ENVIRONMENTAL RISK MANAGEMENT AUTHORITY
DECISION
Amended under s67A on 22 August 2007
26 April 2001
Application code
GMC00002
Application type
To import into containment a genetically modified organism under
section 40(1)(a) of the Hazardous Substances and New Organisms
(HSNO) Act 1996.
Applicant
AgResearch Ltd
Purpose
To import into containment Pseudomonas fluorescens strain SBW
lacZY; Kanr-xylE, genetically modified by marker genes, to be used
as in indicator organism to examine the dispersal and persistence of
formulated micro-organisms.
Date received
03 May 2000
Consideration period
19 March - 24 April 2001
Considered by
The Genetically Modified Organisms (GMO) Standing Committee of
the Environmental Risk Management Authority (the Authority).
Decision
The application to import genetically modified Pseudomonas fluorescens strain SBW lacZY;
Kanr-xylE into containment is approved subject to controls, in accordance with sections
45(1)(a) and 45(2) of the HSNO Act.
Organism Description
The organism has been given the following unique identifier for the ERMA New Zealand
Organism Register: Pseudomonas fluorescens SBW lacZY; Kanr-xylE.
The genetically modified bacterium, P. fluorescens SBW lacZY; Kanr-xylE, is derived from
strain SBW25 which does not contain plasmids. The organism contains two cassettes of
foreign genetic material that are integrated at separate sites in the bacterial chromosome. One
cassette contains two genes from the Escherichia coli lactose operon for utilization of lactose
and colour selection (lacZ [β-galactosidase], and lacY [lactose permease]), under the control
of the iucA promoter from plasmid pMON7117. The other cassette contains a kanamycin
antibiotic resistance gene (Kanr), and the xylE (catechol 2,3 dioxygenase) gene that converts
catechol to a yellow compound. The xylE gene is under the control of a chloramphenicol
resistance gene promoter from plasmid pBR325, while the Kanr gene is under the control of
its own promoter. These cassettes were incorporated into DNA fragments of the P.
fluorescens genome and then introduced into defined sites in the bacterium by homologous
recombination.
The genetically modified P. fluorescens can be distinguished from other P. fluorescens by its
ability to use lactose as a sole food source, by it turning blue when grown with X-gal, and by
creating a yellow colour when grown with catechol.
Application process
The application was formally received on the 03 May 2000, and verified on the 01 March
2001 following an additional information request under section 52(1) of the HSNO Act. The
Committee considered the application did not require to be publicly notified under section 53
of the HSNO Act because the nature of the modifications and the contained use of the
organism, were not deemed to be sufficiently novel compared to other non-notified import
applications to indicate significant public interest.
The documents available for the evaluation and review of the application by ERMA New
Zealand included the application, appendices (including copies of literature cited) and
comments from the Department of Conservation.
The application was considered by the GMO Standing Committee of the Authority appointed
in accordance with section 19(2)(b) of the HSNO Act. The Committee comprised the
following members: Mrs Helen Hughes (Chair), Professor Colin Mantell and Dr Oliver
Sutherland.
Relevant legislative criteria
The application was lodged pursuant to section 40(1)(a) of the HSNO Act. The decision was
determined in accordance with section 45, taking into account additional matters to be
considered under sections 37 and 44 and matters relevant to the purpose of the Act, as
specified under Part II of the Act.
Consideration of the application followed the relevant provisions of the Hazardous
Substances and New Organisms (Methodology) Order 1998, but with particular regard to
clauses 8 (dealing with the scale and significance of the risks, costs and benefits) and 26
(dealing with applications where the risks are negligible).
Reasons for the decision
Purpose
The purpose of the research programme is to use P. fluorescens SBW lacZY; Kanr-xylE as an
indicator organism in contained soil experimental systems, to examine the survival and
release rate of bacteria from various test formulations of beneficial soil microorganisms. The
genetically modified P. fluorescens would be encapsulated with polymers and carriers along
with other (non-modified) bacteria. The foreign genes in the P. fluorescens strain would
permit rapid screening for the release of the bacteria from granular or pelleted formulations
under various soil conditions. In accordance with section 45(1)(a)(i) of the HSNO Act, the
Committee considers that this constitutes an appropriate purpose under section 39(1)(h) of the
HSNO Act: Such other purposes as the Authority thinks fit.
