Risk Assessment and
Risk Management Plan for
DIR 136
Limited and controlled release of cotton genetically
modified for enhanced fibre quality
Applicant: CSIRO
September 2015
PAGE INTENTIONALLY LEFT BLANK
DIR 136 – Risk Assessment and Risk Management Plan (September 2015)
Office of the Gene Technology Regulator
Summary of the Risk Assessment and Risk
Management Plan
for
Licence Application No. DIR 136
Decision
The Gene Technology Regulator (the Regulator) has decided to issue a licence for this
application for a limited and controlled release (field trial) of a genetically modified organism
(GMO) into the environment. A Risk Assessment and Risk Management Plan (RARMP) for
this application was prepared by the Regulator in accordance with the requirements of the Gene
Technology Act 2000 (the Act) and corresponding state and territory legislation, and finalised
following consultation with a wide range of experts, agencies and authorities, and the public.
The RARMP concludes that this field trial poses negligible risks to human health and safety
and the environment and that any risks posed by the dealings can be managed by imposing
conditions on the release.
The application
Application number
DIR 136
Applicant
CSIRO
Project title
Limited and controlled release of cotton genetically modified for
enhanced fibre quality
Parent organism
Cotton (Gossypium hirsutum L.)

Partial and full PME3 (pectin methylesterase enzyme) gene from
cotton – altered fibre quality

