would
therefore receive applications of water, fertilizers, herbicides, insecticides and other
Chapter 1 - Risk assessment context (February 2007)
23
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
agronomic management practices similar to commercially grown non-GM sugarcane.
However, the GM sugarcane lines containing the introduced genes for enhanced WUE may be
trialled under optimal and water stress treatments, and the GM sugarcane lines containing the
introduced gene for improved NUE may be trialled with varying levels of nitrogen
applications.
106. Equipment used on the trial sites would be subject to domestic sugarcane quarantine
legislation and regulations (Plant Protection Act 1989 (Qld) and the Plant Protection
Regulations 2002 (Qld). Therefore, the applicant has indicated that any equipment used will
be routinely cleaned and sterilised.
107. The applicant proposes to use the first season for the planting of up to 2500 individual
lines in a non-replicated trial to allow the phenotypic screening of all transgenic lines in the
field. A selection of these lines will then be used for generating planting material (setts) for
the establishment of a new statistically robust crop trial in the following seasons.
108. The applicant proposes that at the end of each crop cycle all cane will be harvested for
plant yield measurements and all harvested material destroyed except for the ratoons or setts
that will be used to generate the following year’s crop.
109. The applicant proposes that the final data collection and harvest will occur at the end of
each growing season. Commercial sugarcane is routinely harvested before flowering as the
process of flowering leads to a reduction in stem sugar content.
5.3 Presence of related plants in the receiving environment
110. All of the proposed trial sites except the Caboolture site have commercial sugarcane
(S. officinarum x S. spontaneum) growing within 50 kms of their general vicinity. Sugarcane
plants, which may be sexually compatible with the GM sugarcane lines, are maintained on
BSES land nearby to all three of the proposed trial sites.
111. The proposed Cairns trial site would be located in the vicinity (but at least 200m away)
of S. officinarum and S. spontaneum plants, which form part of a BSES germplasm collection.
Both parental species of modern cultivars of sugarcane have also been found naturalised in
other areas in the tropics (Hnatiuk 1990). Australia’s Virtual Herbarium
(<http://www.cpbr.gov.au/avh.html>) also has records of S. spontaneum in the Northern
Territory and Queensland. A few S. officinarum populations have been recorded as escapees
from cultivation and subsequently naturalised in subtropical and tropical regions of Australia.
112. Sugarcane is closely related to the genera Erianthus, Narenga, Miscanthus and
Sclerostachya. These genera and the Saccharum genera are collectively known as the
Saccharum complex. Certain species belonging to different genera within this complex can
interbreed naturally (Bull & Glasziou 1979; Grassl 1980; Daniels & Roach 1987). Some
accessions of these species are maintained as germplasm for breeding programs in sugarcane
experimental stations in Australia.
113. The GM sugarcane may be able cross with other distantly related genera belonging to
tribe Andropogoneae. Crossing has been reported between Saccharum spp. and Imperata
(blady grass), Sorghum (sorghum), Zea (maize) and Bambusa (bamboo) (Thomas &
Venkatraman 1930; Janakiammal 1938; Rao et al. 1967; Nair 1999). However, not all such
crosses have been verified, particularly through molecular techniques.
114. Blady grass is common throughout Queensland coastal areas and would probably be in
the vicinity of the proposed trial sites (information supplied by applicant). Wild Sorghum
species are among the weeds of Australian sugarcane crops (McMahon et al. 2000) and are
widespread in Australia (Hnatiuk 1990). However, commercial sorghum and maize crops are
Chapter 1 - Risk assessment context (February 2007)
24
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
not cultivated near the proposed trial sites (information supplied by applicant). Bambusa
arundinacea is reported in the coastal regions of Queensland, especially at the northern tip of
Queensland, and many species of bamboo are grown as garden plants throughout Australia.
Bamboo is also present at the BSES Meringa research station, location of one of the proposed
trial sites (information supplied by applicant).
5.4 Presence of the introduced genes or similar genes in the environment
115. The majority of the lines containing introduced genes for altered plant architecture,
enhanced WUE and improved NUE are derived from either the crop plants O. sativa, Z. mays,
Saccharum spp., H. vulgare subsp. vulgare, P. coccineus, M. x domestica, or the well
characterised model plant A. thaliana, which is widely used in experimental studies. The
introduced genes for altered plant architecture, enhanced WUE and improved NUE (except
the EcTPSP gene) are highly homologous and most likely orthologous, to genes in most, if
not all, crop and other plants. Therefore, it is expected humans routinely encounter the
introduced genes and their gene products, or their homologs, through contact with plants and
food. This information forms the baseline data for assessing the risks from exposure to these
enzymes as a result of the trial of the GM sugarcane
116. The EcTPSP and nptII genes are derived from E. coli, which is widespread in human
and animal digestive systems as well as in the environment (Blattner et al. 1997). As such, it
is expected humans routinely encounter the encoded protein through contact with plants and
food.
117. The uidA gene, which encodes the GUS enzyme, is also derived from E. coli. GUS
enzyme activity has been detected in numerous microbial, plant and animal species (Flavell et
al. 1992; Gilissen et al. 1998). The GUS protein used in GM plants is 99.8% homologous to
the E. coli GUS protein. GUS is recognised as commonly present on fresh food. The US
Environmental Protection Agency (EPA) does not consider the GUS protein to be toxic to
mammals and has approved its exemption from the requirement to establish tolerance levels
(EPA 2001).
118. The bla (ß-lactamase) gene confers resistance to ß-lactam antibiotics such as ampicillin.
It was isolated from the bacterial Tn3 transposon. The bla gene will not be expressed in the
GM sugarcane lines because the gene’s bacterial (prokaryotic) promoter does not allow the
expression in eukaryotes such as plants.
Section 6
Australian and international approvals
6.1 Australian approvals of the GM sugarcane lines
6.1.1
Previous releases approved by GMAC or the Regulator
119. There has been no previous release of these GM sugarcane lines with altered plant
architecture, enhanced WUE or improved NUE in Australia.
120. In Australia there have been previous small scale field trials of GM sugarcane lines
containing the uidA, nptII (or its homolog gene aphA) or bla genes in trials conducted by both
BSES and the University of Queensland (PR-23, PR-23X, PR-68 and PR-68X), BSES (PR-72
and DIR 019/2002) and CSIRO Tropical Agriculture (PR-73 and PR-136). There is small
scale field trial of GM sugarcane lines containing the nptII and bla genes being conducted
until December 2010 by the University of Queensland (DIR 051/2004).
6.1.2
Approvals by other Australian government agencies
121. The Gene Technology Act 2000 is designed to operate in a cooperative legislative
framework with other regulatory authorities that have complementary responsibilities and
Chapter 1 - Risk assessment context (February 2007)
25
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
specialist expertise. As well as enhancing coordinated decision making, this arrangement
avoids duplication.
122. While the Regulator is responsible for identifying, assessing and managing risks to the
health and safety of people and the environment associated with the use of gene technology,
other government regulatory requirements may also have to be met in respect of release of
GMOs.
123. FSANZ is responsible for human food safety assessment and food labelling, including
GM food. The applicant does not intend to use materials from the GM sugarcane lines in
human food, accordingly an application to FSANZ has not been submitted. FSANZ approval
would need to be obtained before materials from these sugarcane lines could be used in food.
6.2 International approvals
124. There has been no international release of these GM sugarcane lines or other GM plants
containing the introduced genes for altered plant architecture, enhanced WUE or improved
NUE. However, there have been greater than 20 approvals for field trials in the USA of GM
sugarcane lines containing the nptII gene and two approvals for small scale releases of GM
sugarcane lines containing the uidA gene.
Chapter 1 - Risk assessment context (February 2007)
26
DIR 70/2006 – Risk Assessment and Risk Management Plan
Chapter 2
Section 1
Office of the Gene Technology Regulator
Risk assessment
Introduction
125. Risk assessment is the overall process of identifying the sources of potential harm
(hazards) and determining both the seriousness and the likelihood of any adverse outcome
that may arise. The risk assessment (summarised in Figure 2) considers risks from the
proposed dealings with the GMOs that could result in harm to the health and safety of people
or the environment posed by, or as a result of, gene technology.
Figure 2
The risk assessment process.
RISK ASSESSMENT PROCESS *
RISK ASSESSMENT CONTEXT
Evaluation
of events
Consequence
assessment
IDENTIFIED
RISK
HAZARD
IDENTIFICATION
RISK
ESTIMATE
Likelihood
assessment
No identified
risk
* Risk assessment terms are defined in Appendix A.
126. Once the risk assessment context has been established (see Chapter 1) the next step is
hazard identification to examine what harm could arise and how it could happen during a
release of these GMOs into the environment.
127. It is important to note that the word ‘hazard’ is used in a technical rather than a
colloquial sense in this document. The hazard is a source of potential harm. There is no
implication that the hazard will necessarily lead to harm. A hazard can be an event, a
substance or an organism (OGTR 2005).
128. Hazard identification involves consideration of events (including causal pathways) that
may lead to harm. These events are particular sets of circumstances that might occur through
interactions between the GMOs and the receiving environment as a result of the proposed
dealings.
129. A number of hazard identification techniques are used by the Regulator and staff of the
OGTR, including the use of checklists, brainstorming, commonsense, reported international
experience and consultation (OGTR 2005). In conjunction with these techniques, hazards
identified from previous RARMPs prepared for licence applications of the same and similar
GMOs are also considered.
130. The hazard identification process results in the compilation of a list of events. Some of
these events lead to more than one adverse outcome and each adverse outcome can result
from more than one event.
Chapter 2 – Risk assessment (February 2007)
27
DIR 70/2006 – Risk Assessment and Risk Management Plan
Section 2
Office of the Gene Technology Regulator
Hazard characterisation
131. The list of events compiled during hazard identification are characterised to determine
which events represent a risk to the health and safety of people or the environment posed by
or as a result of, gene technology.
132. A risk is identified only when there is some chance that harm will occur. Those events
that do not lead to an adverse outcome or could not reasonably occur do not represent an
identified risk and will not advance in the risk assessment process. Risks associated with the
remaining events are assessed further to determine the seriousness of harm (consequence) and
chance of harm (likelihood). The identified risks must be posed by or result from gene
technology.
133. The criteria used by the Regulator to determine harm are described in Chapter 3 of the
Risk Analysis Framework (OGTR 2005). Harm is assessed in comparison to the parent
organism and in the context of the proposed dealings and the receiving environment. The risk
assessment process focuses on measurable criteria for determining harm.
134. The following factors are taken into account during the analysis of events that may give
rise to harm:
 the proposed dealings, which may include experimentation, development, production,
breeding, propagation, use, growth, importation, possession, supply, transport or
disposal of the GMOs
 the size, duration and regions of the release
 containment measures proposed by the applicant
 characteristics of the non-GM parent (OGTR 2004)
 routes of exposure to the GMOs, the introduced gene(s) and its product(s)
 potential effects of the introduced gene(s) and its product(s) expressed in the GMOs
 potential exposure to the introduced gene(s) and its product(s) from other sources in the
environment
 properties of the biotic and abiotic environment at the site(s) of release
 agronomic management practices for the GMOs.
135. There have been no previous releases of these GMOs in Australia.
136. Events that are discussed in detail later in this Section are summarised below in Table 4.
Events that share a number of common features are grouped together in broader hazard
categories as indicated in the table. Sixteen events were characterised, none of which were
considered to lead to an identified risk that required further assessment.
137. The bla gene is controlled by its own bacterial promoter and is unlikely to be expressed
in the GM sugarcane. In addition, the prevalence of the bla, nptII and uidA genes in the
environment and the lack of evidence for toxicity or allergenicity of the respective encoded
proteins are discussed in Chapter 1, Sections 4.3.8 to 4.3.10. Furthermore, the potential effects
of the bla and nptII and uidA genes and their products were considered in previous
assessments such as DIRs 051/2004, 052/2004, 054/2004, 059/2005, 060/2005 and 066/2006,
some of which are for commercial releases. RARMPs for those DIR licences are available
from the OGTR or from the website <http://www.ogtr.gov.au>. Risks arising from the
introduced bla, nptII or uidA genes in GM sugarcane lines are considered to be negligible.
Chapter 2 – Risk assessment (February 2007)
28
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Therefore, the potential effects of the bla, nptII and uidA genes will not be assessed further for
this application.
Table 4
Hazard category
Section 2.1
Production of a
substance toxic to
people
Summary of events that may give rise to an adverse outcome through the expression of the
introduced genes for altered plant architecture, enhanced WUE or improved NUE.
Event that may give rise to
an adverse outcome
1. Ingestion of GM plant
materials produced by the
sugarcane lines containing
the introduced genes.
Potential adverse
outcome
Toxicity for people.
Identified
risk?
No
Reason
 The introduced genes are widespread in
the environment because of their
presence in many common plants and/or
bacteria. There are no reports of toxicity
associated with the proteins encoded by
the introduced genes.
 Silencing of genes is unlikely to increase
toxicity.
 None of the GM plant materials from the
proposed release would be used as
human food.
2. Contact with, or inhalation
of, GM plant materials
produced by the sugarcane
lines containing the
introduced genes.
Toxicity for people.
No
 The introduced genes are widespread in
the environment because of their
presence in many common plants and/or
bacteria. There are no reports of toxicity
associated with the proteins encoded by
the introduced genes.
 Silencing of genes is unlikely to increase
toxicity.
 Contact with, or inhalation of GM plant
materials would be limited to workers
associated with the proposed field trial.
 Contact would be further limited by the
small size and short duration of the
proposed field trial.
Section 2.2
3. Ingestion of GM plant
Production of a
materials, produced by the
substance
sugarcane lines containing
allergenic to people
the introduced genes.
Allergic reactions in
people.
No
 None of the proteins encoded by the
introduced genes have been listed in
publicly available allergen databases.
 Silencing of genes is unlikely to increase
allergenicity.
 None of the GM plant materials from the
proposed release would be used as
human food.
4. Contact with, or inhalation
of, GM plant materials,
produced by the sugarcane
lines containing the
introduced genes.
Allergic reactions in
people.
No
 None of the proteins encoded by the
introduced genes have been listed in
publicly available allergen databases.
 Contact with, or inhalation of, GM plant
materials would be limited by the use of
standard industry cultivation practices and
the small size and short duration of the
proposed field trial.
 Silencing of genes is unlikely to increase
allergenicity.
 None of the GM plant materials from the
proposed release would be used for the
production of human food, animal feed or
other sugarcane products.
Chapter 2 – Risk assessment (February 2007)
29
DIR 70/2006 – Risk Assessment and Risk Management Plan
Hazard category
Event that may give rise to
an adverse outcome
Potential adverse
outcome
Section 2.3
Production of a
substance toxic to
organisms other
than people
5. Direct or indirect ingestion of
GM plant materials,
produced by the sugarcane
lines containing the
introduced genes, by
vertebrates, invertebrates
and microorganisms
Toxicity for vertebrates,
invertebrates or microorganisms.
Office of the Gene Technology Regulator
Identified
risk?
No
Reason
 Vertebrates, invertebrates and
microorganisms are already widely
exposed to the same or similar proteins.

Exposure of vertebrates, invertebrates
and microorganisms to the GM sugarcane
lines would be limited by the small size
and short duration of the proposed
release.
 None of the GM plant materials from the
proposed release would be used as
animal feed or other sugarcane products.
Section 2.4
Spread and
persistence of the
GM sugarcane in
the environment
6. Expression of the introduced
genes improving the survival
of the GM sugarcane or
volunteers through altered
plant architecture, enhanced
WUE or improved NUE
Weediness.
No
 The main factors limiting the spread and
persistence of sugarcane in the area
proposed for release are likely to include
its low fertility and seed viability, poor
ability to establish and thrive without
human intervention, pests and diseases,
soil type and fertility, and competition from
other plants.
 The release would be of small size and
short duration and the applicant proposes
measures to limit the spread and
persistence of the GM sugarcane lines,
including destroying any GM sugarcane
volunteers.
7. Expression of the introduced
genes improving the survival
of the GM sugarcane or
volunteers through
enhancement of
Weediness.
No
 The release would be of small size and
short duration and the applicant proposes
measures to limit the spread and
persistence of the GM sugarcane lines,
including destroying GM sugarcane
volunteers.
 Abiotic stress tolerances
(other than drought)
 Biotic stress tolerance
8. Dispersal of GM sugarcane
plant materials during
transport, research, storage,
equipment use, flooding or
via animals
 The main factors limiting the spread and
persistence of sugarcane in the area
proposed for release are discussed in
event 6.