Inseparable organisms
In accordance with section 45(a)(ii) of the HSNO Act, the Committee has considered the
effects of inseparable organisms and notes that the genetically modified P. fluorescens would
be imported as pure cultures, provided by the School of Biological Sciences, University of
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Surrey, UK. The Committee, therefore, considers that contamination with other organisms is
very unlikely. Should any inseparable organisms be imported, it is very unlikely that they
would escape containment under the specified containment controls imposed in this decision.
Ability to escape containment
In accordance with section 44(b) of the HSNO Act the Committee has considered the ability
of the organism to escape from containment.
The Committee requires the genetically modified P. fluorescens to be maintained in a facility
approved under MAF/ERMA New Zealand Standard 154.03.02: Containment Facilities for
Microorganisms. The facility will meet physical containment level 2 (PC2) as described in
the Australia/New Zealand Standard (AS/NZS): 2243.3:1995 Safety in Laboratories: Part 3:
(Microbiology), as this organism is not an approved host in Schedule 2 of the HSNO (LowRisk Genetic Modification) Regulations 1998.
Pseudomonas species, except those known to be non-pathogenic, are classified as Risk Group
2 organisms in the AS/NZS 2243.3:1995. However, since P. fluorescens is not reported to be
pathogenic it is classified as a Risk Group 1 organism according to the AS/NZ Microbiology
Standard, which defines Risk Group 1 organisms as:
“(low individual and community risk)- a microorganism that is unlikely to cause
human, plant or animal disease”
The Committee is satisfied that the genetically modified P. fluorescens would be very
unlikely to escape from containment in a viable form, taking into account the laboratory
procedures proposed by the applicant and the containment controls imposed in this decision.
Ability to form self-sustaining populations & ease of eradication
In accordance with sections 37(a) and (b) of the HSNO Act the Committee has considered the
ability of the organism to establish a self-sustaining population and the ease with which any
such populations could be eradicated.
The Committee considered the conditions that P. fluorescens would need to survive, and
notes that the organism is an environmental isolate that does not have specific growth
requirements that would make it less able to survive than wild type P. fluorescens.
Consequently, the modified strain is unlikely to exhibit reduced competitiveness compared
with wild-type P. fluorescens. The Committee therefore considers it is possible that
genetically modified P. fluorescens would survive and reproduce if it escaped containment
into the rhizosphere.
Although the applicant is required as part of this approval to implement procedures for the
immediate attempted retrieval or destruction of any viable material of the organism that has
breached containment, the Committee considers that eradication of a self-sustaining P.
fluorescens population would be difficult. Also, any eradication method is likely to adversely
affect other microorganisms already present in that environment. However, as noted above,
the Committee considers that escape from containment is very unlikely.
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Adverse effects
In accordance with section 45(a)(ii) of the HSNO Act, the Committee has considered the
potential adverse effects associated with the importation and maintenance of the genetically
modified P. fluorescens strain in containment.
Human health effects
The Committee notes that the P. fluorescens strain is non-pathogenic, and that the genetic
modifications do not introduce any pathogenic traits. The Committee therefore considers that
adverse effects to human health are very unlikely.
Environmental effects
The Committee notes that restricted amounts of P. fluorescens SBW lacZY; Kanr-xylE would
be used in individual experiments [maximum of 1 litre culture at a cell density of
approximately 1 x 109 cells ml-1 (ie 1 x 1012 cells L-1)]. In the unlikely event that a liquid
culture were released untreated and undiluted into the environment it could have adverse
local effects on soil or water microorganisms by virtue of the very large numbers of
microorganisms released. However, the Committee is satisfied that given the containment
controls required by this decision, inadvertent release of viable untreated cultures into the
environment is very unlikely.