PME4 (pectin methylesterase enzyme) gene from cotton – enhanced
fibre quality

PME5 (pectin methylesterase enzyme) gene from cotton – enhanced
fibre quality

GAUT1 (galacturonyltransferase) gene from cotton – enhanced fibre
quality

nptII gene from Escherichia coli – antibiotic resistance selectable
marker
Introduced genes and
modified traits
Proposed location
One site at the Australian Cotton Research Institute, Narrabri, NSW
Proposed release size
Up to 1 hectare (ha) per year
Proposed release dates
October 2016 – May 20191
Primary purpose
To assess the agronomic performance and fibre quality of the genetically
modified (GM) cotton lines under field conditions
CSIRO proposes to conduct a field trial of GM cotton lines2 containing introduced cotton
genes, or a fragment of a cotton gene, designed to alter pectin profiles in the cotton fibre to
enhance fibre quality.
The proposed release dates have been changed from October 2015 – May 2018 indicated in the consultation
RARMP, following a request by the applicant noting that water restrictions will delay the trial.
2
The term ‘line’ is used to denote plants derived from a single plant containing a specific genetic modification
resulting from a single transformation event.
1
Summary
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DIR 136 – Risk Assessment and Risk Management Plan (September 2015)
Office of the Gene Technology Regulator
Risk assessment
The risk assessment concludes that there are negligible risks to the health and safety of people,
or the environment, from the proposed release.
The risk assessment process considers how the genetic modifications and proposed activities
conducted with the GMOs might lead to harm to people or the environment. Risks were
characterised in relation to both the seriousness and likelihood of harm, taking into account
current scientific/technical knowledge, information in the application (including proposed
limits and controls), relevant previous approvals and advice received from a wide range of
experts, agencies and authorities consulted on the RARMP. Both the short and long term
impact were considered.
Credible pathways to potential harm that were considered included exposure to the GM plant
material, dispersal of GM seed leading to spread and persistence of the GMOs, and transfer of
the introduced genetic material to sexually compatible cotton plants. Potential harms associated
with these pathways included toxicity or allergenicity to people, toxicity to other desirable
organisms, and environmental harms due to weediness.
The principal reasons for the conclusion of negligible risks are that the proposed limits and
controls effectively contain the GMOs and their genetic material and minimise exposure; the
introduced genetic modifications are unlikely to cause harm to people or the environment; and
the introduced genes are common in the environment.
Risk management plan
The risk management plan describes measures to protect the health and safety of people and to
protect the environment by controlling or mitigating risk. The risk management plan is given
effect through licence conditions.
As the level of risk is considered negligible, specific risk treatment is not required. However,
since this is a limited and controlled release, the licence includes limits on the size, location
and duration of the release, as well as controls to prohibit the use of GM plant material in
human food or animal feed, to minimise dispersal of GM seed or GM pollen from trial sites, to
transport GMOs in accordance with the Regulator’s guidelines, to destroy GMOs not required
for testing or further planting, and to conduct post-harvest monitoring at the trial site to ensure
all GMOs are destroyed.
Summary
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DIR 136 – Risk Assessment and Risk Management Plan (September 2015)
Office of the Gene Technology Regulator
Table of Contents
SUMMARY OF THE RISK ASSESSMENT AND RISK MANAGEMENT PLAN ................................................... I
DECISION
................................................................................................................................................................. I
THE APPLICATION ........................................................................................................................................................... I
RISK ASSESSMENT......................................................................................................................................................... II
RISK MANAGEMENT PLAN ............................................................................................................................................. II
TABLE OF CONTENTS ...............................................................................................................................................III
ABBREVIATIONS ......................................................................................................................................................... IV
CHAPTER 1
RISK ASSESSMENT CONTEXT ...................................................................................................... 1
SECTION 1
SECTION 2
SECTION 3
3.1
3.2
SECTION 4
SECTION 5
5.1
5.2
5.3
5.4
SECTION 6
6.1
6.2
6.3
6.4
SECTION 7
7.1
7.2
BACKGROUND ......................................................................................................................................... 1
REGULATORY FRAMEWORK .................................................................................................................... 1
THE PROPOSED DEALINGS ........................................................................................................................ 2
The proposed limits of the dealings (duration, size, location and people) .............................................. 2
The proposed controls to restrict the spread and persistence of the GMOs in the environment ............. 2
THE PARENT ORGANISM .......................................................................................................................... 3
THE GMOS, NATURE AND EFFECT OF THE GENETIC MODIFICATION......................................................... 3
Introduction to the GMOs ....................................................................................................................... 3
The introduced genes, encoded proteins and their associated effects ..................................................... 4
Toxicity/allergenicity of the proteins associated with the introduced genes ........................................... 6
Characterisation of the GMO .................................................................................................................. 7
THE RECEIVING ENVIRONMENT ............................................................................................................... 8
Relevant abiotic factors .......................................................................................................................... 8
Relevant agricultural practices ................................................................................................................ 8
Presence of related plants in the receiving environment ......................................................................... 8
Presence of similar genes and encoded proteins in the environment ...................................................... 9
RELEVANT AUSTRALIAN AND INTERNATIONAL APPROVALS ................................................................... 9
Australian approvals ............................................................................................................................... 9
International approvals ........................................................................................................................... 9
CHAPTER 2
RISK ASSESSMENT ......................................................................................................................... 10
SECTION 1
SECTION 2
2.1
2.2
2.3
2.4
SECTION 3
SECTION 4
INTRODUCTION ...................................................................................................................................... 10
RISK IDENTIFICATION ............................................................................................................................ 11
Risk source............................................................................................................................................ 11
Causal pathway ..................................................................................................................................... 12
Potential harm ....................................................................................................................................... 13
Postulated risk scenarios ....................................................................................................................... 13
UNCERTAINTY ....................................................................................................................................... 21
RISK EVALUATION ................................................................................................................................ 21
CHAPTER 3
RISK MANAGEMENT PLAN ......................................................................................................... 23
SECTION 1
SECTION 2
SECTION 3
3.1
3.2
SECTION 4
SECTION 5
BACKGROUND ....................................................................................................................................... 23
RISK TREATMENT MEASURES FOR SUBSTANTIVE RISKS ......................................................................... 23
GENERAL RISK MANAGEMENT ............................................................................................................... 23
Licence conditions to limit and control the release ............................................................................... 23
Other risk management considerations ................................................................................................. 26
ISSUES TO BE ADDRESSED FOR FUTURE RELEASES ................................................................................. 27
CONCLUSIONS OF THE CONSULTATION RARMP ................................................................................... 27
REFERENCES .............................................................................................................................................................. 29
APPENDIX A SUMMARY OF SUBMISSIONS FROM PRESCRIBED EXPERTS, AGENCIES AND
AUTHORITIES .................................................................................................................................. 33
APPENDIX B
Table of Contents
SUMMARY OF SUBMISSIONS FROM THE PUBLIC ................................................................ 36
III
DIR 136 – Risk Assessment and Risk Management Plan (September 2015)
Office of the Gene Technology Regulator
Abbreviations
APVMA
CSIRO
DIR
DNA
dpa
FSANZ
GalA
GAUT1
GM
GMO
ha
HG
HGT
km
m
NLRD
NPTII
NSW
OGTR
PC2
PME
PMEI
RARMP
Regulations
Regulator
RNA
RNAi
the Act
Abbreviations
Australian Pesticides and Veterinary Medicines Authority
Commonwealth Scientific and Industrial Research Organisation
Dealings involving Intentional Release
Deoxyribonucleic acid
Days post anthesis
Food Standards Australia New Zealand
Galacturonic acid
Galacturonyltransferase
Genetically modified
Genetically modified organism
Hectare
Homogalacturonan
Horizontal gene transfer
Kilometres
Metres
Notifiable Low Risk Dealing
Neomycin phosphotransferase type II
New South Wales
Office of the Gene Technology Regulator
Physical Containment level 2
Pectin methylesterase
Pectin methylesterase inhibitor
Risk Assessment and Risk Management Plan
Gene Technology Regulations 2001
Gene Technology Regulator
Ribonucleic acid
Ribonucleic acid interference
The Gene Technology Act 2000
IV
DIR 136 – Risk Assessment and Risk Management Plan (September 2015)
Chapter 1
Office of the Gene Technology Regulator
Risk assessment context
Section 1 Background
An application has been made under the Gene Technology Act 2000 (the Act) for
Dealings involving the Intentional Release (DIR) of genetically modified organisms (GMOs)
into the Australian environment.
The Act in conjunction with the Gene Technology Regulations 2001 (the Regulations),
an inter-governmental agreement and corresponding legislation that is being enacted in each
State and Territory, comprise Australia’s national regulatory system for gene technology. Its
objective is to protect the health and safety of people, and to protect the environment, by
identifying risks posed by or as a result of gene technology, and by managing those risks
through regulating certain dealings with GMOs.
This chapter describes the parameters within which potential risks to the health and
safety of people or the environment posed by the proposed release are assessed. The risk
assessment context is established within the regulatory framework and considers applicationspecific parameters (Figure 1).
RISK ASSESSMENT CONTEXT
LEGISLATIVE REQUIREMENTS
(including Gene Technology Act and Regulations)
RISK ANALYSIS FRAMEWORK
OGTR OPERATIONAL POLICIES AND GUIDELINES
PROPOSED DEALINGS
Proposed activities involving the GMO
Proposed limits of the release
Proposed control measures
GMO
Introduced or deleted genes
(genotype)
Novel traits (phenotype)
PREVIOUS RELEASES
Figure 1.
PARENT ORGANISM
Origin and taxonomy
Cultivation and use
Biological characterisation
Ecology
RECEIVING ENVIRONMENT
Environmental conditions
Agronomic practices
Presence of related species
Presence of similar genes
Summary of parameters used to establish the risk assessment context
Section 2 Regulatory framework
Sections 50, 50A and 51 of the Act outline the matters which the Gene Technology
Regulator (the Regulator) must take into account, and the consultation required when preparing
the Risk Assessment and Risk Management Plans (RARMPs) that inform the decisions on
licence applications. In addition, the Regulations outline further matters the Regulator must
consider when preparing a RARMP. In accordance with section 50A of the Act, this
application is considered to be a limited and controlled release application, as its principal
purpose is to enable the applicant to conduct experiments and the applicant has proposed limits
on the size, location and duration of the release, as well as controls to restrict the spread and
persistence of the GMOs and their genetic material in the environment. Therefore, the
Regulator was not required to consult with prescribed experts, agencies and authorities before
preparation of the Risk Assessment and Risk Management Plan.
Chapter 1 – Risk assessment context
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Section 52 of the Act requires the Regulator to seek comment on the RARMP from the
States and Territories, the Gene Technology Technical Advisory Committee, Commonwealth
authorities or agencies prescribed in the Regulations, the Minister for the Environment,
relevant local council(s), and the public.
The Risk Analysis Framework (OGTR 2013a) explains the Regulator’s approach to the
preparation of RARMPs in accordance with the legislative requirements. Additionally, there
are a number of operational policies and guidelines developed by the Office of the Gene
Technology Regulator (OGTR) that are relevant to DIR licences. These documents are
available from the OGTR website.
Any dealings conducted under a licence issued by the Regulator may also be subject to
regulation by other Australian government agencies that regulate GMOs or GM products,
including Food Standards Australia New Zealand (FSANZ), the Australian Pesticides and
Veterinary Medicines Authority (APVMA), the Therapeutic Goods Administration, the
National Industrial Chemicals Notification and Assessment Scheme and the Department of
Agriculture. These dealings may also be subject to the operation of State legislation declaring
areas to be GM, GM free, or both, for marketing purposes.
Section 3 The proposed dealings
CSIRO proposes to release up to 45 lines of genetically modified (GM) cotton into the
environment under limited and controlled conditions. The purpose of the release is to evaluate
the agronomic performance of the GM cotton under Australian field conditions, particularly the
fibre quality characteristics.
The dealings involved in the proposed intentional release include:
conducting experiments with the GMOs
breeding the GMOs
propagating the GMOs
using the GMOs in the course of manufacture of a thing that is not a GMO
growing the GMOs
transporting the GMOs
disposing of the GMOs
possession, supply or use of the GMOs for any of the purposes above.
These dealings are detailed further below.
3.1 The proposed limits of the dealings (duration, size, location and people)
The release is proposed to take place at one site adjacent to the Australian Cotton
Research Institute in Narrabri, New South Wales on a maximum area of 1 hectare per year
between October 2016 and May 2019.
Only trained and authorised staff would be permitted to deal with the GM cotton.
3.2 The proposed controls to restrict the spread and persistence of the GMOs
in the environment
The applicant has proposed a number of controls to restrict the spread and persistence of
the GM cotton and the introduced genetic material in the environment. These include:
 locating the trial site at a location not prone to flooding
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Office of the Gene Technology Regulator
 separating GMOs from other cotton crops by a 20 m wide pollen trap of non-GM cotton
 locating the outer edge of the pollen trap at least 50 m away from cotton lines used for
breeding purposes
 inspecting and cleaning all planting and harvest equipment used at the trial site before
using for other purposes
 cleaning the trial site after harvest
 post-harvest monitoring of the trial site for at least 12 months and destroying any
volunteer cotton until the site has been free of volunteers for six months
 destroying all plant material from the trial not required for analysis or further
experimentation
 transporting and storing the GMOs in accordance with the current Regulator’s
Guidelines for the Transport, Storage and Disposal of GMOs
 not allowing plant material from the GMOs to be used in human food or animal feed.
Section 4 The parent organism
The parent organism is upland cotton (Gossypium hirsutum L.), which is the most
commonly cultivated cotton species worldwide. Cotton is exotic to Australia and is grown as
an agricultural crop in New South Wales and Queensland, with occasional trial or small-scale
cultivation in Victoria, northern Western Australia and in the Northern Territory. In the
2013-14 growing season more than 99% of the Australian cotton crop was GM, with
introduced insect resistance and/or herbicide tolerance GM traits (Roth 2014).
Cotton is grown as a source of textile and industrial fibre, cottonseed oil for food use, and
cottonseed meal for animal feed. Detailed information about the parent organism is contained
in a reference document, The Biology of Gossypium hirsutum L. and Gossypium barbadense L.
(cotton), which was produced to inform the risk assessment process for licence applications
involving GM cotton plants (OGTR 2013b). The document is available from the OGTR
website or on request from the OGTR.
G. hirsutum cultivar Coker 315 has been used to develop the GM cotton lines proposed
for release. This cultivar has been adapted to tissue culture conditions and can be readily
modified by standard techniques. However, it is not commercially grown in Australia.
Section 5 The GMOs, nature and effect of the genetic modification
5.1 Introduction to the GMOs
The applicant proposes to release up to seven categories of cotton genetically modified
for altered fibre quality (Table 1). Categories 1-2 and 4-6 have enhanced expression of one of
the four introduced cotton genes PME3, PME4, PME5 and GAUT1 for better fibre quality.
Category 3 has reduced expression of the PME3 gene and shorter fibre. The last category is
generated by crossing of the fibre-specific PME3 enhanced cotton (Category 1) with any of the
constitutive PME4, PME5 or GAUT1 enhanced cotton lines (Categories 4, 5 and 6), producing
‘stacked’ cotton lines containing genetic modifications from both GM parents. In addition, all
of the GM cotton lines contain an introduced nptII gene, encoding neomycin
phosphotransferase type II, as a selectable marker. More detail on the introduced genes is
provided in Table 1. Regulatory sequence elements have also been introduced to achieve
appropriate expression of the introduced genes; these are described in Table 2.
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Table 1 GM cotton lines proposed for release
Category
Description
Fibre- specific
PME3 enhanced
Constitutive PME3
enhanced
PME3 Reduced
1
2
3
Constitutive PME4
Enhanced
Constitutive PME5
enhanced
Constitutive GAUT1
enhanced
Category 1
combined with
Categories 4, 5 or 6
4
5
6
7
Introduced
Gene*
PME3
PME3
PME3 partial
PME4
PME5
GAUT1
PME3 +PME4
or PME5 or
GAUT1
Protein
produced
Pectin
methylesterase
Pectin
methylesterase
None (RNAi)
Pectin
methylesterase
Pectin
methylesterase
Galacturonyltransferase
PME3 +
PME4 or PME5
or GAUT1
Source
Intended trait
Max No
of lines
G. hirsutum
Longer and finer lint
10
G. hirsutum
Longer and finer lint
5
G. hirsutum
Shorter lint
5
G. hirsutum
Longer lint
5
G. hirsutum
Longer lint
5
G. hirsutum
Longer lint
5
G. hirsutum
Longer and finer lint
10
* Note: all GM cotton plants also contain the nptII gene as a selectable marker.
Table 2 Regulatory sequences introduced into the GM cotton lines proposed for release
Element
Exp1
S1 5’
S7 5’
Intron 1
(PPDK)
Intron 1
(CAT1)
ME 3’
S3 3’
Description
α-expansin gene Gh Exp1
promoter
Subclover stunt virus S1
gene promoter
Subclover stunt virus S7
gene promoter
First intron of pyruvate
dikinase gene
First intron of catalase 1
gene
NADP-malic enzyme gene
terminator
Subclover stunt virus S3
gene terminator
Function
Source
Fibre-specific expression
G. hirsutum
Constitutive expression
Subclover stunt virus
Constitutive expression
Subclover stunt virus
Enhanced expression
Flaveria trinervia
Enhanced expression
Ricinus communis
Transcription stop signal
Flaveria bidentis
Transcription stop signal
Subclover stunt virus
Used in
(Category)
1, 7
1, 2, 3, 4, 5, 6, 7
2, 3, 4, 5, 6, 7
3
3
1, 2, 3, 4, 5, 6, 7
1, 2, 3, 4, 5, 6, 7
All GM cotton lines were produced by Agrobacterium tumefaciens-mediated plant
transformation. Information about this transformation method can be found in the document
Methods of plant genetic modification available from the Risk Assessment References page on
the OGTR website. Stacks between the GM cotton lines were produced by conventional crossbreeding.
5.2 The introduced genes, encoded proteins and their associated effects
5.2.1
Pectin methylesterase (PME) genes
GM cotton plants in Categories 1, 2, 4 and 5 contain one of the three introduced cotton
genes PME3, PME4 and PME5. Cotton plants already contain each of these genes. However,
introduction of a second copy of a gene is expected to increase the expression level of the
protein encoded by that gene. The PME genes encode pectin methylesterases, which catalyse
the de-methylesterification of pectins by converting methoxyl groups into carboxyl groups and
releasing methanol (Jolie et al. 2010).
Pectin is a component of all higher plant cell walls and has diverse functions in plant
growth, morphology, development and plant defence (Mohnen 2008; Pelloux et al. 2007). The
pectin family is a large group of complex polysaccharides mainly consisting of
homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II)
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(Ochoa-Villarreal et al. 2012). HG is the most abundant homopolymer of the pectins,
comprising over 60% of pectins in plant cell walls and is formed by long chains of linear 1,4linked α-D-galacturonic acid (GalA) (Mohnen 2008). HG is synthesised in the Golgi complex
and secreted to the cell wall in a form with a high level of methyl-esterification (OchoaVillarreal et al. 2012). The methylesters are subsequently de-esterified by wall-localised PMEs,
leading to free carboxyl groups and the release of methanol and protons (Wolf et al. 2009). Deesterification of HGs can lead to both cell wall stiffening as well as to enzymatic degradation
of pectin. Thus, the methyl esterification status of HGs is important for the physical properties
of the cell wall and for regulating cell expansion in plants (Held et al. 2011).
PME enzymes in plants play important roles in cell extension and pollen tube growth
(Bosch & Hepler 2005), embryo development (Levesque-Tremblay et al. 2015), seed
germination (Müller et al. 2013) and fibre development (Pinzon-Latorre & Deyholos 2014).
The level of PME activity has also been shown to be positively correlated to dormancy
breakage of yellow cedar (Cupressus nootkatensis) seeds (Ren & Kermode 2000). PME
activity is regulated in part by pectin methylesterase inhibitors (Jolie et al. 2010; Senechal et al.
2015; Wang et al. 2013).
Cotton fibres are highly elongated single-celled seed trichomes that initiate from the seed
coat. The walls of elongating fibre cells are bilayered with the outer layer enriched in deesterified pectins and the inner layer enriched in xyloglucans and cellulose. Like other dicot
plants, cotton has a large PME gene family but only a few are expressed in cotton fibre. PME3,
PME4 and PME5 are the major fibre-expressed PMEs in cotton (Liu et al. 2013). It has been
suggested that pectin methylesterases play an important role in cotton fibre elongation (Pelloux
et al. 2007) and fibre quality (Al-Ghazi et al. 2009).
According to Liu et al. (2013), PME3 is normally expressed during the fibre elongation
stage between 5 and 15 days post anthesis3 (dpa). PME4 shares 78% identity with PME3 at the
amino acid level and is normally expressed at low levels during later fibre elongation stages of
around 15-20 dpa. PME5 is phylogenetically more distant from PME3 and PME4, and shares
only 42% and 43% identity with PME3 and PME4 at the amino acid level, respectively. It is
normally expressed later in fibre development beyond 20 dpa.
GM cotton containing the introduced cotton PME genes are expected to express a higher
level of cell-wall located pectin methylesterases that will affect the expansion of fibre cell
walls and change the final fibre lengths or thickness.
5.2.2
The PME3 RNAi
All Category 3 GM cotton plants contain an RNAi (ribonucleic acid interference)
construct containing fragments of PME3 gene. The construct acts to suppress expression of the
endogenous PME3 gene by a natural regulatory mechanism known as RNAi or gene silencing
(Baykal & Zhang 2010). The mechanism for silencing of genes by RNAi was discussed in
detail in the RARMPs prepared for DIR 112 and DIR 117. The RNAi construct contains a
fragment of the PME3 gene repeated in both sense and antisense orientation, separated by two
plant gene introns. Following transcription, the self-complementary regions of mRNA anneal
and the introns are spliced out, resulting in double-stranded RNA. This is processed by
endogenous cellular RNAi machinery into PME3-specific short interfering RNAs (siRNAs).
The siRNAs then direct the degradation of messenger RNA (mRNA) molecules with matching
sequence, in this case mRNAs transcribed from the endogenous PME3 gene, before they are
translated into proteins. The effect of suppressing expression of PME3 is to reduce the total
PME3 activity in elongating fibres, leading to higher levels of methyl esterified pectin in fibre
cell walls.
3
Anthesis is the period during which a flower is fully open and functional.
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The efficiency of gene silencing is generally determined by the extent of homology
between the silencing construct and the target gene (usually > 95% homology is required) and
the length of the homologous region. In plants, introduced silencing constructs have been
shown to effectively suppress expression of the target genes but can also give rise to silencing
of non-target genes with closely matching sequences (Baykal & Zhang 2010).
5.2.3
The GAUT1 gene
GM cotton plants in Category 6 contain an introduced cotton GAUT1 (GhGAUT1) gene
coding for the pectin synthesis enzyme galacturonyltransferase (GalAT). This protein
synthesises the most abundant pectic polysaccharide HG (Atmodjo et al. 2011). GalAT
catalyses transfer of GalA from uridine-diphosphate-GalA (UDP-GalA) onto the non-reducing
end of polysaccharide and oligosaccharide acceptors to produce polymeric HG (Doong &
Mohnen 1998).
The GAUT1 gene was initially identified in Arabidopsis (Sterling et al. 2006). It belongs
to the carbohydrate-active enzyme glycosyltransferase family 8 (GT8) (Yin et al. 2010). In
Arabidopsis, GAUT1 is a member of the GAUT1-related gene family consisting of 15 GAUT
and 10 GAUT-like genes, which are all involved in the biosynthesis of pectins (Atmodjo et al.
2011). A GAUT1 gene has been isolated from Gossypium barbadense (Chi et al. 2009). This
gene is expressed in the fibres with peak levels at 35 to 40 dpa. It is also expressed in
hypocotyls and leaves, but not in roots.
It has been proposed that there is a positive correlation between pectin synthesis and fibre
elongation (Blamey 2003; Rajasundaram et al. 2014). In cotton, preliminary results showed
that GM plants with over-expression of the GhGAUT1gene under the control of the constitutive
S7 promoter from the subclover stunt virus display higher levels of GAUT protein in leaf
tissues and early fibre elongation compared to control plants (information provided by the
applicant).
5.2.4
The nptII gene
The nptII (also denoted aph(3’)-II) gene was isolated from the common gut bacterium
Escherichia coli and encodes neomycin (or aminoglycoside) phosphotransferase type II, which
inactivates aminoglycoside antibiotics such as kanamycin and neomycin. The nptII gene is
used extensively as a selectable marker in the production of GM plants. Further information
about this gene can be found in the document Marker genes in GM plants available from the
Risk Assessment References page on the OGTR website.
5.3 Toxicity/allergenicity of the proteins associated with the introduced genes
All of the four genes (PME3, PME4, PME5 and GAUT1) used to enhance fibre quality in
the GM cottons are endogenous to cultivated cotton (G. hirsutum). Therefore, humans and
other organisms have a long history of exposure to these genes and their encoded proteins.
Endogenously present plant PMEs can positively or negatively affect the structural
quality of plant-based foods (cloud stability, viscosity, texture, etc). In the food industry, PME
(purified from fungi or other sources) is used as a processing aid in fruit and vegetable juices
(Jolie et al. 2010). PMEs are not known to be toxic. The GAUT1 protein is an important
enzyme in the pectin synthesis pathways with no reported toxicity. The most well-known plant
proteins with toxic properties are lectins and ribosome-inactivating peptides (RIPs) (Wu & Sun
2011), and none of the introduced proteins fall into these categories. In addition, a sequence
similarity search of the introduced proteins against the Toxin and Toxin Target Database
(T3DB) (Wishart et al. 2015) showed that none of the introduced proteins share sequence
similarities with known protein toxins.
Some plant PMEs are known allergens. PMEs in kiwi fruit (Actinidia deliciosa) and olive
tree (Olea europaea) have been identified as allergens (Bublin 2013; Popovic et al. 2013;
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Salamanca et al. 2010). Potential identities between each introduced PME protein and
allergenic proteins listed in the AllergenOnline database from the Food Allergy Research and
Resource Program (FARRP), University of Nebraska (Version 15, updated January 2015) were
evaluated using the FASTA sequence alignment program (Pearson & Lipman 1988). It
revealed that the three cotton PME proteins all showed more than 35% sequence matches over
80 amino acids, which is the threshold for possible cross-reactivity (Codex Alimentarius
Commission 2003; FAO 2001; Goodman 2006; Goodman et al. 2005), to allergenic pectin
methylesterases from kiwi fruit (gi160419153) and olive tree (isoforms: gi269996495 and
gi68270856). This indicates that the introduced PME proteins potentially share immunological
properties with protein allergens.
A similar search conducted for the GAUT1 protein did not yield any sequence matches
with known allergens, indicating a lack of immunological relevant similarities between this
protein and known allergens.
Insertion of the PME3 gene fragment as part of a gene silencing construct does not result
in expression of a novel protein, but in suppression of the expression of endogenous cotton
PME3 protein. This is unlikely to lead to increased toxicity or allergenicity (see more
discussion in Risk Scenario 2).
No studies on the toxicity or allergenicity of the GM cotton plants and their products
have been undertaken to date as the proposed trial is at an early stage.
The antibiotic selectable marker gene nptII has previously been assessed by regulatory
agencies in Australia and in other countries as not posing a risk to human or animal health or to
the environment. FSANZ has approved food derived from GM cotton and GM corn expressing
the NPTII protein as safe for human consumption (FSANZ 2002; FSANZ 2003; FSANZ
2010). Further information on risk assessment of this gene can be found in the document
Marker genes in GM plants available from the Risk Assessment References page on the OGTR
website.
5.4 Characterisation of the GMO
5.4.1
Phenotypic characterisation
The applicant has carried out a preliminary study, under greenhouse conditions, of the
GM cotton lines proposed for release. The applicant has provided information on observed
phenotypes of the GM cotton plants (below).
GM cotton plants in Categories 1 and 2 (enhanced expression of PME3) showed longer
and finer fibres when grown under glasshouse conditions. No other effects on the phenotype of
Category 1 plants (fibre-specific enhanced PME3) were observed. However, GM cotton plants
with constitutively expressed PME3 (Category 2) were observed to have elongated internodes
and to be taller than non-GM cotton plants when grown in the glasshouse.
GM cotton plants in Category 4 with constitutive expression of PME4 were taller than a
non-GM cotton control. They showed longer but not finer fibres when grown under glasshouse
conditions. No other obvious changes to phenotype were observed.
GM cotton plants in Category 5 (constitutive expression of PME5) and Category 6
(constitutive expression of GAUT1) showed longer but not finer fibres. No other effects on the
plant phenotype were observed.
GM cotton plants in Category 3 containing the PME3 RNAi construct had a reduced
PME3 transcription level, reduced total PME enzyme activity in elongating fibres, higher
levels of methyl esterified pectin in the cell walls and shorter fibres. No other obvious
differences in phenotype were observed in comparison to the control non-GM cotton.
Further phenotypic data would be collected during the proposed field trials.
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Section 6 The receiving environment
The receiving environment includes: any relevant biotic/abiotic properties of the
geographic regions where the field trial would occur; intended agricultural 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 2013a).
6.1 Relevant abiotic factors
The abiotic factors relevant to the growth and distribution of commercial cotton in
Australia are discussed in The Biology of Gossypium hirsutum L. and Gossypium barbadense
L. (cotton) (OGTR 2013b). To summarise, factors restricting where cotton can be grown in
Australia are water availability (through rainfall or irrigation), soil suitability and, most
importantly, temperature. Cotton seedlings may be killed by frost, growth and development of
cotton plants below 12°C is minimal, and a long, hot growing season is crucial for achieving
good yields.
The release is proposed to take place at one site in Narrabri, NSW (Section 3.1), which
has a temperate climate as defined by the Köppen Classification system used by the Australian
Bureau of Meteorology.
The proposed site is over 2 km away from the nearest natural waterway and is not prone
to flooding.
6.2 Relevant agricultural practices
The limits and controls of the proposed release are outlined in Sections 3.1 and 3.2 of this
Chapter. It is anticipated that the agronomic practices for the cultivation of the GM cotton by
the applicant will not differ significantly from industry best practices used in Australia. The
GMOs proposed for field release would be planted with a standard cone seeder in rows with
1 m spacing, and the pollen trap would be planted with a commercial seed planter. All cotton
plants would be grown following standard cotton agricultural management practices and would
receive applications of water, fertilisers, herbicides, and insecticides similar to commercially
grown non-GM cotton crops. Conventional cultivation practices for cotton are discussed in
more detail in The Biology of Gossypium hirsutum L. and Gossypium barbadense L. (cotton)
(OGTR 2013b).
The GM cotton seed required for experimentation or future trials will be harvested either
by hand or with modified commercial harvesting equipment used by CSIRO for small plot
breeding experiments. The remaining cotton including plants from the pollen trap will be
picked using a standard picker and destroyed at the site.
After harvest, a cereal crop may be planted within the trial site. The crop will be chosen
from the approved post-harvest crops list on the OGTR website.
6.3 Presence of related plants in the receiving environment
The proposed release site is in Narrabri, NSW, which is a commercial cotton growing
area. Almost 100% of the cotton commercially cultivated in Australia is GM cotton modified
for insect resistance and/or herbicide tolerance (Roth 2014). Commercial cotton grown in
Australia is either Gossypium hirsutum or Gossypium barbadense, with 99% of cotton planted
in 2006 being G. hirsutum (OGTR 2013b). The GM G. hirsutum proposed for release is
capable of crossing with both species of commercially grown cotton. It is worth noting that
improved fibre quality is one of the main characteristics for which both G. hirsutum and
G. barbadense varieties are selected.
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Ephemeral populations of cotton volunteers can be found on cotton farms, by roadsides
where cotton seed is transported, or in areas where cotton seed is used as livestock feed
(Addison et al. 2007; Eastick & Hearnden 2006).
There are 17 native species of Gossypium in Australia, most of which can be found in the
Northern Territory and the north of Western Australia. Generally, they are found in native
vegetation and not in disturbed/modified habitats such as agricultural areas (Groves et al.
2002).
Well established genetic incompatibility prevents crossing of native cotton species with
cultivated cotton in the natural environment (discussed in OGTR 2013b).
6.4 Presence of similar genes and encoded proteins in the environment
The PME3, PME4, PME5 and GAUT1 genes were isolated from cotton. Therefore, these
genes and their encoded proteins are widespread in the Australian environment.
The nptII gene is isolated from the common gut bacteria E. coli (Section 5.2.4). This
gene is also present in GM cotton cultivars already commercially planted in Australia,
including Bollgard II®, Bollgard III®, Roundup Ready Flex®/ Bollgard II®, and Roundup
Ready Flex®/ Bollgard III®.
Section 7 Relevant Australian and international approvals
7.1 Australian approvals
7.1.1
Approvals by the Regulator
None of the GM cotton lines included in this application has previously been approved
by the Regulator for release in Australia.
Information on previous DIR licences for GM cotton is available from the GMO Record
on the OGTR website. The Regulator has previously approved 35 field trials and 9 commercial
releases of GM cotton. There have been no credible reports of adverse effects on human health
or the environment resulting from any of these releases.
7.1.2
Approvals by other government agencies
There are no approvals of these GM cotton lines, including pending approvals, from other
Australian authorities.
7.2 International approvals
None of the GM cotton lines covered in this application has been approved for release in
any other countries.
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Chapter 2
Office of the Gene Technology Regulator
Risk assessment
Section 1 Introduction
The risk assessment identifies and characterises risks to the health and safety of people or to
the environment from dealings with GMOs, posed by or as the result of gene technology (Figure 2).
Risks are identified within the context established for the risk assessment (see chapter 1), taking
into account current scientific and technical knowledge. A consideration of uncertainty, in particular
knowledge gaps, occurs throughout the risk assessment process.
Figure 2 The risk assessment process
Initially, risk identification considers a wide range of circumstances whereby the GMO, or the
introduced genetic material, could come into contact with people or the environment. Consideration
of these circumstances leads to postulating plausible causal or exposure pathways that may give rise
to harm for people or the environment from dealings with a GMO in the short and long term. These
are called risk scenarios.
A number of risk identification techniques are used by the Regulator and staff of the OGTR,
including checklists, brainstorming, reported international experience and consultation (OGTR
2013a). A weed risk assessment approach is used to identify traits that may contribute to risks from
GM plants. In particular, novel traits that may increase the potential of the GMO to spread and
persist in the environment or increase the level of potential harm compared with the parental
plant(s) are used to postulate risk scenarios (Keese et al. 2013). Risk scenarios postulated in
previous RARMPs prepared for licence applications of the same or similar GMOs are also
considered.
Postulated risk scenarios are screened to identify those that are considered to have some
reasonable chance of causing harm. Pathways that do not lead to harm, or could not plausibly occur,
do not advance in the risk assessment process.
Substantive risks (i.e. those identified for further assessment) are characterised in terms of the
potential seriousness of harm (Consequence assessment) and the likelihood of harm (Likelihood
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assessment). Risk evaluation then combines the Consequence and Likelihood assessments to
estimate the level of risk and determine whether risk treatment measures are required. The potential
for interactions between risks is also considered.
Section 2 Risk Identification
Postulated risk scenarios are comprised of three components:
i.
The source of potential harm (risk source).
ii.
A plausible causal linkage to potential harm (causal pathway).
iii.
Potential harm to an object of value, people or the environment.
The risk context, including the following factors, is taken into account when postulating
relevant risk scenarios:
the proposed dealings, which may be to conduct experiments, develop, produce, breed,
propagate, grow, import, transport or dispose of the GMOs, use the GMOs in the course of
manufacture of a thing that is not the GMO, and the possession, supply and use of the
GMOs in the course of any of these dealings
the proposed limits including the extent and scale of the proposed dealings
the proposed controls to restrict the spread and persistence of the GMO and
characteristics of the parent organism(s).
2.1 Risk source
The sources of potential harms can be intended novel GM traits associated with one or more
introduced genetic elements, or unintended effects/traits arising from the use of gene technology.
As discussed in Chapter 1, the GM cotton lines and stacks have been modified by the
introduction of one or two of the four cotton genes or a partial cotton gene sequence. These
introduced genes or partial gene are considered further as potential sources of risk.
In addition, all of the GM cotton lines and stacks contain the nptII antibiotic resistance
selectable marker gene. This gene and its product have already been extensively characterised and
assessed as posing negligible risk to human or animal health or to the environment by the Regulator
as well as by other regulatory agencies in Australia and overseas. Further information about this
gene can be found in the document Marker genes in GM plants available from the Risk Assessment
References page on the OGTR website. As this gene has not been found to pose a substantive risk
to either people or the environment, its potential effects will not be further considered for this
application.
The introduced genes or partial gene are controlled by introduced regulatory sequences. The
regulatory sequences are derived from plants and a plant virus (see Table 2). Regulatory sequences
are naturally present in plants, and the introduced elements are expected to operate in similar ways
to endogenous elements. There is no evidence that regulatory sequences themselves have toxic or
allergenic effects (EPA 1996). Although the viral sequences are derived from a plant pathogen, they
only constitute small fractions of the genomes and cannot themselves cause disease. Hence,
potential harms from the regulatory elements will not be considered further. However, the
introduced regulatory sequences, especially the promoters, control gene expression and hence the
distribution and concentration of the derived proteins and siRNA molecules in the GM plants. The
effects of protein or siRNA molecules and their levels, especially in relation to toxicity and
allergenicity, will be considered below.
The genetic modifications have the potential to cause unintended effects in several ways
including altered expression of endogenous genes by random insertion of introduced DNA in the
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genome, increased metabolic burden due to expression of the introduced proteins, novel traits
arising out of interactions with non-target proteins and secondary effects arising from altered
substrate or product levels in biochemical pathways. However, these types of effects also occur
spontaneously and in plants generated by conventional breeding. Accepted conventional breeding
techniques such as hybridisation, mutagenesis and somaclonal variation can have a much larger
impact on the plant genome than genetic engineering (Schnell et al. 2015). Plants generated by
conventional breeding have a long history of safe use, and there are no documented cases where
conventional breeding has resulted in the production of a novel toxin or allergen in a crop (Steiner
et al. 2013). Therefore, unintended effects resulting from the process of genetic modification will
not be considered further.
2.2 Causal pathway
The following factors are taken into account when postulating plausible causal pathways to
potential harm:













routes of exposure to the GMOs, the introduced gene(s) and gene product(s)
potential effects of the introduced gene(s) and gene product(s) on the properties of the
organism
potential exposure to the introduced gene(s) and gene product(s) from other sources in the
environment
the environment at the site(s) of release
agronomic management practices for the GMOs
spread and persistence (invasiveness) of the GMOs, including establishment and
reproduction
dispersal by natural means and by people
tolerance to abiotic conditions (e.g. climate, soil and rainfall patterns)
tolerance to biotic stressors (e.g. pest, pathogens and weeds)
tolerance to cultivation management practices
gene transfer to sexually compatible organisms
gene transfer by horizontal gene transfer (HGT)
unauthorised activities.
The potential for horizontal gene transfer (HGT) from GMOs to other organisms, and any
possible adverse outcomes, have been reviewed in the literature (Keese 2008) and assessed in many
previous RARMPs. HGT was most recently considered in the RARMP for DIR 108 (available from
the GMO Record on the OGTR website). In previous assessments of HGT no substantive risk was
identified, due to the rarity of these events and because the wild-type gene sequences are already
present in the environment and available for transfer via demonstrated natural mechanisms.
Therefore, HGT will not be further considered for this application.
The potential for unauthorised activities to lead to an adverse outcome has been considered in
many previous RARMPs, most recently in the RARMP for DIR 117 (available from the GMO
Record on the OGTR website). In previous assessments of unauthorised activities, no substantive
risk was identified. The Act provides for substantial penalties for unauthorised dealings with GMOs
or non-compliance with licence conditions, and also requires the Regulator to have regard to the
suitability of an applicant to hold a licence prior to the issuing of the licence. These legislative
provisions are considered sufficient to minimise risks from unauthorised activities. Therefore,
unauthorised activities will not be considered further.
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2.3 Potential harm
Potential harms from GM plants include:






harm to the health of people or desirable organisms, particularly toxicity/allergenicity
reduced establishment or yield of desirable plants
reduced products or services from the land use
restricted movement of people, animals, vehicles, machinery and/or water
reduced quality of the biotic environment (e.g. providing food or shelter for pests or
pathogens) or abiotic environment (e.g. negative effects on fire regimes, nutrient levels, soil
salinity, soil stability or soil water table)
reduced biodiversity for nature conservation.
These harms are based on those used to assess risk from weeds (Keese et al. 2013; Standards
Australia Ltd et al. 2006). Judgements of what is considered harm depend on the management
objectives of the land where the GM plant may spread and persist. A plant species may have
different weed risk potential in different land uses such as dryland cropping or nature conservation.
2.4 Postulated risk scenarios
Four risk scenarios were postulated and screened to identify substantive risk. These scenarios
are summarised in Table 3 and more detail of these scenarios is provided later in this Section.
Postulation of risk scenarios considers impacts of the GM cotton or its products on people
undertaking the dealings, as well as impacts on people and the environment if the GM plants or
genetic material were to spread and/or persist.
In the context of the activities proposed by the applicant and considering both the short and
long term, none of the four risk scenarios gave rise to any substantive risks.
Table 3 Summary of risk scenarios from dealings with GM cotton genetically modified for altered
fibre quality
Risk
Risk source
scenario
1
GM cotton
expressing
introduced
genes for
enhanced
fibre quality
Chapter 2 – Risk assessment
Causal pathway
Potential harm/s
Cultivation of GMOs at
the trial site

Exposure of people who
deal with the GMOs or of
animals at the trial site to
introduced proteins
Toxicity or
allergenicity in
people or toxicity to
desirable organisms
Substantive
risk?
No
Reasons
GM plant material will not be
used in human food or animal
feed.
 The introduced proteins are
endogenous to cultivated cotton
and are not known to be toxic to
people or other organisms.
 The limited scale and short
duration of the trial minimise
exposure of people and other
organisms to the GM plant
material.