Weediness.
No
 The applicant proposes to transport and
store all GM plant materials according to
OGTR guidelines, to clean and sterilise
equipment, and to destroy all harvested
plant materials not required for
propagation or further research.
 Research would be carried out in PC2
facilities and any GM plant waste
materials would be destroyed.
 The proposed sites are at least 50 m
away from natural waterways and are not
prone to flooding.
 Animals are not known to distribute
sugarcane.
Chapter 2 – Risk assessment (February 2007)
30
DIR 70/2006 – Risk Assessment and Risk Management Plan
Hazard category
Event that may give rise to
an adverse outcome
9. Exposure of vertebrates
(including people),
invertebrates and microorganisms to material
produced from the GM
sugarcane lines or
volunteers containing the
introduced genes.
Section 2.5
10.Expression of the
Vertical transfer of
introduced genes in other
genes or genetic
sugarcane or sexually
elements to
compatible plant species.
sexually compatible
plants
Potential adverse
outcome
Office of the Gene Technology Regulator
Identified
risk?
Toxicity for, or allergic
reactions in, people.
Toxicity for other
vertebrates,
invertebrates and/or
micro-organisms.
No
Weediness.
No
Reason
 The potential effects on people are
discussed in Events 1-4.
 The potential effects on organisms other
than people are discussed in Event 5.
 The release would be of small size and
short duration and the applicant proposes
measures to limit the spread and
persistence of the GM sugarcane lines,
including destroying GM sugarcane
volunteers.
 The parent sugarcane cultivar rarely
flowers and both pollen and seed have
low viability.
 Poor sexual compatibility prevents vertical
gene transfer to most sexually compatible
plant species.
 Outcrossing to other sugarcane or
sexually compatible plant species would
be further limited due to the applicant’s
intention to harvest cane before flowering,
the small size, short duration and
containment measures of the trial.
11.Exposure of vertebrates
(including people),
invertebrates and microorganisms to material
produced by other
sugarcane or sexually
compatible plant species
containing the introduced
genes.
Toxicity for, or allergic
reactions in, people.
Toxicity for other
vertebrates,
invertebrates and/or
micro-organisms.
No
12.Presence of the introduced
regulatory sequences in
other sugarcane or sexually
compatible plant species as
a result of gene transfer.
Toxicity for, or allergic
reactions in, people.
Toxicity for other
vertebrates,
invertebrates and/or
micro-organisms.
Weediness.
No
Section 2.6
13.Presence of the introduced
Horizontal transfer
genes, or the introduced
of genes or genetic
regulatory sequences, in
elements to
other organisms as a result
sexually
of gene transfer.
incompatible
organisms
Toxicity for, or allergic
reactions in, people.
Toxicity for other
vertebrates,
invertebrates and/or
micro-organisms.
Weediness.
No
Chapter 2 – Risk assessment (February 2007)
 The limited potential for gene flow is
discussed in Event 10.
 The potential effects of the introduced
genes on people are discussed in Events
1-4.
 The potential effects of the introduced
genes on organisms other than people
are discussed in Event 5.
 The introduced regulatory sequences are
not known to behave any differently than
endogenous regulatory sequences in
plants.
 Outcrossing to other sugarcane or
sexually compatible plant species would
be limited due to the small size, short
duration and containment measures of the
trial.
 The introduced gene or similar genes and
the introduced regulatory sequences are
already present in the environment and
are available for transfer via demonstrated
natural mechanisms.
 Gene transfer from plants to bacteria has
not been demonstrated under natural
conditions.
31
DIR 70/2006 – Risk Assessment and Risk Management Plan
Hazard category
Section 2.7
Unintended
changes in
biochemistry,
physiology or
ecology
Section 2.8
Unauthorised
activities
Office of the Gene Technology Regulator
Event that may give rise to
an adverse outcome
Potential adverse
outcome
Identified
risk?
14.Production of innate toxic or
allergenic compounds as a
result of the expression, or
random insertion of the
introduced genes into the
sugarcane genome during
genetic modification.
Toxicity for, or allergic
reactions in, people.
Toxicity for other
vertebrates,
invertebrates and/or
micro-organisms.
Weediness.
No
15.Altered biochemistry,
physiology or ecology of the
GM sugarcane lines
resulting from expression of
the introduced genes.
Toxicity for, or allergic
reactions in, people.
Toxicity for other
vertebrates,
invertebrates and/or
micro-organisms.
Weediness.
No
16.Use of GMOs outside the
proposed licence conditions
(non-compliance)
Potential adverse
outcomes discussed in
Events 1-15.
No
Reason
 Compositional analysis of the GM
sugarcane lines has not been undertaken,
as the proposed trial represents early
stage research involving phenotypic
screening of multiple transformation
events and selection of lines for possible
further development.
 The small size, short duration and
containment measures of the trial will limit
any possible adverse outcomes.

Unintended adverse effects, if any, would
be limited by the small scale and short
duration of the proposed release.
 Unintended changes typically result in
deleterious effects in the plant so are
unlikely to proceed in the selection
process.
 The Act provides for substantial penalties
for non-compliance and unauthorised
dealings with GMOs and also requires
consideration of the suitability of the
applicant to hold a licence prior to the
issuing of a licence by the Regulator.
2.1 Production of a substance toxic to people
138. Toxicity is the adverse effect(s) of exposure to a dose of a substance as a result of direct
cellular or tissue injury, or through the inhibition of normal physiological processes (Felsot
2000). In order for toxicity to occur, people must be exposed to the substance and at a level
necessary to cause an adverse effect on health. The hallmark of toxicity is the concept of a
dose-response relationship, that is, the severity of the toxic effect is proportional to dose.
Exposure can occur via the oral, dermal or inhalational routes (or a combination of these) and
the rate and degree of absorption, tissue distribution, metabolism and elimination of the
substance all impact on its toxicity.
139. Toxicity may result from a single exposure (acute toxicity) or due to repeated long-term
exposure (chronic toxicity). The adverse health effect(s) may be reversible or irreversible and
there is likely to be variability within the population with regard to how people are affected by
a substance; some people may be relatively sensitive while others may be refractory. Factors
contributing to such variability include genetic polymorphisms, age, gender and pre-existing
diseases (Aldridge et al. 2003).
140. There are various in vitro and in vivo experimental testing methods used to assess
toxicity, with laboratory animals commonly used as models for people. For chemicals and
drugs, a toxicological assessment typically covers acute toxicity, eye and skin irritation, skin
sensitisation, repeat-dose toxicity, reproductive toxicity, developmental toxicity, genotoxicity,
carcinogenicity and neurotoxicity. Human data from clinical trials, occupational exposure
studies and poisoning incidents add further to the toxicological database of a substance.
141. For the risk assessment of naturally-occurring agents (ie those that are endemic in the
environment) consideration needs to be given to the background level of exposure to people
(in addition to the intrinsic toxicity of the substance). Therefore, the risk will actually be
Chapter 2 – Risk assessment (February 2007)
32
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
incremental to this background level of exposure and must take into consideration the range
(variability) of the concentration/amount of the substance in the environment.
142. For the purposes of the risk assessment of GMOs, an assessment of toxicity would take
into consideration the encoded protein from the introduced gene(s), any end-product(s)
produced by the biological activity of the protein(s), the toxicity of the GMO(s) and the
source organisms of the genes.
Event 1: Ingestion of GM plant materials produced by the sugarcane lines containing
the introduced genes
143. The GM sugarcane lines proposed for release differ from non-GM sugarcane in that
they contain up to four introduced genes for altered plant architecture, enhanced WUE or
improved NUE, and marker and reporter genes. As explained in paragraph 137, the marker
and reporter genes bla, nptII and uidA will not be considered further. As the proposal is a
‘proof of concept’ field trial, no toxicity studies have been carried out with the proteins
encoded by the introduced genes or the GM sugarcane lines.
144. However, the introduced genes for altered plant architecture, enhanced WUE or
improved NUE present in the GM sugarcane lines are the same or similar to those present in
many crop plants (sugarcane, maize, barley, apple, soybean and rice), or microorganisms (eg
E. coli). People are exposed to the proteins (and enzymatic products) encoded by the
introduced genes for altered plant architecture, enhanced WUE or improved NUE, or similar
proteins, through normal diet or the environment (see Chapter 1, Section 5.4). There are no
reports of toxicity associated with the proteins encoded by the introduced genes (see
Chapter 1, Sections 4.3.4 to 4.3.7). Furthermore, some of the introduced genes (containing
only partial gene sequences) are intended to silence their endogenous counterparts and
therefore the level of these proteins in plant tissues is likely to be lower than in the non-GM
sugarcane parent. Therefore if these proteins (or enzymatic products) had any toxicity
potential then the respective GM sugarcane lines would be less toxic than the non-GM parent.
145. Food Standards Australia New Zealand (FSANZ) is responsible for human food safety
assessment and FSANZ approval would be required before products from the GM sugarcane
lines could be used in human food.
146. The applicant does not intend to use GM plant materials (including raw sugarcane,
sugar or molasses) from the release in human food or as animal feed, but to destroy any plant
materials remaining following the final harvest after collecting samples for analysis. Ingestion
of materials from the GM sugarcane lines containing the introduced proteins is not expected
to occur from the release.
147. Therefore, no risk is identified. The potential for toxicity for people as a result of
ingestion of GM plant materials produced by the sugarcane lines containing the introduced
genes will not be assessed further.
Event 2: Contact with, or inhalation of, GM plant materials produced by the
sugarcane lines containing the introduced genes
148. Dermal and inhalation toxicity studies have not been conducted with the proteins (or
their enzymatic products) encoded by the introduced genes for altered plant architecture,
enhanced WUE or improved NUE. However, as discussed in Event 1, these or similar
proteins (or their enzymatic products) are widespread in the environment, occurring in many
common plant and bacterial species, and are not known to be toxic to people. Furthermore,
the introduced genes containing partial gene sequences are not expected to increase the
Chapter 2 – Risk assessment (February 2007)
33
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
production of toxic products in the GM sugarcane. Therefore, the introduced genes are not
expected to increase the toxicity of GM sugarcane plant materials.
149. Dermal and inhalation contact with the proteins (or their enzymatic products) encoded
by the introduced genes for altered plant architecture, enhanced WUE or improved NUE may
occur when the plant cells have been damaged or broken, or via pollen. Workers may come
into contact with the protein (and enzymatic products) encoded by the introduced genes
during handling and/or processing the GM sugarcane. However, sugarcane plants possess
leaves with sharp edges and irritating hairs which necessitates appropriate protection for the
handlers, which would significantly reduce potential for dermal contact.
150. While it is possible that people living nearby could come into contact with GM
sugarcane pollen as commercial cultivars rarely flower (Bakker 1999; Berding et al. 2004) the
level of exposure is expected to be negligible. Although sugarcane pollen is very small and
wind dispersed (OGTR 2004), wind is not expected to disperse any pollen more than one
hundred metres (Dr D.M. Hogarth personal communication; Information supplied by the
applicant) and there would be no public access to the trial sites. In addition, the applicant
intends to follow standard agricultural practice and harvest the GM sugarcane before
flowering, to preserve sugar content. This would further reduce the potential for exposure to
the GM sugarcane pollen.
151. Since the release is of small size and short duration, the frequency and duration of
contact with the proteins (and enzymatic products) encoded by the introduced genes is
expected to be limited. In addition, the applicant does not intend to use any sugarcane plant
materials from the release in human food, animal feed or for any other sugarcane products.
152. Therefore, no risk is identified and the potential for toxicity for people as a result of
contact with, or inhalation of, GM plant materials produced by the sugarcane lines containing
the introduced genes will not be assessed further.
2.2 Production of a substance allergenic to people
153. Allergenicity is the potential of a specific substance to elicit an immunological reaction
following its ingestion, dermal contact or inhalation, which may lead to tissue inflammation
and organ dysfunction (Arts et al. 2006). During the induction or sensitisation phase of an
allergic response, a person is exposed for the first time to the substance, which results in a
primary immune response and a persistent state of heightened immune responsiveness to the
substance. Subsequent challenges (ie exposures) result in a powerful and exaggerated immune
response, which can lead to adverse health effects in people. For the purposes of the risk
assessment of GM plants, a potential allergen may be a protein or an end-product generated
through the biological activity of the protein.
154. Allergic reactions following ingestion or inhalation of a substance are mediated by IgE
antibodies (ie they are an immediate-type hypersensitivity reaction). In contrast, allergic
reactions following dermal exposure are associated with a cell-mediated immune response
(type IV hypersensitivity). However, such a reaction following dermal exposure, particularly
to high molecular weight compounds such as proteins, is uncommon as the skin is an effective
barrier to penetration (Arts et al. 2006; Holsapple et al. 2006).
155. There continues to be considerable discussion in the scientific literature on the
characteristics of protein allergens and whether one can predict allergenic potential based on
these characteristics. For the assessment of transgenic food crops, the source of the candidate
gene and whether it is derived from a source known to be allergenic is an important
characteristic as is its reactivity to sera from patients known to be allergic to the source
material (Kimber et al. 1999). Other characteristics commonly used to predict a protein’s
Chapter 2 – Risk assessment (February 2007)
34
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
allergenicity include amino acid sequence homology with known human allergens, the
stability of the protein, resistance to digestion in the gastrointestinal tract and posttranslational glycosylation (Metcalfe et al. 1996; Huby et al. 2000).
Event 3: Ingestion of GM plant materials, produced by the sugarcane lines
containing the introduced genes.
156. The proteins encoded by the introduced genes for altered plant architecture, enhanced
WUE or improved NUE have not been reported to cause allergic reactions in people. As the
proposal is a ‘proof of concept’ field trial, no allergenicity studies have been carried out with
the proteins encoded by the introduced genes or the GM sugarcane lines.
157. All except one of the introduced genes are from the plants barley, rice, sugarcane, bean,
maize, apple and Arabidopsis, which are not known to cause allergies. The final gene, TPSP
is from E.coli, a common gut bacterium. None of the proteins encoded by the introduced
genes appear in publicly available allergen databases.
158. However, some of the introduced genes have some of the characteristics of allergens (as
discussed in Chapter 1). All of the introduced proteins are in the molecular weight range
identified as being typical of allergens (15-70 kDa) and all except the TPSP fusion protein
and the GA oxidase proteins possess at least one putative N-glycosylation site (Asp-XSer/Thr) typical of allergens. Two of the proteins also show some amino acid identity with
known allergens, although these matches are restricted to short amino acid sequences which
are not known to be epitopes.
159. The barley GA20ox-1 protein showed a match of eight identical contiguous amino
acids with a pollen allergen from short ragweed (for details see Chapter 1, Section 4.3.2). The
short ragweed protein is an inhalant allergen and is not known to cause allergy when ingested.
160. The maize DOF-1 protein sequence showed a match of eight identical contiguous amino
acids with a vitellogenin protein from chicken (van het Schip et al. 1987) (for details see
Chapter 1, Section 4.3.4). The homology between this protein and the maize DOF-1 protein is
restricted to a series of eight contiguous amino acids, with no other sequence identity. In
maize the DOF-1 protein is not recognised as being allergenic, suggesting that the short match
with the vitellogenin protein may be merely a chance occurrence unrelated to allergenicity.
161. Taken together, the weight of available evidence suggests that it is unlikely that the
proteins encoded by the introduced genes would be allergenic to people if they were ingested.
Furthermore, some of the introduced genes (containing only partial gene sequences) are
intended to silence their endogenous counterparts and therefore the level of protein in plant
tissues is likely to be lower than in the non-GM sugarcane parent. Therefore, if these proteins
(or enzymatic products) had any allergenic potential then the respective GM sugarcane lines
would be less allergenic than the non-GM parent.
162. None of the GM sugarcane materials from the release would be used in human food or
animal feed. Thus, the potential for allergic reactions in people resulting from exposure to
food is unlikely. Food Standards Australia New Zealand (FSANZ) is responsible for human
food safety assessment and FSANZ approval would be required before products from the GM
sugarcane lines could be used in human food.
163. Therefore, no risk is identified and the potential for production of a substance
allergenic to people will not be assessed further.
Chapter 2 – Risk assessment (February 2007)
35
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Event 4: Contact with, or inhalation of, GM plant materials produced by the
sugarcane lines containing the introduced genes
164. People working with sugarcane plants would be exposed primarily to the outer waxy
cuticle layer at the plant surface, which is essentially free of protein. However, as mentioned
in Event 2, dermal and inhalation exposure to proteins (including the proteins encoded by the
introduced genes for altered plant architecture, enhanced WUE or improved NUE) or to other
cellular components of the sugarcane plants may occur via pollen or if damage to the plants
during handling results in rupture of plant cells. This may result in workers at the trial sites
being exposed to the proteins (and their enzymatic products).