With the exception of the kanamycin resistance gene, the modifications to P. fluorescens are
very unlikely to provide the organism with a selective or competitive advantage, and studies
of this strain of genetically modified P. fluorescens do not reveal greater competitiveness
compared with non-modified strains (de Leij et al 1998). Although the genetically modified
P. fluorescens would have a selective advantage over other strains of this species in the
presence of kanamycin, the Committee considers natural exposure to kanamycin is unlikely,
since kanamycin-sensitive P. fluorescens strains appear to be widespread. The Committee
therefore concludes that the presence of the kanamycin resistance gene would be unlikely to
confer a selective advantage to P. fluorescens in the uncontrolled environment.
The Committee notes that the foreign genetic material is introduced into the P. fluorescens
chromosome and that studies have demonstrated that horizontal transfer of these genes from
these locations is very unlikely (Bailey et al 1995). Consequently, the Committee considers
that horizontal gene transfer of the genetically modified material is very unlikely.
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Effects on the relationship of Māori with their taonga
The Committee notes that P. fluorescens SBW lacZY; Kanr-xylE does not contain genetic
material from native flora or fauna, or from Māori (or human genes in general). The
Committee has not identified any particular adverse cultural effects associated with this
application that differ from genetic modifications on other microorganisms already approved
to be held in containment.
In the unlikely event that the bacterial strain breaches containment, the Committee considers
it would be very unlikely for the organism to pose direct risks to native or valued flora or
fauna or otherwise adversely affect the environment or Māori culture.
Negligible risk
Based on the consideration and analysis of adverse effects on the environment and public
health, and taking into account the containment controls imposed in this decision, the
Committee considers risks associated with the importation of P. fluorescens into containment
are negligible. The Committee has, therefore, considered this application in terms of clause
26 of the Methodology.
Benefits and costs
The Committee considers the primary benefit of the intended research involving genetically
modified P. fluorescens is the acquisition of new scientific knowledge. In particular, the
research is likely to allow a faster and more precise method of determining the survival and
release rate of beneficial bacteria from various test formulations into the surrounding soil.
In the long term, the research may contribute to development of stable long-life formulations
of beneficial microorganisms that may have application in biological control of insect pests
and plant diseases, therefore indirect economic benefits are possible. The information
obtained using a genetically modified strain of P. fluorescens is also likely to be applicable
for non-genetically modified P. fluorescens strains. The Committee therefore considers the
benefits related to biological control are not restricted to use of P. fluorescens SBW lacZY;
Kanr-xylE.
The Committee notes that there may be a scientific cost of not carrying out the research, in
relation to ability to attract research funding and undertake collaborative international studies.
The Committee is satisfied that costs are unlikely to accrue to parties other than the applicant.
Conclusion
The Committee concludes that, taking account of the ability of the organism to escape from
containment (refer section 44(b) of the HSNO Act), the beneficial effects of having the
organism in containment outweigh the likely adverse effects of the organism and any
inseparable organisms, should the organism escape.
Having considered the possible effects of the organism in accordance with sections
45(1)(a)(ii) and (iii) of the HSNO Act, the Committee is satisfied that the proposed
containment regime can adequately contain the organism.
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The application to import into containment the new organism, P. fluorescens strain SBW
lacZY; Kanr-xylE, is thus granted in accordance with section 45(1)(a) of the HSNO Act. As
required under section 45(a)(b) the approval is subject to containment controls, as specified
below.
Containment controls
In order to satisfactorily address the matters detailed in the Third Schedule Part I
Containment Controls for Development, Importations or Field Testing of Genetically
Modified Organisms1 of the HSNO Act, the Authority’s approval of this application is subject
to the following controls:
1.
To limit the likelihood of any accidental release of any organism or any viable
genetic material2:
1.1 The person responsible for a particular research area and/or the person responsible for
the operation of the containment facilities (‘the facility’) shall inform all personnel
involved in the handling of the organism of the Authority’s controls.