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Risk
Risk source
scenario
Causal pathway
Potential harm/s
Office of the Gene Technology Regulator
Substantive
risk?
Reasons
2
GM cotton
expressing
introduced
gene
silencing
construct
Cultivation of GMOs at
the trial site

Exposure of people who
deal with the GMOs or of
animals at the trial site to
introduced siRNA
Toxicity or
allergenicity in
people or toxicity to
desirable organisms
No

3
GM cotton
expressing
introduced
genes for
enhanced
fibre quality
or the gene
silencing
construct
Dispersal of GM seed
outside trial limits

GM seed germinates

Establishment of
populations of the GM
plants
 Toxicity or
allergenicity in
people or toxicity
to desirable
organisms
 Reduced
establishment or
yield of desirable
plants
No

4
GM cotton
expressing
introduced
genes for
enhanced
fibre quality
or the gene
silencing
construct
Pollen from GM plants
fertilise other sexually
compatible plants

GM hybrid seed
germinates

GM hybrids spread and
persist
 Toxicity or
allergenicity in
people or toxicity
to desirable
organisms
 Reduced
establishment or
yield of desirable
plants
No

2.4.1
GM plant material will not be
used in human food or animal
feed.
 The silencing construct does not
lead to expression of a protein.
 The expressed siRNA is unlikely
to affect expression of genes in
animals which ingest the GM
cotton, and even if it did any
effects would be transient.
 The limited scale and short
duration of the trial minimise
exposure of people and other
organisms to the GM plant
material.
The proposed controls minimise
dispersal of GM cotton seeds.
 Cotton has limited ability to
survive outside agricultural
settings and the genetic
modifications are not expected to
increase its weediness.
 The GM cotton is susceptible to
standard weed control
measures.
Cotton has limited ability to
outcross.
 The proposed limits and controls
would minimise pollen flow to
cotton plants outside the trial
site.
 Hybrids between the GMOs and
commercial GM cotton lines are
not expected to have enhanced
toxicity or weediness.
Risk scenario 1
Risk source
Causal
pathway
Potential harm
GM cotton expressing introduced genes for enhanced fibre quality

Cultivation of GMOs at trial sites

Exposure of people who deal with the GMOs or of animals at the trial site to introduced proteins

Toxicity or allergenicity in people or toxicity to desirable organisms
Risk source
The source of potential harm for this postulated risk scenario is GM cotton expressing
introduced genes for enhanced fibre quality.
Causal pathway
Workers who cultivate, harvest, gin, transport, experiment or conduct other dealings with the
GM cotton grown would be exposed to cotton plant material. As the applicant proposes that only
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authorised staff deal with the GM cotton, other people are not expected to be exposed to the GM
plants or plant material. Potential pathways of exposure to the introduced proteins are ingestion,
inhalation or dermal contact. There is little potential for human ingestion of the introduced proteins,
as the applicant proposes that no GM plant material would be used as food. GM plant material that
could potentially be airborne and inhaled includes pollen or cotton dust produced during the
harvesting or ginning processes. However, cotton pollen is heavy, sticky and not easily dispersed by
wind (OGTR 2013b), and people who enter cotton gins typically wear protective face masks
(International Fibre Centre website). Workers could come into skin contact with the introduced
proteins if they touch damaged plants where cell contents have been released. However, no staff
have experienced or observed adverse health effects during development of the GM cotton plant
(information provided by the applicant).
The applicant proposes to test fibre quality of lint from GM cotton, spun into yarn and woven
into fabric at a non-certified facility. Processed cotton lint contains over 99% cellulose and does not
contain detectable protein (OGTR 2013b). Therefore, people handling lint from GM cotton would
not be exposed to the introduced proteins.
Non-human organisms may be exposed directly to the introduced proteins through ingesting
the GM plants, or exposed indirectly through the food chain, or exposed through contact with dead
plant material (soil organisms). Livestock would not be expected to ingest the introduced proteins
as the GM plant material is not to be used as animal feed. Wild mammals and birds generally avoid
feeding on cotton plants, in particular finding the seed unpalatable because of its high gossypol
content (OGTR 2013b). A range of invertebrates would be expected to ingest GM cotton plant
material. The small scale and short duration of the proposed field trial would restrict the total
number of invertebrates exposed to the introduced proteins.
Potential harm
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).
Allergenicity is the potential of a 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).
Non-GM cotton produces natural toxins for defence against herbivory including gossypol and
cycloprenoid fatty acids (OGTR 2013b). The introduced genes are not involved in the metabolic
pathways associated with these toxins and are therefore unlikely to affect the levels of endogenous
toxins in cotton.
The introduced genes for fibre quality (PME3, PME4, PME5 and GAUT1) encode enzymes
that are involved in pectin synthesis (GAUT1) and modification (PME3, PME4 and PME5)
(Chapter 1, Section 5.2). These genes are endogenous cotton genes but their expression levels and
spatial distributions in GM cotton are expected to be different when they are under the control of a
non-endogenous promoter. As discussed in Chapter 1 Section 5.3, the proteins encoded by these
genes are not known to be toxic to people or to other organisms. The GAUT1 protein has no
similarity to known allergens. However, the PME proteins have sequence similarity to known
allergens present in kiwifruit and olive pollen. Increased expression levels and expression of these
proteins in additional tissues may lead to increased allergenicity towards people who are sensitive to
kiwifruit or olive pollen. However, cotton pollen is not usually airborne and the GMOs would not
be used in human food.
The GAUT1 enzyme is involved in pectin synthesis. The expression level of this enzyme is
expected to increase the amount of pectin in the cell wall. Pectin is a food additive approved by
FSANZ (code number 440 under Schedule 8 of the Australia New Zealand Food Standards Code).
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When a fruit (such as tomato) is homogenised for preparing fruit juice, the PME activity can
cause rapid pectin breakdown by hydrolysing pectin methylesters to produce polygalacturonic acid
and methanol (Anthon & Barrett 2012). Human consumption of high levels of methanol can be
toxic to the nervous system, particularly affecting the eyesight. Overexpression of the introduced
cotton PME genes may lead to higher levels of PME activities in the GM cotton, which could result
in increased methanol levels in the GM cotton tissues if natural mechanisms for removing methanol
from cells are overloaded. However, the GMOs would not be used in human food or animal feed.
Conclusion
Risk scenario 1 is not identified as a substantive risk because the GM plant material will not
be used in human food or animal feed; the introduced proteins are endogenous to cultivated cotton
and not known to be toxic; and the limited scale, short duration and the proposed controls of the
trial minimise exposure of people and other organisms to the GM plant material. Therefore, this risk
could not be greater than negligible and does not warrant further detailed assessment.
2.4.2
Risk Scenario 2
Risk source
Causal
pathway
Potential harm
GM cotton expressing introduced gene silencing construct

Cultivation of GMOs at trial sites

Exposure of people who deal with the GMOs or of animals at the trial site to introduced siRNA

Toxicity or allergenicity in people or toxicity to desirable organisms
Risk source
The source of potential harm for this postulated risk scenario is the GM cotton expressing the
introduced gene silencing construct (Category 3). The silencing construct containing PME3 gene
fragments is designed to produce siRNAs that reduce or suppress expression of the PME3 gene,
thus reducing PME3 activity in developing cotton fibre.
Causal pathway
People and other organisms may be exposed to this GM cotton in the same way as described
in Risk Scenario 1. Exposure is restricted by the same factors discussed in Risk Scenario 1.
Potential harm
The potential harms arising from this risk scenario are toxicity or allergenicity in people, or
toxicity to desirable organisms, as a result of exposure to the GM cotton. Transcription of the gene
fragment in the silencing construct results in hairpin RNA, which enters the RNAi pathway rather
than being translated into a protein. Therefore, the introduction of the silencing construct does not
lead to expression of a novel protein that could potentially be toxic or allergenic. All known food
allergens are proteins, those derived from plants coming chiefly from peanut, tree nuts, wheat and
soybean (Delaney et al. 2008; Herman & Ladics 2011).
The possibility exists that, rather than direct toxicity, RNAi constructs may give rise indirectly
to altered toxicity or allergenicity. The gene silencing activity of RNAi in plants requires a high
level of sequence complementarity. However, in the unlikely event that short sequences from the
RNAi construct were a sufficiently close match to expressed non-target sequences, RNAi constructs
could potentially give rise to off-target silencing effects within the plant. Silencing of non-target
genes may lead to changes other than the intended effects, potentially including changes to levels of
endogenous toxins or allergens. However, as discussed in Section 2.1, these types of effects also
occur spontaneously and in plants generated by conventional breeding. Any off-target effects are
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not expected to influence the expression of any endogenous compounds that have toxic or allergenic
properties to a greater extent than could occur through conventional breeding between different
cotton varieties.
By targeting the PME3 gene, the silencing construct reduces PME3 activity in elongating
fibre cells. As a result, the GM cotton has higher levels of methyl esterified pectin in the cell walls
and shorter fibres. No other phenotypic changes were observed when compared to non-GM cotton
(Chapter 1, Section 5.4.1). Since the endogenous PME3 gene is expressed specifically in
developing fibre, it is expected that the genetic modification will only change the pectin profiles in
fibres and not affect any other metabolic pathways.
Hairpin RNA transcribed from the silencing construct is processed into siRNAs (short
interfering RNAs), which fall under a general category of small RNAs that also includes
microRNAs (miRNAs). siRNAs and miRNAs are common in both plants and animals and are
believed to play regulatory roles in many biological processes. Animals and plants naturally
produce thousands of different siRNA molecules and these are consumed by humans and other
organisms whenever they eat plant or animal cells. Zhang et al. (2011) reported that natural plant
miRNAs can be absorbed by mammals through food intake, and have the potential to modulate
gene expression in animals. This risk has been further analysed by other researchers but remains
controversial (Liang et al. 2014; Petrick et al. 2013). More detailed discussion of this issue can be
found in the RARMP for DIR 131.
The possibility exists that siRNAs produced in GM cotton lines could, after ingestion,
modulate expression of human or animal genes, with unknown physiological effects. The siRNAs
would need to be produced at high levels in GM cotton, a large amount of the GM cotton would
need to be consumed, the siRNA would need to match a target sequence in a human or animal gene,
and be taken up by cells expressing that gene. As noted above, the GM cotton will not be used for
human food or animal feed. Mammals do not have genes that are homologous to the cotton PME3
gene targeted by the introduced silencing construct (ExPASy, NCBI). Even if siRNAs were
acquired through eating GM cotton and did affect gene expression, it is expected that any effect
would be transient as described in Zhang et al (2011). None of these GM cotton lines will be used
for human food or animal feed, and the trial is of small scale and short duration.
Conclusion
Risk scenario 2 is not identified as a substantive risk due to the introduced gene fragment not
coding for any protein; mammals do not have genes homologous to the cotton PME3 gene targeted
by the introduced silencing construct; the siRNAs produced are unlikely to affect expression of
genes in animals which ingest the GM cotton (and any such effects would be transient); and the
proposed limits and controls would minimise exposure of people and other organisms to the GM
plant material. Therefore this risk could not be greater than negligible and does not warrant further
detailed assessment.
2.4.3
Risk Scenario 3
Risk source
Causal
pathway
Potential
harms
Chapter 2 – Risk assessment
GM cotton expressing introduced genes for enhanced fibre quality or gene silencing construct