165. There are no reports of the proteins encoded by the introduced genes for altered plant
architecture, enhanced WUE or improved NUE causing allergic reactions in people, however
two of the proteins do have some characteristics associated with known allergens (for details
see Event 3, and Section 4.3 in Chapter 1).
166. The barley GA20ox-1 protein showed a match of eight identical contiguous amino acids
with a pollen allergen (Amb a1) from short ragweed (for details see Chapter 1, Section 4.3.2).
Although the ragweed allergen it is a potent inhalant allergen (King et al. 1964), the sequence
similarity with barley GA20ox-1 is confined to the eight amino acid match with no additional
homology in the remaining protein sequence.
167. Inhalation of pollen by workers could potentially result in allergic reactions if this
protein was allergenic and was expressed in the pollen. However, due to the very limited
quantities of pollen produced by sugarcane, the applicant’s intention to harvest before
flowering and the small size of the trial, it is expected that people would be exposed to very
small quantities of pollen.
168. As a result, it is unlikely that contact with GM plant materials would cause allergic
reactions in people due to the presence of proteins encoded by the genes for altered plant
architecture, enhanced WUE or improved NUE. Furthermore, the introduced genes containing
partial gene sequences are not expected to increase the allergenicity of GM sugarcane plant
materials.
169. Human contact with the GM plant materials containing the proteins (and enzymatic
products) encoded by the introduced genes would be limited because the small scale and short
duration of the release and none of the materials would be used in human food, animal feed or
in the production of other sugarcane products.
170. Therefore, no risk is identified. The potential for allergic reactions in people, resulting
from the use or contact with GM plant materials produced by the sugarcane lines containing
the introduced genes, will not be assessed further.
2.3 Production of a substance toxic to organisms other than people
171. A range of organisms may be exposed directly or indirectly to GM plant materials
containing the introduced genes for altered plant architecture, enhanced WUE or improved
NUE. Organisms may be exposed directly through biotic interactions with GM sugarcane
plants (vertebrates, insects, symbiotic microorganisms and/or pathogenic fungi) or through
contact with root exudates or dead plant materials (soil biota). Indirect exposure would
include organisms that feed on organisms that feed on GM sugarcane or degrade it
(vertebrates, insects, fungi, Oomycetes and/or bacteria).
Chapter 2 – Risk assessment (February 2007)
36
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Event 5: Direct or indirect ingestion of GM plant materials, produced by the
sugarcane lines containing the introduced genes, by vertebrates, invertebrates and
microorganisms
172. Vertebrates such as livestock and wildlife (including mammals) may be exposed to the
GM sugarcane lines containing the proteins (and enzymatic products) encoded by the
introduced genes for altered architecture, enhanced WUE or improved NUE through direct
feeding or indirectly through consumption of other organisms which have fed on the GM
sugarcane plants.
173. Invertebrates, including beneficial insects, could be exposed directly to the GM
sugarcane lines containing the proteins (and enzymatic products) encoded by the introduced
genes through feeding on the plants, or via the soil when sugarcane tissues decompose.
Exposure of soil invertebrates to the proteins (and enzymatic products) encoded by the
introduced genes could also occur as a result of root exudations. Exposure could also occur
indirectly, through consumption of other organisms that have fed on the GM sugarcane plants.
174. Microorganisms, particularly soil micro-organisms, will be exposed to the GM
sugarcane lines containing the proteins (and enzymatic products) encoded by the introduced
genes during growth and decomposition of plant materials, possibly as a result of root
exudations. Root exudation has been observed in GM maize (Saxena et al. 1999; Stotzky
2000) and GM cotton (Gupta et al. 2002). Root breakage could also lead to the release of the
introduced proteins into the soil. After harvest of GM sugarcane, the remaining plant material
may be tilled into the soil, leading to exposure of soil microorganisms to GM plant residues.
175. Contact with or ingestion by vertebrates, invertebrates and microorganisms of GM plant
materials containing the proteins (and enzymatic products) encoded by the introduced genes
would be limited because of the small scale and short duration of the release. In addition, after
harvest the applicant proposed to destroy GM sugarcane materials produced, apart from
retaining some plant materials for research purposes and for new plantings within the trial,
and none of the materials would be used as animal feed. There are no reports of toxicity
associated with the proteins (and enzymatic products) encoded by the introduced genes (see
Chapter 1, Sections 4.3.4 to 4.3.7). Furthermore, vertebrates, invertebrates and microorganisms would already be exposed to the introduced proteins (and enzymatic products) or
very similar proteins in the environment (see Chapter 1, Section 5.4). As mentioned in
Event 1, the introduced genes containing partial gene sequences are not expected to increase
the toxicity of GM sugarcane plant materials.
176. Therefore, no risk is identified. The potential for toxicity for organisms other than
people as a result of direct or indirect ingestion of the GM plant materials produced by the
sugarcane lines containing the introduced genes will not be assessed further.
2.4 Spread and persistence of the GM sugarcane in the environment
177. Information on non-GM sugarcane is included here to establish a baseline for
comparison with the GM sugarcane being considered in this risk assessment. Attributes of
non-GM sugarcane associated with potential weediness are discussed in the document The
Biology and Ecology of Sugarcane (Saccharum spp. hybrids) in Australia (OGTR 2004). This
document concludes that non-GM sugarcane is not a problematic weed in Australia and
occurs almost exclusively as a managed agricultural crop. In sugarcane districts, volunteer
sugarcane plants may occur along roadsides or railways where they may establish after
displacement during transport, but there is no indication that these plants become selfperpetuating populations. Sugarcane has been grown for centuries throughout the world
without any reports that it is a serious weed.
Chapter 2 – Risk assessment (February 2007)
37
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
178. Characteristics of sugarcane that may contribute to its potential to spread and persist
include its ability to regenerate by re-sprouting from underground buds or from vegetative
cuttings containing viable buds. However, stem cuttings are unlikely to survive very long in
the field as they are degraded rapidly by soil micro-organisms in tropical conditions.
Normally, stem cuttings need to be dipped in fungicide for survival to germination (FAO
2001b; FAO 2004).
179. Sugarcane is almost exclusively propagated asexually but it may reproduce sexually.
However, due to breeding (Berding et al. 2004), climatic conditions (Bakker 1999), and
industry agricultural practices, commercial sugarcane cultivars rarely flower. Moreover, if
sugarcane does flower it generally exhibits poor fertility (Bakker 1999). In cooler climates
such as areas from the Burdekin region and further south, the parent cultivar Q117 has been
shown to have very low pollen viability (2-3%)(Bonnett et al. 2007). Furthermore, sugarcane
pollen rapidly desiccates and is not viable beyond 35 minutes at 26.5ºC at 65% relative
humidity (Venkatraman 1922; Moore 1976). Additionally, sugarcane seed is short lived,
losing 90% of its viability in 80 days at 28°C if not treated to enhance its survival (Rao 1980).
180. Modern sugarcane cultivars (Saccharum spp. hybrid) are not invasive in natural
undisturbed environments. The inability of sugarcane to establish, spread and persist is likely
to be due to factors including competition with other plants, pests and diseases, soil type and
fertility, moisture stress, sunlight requirements and low temperatures (Bakker 1999; Hogarth
& Allsopp 2000; OGTR 2004).
181. The weed status of sugarcane has also been considered previously in RARMPs
produced during the assessment of other GM sugarcane lines (DIRs 019/2002 and 051/2004)
and very low risks were identified.
Event 6: Expression of the introduced genes improving the survival of the GM
sugarcane or volunteers through altered plant architecture, enhanced WUE (drought
tolerance) or improved NUE
182. Up to 1400 of the GM sugarcane lines contain introduced gene constructs for altered
plant architecture (see Table 1), which if successful could result in plants with different stalk
heights, weights and numbers. The GM sugarcane lines may also have increased suckering
and resistance to lodging.
183. Many studies in potato, pumpkin, Arabidopsis, aspen, tobacco, rice and lettuce have
examined the effects of introducing or silencing genes encoding gibberellin oxidases
homologous to those introduced into the proposed GM sugarcane lines (Coles et al. 1999;
Carrera et al. 2000; Curtis et al. 2000; Eriksson et al. 2000; Niki et al. 2001; Vidal et al. 2001;
Sakamoto et al. 2003; Biemelt et al. 2004; Israelsson et al. 2004; Lee & Zeevaart 2005; Radi
et al. 2006). The resulting phenotypes were varied and included increases in plant height,
decreased time to flowering, earlier tuber formation, increased lateral root formation and
thicker roots, and an increase in fruit and seed numbers. However, some phenotypes were
dwarfed, had delayed flowering, were less fertile or sterile, and had smaller roots. The GM
sugarcane lines containing the introduced genes HvGA20ox-1 or 2 for altered plant
architecture appear to produce stalks more rapidly (1-2 months earlier), and GM sugarcane
lines containing the introduced gene PcGA2ox-1 grow more slowly and are shorter, than their
non-GM sugarcane parent under glasshouse conditions (see Chapter 1, Section 4.5.2).
184. The other genes for altered plant architecture introduced into the GM sugarcane lines
include the MAX and TB1 gene homologs. Studies in Arabidopsis with the endogenous MAX3
and MAX4 genes have shown that when the expression of these genes is suppressed, or is nonexistent, phenotypes included dwarfing, more branching and higher inflorescence numbers
Chapter 2 – Risk assessment (February 2007)
38
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
(Seregélyes et al. 2003; Booker et al. 2004; Bainbridge et al. 2005). In petunia, silencing of
the endogenous MAX4 gene resulted in plants with a similar phenotype to that reported in the
A. thaliana studies but with fewer roots and small flowers and an increased flowering time
(Snowden et al. 2005). Over-expression of the endogenous OsTB1 gene in rice led to a
reduction in tillers while the converse phenotype resulted from the silencing of the
endogenous gene (Takeda et al. 2003).
185. Although alteration of plant architecture by the introduced genes could potentially result
in weediness of the GM sugarcane lines, it could also result in GM plants of lower fitness than
other commercially available sugarcane varieties because of the potential
metabolic/physiological burdens. For example, the sugarcane may have stunted growth, be
too tall and fall over, produce less seeds, and have a decreased ability to tolerate competition
from other plants. The trial will enable the applicant to assess the effect of the introduced
genes on plant physiology and agronomic performance.
186. Up to 300 of the GM sugarcane lines contain introduced gene constructs for enhanced
WUE (see Table 1), which if successful would confer enhanced drought stress tolerance. The
introduced genes have demonstrated in plants, other than sugarcane, the capacity to produce a
water-efficient phenotype (Garg et al. 2002) or are involved in the response to dehydration
stress (Urao et al. 1993; Gao et al. 2001). In an environment in which water availability was
the main factor limiting the spread and persistence of sugarcane, expression of the genes for
WUE could result in weediness of the GM sugarcane lines. However, under glasshouse
conditions, necrosis is observed on leaves of the GM sugarcane expressing the introduced
MdS6PDH gene for enhanced WUE. The necrosis is more noticeable at the leaf apex where
the sorbitol content is the highest. The condition is more severe in the plants producing larger
amounts of sorbitol. These GM sugarcane lines are up to 30% shorter compared to non-GM
parent (see Chapter 1, Section 4.5.2). Therefore, expression of the MdS6PDH gene for
enhanced water use efficiency in the GM sugarcane lines would probably result in less fit
plants, which may be more vulnerable to attack by fungal and/or bacterial pathogens, because
of an increase in sorbitol content. These necrotic lesions have also been reported on some GM
tobacco plants expressing MdS6PDH (Sheveleva et al. 1998).
187. Up to 100 of the GM sugarcane lines contain an introduced DOF1 gene for improved
NUE (see Table 1), which if successful would confer improved growth in soil with low
nitrogen levels. The ZmDOF1 gene has demonstrated the capacity in the plant A. thaliana to
produce a phenotype with improved growth under low nitrogen conditions in the laboratory
(Yanagisawa et al. 2004). In an environment in which nitrogen availability was the main
factor limiting the spread and persistence of sugarcane, expression of the genes for NUE
could result in weediness of the GM sugarcane lines.
188. As discussed above, multiple factors limit the distribution and growth of sugarcane in
the areas proposed for release including its low fertility and seed viability, poor ability to
establish and thrive without human intervention, competition with other plants, soil type and
fertility, and pests and diseases (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2004).
Alteration of one factor such as WUE or NUE is unlikely to increase the survival of sugarcane
enough to lead to an increase in weediness and in some cases the modification may have a
negative impact of the plants’ fitness levels.
189. In addition, the release would be of limited size and short duration and the applicant
proposed a number of measures to limit the spread and persistence of the GM sugarcane lines
(see Chapter 1, Section 2.3).
Chapter 2 – Risk assessment (February 2007)
39
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
190. Therefore, no risk is identified and the potential for the spread and persistence of
sugarcane outside of the trial site leading to weediness through altered plant architecture,
enhanced WUE (drought tolerance) or improved NUE as a result of the expression of the
introduced genes will not be assessed further.
Uncertainty
This is a ‘proof of concept’ field trial and therefore, the effect of the introduced genes for
altered plant architecture on the GM sugarcane lines is unknown in the field. Also, the ability
of the GM sugarcane plants to withstand drought stress or have improved NUE throughout
different stages of their lifecycle, as compared to commercially available sugarcane cultivars,
is unknown. Data on the unintentional effects on plant architecture relating to weediness, or
enhanced WUE and improved NUE, may be required to assess possible future applications
involving larger scale or commercial releases of any of these GM sugarcane lines. However,
the data are not required for this release because the trial is limited in locations, size and
duration.
Event 7: Expression of the introduced genes improving the survival of the GM
sugarcane or volunteers through enhancement of abiotic stress tolerances (other than
drought stress) or biotic stress tolerances
Abiotic stress tolerance
191. The GM sugarcane lines express genes that may alter plant architecture, enhance WUE
or improve NUE. It is possible that the introduced genes could potentially confer enhanced
tolerances to other abiotic stresses, other than those associated with water or nitrogen
deficiencies, which could lead to increased spread and persistence of the GM sugarcane
plants.
192. Other enhanced abiotic stress tolerances may improve the GM sugarcane lines’ ability
to survive, or have improved performance, under lower and higher temperatures. It is also
possible the plants could tolerate higher soil salinity, or have increased seed dormancy, seed
or pollen viability, and/or improved seedling germination rates under various abiotic stresses
(other than drought stress). For example, the MdS6PDH gene is known to confer enhanced
salt stress tolerance to transgenic Japanese persimmon plants (Deguchi et al. 2004) and the
introduction of the EcTPSP gene into rice helped the plants withstand salt and low
temperature stresses, as well as drought stress (Garg et al. 2002; Jang et al. 2003). In
Arabidopsis plants, the expression of AtMYB2 is induced by drought and salt stress,
suggesting its involvement in responses to these stresses (Urao et al. 1993).
193. It should be noted that when a gene is expressed in different plant species the same
effect(s) on phenotype does not always eventuate (Oh et al. 2005). Therefore, the effect on
abiotic stress response as a result of the expression of the introduced genes may not eventuate
in the GM sugarcane lines.
194. This is a ‘proof of concept’ field trial and therefore, the ability of the GM sugarcane
plants to withstand abiotic stress tolerances throughout different stages of their lifecycle as
compared to commercially available sugarcane cultivars is unknown.
Biotic stress tolerance
195. The genes for altered plant architecture, enhanced WUE or improved NUE could
potentially enhance resistance of the GM sugarcane lines to pests or pathogenic
microorganisms. This could lead to spread and persistence of the GM sugarcane lines if pests
or diseases were the main limiting factors.
Chapter 2 – Risk assessment (February 2007)
40
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
196. Most diseases of sugarcane are controlled by planting resistant clones (Berding et al.
2004). The most economically important disease of sugarcane is probably ratoon stunting
disease, caused by a bacterium Clavibacter xyli subsp xyli (McLeod et al. 1999). Other
important diseases include orange rust (Puccinia kuehnii), common rust (Puccinia
melanocephala), pachymetra root rot (Pachymetra chaunorhiza) and sugarcane smut
(Ustilago scitaminea) (Bakker 1999). The major pests of sugarcane are the larvae of cane
beetles which destroy the roots of the cane (Berding et al. 2004). Other insect pests such as
plant hopper (Perkinsiella saccharicida) do not cause a significant yield loss but do transmit
viral diseases such as Fiji virus disease or sugarcane mosaic virus. Sugarcane yields can also
be reduced by vertebrate pests such as ground rats (Rattus sordidus), climbing rats (Melomys
burtoni) and feral pigs (Sus scrofa) (Agnew 1997).