1.2 The facility shall be approved by Ministry of Agriculture and Forestry (MAF) in
accordance with the MAF Biosecurity Authority/ERMA New Zealand Standard
154.03.023 Containment Facilities for Microorganisms and the controls of the Authority.
1.3 The construction and operation of the facility in which the organism is maintained, shall
be in accordance with the:
a) MAF Biosecurity Authority/ERMA New Zealand Standard 154.03.023: Containment
Facilities for Microorganisms, and
b) Australian New Zealand Standard AS/NZS 2243.3:19953 Safety in Laboratories: Part
3: (Microbiology), at Physical Containment Level 2 (PC2).
2.
To exclude unauthorised people from the facility:
2.1 The identification of entrances, numbers of and access to entrances, and security
requirements for the entrances and the facility shall be in compliance with the
requirements of the standards listed in control 1.3.
1
Bold headings refer to Matters to be Addressed by Containment Controls for Development and Field Testing of
Genetically Modified Organisms, specified in the Third Schedule of the HSNO Act 1996.
2
Viable Genetic Material is biological material that can be resuscitated to grow into tissues or organisms. It can
be defined to mean biological material capable of growth even though resuscitation procedures may be required,
eg when organisms or parts thereof are sublethally damaged by being frozen, dried, heated, or affected by
chemical.
3
Any reference to this standard in these controls refers to any subsequent version approved or endorsed by
ERMA New Zealand
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3.
To exclude other organisms from the facility and to control undesirable and
unwanted organisms within the facility:
3.1 The exclusion of other organisms from the facility and the control of undesirable and
unwanted organisms within the facility shall be in compliance with the standards listed in
control 1.3.
4.
To prevent unintended release of the organism by experimenters working with the
organism:
4.1 The prevention of unintended release of the organism by experimenters working with the
organism shall be in compliance with the standards listed in control 1.3.
5.
To control the effects of any accidental release or escape of an organism:
5.1 Control of the effects of any accidental release or escape of an organism shall be in
compliance with the standards listed in control 1.3.
5.2 If a breach of containment occurs, the facility operator must ensure that the MAF
Inspector responsible for supervision of the facility has received notification of the
breach within 24 hours.
5.3 In the event of any breach of containment the contingency plan for the attempted
retrieval or destruction of any viable material of the organism that has escaped shall be
implemented immediately. The contingency plan shall be included in the containment
manual in accordance with MAF Biosecurity Authority/ERMA New Zealand Standard
154.03.023: Containment Facilities for Microorganisms.
6.
Inspection and monitoring requirements for containment facilities:
6.1 The inspection and monitoring requirements for containment facilities shall be in
compliance with the standards listed in control 1.3.
6.2 The containment manuals shall be updated, as necessary, to address the implementation
of the controls imposed by this approval, in accordance with MAF Biosecurity
Authority/ERMA New Zealand Standard 154.03.023: Containment Facilities for Microorganisms.
7.
Qualifications required of the persons responsible for implementing those
controls:
7.1 The training of personnel working in the facility shall be in compliance with the
standards listed in control 1.3.
_____________________________
Date: 26 April 2001
Mrs Helen Hughes Chair,
GMO Standing Committee of the Authority
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Amendment: November 2006
Changes to controls:



Addition of footnotes to the containment facility references and the Australian/New
Zealand containment facility references to “future proof” the decision
Standardise the wording of the breach of containment control
Removal of the control regarding inspection of facilities by the Authority, its agent or
enforcement officers
____________________________
Date: 22 August 2007
Dr Kieran Elborough
Chair, GMO Standing Committee
References
Bailey, M. J., Lilley, A. K., Thompson, I. P., Rainy, P. B, and Ellis, R. J. 1995. Site directed
chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar
beet; stability and potential for market gene transfer. Molecular Ecology 4: 755-763.
de Leij, F. A. A. M., Thomas, C. E., Bailey, M. J., Whipps, J. M. and Lynch, J. M. 1998.
Effect of insertion site on metabolic load on the environmental fitness of a genetically
modified Pseudomonas fluorescens isolate. Appl. Env. Microbiol 64: 2634-2638.
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