Dispersal of GM seed outside trial limits

GM seed germinates

Establishment of populations of the GM plants

Toxicity or allergenicity in people or toxicity to desirable organisms
or
Reduced establishment or yield of desirable plants
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Risk source
The source of potential harm for this postulated risk scenario is GM cotton expressing
introduced genes for enhanced fibre quality or the gene silencing construct.
Causal Pathway
The first step in the causal pathway for this risk scenario is dispersal of GM seed outside the
trial limits. This could occur due to persistence of viable GM seeds at the trial site after the intended
duration of the trial, or through physical movement of GM seeds to areas outside the trial site.
The applicant proposes a number of control measures to prevent persistence of GM seeds in
the seed bank at the trial site. These include destroying GMOs that remain in the trial site after
harvest, cultivating the site after harvest to encourage decomposition or germination of remaining
seed, destroying any volunteers found prior to flowering, and post-harvest monitoring of the trial
site for at least twelve months and until the site has been clear of volunteers for six months. It is not
expected that expression of the introduced genes for enhanced fibre quality or the gene silencing
construct would increase the ability of the GMOs to survive these standard control measures.
Cotton seeds are enclosed in large, heavy bolls that remain attached to the plant. At maturity
the bolls split open and the fibres can facilitate seed dispersal by wind over distances less than
100 m (OGTR 2013b). Cotton seed is not normally physically transported by runoff after rainfall or
irrigation. Extreme weather conditions such as flooding or high winds may cause dispersal of plant
parts. The proposed release site is located at least 2 km from the nearest river and protected by flood
levee banks. Although there are irrigation channels in the proximity, these channels do not flow into
natural waterways and the applicant proposes to monitor the channels post-harvest for cotton
volunteers.
Wild mammals and birds generally avoid feeding on cotton plants, in particular finding the
seed unpalatable because of its high gossypol content (OGTR 2013b). Therefore, wild animals are
unlikely to disperse GM cotton seeds from the trial site. GM cotton seeds would not be used as
stock feed, so would not be dispersed by stock.
Dispersal of cotton seeds by authorised people entering the trial site would be minimised by
cleaning all equipment, including clothing, used with the GM cotton before using it for any other
purpose. GM seed cotton would be ginned separately from any other cotton crop in an approved
facility to avoid accidentally mixing GM cotton seed with other cotton seed, then dispersing the
mixed cotton seed. The applicant proposes to contain GM plant materials during transportation and
storage in accordance with the Regulator’s Guidelines for the Transport, Storage and Disposal of
GMOs. Only plant material needed for experimentation would be transported outside the site.
Spillage of GM seed during transport to and from the release site would be rare and could be readily
controlled through cleaning and monitoring of the site of the spill.
The spread and persistence of non-GM cotton plants are limited by a number of biotic and
abiotic factors, especially cold stress in southern Australia and water stress in non-irrigated
environments throughout almost all of Australia. Feral cotton populations are sparse and ephemeral
in all current cotton growing regions of Australia (OGTR 2013b). A study found that even when
cotton was sown in cleared sites in northern Australian with high water availability, the cotton
plants did not establish stable populations (Eastick & Hearnden 2006). Modelling of climactic
factors limiting cotton persistence indicate that cotton has naturalisation potential only in the coastal
regions of north-east Australia (Rogers et al. 2007), which are distant from the proposed trial site. A
few small populations of naturalised cotton are reported in northern Australia, but these are not
derived from modern cultivars (OGTR 2013b), and may have a greater ability to survive outside
agricultural settings than modern cotton cultivars.
The introduced PME and GAUT1 genes are endogenous to cotton and are primarily involved
in fibre development. Over-expression of these genes or the gene silencing construct is expected to
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alter the pectin profiles in cell walls. As pectin has diverse functions in plant growth, morphology,
defence and seed dormancy, as discussed in Chapter 1, Section 5.2, these characteristics may be
altered in the GM cotton lines. However, preliminary studies by the applicant showed that, under
glasshouse conditions, all GM cotton lines in Categories 1, 3, 5 and 6 displayed normal agronomic
characteristics apart from expected changes in lint fibres. GM cotton plants in Categories 2 and 4
with over-expression of the PME3 gene and PME4 gene, respectively, showed increased height
relative to non-GM cotton (Chapter 1, Section 5.4.1). There is no evidence suggesting that the
genetic modifications have altered seed production characteristics or tolerance to abiotic or biotic
stresses that could enhance the potential for dispersal or persistence of the GM cotton lines
proposed for release. Fibre quality is one of the main characteristics for which cotton is bred. NonGM cotton varieties with enhanced fibre quality have not been reported to spread more easily or
persist longer than other cotton varieties.
Potential harms
The potential harms from this risk scenario are toxicity or allergenicity in people or toxicity to
desirable organisms, or reduced establishment or yield of desirable plants.
Risk Scenarios 1 and 2 considered the potential for the introduced genes or gene silencing
construct to lead to toxicity or allergenicity, and did not identify any substantive risks.
The GM cotton could reduce the establishment or yield of desirable plants in agricultural
settings if GM cotton volunteers grew in other crops. If this happened, the GM cotton volunteers
could easily be controlled by standard measures such as application of herbicides or cultivation.
The GM cotton could reduce the establishment or yield of desirable plants in the natural
environment if the GM cotton spread and persisted as a weed in nature reserves, displacing native
vegetation. However, as discussed above, cotton has limited potential to survive outside agricultural
settings, and the introduced proteins or the gene silencing construct are not expected to increase its
ability to spread and persist. In the unlikely event of GM cotton plants establishing themselves in
nature reserves, small and ephemeral feral GM cotton populations would be unlikely to cause harms
associated with weediness such as reducing desired plants or restricting physical movement.
Conclusion
Risk scenario 3 is not identified as a substantive risk because the proposed controls minimise
dispersal of GM cotton seeds, cotton has limited ability to survive outside agricultural settings and
the introduced proteins or gene silencing construct are not expected to increase its weediness, and
the GM cotton is susceptible to standard weed control measures. Therefore, this risk could not be
greater than negligible and does not warrant further detailed assessment.
2.4.4
Risk Scenario 4
Risk source
Causal
pathway
Potential
harms
Chapter 2 – Risk assessment
GM cotton expressing introduced genes for enhanced fibre quality or gene silencing construct

Pollen from GM plants fertilising other sexually compatible plants

GM hybrid seed germinates

GM hybrids spread and persist

Toxicity or allergenicity in people or toxicity to desirable organisms
or
Reduced establishment or yield of desirable plants
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Risk source
The source of potential harm for this postulated risk scenario is GM cotton expressing
introduced genes for enhanced fibre quality or a gene silencing construct.
Causal pathway
The first step in the causal pathway for this risk scenario is pollen from GM plants fertilising
other sexually compatible plants. Cotton is predominantly self-pollinating, with pollen that is large,
sticky and heavy and generally not dispersed by wind. Pollen can be transported by insect
pollinators, chiefly honeybees, but gene flow studies have shown that outcrossing occurs at low
levels and decreases rapidly with distance (OGTR 2013b). The only sexually compatible plants are
G. hirsutum or G. barbadense plants, as native Gossypium species are not sexually compatible with
cotton (Chapter 1, Section 6.3). It is not expected that the introduced genes for enhanced fibre
quality or the gene silencing construct would alter the pollen dispersal characteristics of the GM
cotton.
The applicant has proposed to restrict pollen flow by surrounding the trial site with a 20 m
pollen trap of non-GM cotton. In addition, the applicant has proposed to destroy any post-harvest
cotton volunteers on the trial site before flowering. These controls would minimise the potential for
pollinators to transfer pollen from GMOs to related plants outside the trial sites.
Some outcrossing is expected to occur between the GMOs and other cotton plants grown at
the trial site, i.e. non-GM comparator cotton plants and cotton plants in the pollen trap. As non-GM
cotton plants grown in the trial site and pollen traps are expected to produce a small proportion of
hybrid seeds, the applicant has proposed that non-GM cotton planted in the trial site and/or in the
pollen trap will be handled as if they are the GMOs. The limits and controls proposed for the GMOs
would minimise dispersal of hybrid seeds (see Risk Scenario 3).
Any hybrid seeds that did occur outside of the trial limits would be unlikely to establish a
stable population, as discussed in Risk Scenario 3. If the GMOs pollinated a commodity cotton
crop, a small amount of hybrid seed containing the introduced proteins could enter human food and
animal feed. If the GMOs pollinated a cotton crop intended for seed production, hybrid seed could
be purposefully dispersed and grown, leading to wider exposure to the introduced proteins. The
applicant has proposed locating the outer edge of the pollen trap at least 50 m away from cotton
lines used for breeding purposes, to further minimise gene flow.
Potential harms
The potential harms from this risk scenario are toxicity or allergenicity in people or toxicity in
desirable organisms, or reduced establishment or yield of desirable plants.
Risk Scenarios 1 and 2 considered the potential for the introduced genes or gene silencing
construct to lead to toxicity or allergenicity, and did not identify any substantive risks. This is also
expected to be the case if the introduced proteins are expressed in hybrids with other cotton.
The potential for the GMOs to reduce establishment or yield of desirable plants was discussed
in Risk Scenario 3. Cotton plants expressing the introduced proteins or the gene silencing construct
are unlikely to spread and persist in nature reserves or to survive standard weed management
practices for cotton volunteers in agricultural settings.
Conclusion
Risk scenario 4 is not identified as a substantive risk because cotton has limited ability to
outcross, the proposed limits and controls would minimise pollen flow to cotton plants outside the
trial site, and hybrids between the GMOs and commercial GM cotton lines are not expected to have
enhanced toxicity or weediness. Therefore, this risk could not be greater than negligible and does
not warrant further detailed assessment.
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Section 3 Uncertainty
Uncertainty is an intrinsic property of risk and is present in all aspects of risk analysis 4.
Uncertainty in risk assessments arises from sources such as incomplete knowledge and inherent
biological variability. Uncertainty is addressed by approaches including balance of evidence,
conservative assumptions, and applying risk management measures that reduce the potential for risk
scenarios involving uncertainty to lead to harm. If there is residual uncertainty that is important to
estimating the level of risk, the Regulator will take this uncertainty into account in making
decisions.
As field trials of GMOs are designed to gather data, there are generally data gaps when
assessing the risks of a field trial application. However, field trial applications are required to be
limited and controlled. Even if there is uncertainty about the characteristics of a GMO, limits and
controls restrict exposure to the GMO, and thus decrease the likelihood of harm.
For DIR 136, uncertainty is noted particularly in relation to:
Potential for increased allergenicity due to higher concentrations of PME proteins
Potential for altered levels of endogenous toxic metabolites
Potential effects of the genetic modifications on weediness characteristics.
Additional data, including information to address these uncertainties, may be required to
assess possible future applications with reduced limits and controls, such as a larger scale trial or
the commercial release of these GMOs.
Chapter 3, Section 4, discusses information that may be required for future release.
Section 4 Risk Evaluation
Risk is evaluated against the objective of protecting the health and safety of people and the
environment to determine the level of concern and, subsequently, the need for controls to mitigate
or reduce risk. Risk evaluation may also aid consideration of whether the proposed dealings should
be authorised, need further assessment, or require collection of additional information.
Factors used to determine which risks need treatment may include:




risk criteria
level of risk
uncertainty associated with risk characterisation
interactions between substantive risks.
Four risk scenarios were postulated whereby the proposed dealings might give rise to harm to
people or the environment. In the context of the control measures proposed by the applicant, and
considering both the short and long term, none of these scenarios were identified as substantive
risks. The principal reasons for these conclusions are summarised in Table 3 and include:




none of the GM plant material or products will enter human food or animal feed supply
chains
widespread presence of the introduced genes and their encoded proteins in the environment
and lack of known toxicity
limited ability of the GM cotton plants to establish populations outside cultivation
limited ability of the GM cotton plants to transfer the introduced genetic material to other
cotton plants
4
A more detailed discussion of uncertainty is contained in the Regulator’s Risk Analysis Framework available from the
Risk Assessment References page on the OGTR website or via Free call 1800 181 030.
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