197. The introduced genes (or orthologs) for altered plant architecture, enhanced WUE or
improved NUE are not known to confer enhanced biotic stress tolerance.
198. The high sugar content of sugarcane makes it an ideal host for a number of
microorganisms including bacteria and fungi leading to its degradation. It is not expected that
the introduced genes will alter this.
199. This is a ‘proof of concept’ field trial and therefore, the ability of the GM sugarcane
plants to withstand biotic stress tolerances throughout different stages of their lifecycle as
compared to commercially available sugarcane cultivars is unknown.
Conclusion
200. As discussed above, multiple factors limit the distribution and growth of sugarcane in
the areas proposed for release including its low fertility and seed viability, poor ability to
establish and thrive without human intervention, competition with other plants, soil type and
fertility, and pests and diseases (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2004).
201. In addition, the release would be of limited size and short duration and the applicant
proposed a number of measures to limit the spread and persistence of the GM sugarcane lines
(see Chapter 1, Section 2.3).
202. Therefore, no risk is identified and the potential for the spread and persistence
(weediness) of sugarcane as a result of the expression of the introduced genes for altered plant
architecture, enhanced WUE or improved NUE will not be assessed further.
Uncertainty
203. Currently, there is no direct evidence that GM sugarcane lines may have enhanced
tolerances to a number of abiotic and biotic stresses as compared to their non-GM parent.
However, in the highly unlikely instance where the plants may have enhanced tolerances to
several environmental stresses, the GM plants will most likely be less fit as compared to other
commercially available sugarcane varieties because of the potential metabolic/physiological
burdens. For example, the sugarcane may have stunted growth, be too tall and fall over,
produce less seeds, and have a decreased ability to tolerate competition from other plants.
204. Data on tolerances to abiotic/biotic factors, additional to that already discussed for
enhanced drought tolerance of the GM sugarcane lines may be required to assess possible
future applications involving larger scale or commercial releases of any of these GM
sugarcane lines. However, the data are not required for this release because the trial is limited
in locations, size and duration.
Chapter 2 – Risk assessment (February 2007)
41
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Event 8: Dispersal of GM plant materials during transport, research, storage,
equipment use, flooding or via animals
205. Commercial sugarcane is propagated vegetatively. A stem cutting called a ‘sett’
contains one or more buds. Usually one bud per cutting is developed into a primary stalk
which then gives rise to tillers from underground buds (see more details in Biology and
Ecology of Sugarcane (Saccharum spp. hybrids) in Australia (OGTR 2004).
206. It is feasible the GM sugarcane could be dispersed through seeds. However, as
discussed in Event 10 seed production is rare and sugarcane seed has low viability (Rao 1980;
OGTR 2004), information supplied by the applicant) and does not germinate readily (Breaux
& Miller 1987). Therefore, any dispersed seed may not germinate, and if it does, the spread
and persistence of any new GM sugarcane plants outside of the trial sites would be limited by
multiple factors including its low fertility and seed viability, poor ability to establish and
thrive without human intervention, competition with other plants, soil type and fertility, and
pests and diseases (Bakker 1999; Hogarth & Allsopp 2000; OGTR 2004). In addition, the
applicant intends to harvest the majority of the GM sugarcane before flowering, and this
would limit seed production.
Dispersal by spillage during transport, research, storage or from equipment
207. As discussed in the assessment for DIR 051/2004, viable sugarcane stems could be
unintentionally dispersed during transportation. Some sugarcane volunteers have been found
growing along roadsides or railways in sugarcane cultivation areas. These volunteers are
believed to have originated from stem cuttings displaced or fallen from vehicles. These
volunteers generally consist of only a few stools (a group of stems growing from a single
original plant base) and do not become self perpetuating or result in further spread.
208. In the course of the dealings the applicant proposed to transport GM sugarcane setts
(stem cuttings used for vegetative propagation) to the release sites, cultivate GM sugarcane
plants and collect GM plant materials for research purposes, laboratory research or new
plantings within the trial. Accidental spillage or dispersal of GM plant materials, especially
setts, in the course of these dealings could allow the GM sugarcane plants to spread and
persist in the environment.
209. The applicant proposed to transport the GM sugarcane setts to the trial sites according
to OGTR transport guidelines (Guidelines for the transport of GMOs, June 2001; Policy on
transport and supply of GMOs, July 2005). Therefore, any spillage of setts during transport to
and from the release sites would be rare. Moreover, the movement of sugarcane and
sugarcane machinery is restricted in Queensland by the Plant Protection Act 1989 (Qld) and
the Plant Protection Regulations 2002 (Qld). Any incident involving spillage of GM setts is
expected to be readily controlled through cleaning and monitoring of the site of the spill. In
addition, the opportunity for any adverse outcome from any such rare occurrence is further
diminished by the need for appropriate environmental conditions for survival and persistence
of any escaped setts.
210. The applicant proposed to thoroughly clean equipment used in the harvest of the GM
sugarcane (the cane may be hand harvested for laboratory samples) to prevent dispersal of
GM plant materials to other locations and to meet domestic quarantine requirements. The
applicant proposed to destroy all plant materials (including materials from the non-GM guard
row) other than materials collected for future research or for new plantings within the trial.
The sites would be monitored for volunteers after the final harvest and any volunteer
sugarcane plants would be destroyed.
Chapter 2 – Risk assessment (February 2007)
42
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
211. The applicant proposed to conduct any lab research in PC2 facilities. Any plant material
collected for this purpose and not required for further study will be destroyed in the PC2
facility.
Dispersal by flooding of the release site
212. Severe weather conditions (eg flooding) could lead to the dispersal of GM plant
materials. The applicant proposed that the trial sites will be at least 50 m away from natural
waterways, on land not prone to flooding, and surrounded by a guard row of non-GM
sugarcane and a 6 m isolation zone. In case of heavy rain, any GM plant materials on the
ground are therefore not expected to be dispersed beyond the area of the release (including the
guard row, which would act as a physical barrier once the plants were established). Dispersal
of the GM sugarcane would be highly unlikely as sugarcane does not generally propagate
vegetatively without human intervention (OGTR 2004).
Dispersal by animals
213. As sugarcane can reproduce through vegetative cuttings, it is theoretically possible that
pests such as feral pigs or other large animals could disperse viable materials. However, as
discussed in the assessment for DIR 051/2004 there are no reports of this occurring.
214. Numerous animal pests of sugarcane, including mammals, birds and insects, are known
who could eat GM sugarcane plant materials, including flowers, pollen and seed. Of those,
seed has a potential to produce a new GM sugarcane plant if it were still viable after
excretion. However, the seed needs appropriate environmental conditions for germination and
these are seldom present without human intervention. The applicant proposed to surround GM
sugarcane with a row of non-GM sugarcane guard plants, which are expected to minimise
dispersal by large mammals. The applicant also intends to harvest the GM sugarcane lines
before flowering and this would limit the production of seed. In addition, the animal pests of
sugarcane in the release areas are expected to be controlled (as in commercial plantations),
further reducing the potential for dispersal by animals.
215. Therefore, no risk is identified and the potential for an adverse outcome as a result of
dispersal of GM plant materials during transport, research, storage, equipment use, flooding
or via animals will not be assessed further.
Event 9: Exposure of vertebrates (including people), invertebrates and
microorganisms to material produced by the GM sugarcane lines or volunteers
expressing the introduced genes
216. The potential of increased spread and persistence of the GM sugarcane lines in the
environment was addressed in Events 6, 7 and 8 no risk was identified. However, in the
highly unlikely instance of any of these events occurring, spread and persistence of the GM
sugarcane plants in the environment could lead to increased exposure of vertebrates
(including people), invertebrates and microorganisms to the proteins (and enzymatic
products) encoded by the introduced genes for altered plant architecture, enhanced WUE or
improved NUE.
217. An adverse outcome could occur, if the proteins (and enzymatic products) encoded by
the introduced genes for altered plant architecture, enhanced WUE or improved NUE were
toxic or allergenic for people. The potential for the proteins causing toxic or allergic reactions
in people was assessed in Events 1 to 4 and no risk was identified.
218. Organisms other than people may be exposed directly, through feeding on the GM
sugarcane plants or indirectly through eating organisms that have fed on or degraded the GM
sugarcane plants as a result of spread and persistence of the GM sugarcane in the
Chapter 2 – Risk assessment (February 2007)
43
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
environment. These organisms include vertebrates, invertebrates and microorganisms. The
toxicity potential of proteins encoded by the genes for altered plant architecture, enhanced
WUE and improved NUE to organisms other than people was considered in Event 5 and no
risk was identified.
219. In addition, the chain of events that would lead to increased exposure of vertebrates,
invertebrates and microorganisms depends on the least likely event to occur, which is an
enhanced capacity for spread and persistence in the GM sugarcane lines proposed for release.
220. The release would be of limited size and short duration and the applicant proposed a
number of measures to limit the spread and persistence of the GM sugarcane lines in the
environment (for details see Chapter 1, Section 2.3).
221. Therefore, no risk is identified and the potential for toxicity or allergic reactions in
people or other organisms as a result of spread and persistence of the GM sugarcane lines in
the environment will not be assessed further.
2.5 Vertical transfer of genes or genetic elements to sexually compatible
plants
222. Transfer of genetic materials to offspring by reproduction, either asexual or sexual
(vertical gene transfer) could result in the transfer of the introduced genes altered plant
architecture, enhanced WUE or improved NUE, or their associated regulatory elements to
other plants. The sexually compatible species present in Australia that could receive genes
from the GM sugarcane lines are commercial sugarcane and other Saccharum spp. (S.
spontaneum and S. officinarum). Other genera within the tribe Andropogoneae may also be
able to receive genes from the GM sugarcane lines, although some level of sexual
incompatibility will limit this transfer.
223. Weediness resulting from an increase in the spread and persistence of other sugarcane
plants is contingent on both of the following steps:
 transfer of the introduced gene construct to other sugarcane plants
 weediness of the recipient plants as a result of expression of the introduced gene.
Event 10: Expression of the introduced genes in other sugarcane or sexually
compatible plant species
Outcrossing to other sugarcane plants
224. All of the proposed trial sites except the Caboolture site have commercial sugarcane
(S. officinarum x S. spontaneum) growing within 50 kms of their general vicinity. In one of
the areas proposed for release (BSES Meringa research station) Saccharum spp. (commercial
sugarcane, S. spontaneum and S. officinarum) are grown as germplasm collections (see
Chapter 1, Section 5.3). The genes for altered plant architecture, enhanced WUE or improved
NUE could be transferred to these sexually compatible sugarcane plants resulting in an
increased potential for weediness.
225. Sugarcane is typically vegetatively propagated but may reproduce sexually. Many
sugarcane cultivars do not flower reliably which is reported to interrupt cross-breeding
programs (Berding et al. 2004). Observations of the breeding collection at BSES Meringa
research station indicated that an average of 38.2% of clones in the breeding collection
flowered from 1978 to 1996 and the flowering was variable, ranging from 16% of clones in
1993 to 66% in 1984. Cool or dry climatic conditions negatively impact on the development
of the flowers (Bakker 1999).
Chapter 2 – Risk assessment (February 2007)
44
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
226. However, even if sugarcane does flower, it generally exhibits poor fertility (Bakker
1999). The non-GM parent cultivar Q117 produces pollen which is only 10-20% viable
(McIntyre & Jackson 2001) or less (2-3%) in cooler regions such as Burdekin (Bonnett et al.
2007). As such, Q117 is only used as a female in breeding programs (McIntyre & Jackson
2001); information supplied by the applicant).
227. Under the average temperature and humidity expected for the areas proposed for the
release sugarcane pollen would not be expected to travel further than 100 metres (Dr D. M.
Hogarth, personal communication; information supplied by applicant). Sugarcane pollen
rapidly desiccates and is not viable beyond 35 minutes at 26.5ºC at 65% relative humidity
(Venkatraman 1922; Moore 1976). Any seed that may result from a cross would most likely
have limited viability and dormancy (OGTR 2004);information supplied by applicant).
Furthermore, sugarcane seeds are notoriously hard to germinate and the seedlings are very
delicate and vulnerable for the first three to four weeks after germination (Breaux & Miller
1987) and therefore require careful nurturing.
228. Standard cultivation practices for commercial sugarcane cultivation include harvesting
prior to flowering to maximise sugar content. Given that this is likely to occur for any nearby
commercial crops and the applicant intends to follow the same procedures, the opportunity for
gene flow would be limited.
Outcrossing to other sexually compatible plants
229. Sugarcane may also be sexually compatible with genera, within the tribe
Andropogoneae, outside Saccharum genus (for more detail see Chapter 1, Section 5.3)
(OGTR 2004). These genera include Erianthus, Narenga, Miscanthus, Sclerostachya,
Imperata (blady grass), Sorghum (sorghum), Zea (maize) and Bambusa (bamboo).
230. Commercial sorghum and maize crops are not cultivated near the proposed trial sites
(information supplied by applicant), however, other compatible species may be present in the
vicinity of the trial site.
231. Blady grass is common throughout Queensland coastal areas and would probably be in
the vicinity of the proposed trial sites (information supplied by applicant). Wild Sorghum
species are among the weeds of Australian sugarcane crops (McMahon et al. 2000) and are
widespread in Australia (Hnatiuk 1990).
232. Bambusa arundinacea is reported in the coastal regions of Queensland, especially at the
northern tip of Queensland, and many species of bamboo are grown as garden plants
throughout Australia. Bamboo is also present at the BSES Meringa research station, one of
the proposed trial sites (information supplied by applicant).
233. However, crosses of sugarcane with genera outside the Saccharum genus are extremely
difficult even under experimental conditions. The extremely low numbers of known genuine
progeny obtained in any crosses have been of low vigour and sterile. For example, out of 42
crosses made between Erianthus arundinaceus and hybrid Saccharum spp., 17 produced
seedlings, none of which were genuine hybrids (Piperidis et al. 2000). Crosses using
Saccharum officinarum as the female parent produced some hybrids but these were sterile.
Saccharum officinarum x Sorghum bicolor crosses have been reported, but these lack
vegetative vigour (Nair 1999).
234. Although potentially sexually compatible genera may be growing adjacent to the trial
sites, they might not flower at the same time, and outcrossing would be limited due to very
low pollen viability, poor sexual compatibility and the containment measures proposed by the
applicant (see Chapter 1, Section 2.3). This would make any gene flow highly unlikely.
Chapter 2 – Risk assessment (February 2007)
45
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Potential for weediness in plants resulting from outcrossing
235. In the unlikely instance of outcrossing to a commercially grown non-GM sugarcane, the
potential for weediness would be the same as for the GM sugarcane lines themselves, which
were assessed (Events 6-8, this Chapter) and no risk was identified. The spread and
persistence of sugarcane in the areas proposed for release is limited by multiple factors
including its low fertility and seed viability, poor ability to establish and thrive without human
intervention, competition with other plants, soil type and fertility, and pests and diseases
(Bakker 1999; Hogarth & Allsopp 2000; OGTR 2004).
236. In the highly unlikely instance of outcrossing to a plant sexually compatible with
sugarcane the hybrid will tend to be infertile or exhibit low vigour. This would make
enhanced spread and persistence of this plant also highly unlikely.
237. The limited area and duration of this proposed release would further reduce the
likelihood of weediness occurring as a result of gene transfer from the GM sugarcane plants
to other sugarcane or related plants.
Conclusion
238. As discussed above, multiple factors would limit the transfer of genes from the GM
sugarcane lines to other sugarcane plants or sexually related species and the potential for
weediness of these plants.
239. In addition, the applicant proposed a release of limited size and short duration and a
number of measures to limit the spread and persistence of the GM sugarcane lines. These
include post harvest monitoring of the trial sites for at least 12 months following the final
harvest and the destruction of any volunteers.
240. Therefore, no risk is identified and the potential of expression of the introduced genes
for altered plant architecture, enhanced WUE and improved NUE in other sugarcane and
sexually compatible plant species leading to weediness will not be assessed further.
Event 11: Exposure of vertebrates (including people), invertebrates and microorganisms to material produced by other sugarcane or sexually compatible plant
species containing the introduced genes
241. Transfer of the genes for altered plant architecture, enhanced WUE or improved NUE to
other sugarcane and sexually compatible plants could lead to increased exposure of
vertebrates (including people), invertebrates and microorganisms to the proteins and
enzymatic products encoded by the introduced genes.
242. The transfer of the introduced genes to other sugarcane and sexually compatible plants
was assessed in Event 10 and no risk was identified. In the highly unlikely event of this
occurring the potential adverse outcomes arising from these sexually compatible plants would
be highly similar to the GM sugarcane lines for release, which were assessed in Events 1 to 5
and no risk was identified.
243. In addition to the proposed measures to limit the spread and persistence of the GM
sugarcane lines, the applicant proposed a release of limited size and short duration. The
applicant also proposed to monitor the GM sugarcane trial sites for at least 12 months after
the final harvest and destroy any volunteers.
244. Therefore, no risk is identified. The potential for toxicity or allergenicity to vertebrates
(including people), invertebrates and microorganisms, through increased exposure to material
produced by other sugarcane or sexually compatible plant species containing the introduced
genes will not be assessed further.
Chapter 2 – Risk assessment (February 2007)
46
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Event 12: Presence of the introduced regulatory sequences in other sugarcane or
sexually compatible plant species as a result of gene transfer
245. All of the introduced regulatory sequences operate in the same manner as regulatory
elements endogenous to sugarcane plants. The transfer of either endogenous or introduced
regulatory sequences could result in unpredictable effects. The impacts from the introduced
regulatory elements are equivalent and no greater than the endogenous regulatory elements.
246. The transfer of genetic material to other sugarcane or sexually compatible plant species
was assessed in Event 10 and considered highly unlikely.
247. The applicant proposed to monitor the GM sugarcane trial sites for at least 12 months
after the final harvest and to destroy any volunteers.
248. Therefore, no risk is identified and the potential for an adverse outcome as a result of
vertical gene transfer of introduced regulatory sequences will not be assessed further.
2.6 Horizontal transfer of genes or genetic elements to sexually incompatible
organisms.
Event 13: Presence of the introduced genes, or the introduced regulatory sequences,
in other organisms as a result of gene transfer
249. Transfer of the introduced regulatory sequences and/or the introduced genes for altered
plant architecture, enhanced WUE or improved NUE from the GM sugarcane plants to
sexually incompatible plants, animals or microorganisms (horizontal gene transfer) could
occur only rarely without human intervention.
250. Most gene transfers have been identified through analyses of gene sequences (Worobey
& Holmes 1999; Ochman et al. 2000). In general, gene transfers are detected over
evolutionary time scales of millions of years (Lawrence 1999). Most gene transfers have been
from virus to virus (Lai 1992), or between bacteria (Ochman et al. 2000). In contrast, transfers
of plant genetic materials to other microorganisms such as bacteria, viruses or fungi have
been exceedingly rare.
251. Transfer of the regulatory sequences to other organisms could alter the expression of
endogenous genes in unpredictable ways. However, all of the introduced regulatory sequences
operate in the same manner as regulatory elements endogenous to sugarcane plants. The
transfer of either endogenous or introduced regulatory sequences could result in adverse
unpredictable effects. As there is no difference between those two events, this does not
represent a novel adverse outcome as a result of the genetic modification.
252. Horizontal gene transfer has been examined in detail in a number of other RARMPs
(most recently DIR 057/2004), which are available from the OGTR website
<http://www.ogtr.gov.au> or by contacting the Office. These assessments have concluded that
horizontal gene transfer from plants to other sexually incompatible organisms occurs rarely
and usually only on evolutionary timescales. Reports of horizontal gene transfer from plants
to bacteria occurring during laboratory experiments have not only relied on the use of highly
similar sequences to allow homologous recombination to occur, but also on conditions
designed to enhance the selective advantage of gene transfer events (Nielsen et al. 2000;
Gebhard & Smalla 1998; Mercer et al. 1999; Nielsen 1998; De Vries et al. 2001). Horizontal
gene transfer is not expected to produce any adverse outcomes during this limited and
controlled release.
253. Therefore, no risk is identified. The potential for an adverse outcome as a result of
horizontal gene transfer will not be assessed further.
Chapter 2 – Risk assessment (February 2007)
47
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
2.7 Unintended changes in biochemistry, physiology or ecology
254. A single plant gene can have an influence on multiple, sometimes unrelated, plant traits.
This phenomenon is known as pleiotropy and all such multiple effects are sometimes
unknown. Certain types of genes have a higher likelihood of causing pleiotropic effects.
These genes include those that encode proteins that can regulate the transcription of a number
of genes (eg transcription factors).
255. Gene technology has the potential to cause unexpected and unintended pleiotropic
effects due to the process used to insert new genetic material or by producing a gene product
that affects multiple traits. Such effects may include:
 altered expression of an unrelated gene at the site of insertion
 altered expression of an unrelated gene distant to the site of insertion, for example, due
to the encoded protein of the introduced gene changing chromatin structure, affecting
methylation patterns, or regulating signal transduction and transcription
 increased metabolic burden associated with high level expression of the introduced gene
 novel traits arising from interactions of the protein encoded by the introduced gene
product with endogenous non-target molecules
 secondary effects arising from altered substrate or product levels in the biochemical
pathway incorporating the protein encoded by the introduced gene.
256. Unintended pleiotropic effects might result in adverse outcomes such as toxicity or
allergenicity; weediness, pest or disease burden; or reduced nutritional value as compared to
the parent organism. However, accumulated experience with genetic modification of plants
indicates that the process has little potential for unexpected outcomes that are not detected and
eliminated during the routine process of selecting plants that are morphologically similar to
the conventional plant except for the new properties (Bradford et al. 2005). Additionally,
unintended changes that occur as a result of gene insertions are rarely advantageous to the
plant (Kurland et al. 2003).
257. All methods of plant breeding can induce unanticipated changes in plants, including
pleiotropic effects (Haslberger 2003). Most often the important nutrients, toxicants, and other
components are present in the new plant variety at levels that are within the range expected
for commercial varieties.
Event 14: Altered levels of innate toxic or allergenic compounds as a result of the
expression, or random insertion of the introduced genes into the sugarcane genome
during genetic modification
258. Sugarcane products (from either GM or non-GM plants) can be detrimental if fed to
animals in large quantities due to the presence of anti-nutritional compounds including
hydrocyanic acid and lignin (Leng 1991, OGTR 2004). Sugarcane pollen may also be an
allergen (Chakraborty et al. 2001), although allergic responses to the commercial hybrid
cultivars of sugarcane have not been reported in Australia. Further discussion regarding the
toxicity and allergenicity of sugarcane is provided in the Biology and Ecology of Sugarcane
(Saccharum spp. hybrids) in Australia (OGTR 2004).
259. There is potential for the GM sugarcane plants for release to produce toxic or allergenic
compounds as a result of the expression of the genes for altered plant architecture, enhanced
WUE or improved NUE. Additionally, random insertion of the gene construct during the
genetic modification process could potentially result in the production of toxic or allergenic
compounds in the GM sugarcane lines.
Chapter 2 – Risk assessment (February 2007)
48
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
260. Thus far, exposure to plant materials from the GM sugarcane lines has been limited to a
few workers maintaining these plants in the glasshouse. The applicant has reported no adverse
outcomes from exposure to the GM sugarcane plant materials, suggesting that expression of
the introduced genes has not resulted in a change in the level of toxicity or allergenicity
associated with the non-GM parent sugarcane.
261. Furthermore, the introduced genes containing partial gene sequences are not expected to
increase the toxicity and/or allergenicity of GM sugarcane plant materials.
262. In addition, the release would be of limited size and short duration and the applicant
proposed a number of measures to limit the spread and persistence of the GM sugarcane lines.
263. Therefore, no risk is identified and the potential for production of toxic or allergenic
compounds as a result of random insertion of the gene construct into the GM sugarcane lines
will not be assessed further.
Uncertainty
264. Data on the potential toxicity or allergenicity of the proteins encoded by the introduced
genes, and plant materials may be required for risk assessments of applications for larger
scale or commercial release of these GM sugarcane lines. Further information is not required
for assessing the risks of this release because the trial is limited in locations, size and duration
and none of the GM sugarcane plant materials are intended for use in human food, animal
feed or in the production of other sugarcane products
Event 15: Altered biochemistry, physiology or ecology of the GM sugarcane lines
resulting from expression of the introduced genes
265. Biochemical, physiological or ecological changes to the GM sugarcane lines for release
could occur either as a result of the expression of the introduced genes for altered plant
architecture, enhanced WUE or improved NUE, or of the transformation process itself. The
applicant has reported that GM sugarcane lines containing the MdS6PDH gene had leaf
necrosis (see Chapter 1, Section 4.5.2). The trial will enable the in field agronomic
performance of the GM sugarcane lines to be assessed. Unintended changes typically result in
deleterious effects in the plant so are unlikely to proceed in the selection process. However,
this information is not required for assessing the risks of this release because the trial is
limited in locations, size and duration. Furthermore, none of the GM sugarcane plant
materials are intended for use in human food, animal feed or in the production of other
sugarcane products.
266. Therefore, no risk is identified. The potential for weediness and/or toxicity and/or
allergenicity to people and other organisms, as a result of unintended changes in
biochemistry, physiology or ecology will not be assessed further.
Uncertainty
267. Insertion of new genes and traits by conventional breeding or genetic modification can
result in unintended and unexpected changes. Therefore, more data on the potential
pleiotropic effects of the genetic modifications on GM sugarcane lines selected for further
development, and how these may affect potential weediness, toxicity and allergenicity, may
be required to assess any future applications for a larger scale or commercial release of any of
these GMOs.
Chapter 2 – Risk assessment (February 2007)
49
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
2.8 Unauthorised activities
Event 16: Use of GMOs outside the proposed licence conditions (non-compliance)
268. If a licence were to be issued, non-compliance with the conditions of the licence could
lead to spread and persistence of the GM sugarcane lines outside of the proposed release
areas. The adverse outcomes that this event could cause were assessed in Events 1-15. The
Act provides for substantial penalties for non-compliance and unauthorised dealings with
GMOs. The Act also requires that the Regulator has regard for the suitability of the applicant
to hold a licence prior to the issuing of a licence. These legislative provisions are considered
sufficient to minimise risks from unauthorised activities.
269. Therefore, no risk is identified and the potential for an adverse outcome as a result of
unauthorised activities will not be assessed further.
Section 3
Risk estimate process for identified risks
270. The hazard identification process considered the circumstances by which people or the
environment may be exposed to the GMOs, GM plant materials, GM plant by-products, the
introduced genes, or products of the introduced genes.
271. Sixteen events were identified and assessed whereby the proposed release of the GM
sugarcane lines might give rise to harm to people or the environment.
272. These 16 events included consideration of whether, or not, expression of the introduced
genes could result in products that are toxic or allergenic to people or other organisms,
produce unintended changes in the biochemistry, physiology or ecology of the GM sugarcane
lines, or alter characteristics that may impact on spread and persistence of the GMOs. In
addition, consideration was given to the opportunity for gene flow to other organisms, and its
effects.
273. All events were characterised in relation to both the magnitude and probability of harm
in the context of proposed controls to limit the spread and persistence of the GMOs. Detailed
consideration of the sixteen events for this particular field trial demonstrated that none gave
rise to an identified risk that required further assessment. The principle reasons include:
 small scale of the trial is limited in both area and duration
 suitability of containment and disposal measures proposed by the applicant to limit the
spread and persistence of the GM plants
 none of the GM plant materials will be used for any other purpose
 widespread presence of the same or similar proteins (and enzymatic products) in the
environment and lack of evidence of harm from these proteins and their products
 the lack of known toxicity or allergenicity of the proteins (and enzymatic products)
encoded by the introduced genes, and the unlikely potential for gene suppression to
increase toxicity or allergenicity
 limited capacity of the GM sugarcane lines to spread and persist in the areas proposed
for release
 limited ability and opportunity for the GM sugarcane lines to transfer the introduced
genes to commercial sugarcane crops or other sexually compatible species.
274. Therefore, as no risks to the health and safety of people or the environment were
identified from the proposed limited and controlled release of GM sugarcane lines, the level
of risk is considered to be negligible.
Chapter 2 – Risk assessment (February 2007)
50
DIR 70/2006 – Risk Assessment and Risk Management Plan
Chapter 3
Office of the Gene Technology Regulator
Risk management
275. Risk management includes evaluation of risks identified in Chapter 2 to determine
whether or not specific treatments are required to mitigate harm to human health and safety,
or the environment, that may arise from the release. Other risk management considerations
required under the Act are also addressed in this chapter. Together, these risk management
measures are used to inform the decision-making process and determine licence conditions
that are imposed by the Regulator under the Act. In addition, the roles and responsibilities of
other regulators under Australia’s integrated regulatory framework for gene technology are
also explained.
Section 1
Background
276. Under section 56 of the Act, the Regulator must not issue a licence unless satisfied that
any risks posed by the dealings proposed to be authorised by the licence are able to be
managed in a way that protects the health and safety of people and the environment. All
licences are required to be subject to three conditions prescribed in the Act.
277. Section 63 requires that each licence holder inform relevant people of their obligations
under the licence. Other mandatory statutory conditions contemplate the Regulator
maintaining oversight of licensed dealings. For example section 64 requires the licence holder
to provide access to premises to OGTR monitors, and section 65 requires the licence holder to
report any information about risks or unintended effects of the dealing to the Regulator on
becoming aware of them. Matters related to the ongoing suitability of the licence holder are
also required to be reported to the Regulator.
278. It is a further requirement that the licence be subject to any conditions imposed by the
Regulator. Examples of the matters to which conditions may relate are listed in section 62 of
the Act. Licence conditions can be imposed to limit and control the scope of the dealings and
the possession, supply, use, transport or disposal of the GMO for the purposes of, or in the
course of, a dealing. In addition, the Regulator has extensive powers to monitor compliance
with licence conditions under section 152 of the Act.
Section 2
Other Australian regulators
279. Australia's gene technology regulatory system operates as part of an integrated
legislative framework. Other agencies that also regulate GMOs or GM products include
FSANZ, APVMA, Therapeutic Goods Administration (TGA), National Industrial Chemicals
Notification and Assessment Scheme (NICNAS), National Health and Medical Research
Council (NHMRC) and Australian Quarantine and Inspection Service (AQIS). Dealings
conducted under any licence issued by the Regulator may also be subject to regulation by one
or more of these agencies18.
280. The Gene Technology Act 2000 requires the Regulator to consult these agencies during
the assessment of DIR applications. The Gene Technology (Consequential Amendments) Act
2000 requires the agencies to consult the Regulator for the purpose of making certain
decisions regarding their assessments of products that are, or contain a product from, a GMO.
281. FSANZ is responsible for human food safety assessment, including GM food. As the
trial involves early stage research the applicant does not intend any material from these GM
More information on Australia’s integrated regulatory framework for gene technology is contained in the Risk
Analysis Framework available from the Office of the Gene Technology Regulator (OGTR). Free call 1800 181
030 or at <http://www.ogtr.gov.au/pdf/public/raffinal2.2.pdf>.
18
Chapter 3 – Risk management (February 2007)
51
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
sugarcane lines to be used in human food. Accordingly, the applicant has not applied to
FSANZ for evaluation of any of the GM sugarcane lines for use in human food. FSANZ
approval would need to be obtained before they could be used in this way.
282. No other approvals are required.
Section 3
Risk treatment measures for identified risks
283. The risk assessment of events listed in Chapter 2 concluded that there are negligible
risks to people and the environment from the proposed trial of GM sugarcane. The Risk
Analysis Framework, which guides the risk assessment and risk management process, defines
negligible risks as insubstantial with no present need to invoke actions for their mitigation.
284. These events were considered in the context of the scale of the release (up to 3 sites of
up to 2 hectares, over the three cropping cycles between February 2007 and November 2010,
in the Queensland local government areas of Bundaberg, Caboolture and Cairns), the
containment measures and agricultural practices proposed by the applicant and the receiving
environment (see Section 5, Chapter 1).
Section 4
General risk management
285. Containment measures consistent with the risk assessment context have been imposed
to limit the trial to the locations, size and duration requested by the applicant, which are
summarised below (Section 4.1).
4.1 Summary of proposed licence conditions
4.1.1
Measures to limit and control the proposed trial
286. A number of licence conditions19 have been imposed to limit the spread and persistence
of the GMOs, including requirements to:
 surround the trial sites by one guard row of non-GM sugarcane and an isolation zone of
at least 6 metres
 locate the trial sites at least 50 m away from natural waterways
 harvest and process sugarcane from the trial separately from any commercial sugarcane
 destroy all plant materials not required for experimentation or new plantings
 following cleaning of sites, monitor for and destroy any GM sugarcane that may grow
for at least 12 months until the site is clear of volunteers for a continuous 6 month
period.