Office of the Gene Technology Regulator
limits on the size, location and duration of the release proposed by CSIRO
suitability of controls proposed by CSIRO to restrict the spread and persistence of the GM
cotton plants and their genetic material.
Therefore, risks to the health and safety of people, or the environment, from the proposed
release of the GM cotton plants into the environment are considered to be negligible. The Risk
Analysis Framework (OGTR 2013a), which guides the risk assessment and risk management
process, defines negligible risks as risks of no discernible concern with no present need to invoke
actions for mitigation. Therefore, no controls are required to treat these negligible risks. Hence, the
Regulator considers that the dealings involved in this proposed release do not pose a significant risk
to either people or the environment.
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Chapter 3
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Risk management plan
Section 1 Background
Risk management is used to protect the health and safety of people and to protect the
environment by controlling or mitigating risk. The risk management plan addresses risks evaluated
as requiring treatment and considers limits and controls proposed by the applicant, as well as
general risk management measures. The risk management plan informs the Regulator’s decisionmaking process and is given effect through licence conditions.
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 subject to three conditions prescribed in the Act. Section 63 of the Act
requires that each licence holder inform relevant people of their obligations under the licence. The
other statutory conditions allow the Regulator to maintain oversight of licensed dealings: section 64
requires the licence holder to provide access to premises to OGTR inspectors 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.
The licence is also 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. In addition, the Regulator has extensive
powers to monitor compliance with licence conditions under section 152 of the Act.
Section 2 Risk treatment measures for substantive risks
The risk assessment of risk scenarios listed in Chapter 2 concluded that there are negligible
risks to people and the environment from the proposed field trial of GM cotton. These risk scenarios
were considered in the context of the scale of the proposed release (Chapter 1, Section 3.1), the
proposed containment measures (Chapter 1, Section 3.2), and the receiving environment (Chapter 1,
Section 6), and considering both the short and the long term. The risk evaluation concluded that no
controls are required to treat these negligible risks.
Section 3 General risk management
The limits and controls proposed in the application were important in establishing the context
for the risk assessment and in reaching the conclusion that the risks posed to people and the
environment are negligible. Therefore, to maintain the risk context, licence conditions have been
imposed to limit the release to the proposed size, location and duration, and to restrict the spread
and persistence of the GMOs and their genetic material in the environment. The conditions are
discussed and summarised in this Chapter and listed in the licence.
3.1 Licence conditions to limit and control the release
3.1.1
Consideration of limits and controls proposed by CSIRO
Sections 3.1and 3.2 of Chapter 1 provide details of the limits and controls proposed by CSIRO
in their application. These are taken into account in the four risk scenarios postulated for the
proposed release in Chapter 2. Many of the proposed control measures are considered standard for
GM crop trials and have been imposed by the Regulator in previous DIR licences. The
appropriateness of these controls is considered further below.
The applicant proposes that the release will take place at one site in Narrabri, NSW. The total
planting area each year will not exceed 1 ha and the duration of the proposed release would be
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limited to three growing seasons. The small size and short duration of the trial would limit exposure
to the GMOs (Risk Scenarios 1 and 2).
The applicant proposes that only authorised and trained personnel would be permitted to deal
with the GMOs. A standard licence condition requires that all persons dealing with the GMOs must
be informed of any applicable licence conditions. These measures would limit the potential
exposure of humans to the GMOs (Risk Scenarios 1 and 2).
The applicant does not propose using any of the GM plant material for human or animal
consumption. Therefore, a condition in the licence prohibits material from the trial from being used
for human food or animal feed. This control will restrict exposure of humans and other organisms to
the GMOs (Risk Scenarios 1 and 2) and the potential for GM cotton seed to be dispersed outside the
trial limits (Risk Scenario 3).
The applicant proposes to test the fibre quality of lint from GM cotton grown in the trial at a
non-certified facility. At the end of the experimentation, all material derived from the lint will either
be destroyed or stored in certified PC2 laboratories. As discussed in Risk Scenario 1, cotton lint is
free of DNA and protein so is not a means of exposure to GM material. Therefore, the licence does
not impose conditions on experimentation, transport and sale of lint from GM cotton, other than the
prohibition of use for food or feed as described above.
The applicant proposes to clean all equipment used with the GMOs before using the
equipment for other purposes. Equipment used on the trial site would be cleaned on site. The GM
cotton would be ginned separately from other cotton crops and the gin would be cleaned after use to
prevent GM cotton seed mixing with other seed. These measures are appropriate to restrict potential
dispersal of GM cotton seed outside the trial sites (Risk Scenario 3).
After the trial site has been harvested, the applicant proposes to destroy all GMOs except for
plant material required for testing and cotton seed required for further authorised planting. Cotton
seeds have low dormancy levels and do not generally form a viable seed bank, however, dormancy
can be induced in cotton seeds by low soil temperature and/or soil moisture (OGTR 2013b). The
applicant proposes post-harvest cultivation of the trial sites to promote cotton seed germination or
decomposition. A licence condition requires tillage in the spring or summer following the harvest,
and provision of adequate soil moisture, so that soil temperature and moisture will be suitable for
cotton seed germination. These measures would restrict the persistence of a GM cotton seed bank
after the duration of the trial (Risk Scenario 3).
The applicant proposes that each trial site will be monitored post-harvest at least every two
months for a minimum of twelve months and until the site has been clear of volunteers for at least
six months. During this period any cotton volunteers will be destroyed before flowering. However,
the proposed frequency of monitoring may not be sufficient. The OGTR’s experience from previous
GM cotton trials indicates that if there is vigorous growth of a post-harvest crop, cotton volunteers
in the 2-4 leaf stage may be missed during inspections, and also that in particularly favourable
weather conditions the period between the 4 leaf stage and the beginning of flowering can be less
than 40 days. Therefore, a licence condition requires post-harvest monitoring at least every 35 days,
with destruction of any cotton volunteers, for a period of at least 12 months and until no volunteers
are detected for a continuous 6 month period. These measures would restrict the persistence of
GMOs after completion of the trial (Risk Scenario 3).
The applicant proposes that GMOs will be transported and stored according to the Regulator’s
current Guidelines for the Transport, Storage and Disposal of GMOs (OGTR website). These
protocols would restrict the potential for dispersal of GM seeds outside the trial sites (Risk
Scenario 3). This is included as a licence condition.
The applicant proposes to locate the trial sites more than two km away from the nearest river.
The land is protected by flood levees. The applicant has also proposed to monitor the adjacent
irrigation channels after the trial and destroy any volunteers. The licence includes standard DIR
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licence conditions that require: that the site be at least 50 m from the nearest natural waterway or
man-made waterway that flows into a natural waterway; that any area where the GMOs may have
dispersed, including irrigation channels, be monitored after harvest; and immediate notification of
any extreme weather conditions affecting the site during the proposed release. These measures will
minimise likelihood for the GM cotton establishing outside the proposed release site, including as a
result of extreme weather events (Risk Scenario 3).
The applicant proposes that the trial site will be surrounded by a 20 m wide pollen trap to
control pollen flow from the GMOs to cotton plants outside the trial site. The plants within the
pollen trap would be non-GM cotton and would be managed so as to flower at the same time as the
GMOs. As discussed in Risk Scenario 4, cotton is predominantly self-pollinating and outcrossing
rates decrease rapidly with distance. A 20 m pollen trap around GM cotton was found to be an
effective buffer under Australian conditions (Llewellyn et al. 2007). Therefore, using a 20 m pollen
trap would minimise gene transfer to cotton plants outside the trial sites (Risk Scenario 4).
Although the applicant proposes that the plants in the pollen trap will be non-GM, the licence
permits pollen trap plants to be either non-GM or GM cotton approved for commercial release in
Australia. The applicant proposes to maintain a path of up to 3 m in width through the pollen trap to
allow access of vehicles and equipment to the GMO planting area. Since previous cotton licences
have allowed the access path to be up to 2.5 m, which has been proven to be adequate for access,
the licence specifies a path of up to 2.5 m wide.
The applicant proposes to locate the outer edge of the pollen trap at least 50 m away from
cotton lines used for breeding purposes, to further minimise gene flow. As discussed above, a 20 m
pollen trap is considered adequate to control pollen flow; this is not included as a licence condition.
The applicant intends to plant both GM and non-GM cotton in the trial site. The applicant
proposes to treat all non-GM cotton from the trial site or cotton from the pollen trap as if it were the
GM cotton in this application. This measure would minimise exposure to or dispersal of hybrid seed
resulting from outcrossing between the GMOs and other cotton (Risk Scenario 4).
The applicant proposes that GMOs may be transported, stored or subjected to experiments in
certified physical containment facilities under a Notifiable Low Risk Dealing (NLRD) authorisation
in accordance with applicable requirements of the Gene Technology Regulations 2001. This is
considered appropriate but is not included in the licence as it would be conducted under a separate
valid authorisation.
3.1.2
Summary of licence conditions to be implemented to limit and control the release
A number of licence conditions have been imposed to limit and control the proposed release,
based on the above considerations. These include requirements to:

limit the duration of the field trial to between October 2016 and May 2019

limit the field trial to a maximum area of 1 ha per year at one site in Narrabri, NSW

locate the trial site at least 50 m away from waterways

restrict gene flow via pollen from the field trial site by surrounding the trial site with a
20 m pollen trap of non-GM cotton or GM cotton approved for commercial release

ensure that pollen trap plants flower for the same period of time as the GMOs

treat any non-GM cotton planted in the planting area or pollen trap plants as if they were
the GMOs