4.1.2
Measures to control other activities associated with the trial
287. The Regulator has issued guidelines and policies for the transport and supply of GMOs
(Guidelines for the transport of GMOs, June 2001; Policy on transport and supply of GMOs,
19
BSES intends to follow standard industry cultivation protocols, including harvesting before flowering to
preserve the sugar content of the GM lines. In addition, the applicant proposed the removal of sugarcane flowers
at the trial sites prior to anthesis as an additional containment measure to prevent the possibility of gene flow. No
risks were identified even if harvesting of the GM sugarcane lines did not occur and flowers were not removed
prior to anthesis, primarily because the parent cultivar has low fertility (ie rarely flowers; low pollen production
and poor seed viability), is unlikely to flower synchronously with other sexually compatible plants, outcrossing
to related species would be limited due to poor sexual compatibility and progeny are likely to have limited
viability. In addition, the applicant proposed other suitable measures to contain the release which are required by
the licence conditions. Therefore, neither harvesting before flowering nor the removal of sugarcane flowers prior
to anthesis have been included as licence conditions.
Chapter 3 – Risk management (February 2007)
52
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
July 2005). Licence conditions based on these guidelines and policies have been imposed
regarding transportation and storage, and to control possession, use or disposal of the GMOs
for the purposes of, or in the course of, the authorised dealings.
4.2 Other risk management considerations
288. All DIR licences issued by the Regulator contain a number of general conditions that
relate to general risk management. These include, for example:
 applicant suitability
 contingency and compliance plans
 identification of the persons or classes of persons covered by the licence
 reporting structures, including a requirement to inform the Regulator if the applicant
becomes aware of any additional information about risks to the health and safety of
people or the environment
 monitoring for compliance.
4.2.1
Applicant suitability
289. In making a decision whether or not to issue a licence, the Regulator must have regard
to the suitability of the applicant to hold a licence. Under section 58 of the Act matters that
the Regulator must take into account include:
 any relevant convictions of the applicant (both individuals and the body corporate)
 any revocation or suspension of a relevant licence or permit held by the applicant under
a law of the Commonwealth, a State or a foreign country
 the applicant's history of compliance with previous approved dealings
 the capacity of the applicant to meet the conditions of the licence.
290. Before making the decision whether or not to issue a licence for this application
(DIR 070/2006), the Regulator determined that BSES is suitable to hold a licence.
291. Conditions in the licence include a requirement for the licence holder to inform the
Regulator of any circumstances that would affect their suitability or their capacity to meet the
conditions of the licence.
292. In addition, any applicant organisation must have access to a properly constituted
Institutional Biosafety Committee and be an accredited organisation under the Act.
4.2.2
Contingency and compliance plans
293. The licence requires BSES to submit a plan detailing how it intends to ensure
compliance with the licence conditions and document that compliance. This plan is required
before the planting of the GM sugarcane lines commences.
294. BSES is also required to submit a contingency plan to the Regulator within 30 days of
the issue date of the licence. This plan must detail measures to be undertaken in the event of
any unintended presence of the GM sugarcane lines outside of the permitted areas.
295. BSES is also required to provide a method to the Regulator for the reliable detection of
the presence of the GMOs and the introduced genetic materials in a recipient organism. This
instrument would be required within 30 days of the issue date of the licence.
4.2.3
Identification of the persons or classes of persons covered by the licence
Chapter 3 – Risk management (February 2007)
53
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
296. The persons covered by this licence are the licence holder and employees, agents or
contractors of the licence holder and other persons who are, or have been, engaged or
otherwise authorised by the licence holder to undertake any activity in connection with the
dealings authorised by this licence.
4.2.4
Reporting structures
297. The licence obliges the licence holder to immediately report any of the following to the
Regulator:
 any additional information regarding risks to the health and safety of people or the
environment associated with the trial
 any contraventions of the licence by persons covered by the licence
 any unintended effects of the trial.
298. The licence holder is also obliged to submit an Annual Report within 90 days of the
anniversary of the licence containing any information required by the licence, including the
results of inspection activities.
299. A number of written notices are also required under the licence that would assist the
OGTR in designing and implementing a monitoring program for all licensed dealings. The
notices include:
 expected and actual dates of planting
 expected and actual dates of final destroying and cleaning at the end of the trial.
4.2.5
Monitoring for compliance
300. The Act stipulates, as a condition of every licence, that a person who is authorised by
the licence to deal with a GMO, and who is required to comply with a condition of the
licence, must allow inspectors and other persons authorised by the Regulator to enter premises
where a dealing is being undertaken for the purpose of monitoring or auditing the dealing.
Post-release monitoring continues until the Regulator is satisfied that all the GMOs resulting
from the authorised dealings have been removed from the release sites.
301. If monitoring activities identify changes in the risks associated with the authorised
dealings, the Regulator may also vary licence conditions, or if necessary, suspend or cancel
the licence.
302. In cases of non-compliance with licence conditions, the Regulator may also instigate an
investigation to determine the nature and extent of non-compliance. These include the
provision for criminal sanctions of large fines and/or imprisonment for failing to abide by the
legislation, conditions of the licence or directions from the Regulator, especially where
significant damage to health and safety of people or the environment could result.
Section 5
Issues to be addressed for future releases
303. In view of the early stage research involved in the proposed trial, the risk assessment
identified additional information that may be required to assess an application for a larger
scale trial, reduced containment conditions or a commercial release of any of these GM
sugarcane lines. This would include:
 molecular characterisation of GM sugarcane lines selected for possible future releases
Chapter 3 – Risk management (February 2007)
54
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
 additional data on the potential toxicity or allergenicity of proteins encoded by the
introduced genes for altered plant architecture, enhanced WUE and improved NUE, and
of plant materials from the GM sugarcane lines selected for further research
 altered biochemical, physiological and agronomic characteristics indicative of
weediness in the selected GM sugarcane lines including measurement of tolerance to
environmental stresses and reproductive capacity.
Section 6
Conclusions of the RARMP
304. The risk assessment concludes that this limited and controlled release of up to 2500 GM
sugarcane lines on a maximum area of 18 ha over 3 cropping cycles in the Queensland local
government areas of Bundaberg, Caboolture and Cairns poses negligible risks to the health
and safety of people and the environment.
305. The risk management plan concludes that these negligible risks do not require specific
risk treatment measures. However, licence conditions have been imposed to contain the trial
to the locations, size and duration requested by the applicant.
Section 7
DIR 070/2006 Licence
306. The licence DIR 070/2006 is available on the OGTR website
<http://www.ogtr.gov.au/gmorec/ir.htm#table>, following the path to DIR 070/2006.
Chapter 3 – Risk management (February 2007)
55
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
References
Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K., Yamaguchi-Shinozaki, K. (2003).
Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) Function as Transcriptional Activators
in Abscisic Acid Signaling. The Plant Cell 15: 63-78.
Addiscott, T.M., Whitmore, A.P., Powlson, D.S. (1991). Farming, fertilizers and the nitrate
problem. C.A.B. International, Wallingford.
Agnew, J.R. (1997). Australian sugarcane pests. Bureau of Sugar Experiment Stations,
Brisbane.
Ahmad, I., Larher, F., Stewart, G.R. (1979). Sorbitol, a compatible osmotic solute in Plantago
maritima. New Phytologist 82: 671-678.
Aldridge, J., Gibbons, J., Flaherty, M., Kreider, M., Romano, J., Levin, E. (2003).
Heterogeneity of Toxicant Response: Sources of Human Variability. Toxicological Sciences
76: 3-20.
ANZFA (2001a). Final assessment report - Application A379: Oil and linters from
Bromoxynil tolerant cotton transformation events 10211 and 10222. Report No. A379, Food
Standards Australia New Zealand, Canberra, Australia.
ANZFA (2001b). Final assessment report - Application A382: Food derived from insectprotected potato lines BT-06, ATBT04-06, ATBT04-31, ATBT04-36, and SPBT02-05.
Report No. A382, Food Standards Australia New Zealand, Canberra, Australia.
ANZFA (2001c). Final assessment report - Application A383: Food derived from insect and
potato leafroll virus-protected potato lines RBMT21-129, RBMT21-350, and RBMT22-82.
Report No. A383, Food Standards Australia New Zealand, Canberra, Australia.
ANZFA (2001d). Final assessment report - Application A384: Food derived from insect and
potato virus Y-protected potato lines RBMT15-101, SEMT15-02 and SEMT15-15. Report
No. A384, Food Standards Australia New Zealand, Canberra, Australia.
ANZFA (2001e). Final assessment report application A378: Food derived from glyphosatetolerant sugarbeet line 77 (GTSB77). Report No. A378, Australia New Zealand Food
Authority, Canberra, Australia.
ANZFA (2001f). Food derived from glyphosate-tolerant cotton line 1445 - A safety
assessment. Australia New Zealand Food Authority, Canberra, Australia.
Arencibia, A.D., Carmona, E.R., Tellez, P., Chan, MT., Yu, SM., Trujillo, L.E., Oramas, P.
(1998). An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by
Agrobacterium tumefaciens. Transgenic Research 7: 213-222.
Arts, J., Mommers, C., de Heer, C. (2006). Dose-response relationships and threshold levels
in skin and respiratory allergy. Critical review in Toxicology 36: 219-251.
References (February 2007)
56
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Bainbridge, K., Sorefan, K., Ward, S., Leyser, O. (2005). Hormonally controlled expression
of the Arabidopsis MAX4 shoot branching regulatory gene. Plant Journal 44: 569-580.
Bakker, M. (1999). Sugarcane cultivation and management. Kluwer Academic/Plenum
Publishers, New York.
Beck, E., Ludwig, G., Auerswald, E.A., Reiss, B., Schaller, H. (1982). Nucleotide sequence
and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene
19: 327-336.
Bell, M.J., Garside, A.L. (2005). Shoot and stalk dynamics and the yield of sugarcane crops in
tropical and subtropical Queensland, Australia. Field Crops Research 92: 231-248.
Bellaloui, N., Brown, P.H., Dandekar, A.M. (1999). Manipulation of in vivo sorbitol
production alters boron uptake and transport in tobacco. Plant Physiol 119: 735-742.
Bennett, T., Sieberer, T., Willett, B., Booker, J., Luschnig, C., Leyser, O. (2006). The
Arabidopsis MAX pathway controls shoot branching by regulating auxin transport. Current
Biology 16: 553-563.
Berberich, S.A., Leimgruber, R.M., Regan, G.J. (1993). Preparation and verification of dose
for a mouse acute oral toxicity study with neomycin phosphotransferase II protein (NPTII),
study ML-91-409. Unpublished. Monsanto Report No. MSL:13277. Monsanto Technical
Report.
Berding, N., Hogarth, M., Cox, M. (2004). Plant improvement of sugarcane. Chapter 2. In: G
James, ed. Sugarcane, Edition Second. Blackwell Science Ltd, pp 20-47.
Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucleic Acids
Research 12: 8711-8721.
Biemelt, S., Tschiersch, H., Sonnewald, U. (2004). Impact of altered gibberellin metabolism
on biomass accumulation, lignin biosynthesis, and photosynthesis in transgenic tobacco
plants. Plant Physiology 135: 254-265.
Birch, R.G., Bower, R., Elliot, A., Hansom, S., Basnayake, S., Zhang, L. (2000). Regulation
of transgene expression: progress towards practical development in sugarcane and
implications for other plant species. In: AD Arencibia, ed. Plant genetic engineering:
towards the third millenium. Elsevier, Amsterdam.
Blattner, F.R., Plunkett, G., Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides,
J., Glasner, J.D., Rode, C.K., Mayhew, G.F., Gregor, J., Davis, N.W., Kirkpatrick, H.A.,
Goeden, M.A., Rose, D.J., Mau, B., Shao, Y. (1997). The complete genome sequence of
Escherichia coli K-12. Science 277: 1453-1462.
Bonnett, G. D, Berding, N, Morgan, T., and Fitzgerald, P. (2007). Implementation of
gentically modified sugarcane - the need for a better understanding of sexual reproduction.
pp. In press.
Bonnett, G. D, Berding, N, Salter, B, and Hurney, A. P (2004). Suckering in the wet tropics:
consequences, environmental stimuli and potential solutions. In "2004 Conference of the
References (February 2007)
57
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Australian Society of Sugar Cane Technologists", Hogarth-D-M eds, PK Editorial Services
Pty Ltd, Brisbane, Australia., Brisbane, Queensland, Australia. pp. 1-10.
Bonnett, G.D., Salter, B., Albertson, P.L. (2001). Biology of suckers: late-formed shoots in
sugarcane. Annals of Applied Biology 138: 17-26.
Booker, J., Auldridge, M., Wills, S., McCarty, D., Klee, H., Leyser, O. (2004). MAX3/CCD7
is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling
molecule. Current Biology 14: 1232-1238.
Bower, R., Birch, R.G. (1992). Transgenic sugarcane plants via microprojectile
bombardment. Plant Journal 2: 409-416.
Bower, R., Elliott, A.R., Potier, B.A.M., Birch, R.G. (1996). High-efficiency, microprojectilemediated cotransformation of sugarcane, using visible or selectable markers. Molecular
Breeding 2: 239-249.
Bradford, K.J., van Deynze, A., Gutterson, N., Parrott, W., Strauss, S.H. (2005). Regulating
transgenic crops sensibly: lessons from plant breeding, biotechnology and genomics. Nature
Biotechnology 23: 439-444.
Breaux, R.D., Miller, J.D. (1987). Seed handling, germination and seedling propogation.
Chapter 10. In: DJ Heinz, ed. Sugarcane improvement through breeding, Volume 11.
Elsevier, Amsterdm. pp 385-407.
Brown, P.H., Bellaloui, N., Hu, H., Dandekar, A. (1999). Transgenically enhanced sorbitol
synthesis facilitates phloem boron transport and increases tolerance of tobacco to boron
deficiency. Plant Physiol 119: 17-20.
Buchanan, B.B. (2001). Genetic Engineering and the Allergy Issue. Plant Physiology 126: 57.
Bull, T.A., Glasziou, K.T. (1979). Sugarcane. Chapter 4. In: JV Lovett, A Lazenby, eds.
Australian field crops. Angus and Robertson Publishers, pp 95-113.
Calgene Inc. (1990). Request for advisory opinion - KanR gene: safety and use in the
production of genetically engineered plants. Report No. FDA Docket Number 90A-0416,
USA.
Carrera, E., Bou, J., Garcia-Martinez, J.L., Prat, S. (2000). Changes in GA 20-oxidase gene
expression strongly affect stem length, tuber induction and tuber yield of potato plants. Plant
Journal 22: 247-256.
Cellini, F., Chesson, A., Colquhoun, I., Constable, A., Davies, H.V., Engel, K.H., Gatehouse,
A.M., Karenlampi, S., Kok, E.J., Leguay, J.J., Lehesranta, S., Noteborn, H.P., Pedersen, J.,
Smith, M. (2004). Unintended effects and their detection in genetically modified crops. Food
and Chemical Toxicology 42: 1089-1125.
Chakraborty, P., Gupta-Bhattacharya, S., Chowdhury, I., Majumdar, M.R., Chanda, S. (2001).
Differences in concentrations of allergenic pollens and spores at different heights on an
agricultural farm in West Bengal, India. Annals of Agricultural and Environmental Medicine
8: 123-130.
References (February 2007)
58
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Chaves, M.M., Oliveira, M.M. (2004). Mechanisms underlying plant resilience to water
deficits: prospects for water-saving agriculture. Journal of Experimental Botany 55: 23652384.
Chinnusamy, V., Schumaker, K., Zhu, J.K. (2004). Molecular genetic perspectives on crosstalk and specificity in abiotic stress signalling in plants. Journal of Experimental Botany 55:
225-236.
Chinnusamy, V., Zhu, J., Zhu, J.-K. (2006). Gene regulation during cold acclimation in
plants. Physiologia Plantarum 126: 52-61.
Chollet, R., Vidal, J., O'Leary, M.H. (1996). Phosphoenolpyruvate carboxylase: a ubiquitous,
highly regulated enzyme in plants. Annual Review of Plant Physiology and Plant Molecular
Biology 47: 273-298.
Christensen, A.H., Sharrock, R.A., Quail, P.H. (1992). Maize polyubiquitin genes: structure,
thermal perturbation of expression and transcript splicing, and promoter activity following
transfer to protoplasts by electroporation. Plant Molecular Biology 18: 675-689.
Codex Alimentarius Commission (2003). Report of the third session of the codex ad hoc
intergovernmental task force on foods derived from biotechnology (Alinorm 03/34), 25th
session, Rome, Italy. 30 June-5 July, 2003. Joint FAO/WHO Food Standard Programme.
Coles, J.P., Phillips, A.L., Croker, S.J., Garcia-Lepe, R., Lewis, M.J., Hedden, P. (1999).
Modification of gibberellin production and plant development in Arabidopsis by sense and
antisense expression of gibberellin 20-oxidase genes. Plant Journal 17: 547-556.
Curtis, I.S., Ward, D.A., Thomas, S.G., Phillips, A.L., Davey, M.R., Power, J.B., Lowe, K.C.,
Croker, S.J., Lewis, M.J., Magness, S.L., Hedden, P. (2000). Induction of dwarfism in
transgenic Solanum dulcamara by over-expression of a gibberellin 20-oxidase cDNA from
pumpkin. Plant Journal 23: 329-338.
Daniels, J., Roach, B.T. (1987). Taxonomy and evolution. Chapter 2. In: DJ Heinz, ed.
Sugarcane improvement through breeding, Volume 11. Elsevier, Amsterdam, Netherlands. pp
7-84.
Davies, P.J. (2004). Plant Hormones: Biosynthesis, Signal Transduction, Action!. Davies-P-J.
(eds). Cornell University, Ithaca, NY, USA., pp xii-750.
De Vries, J., Meier, P., Wackernagel, W. (2001). The natural transformation of the soil
bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly
depends on homologous sequences in the recipient cells. FEMS Microbiology Letters 195:
211-215.
Deguchi, M., Koshita, Y., Gao, M., Tao, R., Tetsumura, T., Yamaki, S., Kanayama, Y.
(2004). Engineered sorbitol accumulation induces dwarfism in Japanese persimmon. J Plant
Physiol 161: 1177-1184.
Depicker, A., Stachel, S., Dhaese, P., Zambryski, P., Goodman, H.M. (1982). Nopaline
synthase: transcript mapping and DNA sequence. Journal of Molecular and Applied Genetics
1: 561-573.
References (February 2007)
59
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Doebley, J., Stec, A., Hubbard, L. (1997). The evolution of apical dominance in maize.
Nature 386: 485-488.
Edwards, G.E., Nakamoto, H., Burnell, J.N., Hatch, M.D. (1985). Pyruvate, Pi dikinase and
NADP-malate dehydrogenase in C4 photosynthesis: properties and mechanism of light/dark
regulation. Annual Review of Plant Physiology 36: 255-286.
EFB (2001). Antibiotic resistance markers in genetically modified (GM) crops. Report No.
Briefing Paper no. 10, European Federation of Biotechnology, Barcelona, Spain.
EPA (2001). -D Glucuronidase from E. coli and the genetic material necessary for its
production as a plant pesticide inert ingredient: exemption from the requirement of a
tolerance. Federal Register 66: 42957-42962.
Eriksson, M.E., Israelsson, M., Olsson, O., Moritz, T. (2000). Increased gibberellin
biosynthesis in transgenic trees promotes growth, biomass production and xylem fiber length.
Nature Biotechnology 18: 784-788.
FAO (2001a). Evaluation of allergenicity of genetically modified foods. Report of a joint
FAO/WHO expert consultation on allergenicity of foods derived from biotechnology. Rome,
Italy.
FAO (2001b). Safe movement of sugarcane germplasm. Autrey, L., Frison, E., Putter, C.
(eds). The International Board for Plant Genetic Resources FAO in collaboration with The
International Society of Sugarcane Technologists,
FAO (2004). Species description Oryza sativa L.
http://www.fao.org/ag/AGP/AGPC/doc/GBASE/data/pf000274.htm.
FDA (1994). Secondary food additives permitted in food for human consumption; food
additives permitted in feed and drinking water of animals; aminoglycoside 3'phosphotransferase II; final rule. Report No. 59, United States Food and Drug Administration,
Washington, USA.
Felsot, A.S. (2000). Insecticidal genes. Part 2: Human health hoopla. Agrichemical &
Environmental News 168: 1-7.
Flavell, R.B., Dart, E., Fuchs, R.L., Fraley, R.T. (1992). Selectable marker genes: safe for
plants? Biotechnology (N Y ) 10: 141-144.
FSANZ (2003). Final Assessment Report - Application A484: Food from Insect Protected
MON863 Corn.
Fuchs, R.L., Astwood, J.D. (1996). Allergenicity assessment of foods derived from
genetically modified plants. Food Technology 50: 83-88.
Fuchs, R.L., Heeren, R.A., Gustafson, M.E., Rogan, G.J., Bartnicki, D.E., Leimgruber, R.M.,
Finn, R.F., Hershman, A., Berberich, S.A. (1993a). Purification and characterization of
microbially expressed neomycin phosphotransferase II (NPTII) protein and its equivalence to
the plant expressed protein. Biotechnology (NY) 11: 1537-1542.
References (February 2007)
60
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Fuchs, R.L., Ream, J.E., Hammond, B.G., Naylor, M.W., Leimgruber, R.M., Berberich, S.A.
(1993b). Safety assessment of the neomycin phosphotransferase II (NPTII) protein.
Biotechnology (N Y ) 11: 1543-1547.
Galinat, W.C. (1998). The origin of corn. In: GF Sprague, JW Dudley, eds. Corn and Corn
Improvement, Edition 3rd pp 1-31.
Gao, M., Tao, R., Miura, K., Dandekar, A.M., Sugiura, A. (2001). Transformation of
Japanese persimmon (Diospyros kaki Thunb.) with apple cDNA encoding NADP-dependent
sorbitol-6-phosphate dehydrogenase. Plant Sci 160: 837-845.
Garg, A., Kim, J., Owens, T., Ranwala, A., Choi, Y., Kochian, L., Wu, RJ. (2002). Trehalose
accumulation in rice plants confers high tolerance levels to different abiotic stresses.
Proceeding of the National Academic of Science, USA 99: 15898-15903.
Gebhard, F., Smalla, K. (1998). Transformation of Acinetobacter sp. strain BD413 by
transgenic sugar beet DNA. Applied and Environmental Microbiology 64: 1550-1554.
Gilissen, L.J.W., Metz, P.L.J., Stiekema, W.J., Nap, J.P. (1998). Biosafety of E.coli glucuronidase (GUS) in plants. Transgenic Research 7: 157-163.
Good, A.G., Shrawat, A.K., Mueth, M. (2004). Can less yield more? Is reducing nutrient
input into the environment compatible with maintaining crop production? Trends in Plant
Science 9: 597-605.
Grassl, C.O. (1980). Breeding Andropoganeae at the generic level for biomass. Sugarcane
Breeders' Newsletter 41-57.
Gupta, V. V. S. R., Roberts, G. N., Neate, S. M., McClure, S. G., Crisp, P., and Watson, S. K.
(2002). Impact of Bt-cotton on biological processes in Australian soils.Akhurst, R. J., Beard,
C. E., and Hughes, P. A. eds, CSIRO, pp. 191-194.
Haslberger, A.G. (2003). Codex guidelines for GM foods include the analysis of unintended
effects. Nature Biotechnology 21: 739-741.
Hedden, P., Kamiya, Y. (1997). Gibberellin biosynthesis: enzymes, genes and their
regulation. Annual Review of Plant Physiology and Plant Molecular Biology 48: 431-460.
Hedden, P., Phillips, A.L. (2000). Gibberellin metabolism: new insights revealed by the
genes. Trends in Plant Science 5: 523-530.
Herouet, C., Esdaile, D.J., Mallyon, B.A., Debruyne, E., Schulz, A., Currier, T., Hendrickx,
K., van der Klis, R.-J., Rouan, D. (2005). Safety evaluation of the phosphinothricin
acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to
glufosinate-ammonium herbicide in transgenic plants. Regulatory Toxicology and
Pharmacology 41: 134-149.
Hileman, R.E., Silvanovich, A., Goodman, R.E., Rice, E.A., Holleschak, G., Astwood, J.D.,
Hefle, S.L. (2002). Bioinformatic methods for allergenicity assessment using a
comprehensive allergen database. International Archives of Allergy and Immunology 128:
280-291.
References (February 2007)
61
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Hnatiuk, R.J. (1990). Census of Australian vascular plants. Australian Government
Publishing Service, Canberra.
Hogarth, D.M., Allsopp, P.G. (2000). Manual of Canegrowing. Bureau of Sugar Experiment
Stations, Brisbane.
Holden, J.R. (1998). Irrigation of sugarcane. Holden, J.R. (eds). BSES, Brisbane.
Holsapple, M., Jones, D., Kawabata, T., Kimber, I., Sarlo, K., Selgrade, M., Shah, J.,
Woolhiser, M. (2006). Assessing the potential to induce respiratory hypersensitivity.
Toxicological Sciences 91: 4-13.
Hubbard, L., McSteen, P., Doebley, J., Hake, S. (2002). Expression patterns and mutant
phenotype of teosinte branched1 correlate with growth suppression in maize and teosinte.
Genetics 16: 1927-1935.
Huby, R.D.J., Dear, n, RJ., Kimber, I. (2000). Why are some proteins allergens?
Toxicological Sciences 55: 235-246.
Iida, K., Seki, M., Sakurai, T., Satou, M., Akiyama, K., Toyoda, T., Konagaya, A., Shinozaki,
K. (2005). RARTF: Database and tools for complete sets of Arabidopsis transcription factors.
DNA Research 12: 247-256.
Inman-Bamber, N. G., Zund, P. R., and Muchow, R. C. (28-4-2000). Water use efficiency and
soil water availability for sugarcane. In "Proceedings of the Conference of the Australian
Society of Sugar Cane Technologists", Hogarth, D. M. eds, Watson Ferguson and Company,
Brisbane, Australia. pp. 264-269.
Israelsson, M., Mellerowicz, E., Chono, M., Gullberg, J., Moritz, T. (2004). Cloning and
overproduction of gibberellin 3-oxidase in hybrid aspen trees. Effects on gibberellin
homeostasis and development. Plant Physiol 135: 221-230.
Janakiammal, E.K. (1938). A Saccharum-Zea cross. Nature 142: 618-619.
Jang, I., Oh, S., Seo, J., Choi, W., Song, S., Kim, C., Kim, Y., Seo, H., Choi, Y., Nahm, B.,
Kim, JK. (2003). Expression of a bifunctional fusion of the Escherichia coli genes for
trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice
plants increases trehalose accumulation and abiotic stress tolerance without stunting growth.
Plant Physiology 131: 516-524.
Jefferson, R.A., Burgess, S.M., Hirsh, D. (1986). -Glucuronidase from Escherichia coli as a
gene-fusion marker. Proceedings of the National Academy of Sciences of the United States of
America 83: 8447-8451.
Kimber, I., Kerkvliet, N.L., Taylor, S.L., Astwood, J.D., Sarlo, K., Dearman, R.J. (1999).
Toxicology of protein allergenicity: prediction and characterization. Toxicological Sciences
48: 157-162.
King, T.P., Norman, P.S., Connell, J.T. (1964). Isolation and Characterization of Allergens
from Ragweed Pollen. II. Biochemistry 3: 458-468.
References (February 2007)
62
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Klee, H.J., Rogers, S.G. (1989). Plant gene vectors and genetic transformation: plant
transformation systems based on the use of Agrobacterium tumefaciens. Cell Culture and
Somatic Cell Genetics of Plants 6: 1-23.
Kurland, C.G., Canback, B., Berg, O.G. (2003). Horizontal gene transfer: a critical view.
Proceedings of the National Academy of Science of the United States of America 100: 96589662.
Lai, M.M.C. (1992) RNA recombination in animal and plant viruses. Microbiol Rev 56 (1):
61-79.
Lawrence, J.G. (1999). Gene transfer, speciation, and the evolution of bacterial genomes.
Current Opinion in Microbiology 2: 519-523.
Lee, D.J., Zeevaart, J.A. (2005). Molecular cloning of GA 2-oxidase3 from spinach and its
ectopic expression in Nicotiana sylvestris. Plant Physiol 138: 243-254.
Leng, R.A. (1991). Application of biotechnology to nutrition of animals in developing
countries. Report No. FAO Technical papers, FAO, Rome.
Levy, S.B., Marshall, B., Schluederberg, S., Rowse, D., Davis, J. (1998). High frequency of
antimicrobial resistance in human fecal flora. Antimicrobial Agents and Chemotherapy 32:
1801-1806.
Lewin, B. (1994a). Building the transcription complex: promoters, factors, and the RNA
polymerases. Chapter 29. In: Genes V. Oxford University Press, New York. pp 847-877.
Lewin, B. (1994b). Regulation of transcription: factors that activate the basal apparatus.
Chapter 30. In: Genes V. Oxford University Press, New York. pp 879-909.
McIntyre, C.L., Jackson, P.A. (2001). Low level of selfing found in a sample of crosses in
Australian sugarcane breeding programs. Euphytica 117: 245-249.
McLeod, R.S., McMahon, G.G., Allsopp, P.G. (1999). Costs of major pests and diseases to
the Australian sugar industry. Plant Protection Quarterly 14: 42-46.
McMahon, G., Lawrence, P., O'Grady, T. (2000). Weed control in sugarcane. Chapter 12. In:
DM Hogarth, P Allsopp, eds. Manual of cane growing. Bureau of Sugar Experiment Stations,
Indooroopilly. pp 241-261.
McSteen, P., Leyser, O. (2005). Shoot branching. Annual Review of Plant Biology 56: 353374.
Mercer, D.K., Scott, K.P., Bruce-Johnson, W.A., Glover, L.A., Flint, H.J. (1999). Fate of free
DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA
in human saliva. Applied and Environmental Microbiology 65: 6-10.
Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L., Fuchs, R.L.
(1996). Assessment of the allergenic potential of foods derived from genetically engineered
crop plants. Critical Reviews in Food Science and Nutrition 36(S): S165-S186.
References (February 2007)
63
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Miki, B., McHugh, S. (2004). Selectable marker genes in trangenic plants: applications,
alternatives and biosafety. Journal of Biotechnology 107: 193-232.
Milligan, S.B., Gravois, K.A., Bischoff, K.P., Martin, F.A. (1990). Crop effects on genetic
relationships among sugarcane traits. Crop Science 30: 927-931.
Moore, P.H. (1976). Studies on sugarcane pollen. II. Pollen storage. Phyton, Argentina 34:
71-80.
Nair, N.V. (1999). Production and cyto-morphological analysis of intergeneric hybrids of
Sorghum x Saccharum. Euphytica 108: 187-191.
Nardelli, D., het Schip, F.D., Gerber-Huber, S., Haefliger, J.A., Gruber, M., Ab, G., Wahli,
W. (1987). Comparison of the organization and fine structure of a chicken and a Xenopus
laevis vitellogenin gene. Journal of Biological Chemistry 262: 15377-15385.
Naylor, M.W. (1992). Acute oral toxicity study of beta-glucuronidase (GUS) protein in albino
mice. Unpublished. Report No. Monsanto Report No. MSL:12485, Monsanto Company, St.
Louis.
Nielsen, K.M. (1998). Barriers to horizontal gene transfer by natural transformation in soil
bacteria. Acta pathologica, microbiologica, et immunologica Scandinavica 106: 77-84.
Nielsen, K.M., Smalla, K., van Elsas, J.D. (2000) Natural transformation of Acinetobacter sp.
strain BD413 with cell lysates of Acinetobacter sp., Pseudomonas fluorescens, and
Burkholderia cepacia in soil microcosms. Appl Environ Microbiol 66 (1): 206-212.
Niki, T., Nishijima, T., Nakayama, M., Hisamatsu, T., Oyama-Okubo, N., Yamazaki, H.,
Hedden, P., Lange, T., Mander, L.N., Koshioka, M. (2001). Production of dwarf lettuce by
overexpressing a pumpkin gibberellin 20-oxidase gene. Plant Physiol 126: 965-972.
Ochman, H., Lawrence, J.G., Grolsman, E. (2000). Lateral gene transfer and the nature of
bacterial innovation. Nature 405: 299-304.
OGTR (2002). The Biology and Ecology of Cotton (Gossypium hirsutum) in Australia.
OGTR (2004). The Biology and Ecology of Sugarcane (Saccharum spp. L.) in Australia.