clean all equipment used with the GMOs before using it for any other purpose

gin the GMOs separately from any other cotton crop
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
use tillage and irrigation to promote germination of any cotton seeds remaining in the
trial site after harvest

monitor the trial site for at least 12 months after harvest and destroy any cotton volunteers
until no volunteers are detected for a continuous 6 month period

destroy all GMOs from the trial that are not required for testing or future planting

transport and store the GMOs in accordance with the Regulator’s Guidelines for the
Transport, Storage and Disposal of GMOs

not allow GM plant material to be used for human food or animal feed.
3.2 Other risk management considerations
All DIR licences issued by the Regulator contain a number of conditions that relate to general
risk management. These include conditions relating to:
applicant suitability
contingency plans
identification of the persons or classes of persons covered by the licence
reporting requirements
access for the purpose of monitoring for compliance.
3.2.1
Applicant suitability
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, for either an individual applicant or a body corporate, include:
any relevant convictions of the applicant
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 capacity of the applicant to meet the conditions of the licence.
On the basis of information submitted by the applicant and records held by the OGTR, the
Regulator considers CSIRO suitable to hold a licence. The licence includes a requirement for the
licence holder to inform the Regulator of any information that would affect their suitability.
In addition, any applicant organisation must have access to a properly constituted Institutional
Biosafety Committee and be an accredited organisation under the Act.
3.2.2
Contingency plan
CSIRO is required to submit a contingency plan to the Regulator before planting the GMOs.
This plan will detail measures to be undertaken in the event of any unintended presence of the GM
cotton outside permitted areas.
CSIRO is also required to provide the Regulator with a method to reliably detect the GMOs or
the presence of the genetic modifications in a recipient organism. This methodology is required
before planting the GMOs.
3.2.3
Identification of the persons or classes of persons covered by the licence
The persons covered by the 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 the licence. Prior to growing the GMOs, CSIRO is required to provide a list of people and
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organisations that will be covered by the licence, or the function or position where names are not
known at the time.
3.2.4
Reporting requirements
The licence requires 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.
A number of written notices are also required under the licence to assist the Regulator in
designing and implementing a monitoring program for all licensed dealings. The notices include:
expected and actual dates of planting
details of areas planted to the GMOs
expected dates of flowering
expected and actual dates of harvest and cleaning after harvest
details of inspection activities.
3.2.5
Monitoring for compliance
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 site.
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.
In cases of non-compliance with licence conditions, the Regulator may instigate an
investigation to determine the nature and extent of non-compliance. The Act provides 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 4 Issues to be addressed for future releases
Additional information has been identified that may be required to assess an application for a
commercial release of these GM cotton lines, or to justify a reduction in limits and controls. This
includes:
additional molecular and biochemical characterisation of the GM cotton plants including
potential for increased toxicity and allergenicity
additional phenotypic characterisation of the GM cotton plants, particularly with respect
to traits such as seed dormancy, which may contribute to weediness.
Section 5 Conclusions of the consultation RARMP
The RARMP concludes that the proposed limited and controlled release of GM cotton poses
negligible risks to the health and safety of people or the environment as a result of gene technology,
and that these negligible risks do not require specific risk treatment measures.
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However, conditions have been imposed to limit the release to the proposed size, location and
duration, and to restrict the spread and persistence of the GMOs and their genetic material in the
environment, as these were important considerations in establishing the context for assessing the
risks.
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References
Addison, S.J., Farrell, T., Roberts, G.N., Rogers, D.J. (2007) Roadside surveys support
predictions of negligible naturalisation potential for cotton (Gossypium hirsutum) in north-east
Australia. Weed Research 47: 192-201.
Al-Ghazi, Y., Bourot, S., Arioli, T., Dennis, E.S., Llewellyn, D.J. (2009) Transcript profiling
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DIR 136 – Risk Assessment and Risk Management Plan (September 2015)
Office of the Gene Technology Regulator
Appendix A Summary of submissions from
prescribed experts, agencies and
authorities5
Advice received by the Regulator from prescribed experts, agencies and authorities on the
consultation RARMP is summarised below. All issues raised in submissions that related to
risks to the health and safety of people and the environment were considered in the context of
the currently available scientific evidence and were used in finalising the RARMP that formed
the basis of the Regulator’s decision to issue the licence.
Sub. No.
1
2
Summary of issues raised
Comment
Agrees with the overall conclusions of the
RARMP.
Noted
Suggests considering the effectiveness of
proposed use of personal protective equipment
(PPE) in limiting exposure to GM material.
The applicant reported no staff experiencing or observing
adverse health effects during development of the GM cotton
plant. Text has been added to Risk Scenario 1 to note this. The
risk of increased toxicity or allergenicity of the GM cotton has
been assessed as negligible in the RARMP. Therefore, no
prescriptive conditions regarding PPE are imposed in the
licence. However, licence conditions require the licence holder
to report any adverse effects of the GM cotton on human health.
Agrees with the conclusion of the RARMP that
the risk of toxicity by expression of the proteins
in the GM cotton, or in any other plant they may
be transferred to by hybridisation, is negligible.
Notes that GM plant material will not be used for
animal feed.
Conclusion of negligible risk of toxicity can be
further supported by indicating that only a small
number of plant proteins have ever been shown
to be toxic, the most well-known being lectins
and ribosome-inactivating peptides (RIPs) (Wu
& Sun 2011), and none of the proteins
expressed from the introduced genes fall into
these categories.
Also, mentioned in Paragraph 32 of using a
BLAST search against the Entrez Protein
dataset needs clarification; it is not obvious why
such a search would necessarily identify
homology with any known toxin.
Text and references on toxic proteins have been added to
Chapter 1, Section 5.3. Text in Paragraph 32 regarding the
BLAST search against the Entrez Protein dataset has been
removed.
5
Prescribed agencies include GTTAC, State and Territory Governments, relevant local governments, Australian
Government agencies and the Minister for the Environment.
Appendix A
33
DIR 136 – Risk Assessment and Risk Management Plan (September 2015)
Sub. No.
Summary of issues raised
Office of the Gene Technology Regulator
Comment
Text to this effect has been added to Chapter 1 Section 6.3 and
Given the nature of the genetic modifications
Risk Scenario 3.
and trait, there is no reason to believe that the
GM plants, or any other plant to which they may
be transferred by hybridisation, will possess an
increased ability to spread and persist and
induce environmental harms, such as reducing
the establishment or yield of desired plants,
beyond those current cotton varieties.
Although this is a limited and controlled field
trial, the RARMP would benefit from considering
the environmental harms of the GM plants in the
context of available varieties that have been
selected to have improved fibre quality.
Cumulative knowledge suggests that the
environmental management of the GM plants in
this application will be no different from currently
available varieties that have been selected for
the properties of their fibre.
The risk of unintended release into the
environment of GM plant material, or gene
transfer to non-GM cotton or related species, is
appropriately minimised by the proposed limits
and controls.
Noted
Generally agrees with Paragraph 161 for issues
to be addressed for future releases, but
suggests that the risk of toxic effects on native
animals is so low that this would unlikely
constitute a reason to seek additional molecular
and biochemical characterisation of the GM
plants.
If the GM cotton were to be commercially released, planting may
occur on a very large scale, exposing many animals and people
to the GM cotton and its products. Therefore, some additional
molecular and biochemical characterisation of the GM plants is
considered appropriate.
Although the role of PME in seed dormancy is
Text regarding seed dormancy has been added in Chapter 3,
not recorded in a recent review (Graeber et al.
Section 4.
2012), it is noted that an increase in the activity
of PME has been correlated with the cessation
of seed dormancy and germination in yellow
cedar (Paragraph 21). It is possible that if the
expression of the introduced genes affects seed
dormancy, then this in-turn may affect the
persistence of populations of cotton. This aspect
should be addressed if the plants are ever to be
commercialised.
Appendix A
Suggests that if the GM plants ever reach the
point of commercialisation, a thorough contrast
and comparison of the GM plants with those
commercialised varieties which have been bred
to have improved fibre quality should be
provided.
Noted
Paragraph 76 identifies one of the potential
harms as to the ‘health of people or desirable
organisms, including toxicity/allergenicity’. As
toxicity and allergenicity are referred to in the
RARMP, and indeed are likely the most
prominent health issues, it is suggested that the
word ‘including’ be replaced with ‘particularly’.
Text in paragraph 76 has been modified accordingly.
34
DIR 136 – Risk Assessment and Risk Management Plan (September 2015)
Sub. No.
Summary of issues raised
The RARMP (Paragraphs 31-37 and 84-88) fails
to mention that the ingestion of food can also be
associated with other adverse health effects,
such as food intolerance and autoimmune
diseases. Some discussion of these health
issues would improve the RARMP.
Office of the Gene Technology Regulator
Comment
Since the GM cotton material is not permitted in human food,
the other adverse health effects associated with ingestion of
food are not considered in this RARMP. FSANZ is responsible
for assessing food for human consumption.
3
Noted
Noted that the RARMP was circulated for
comment and no adverse comments were
received. Indicated no objection to the issue of a
licence for DIR 136.
4
Notes that the licence will prohibit the use of
material from the trial for human or animal
consumption. No further comments on the
licence application at this stage.
Noted
5
Supported the conclusions of the RARMP that
the proposed dealings pose negligible risk of
harm to human health and the environment.
Noted
6
No concerns. Cannot see any material risks with Noted
this work both in the nature of what is being
tested and the control measures that will be in
place. Considers this exercise to have negligible
biological, human, animal or ecological risks.
Graeber, K., Nakabayashi, K., Miatton, E., Leubner-Metzger, G., Soppe, W.J. (2012) Molecular
mechanisms of seed dormancy. Plant Cell Environ 35: 1769-1786.
Wu, W., Sun, R. (2011) Toxicological studies on plant proteins: a review. Journal of Applied Toxicology
32: 377-386.
Appendix A
35
DIR 136 – Risk Assessment and Risk Management Plan (September 2015)
Office of the Gene Technology Regulator
Appendix B Summary of submissions from the public
The Regulator received one submission from the public on the consultation RARMP. The issues
raised in this submission are summarised in the table below. All issues raised in the submission
that related to risks to the health and safety of people and the environment were considered in the
context of currently available scientific evidence in finalising the RARMP that formed the basis of
the Regulator’s decision to issue the licence.
Issues raised: MT: Marketing and trade.
Sub. No. Issue
1
Appendix B
Summary of issues raised
Comment
None
Supports the conclusion of the RARMP that
risks to the health and safety of people, or the
environment from the proposed release are
negligible.
Noted
MT
Views maintenance and improvement of cotton
fibre quality as critical to the future of the cotton
industry. Lint quality is one of the main
economic drivers behind the profitability of
cotton. The cotton industry is one of Australia’s
largest rural export earners. Australia’s
international reputation as a reliable supplier of
very high quality cotton can be supported and
sustained through responsible
commercialisation of these fibre quality traits
developed by CSIRO.
When deciding whether or not to issue a licence,
matters that relate to marketing and trade are
outside the legislative responsibility of the
Regulator.
36