OGTR, Canberra.
OGTR (2005). Risk Analysis Framework. Australian Government, Canberra, ACT.
Oh, S.J., Song, S.I., Kim, Y.S., Jang, H.J., Kim, S.Y., Kim, M., Kim, Y.K., Nahm, B.H., Kim,
J.K. (2005). Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to
abiotic stress without stunting growth. Plant Physiology 138: 341-351.
Pawlowski, W.P., Somers, D.A. (1996). Transgene inheritance in plants genetically
engineered by microprojectile bombardment. Mol Biotechnol 6: 17-30.
Piperidis, G., Christopher, M.J., Carroll, B.J., Berding, N., D'Hont, A. (2000). Molecular
contribution to selection of intergeneric hybrids between sugarcane and the wild species
Erianthus arundinaceus. Genome 43: 1033-1037.
References (February 2007)
64
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Radi, A., Lange, T., Niki, T., Koshioka, M., Lange, M.J. (2006). Ectopic expression of
pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic
Arabidopsis plants. Plant Physiol 140: 528-536.
Rafnar, T., Griffith, I.J., Kuo, M.C., Bond, J.F., Rogers, B.L., Klapper, D.G. (1991). Cloning
of Amb a I (antigen E), the major allergen family of short ragweed pollen. Journal of
Biological Chemistry 266: 1229-1236.
Ramanjulu, S., Bartels, D. (2002). Drought- and desiccation-induced modulation of gene
expression in plants. Plant, Cell and Environment 25: 141-151.
Rao, J.T., Alexander, M.P., Kandaswami, P.A. (1967). Intergeneric hybridisation studies
between Saccharum (sugarcane) and Bambusa (bamboo). Journal of the Indian Botanical
Society 46: 199-208.
Rao, P. S. (1980). Fertility, seed storage and seed viability in sugarcane. In "Proceedings of
the International Society of Sugar Cane Technologists", pp. 1236-1240.
Rogers, S.G., O'Connell, K., Horsch, R.B., Fraley, R.T. (1985). Biotechnology in plant
science. Zaitlin, M., Day, P., Hollaender, A., Wilson, C.A. (eds). Academic Press Inc, New
York. pp 219-226.
Sakamoto, T., Morinaka, Y., Ishiyama, K., Kobayashi, M., Itoh, H., Kayano, T., Iwahori, S.,
Matsuoka, M., Tanaka, H. (2003). Genetic manipulation of gibberellin metabolism in
transgenic rice. Nature Biotechnology 21: 909-913.
Saxena, D., Flores, S., Stotzky, G. (1999). Insecticidal toxin in root exudates from Bt corn.
Nature 402: 480.
Schmitz, G., Theres, K. (2005). Shoot and inflorescence branching. Current Opinion in Plant
Biology 8: 506-511.
Schwartz, S.H., Qin, X., Loewen, M.C. (2004). The biochemical characterization of two
carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound
inhibits lateral branching. Journal of Biological Chemistry 279: 46940-46945.
Seki, M., Narusaka, M., Ishida, J., Nanjo, T., Fujita, M., Oono, Y., Kamiya, A., Nakajima,
M., Enju, A., Sakurai, T., Satou, M., Akiyama, K., Taji, T., Yamaguchi-Shinozaki, K.,
Carninci, P., Kawai, J., Hayashizaki, Y., Shinozaki, K. (2002). Monitoring the expression
profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a fulllength cDNA microarray. Plant Journal 31: 279-292.
Seo, H., Koo, Y., Lim, J., Song, J., Kim, C., Kim, J., Lee, J., Choi, YD. (2000).
Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and
trehalose-6-phosphate phosphatase of Escherichia coli. Applied & Environmental
Microbiology 66: 2484-2490.
Seregélyes, C., Barna, B., Hennig, J., Konopka, D., Pasternak, T.P., Lukács, N., Fehér, A.,
Horváth, G., V, Dudits, D. (2003). Phytoglobins can interfere with nitric oxide functions
during plant growth and pathogenic responses: a transgenic approach. Plant Science 165: 541550.
References (February 2007)
65
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Sheveleva, E.V., Marquez, S., Chmara, W., Zegeer, A., Jensen, R.G., Bohnert, H.J. (1998).
Sorbitol-6-Phosphate Dehydrogenase Expression in Transgenic Tobacco. High Amounts of
Sorbitol Lead to Necrotic Lesions. Plant Physiology 117: 831-839.
Shinozaki, K., Yamaguchi-Shinozaki, K., Seki, M. (2003). Regulatory network of gene
expression in the drought and cold stress responses. Current Opinion in Plant Biology 6: 410417.
Silvanovich, A., Nemeth, M.A., Song, P., Herman, R., Tagliani, L., Bannon, G.A. (2006). The
value of short amino acid sequence matches for prediction of protein allergenicity.
Toxicological Sciences 90: 252-258.
Singh, G., Chapman, S. C., Jackson, P. A., and Lawn, R. J. (2000). Lodging - a major
constraint to high yield and CCS in the wet and dry tropics. In "Proceedings of the
Conference of the Australian Society of Sugar Cane Technologists", Hogarth, D. M. eds,
Watson Ferguson and Company, Brisbane, Australia. pp. 315-321.
Singh, K.B. (1998). Transcriptional regulation in plants: The importance of combinatorial
control. Plant Physiology 118: 1111-1120.
Smalla, K., Van Overbeek, L.S., Pukall, R., van Elsas, J.D. (1993). Prevalence of npt II and
Tn5 in kanamycin-resistant bacteria from different environments. FEMS Microbiology
Ecology, 13: 47-58.
Snowden, K.C., Simkin, A.J., Janssen, B.J., Templeton, K.R., Loucas, H.M., Simons, J.L.,
Karunairetnam, S., Gleave, A.P., Clark, D.G., Klee, H.J. (2005). The Decreased apical
dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 gene affects
branch production and plays a role in leaf senescence, root growth, and flower development.
Plant Cell 17: 746-759.
Sorefan, K., Booker, J., Haurogne, K., Goussot, M., Bainbridge, K., Foo, E., Chatfield, S.,
Ward, S., Beveridge, C., Rameau, C., Leyser, O. (2003). MAX4 and RMS1 are orthologous
dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea. Genes and
Development 17: 1469-1474.
Spanu, T., Luzzaro, F., Perilli, M., Amicocante, G., Toniolo, A., Fadda, G., The Italian ESBL
Study Group (2002). Occurrence of extended-spectrum beta-lactamases in members of the
family Enterobacteriaceae in Italy: Implications for resistance to beta-lactams and other
antimicrobial drugs. Antimicrobial Agents and Chemotherapy 46: 196-202.
Stadler, M.B., Stadler, B.M. (2003). Allergenicity prediction by protein sequence. FASEB
Journal 17: 1141-1143.
Stotzky, G. (2000). Microorganisms in soil: Factors influencing their activity. Microsporidia:
Basic Biology 2057-2065.
Takeda, T., Suwa, Y., Suzuki, M., Kitano, H., Ueguchi-Tanaka, M., Ashikari, M., Matsuoka,
M., Ueguchi, C. (2003). The OsTB1 gene negatively regulates lateral branching in rice. Plant
Journal 33: 513-520.
The Royal Society (2002). Genetically modified plants for food use and human health—an
update. Report No. Policy document 4/02, The Royal Society, UK.
References (February 2007)
66
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Thomas, K., Bannon, G., Hefle, S.L., Herouet, C., Holsapple, M., Ladics, G., MacIntosh, S.,
Privalle, L. (2005). In Silico Methods for Evaluating Human Allergenicity to Novel Proteins:
International Bioinformatics Workshop Meeting Report, 23-24 February 2005. Toxicological
Sciences 88: 307-310.
Thomas, R., Venkatraman, T.S. (1930). Sugarcane-Sorghum hybrids. Indian Journal of
Agricultural Sciences 6: 1105-1106.
Umemura, Y., Ishiduka, T., Yamamoto, R., Esaka, M. (2004). The Dof domain, a zinc finger
DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn
finger domain. Plant Journal 37: 741-749.
Urao, T., Yamaguchi-Shinozaki, K., Urao, T., Shinozaki, K. (1993). An Arabidopsis myb
homolog is induced by dehydration stress and its gene product binds to the conserved MYB
recognition sequence. Plant Cell 5: 1529-1539.
US FDA (1998). Guidance for industry: use of antibiotic resistance marker genes in
transgenic plants.1-26. http://vm.cfsan.fda.gov/~dms/opa-armg.html.
van het Schip, F.D., Samallo, J., Broos, J., Ophuis, J., Mojet, M., Gruber, M., Geert, A.B.
(1987). Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence
of the encoded yolk precursor protein. Journal of Molecular Biology 196: 245-260.
Van Larebeke, N., Engler, G., Holsters, M., Van den Elsacker, S., Zaenen, I., Schilperoort,
R.A., Schell, J. (1974). Large plasmid in Agrobacterium tumefaciens essential for crown gallinducing ability. Nature 252: 169-170.
Venkatraman, R.S.T.S. (1922). Germination and preservation of sugarcane pollen.
Agricultural Journal of India 17: 127-132.
Vidal, A.M., Gisbert, C., Talon, M., Primo-Millo, E., Lopez-Diaz, I., Garcia-Martinez, J.L.
(2001). The ectopic overexpression of a citrus gibberellin 20-oxidase enhances the non-13hydroxylation pathway of gibberellin biosynthesis and induces an extremely elongated
phenotype in tobacco. Physiol Plant 112: 251-260.
Vinocur, B., Altman, A. (2005). Recent advances in engineering plant tolerance to abiotic
stress: achievements and limitations. Current Opinion in Biotechnology 16: 123-132.
Wang, K., Herrera-Estrella, L., Van Montagu, M., Zambryski, P. (1984). Right 25 bp
terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA
transfer from Agrobacterium to the plant genome. Cell 38: 455-462.
Wang, W., Vinocur, B., Altman, A. (2003). Plant responses to drought, salinity and extreme
temperatures: towards genetic engineering for stress tolerance. Planta 218: 1-14.
Worobey, M., Holmes, E.C. (1999). Evolutionary aspects of recombination in RNA viruses.
Journal of General Virology 80: 2535-2543.
Xiong, L., Zhu, J.K. (2001). Abiotic stress signal transduction in plants: Molecular and
genetic perspectives. Physiologia Plantarum 112: 152-166.
References (February 2007)
67
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Yanagisawa, S. (1995). A novel DNA-binding domain that may form a single zinc finger
motif. Nucleic Acids Research 23: 3403-3410.
Yanagisawa, S. (2000). Dof1 and Dof2 transcription factors are associated with expression of
multiple genes involved in carbon metabolism in maize. Plant Journal 21: 281-288.
Yanagisawa, S. (2001). The transcriptional activation domain of the plant-specific Dof1 factor
functions in plant, animal, and yeast cells. Plant Cell Physiology 42: 813-822.
Yanagisawa, S. (2002). The Dof family of plant transcription factors. Trends in Plant Science
7: 555-560.
Yanagisawa, S. (2004). Dof domain proteins: plant-specific transcription factors associated
with diverse phenomena unique to plants. Plant Cell Physiology 45: 386-391.
Yanagisawa, S., Akiyama, A., Kisaka, H., Uchimiya, H., Miwa, T. (2004). Metabolic
engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and
growth under low-nitrogen conditions. Proceeding of the National Academic of Science, USA
101: 7833-7838.
Yanagisawa, S., Schmidt, RJ. (1999). Diversity and similarity among recognition sequences
of Dof transcription factors. Plant Journal 17: 209-214.
Yanagisawa, S., Sheen, J. (1998). Involvement of maize Dof zinc finger proteins in tissuespecific and light-regulated gene expression. Plant Cell 10: 75-89.
Zhu, J.K. (2002). Salt and drought stress signal transduction in plants. Annual Review of Plant
Biology 53: 247-273.
References (February 2007)
68
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Appendix A Definitions of terms in the Risk Analysis
Framework used by the Regulator
(* terms defined as in Australia New Zealand Risk Management Standard AS/NZS
4360:2004)
Consequence
outcome or impact of an adverse event
Marginal: there is minimal negative impact
Minor: there is some negative impact
Major: the negative impact is severe
Event*
occurrence of a particular set of circumstances
Hazard*
source of potential harm
Hazard identification
the process of analysing hazards and the events that may give rise to harm
Intermediate
the negative impact is substantial
Likelihood
chance of something happening
Highly unlikely: may occur only in very rare circumstances
Unlikely: could occur in some circumstances
Likely: could occur in many circumstances
Highly likely: is expected to occur in most circumstances
Quality control
to check, audit, review and evaluate the progress of an activity, process or system on an
ongoing basis to identify change from the performance level required or expected and
opportunities for improvement
Risk
the chance of something happening that will have an undesired impact
Negligible: risk is insubstantial and there is no present need to invoke actions for mitigation
Low: risk is minimal but may invoke actions for mitigation beyond normal practices
Moderate: risk is of marked concern requiring mitigation actions demonstrated to be effective
High: risk is unacceptable unless actions for mitigation are highly feasible and effective
Appendix A (February 2007)
69
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Risk analysis
the overall process of risk assessment, risk management and risk communication
Risk analysis framework
systematic application of legislation, policies, procedures and practices to analyse risks
Risk assessment
the overall process of hazard identification and risk estimation
Risk communication
the culture, processes and structures to communicate and consult with stakeholders about
risks
Risk Context
parameters within which risk must be managed, including the scope and boundaries for the
risk assessment and risk management process
Risk estimate
a measure of risk in terms of a combination of consequence and likelihood assessments
Risk evaluation
the process of determining risks that require treatment
Risk management
the overall process of risk evaluation, risk treatment and decision making to manage potential
adverse impacts
Risk management plan
integrates risk evaluation and risk treatment with the decision making process
Risk treatment*
the process of selection and implementation of measures to reduce risk
Stakeholders*
those people and organisations who may affect, be affected by, or perceive themselves to be
affected by a decision, activity or risk
States
includes all State governments, the Australian Capital Territory and the Northern Territory
governments
Uncertainty
imperfect ability to assign a character state to a thing or process; a form or source of doubt
Appendix A (February 2007)
70
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Appendix B Summary of issues raised in
submissions received from prescribed
experts, agencies and authorities20 on
application DIR 070/2006
All issues raised in submissions relating to risks to the health and safety of people and the
environment were considered in the context of the currently available scientific evidence that
was used in the preparation of the consultation RARMP.
Issues raised relating to the Risk Assessment (considered in Ch 2):
 Enhanced spread and persistence (weediness) of the GM sugarcane (Events 6, 7 and 8).
 Gene flow to other commercial sugarcane crops, related species or unrelated species
(Events 10 to 13).
 Toxicity and/or allergenicity of the proteins (and enzymatic products) encoded by the
introduced genes (Events 1 to 5, 9, and 11 to 15).
 Dissemination of the GM sugarcane material beyond the intended areas (Events 10 to
13, and 16).
 Potential pleiotropic effects of the introduced genes (Events 14 and 15).
Issues raised relating to the Risk Management Plan (considered in Ch 3 and licence):
 Containment measures
 Transport procedures for the GM sugarcane
 Post-harvest monitoring and practices
 Disposal of GM plant materials not required for further research or approved plantings.
20
GTTAC, State and Territory governments, Australian Government agencies, the Minister for Environment and
Heritage and Local councils where the release may occur.
Appendix B (February 2007)
71
DIR 70/2006 – Risk Assessment and Risk Management Plan
Office of the Gene Technology Regulator
Appendix C Summary of issues raised in
submissions received from the public
on application DIR 070/2006
Two submissions received from the public are summarised in the table below.
All issues relating to risks to the health and safety of people and the environment were
considered in the context of the currently available scientific evidence.
Issues raised: EN: environmental risks; H: human health and safety.
Other abbreviations: OGTR: The Office of the Gene Technology Regulator
Submission
no.
Issue
Summary or issues raised
Consideration
in RARMP
Comment
1
None
-
Noted
2
H, EN
Fully supports the proposed
application after considering the
transgenic phenotypes proposed
for release and has confidence in
the expertise of BSES staff to
comply with OGTR requirements
and licence conditions.
Such a release could not be
controlled. The area could not be
de-contaminated, and release to
the surrounding environment
could not be controlled for very
long.
Human health and well being are
put at risk.
Ch 2
Licence conditions have been
proposed to contain the small
scale release.
Ch 2
None of the GM plant material
from the proposed trial will be
used in human food or animal
feed.
H
Appendix C (February 2007)
72