Version 1
1 June 2017
RISK ANALYSIS
Inadvertent dealings with petunia genetically modified for
altered flower colour
1. Introduction to the GMOs
Petunias (
Petunia hybrida) are widely grown in Australia as ornamental plants (Harden
1992). Petunia cultivars African Sunset, Trilogy Red, Trilogy Deep Purple and Trilogy Mango
were imported into Australia between 2013-2017, and were recently discovered to be
genetically modified. Testing has found that the GM petunia cultivars contain an introduced
gene conferring altered flower colour and an introduced antibiotic resistance marker gene
(Table 1).
Table 1: Genetic elements detected in the GM petunias
Genetic
Encoded protein
Function
Source
element
p35S
N/A
Constitutive promoter Cauliflower mosaic virus
A1
Dihydroflavonol 4-reductase Altered flower colour
Zea mays
(DFR)
pNOS
N/A
Constitutive promoter
Agrobacterium
tumefaciens
nptI
Neomycin
Antibiotic resistance
Escherichia coli
phosphotransferase II (NPTII) marker gene
Source: testing by university researchers (Bashandy & Teeri 2017) and Finnish Food Safety
Authority.
The cultivars Raspberry Blast, Candy Blast (also known as Rose Blast Charm) and
Colourworks Homare were also imported. Cultivars Raspberry Blast and Candy Blast have
been tested by the United States Department of Agriculture and found to be GMOs (APHIS
guidance GE petunias accessed on 25/5/17). Cultivar Colourworks Homare is a suspected
GMO. USDA testing covers the p35S, pNOS and
nptI genetic elements (APHIS guidance
petunia testing accessed on 26/5/17). It is assumed, as cultivars Raspberry Blast and Candy
Blast share three introduced genetic elements with the GM petunias African Sunset, Trilogy
Red, Trilogy Deep Purple and Trilogy Mango, that they have been genetically modified in the
same way. This risk analysis document is based on currently available information regarding
the introduced genetic modifications as outlined above in Table 1.
Altered flower colour trait
Petunia flower colour is due to anthocyanin pigments. The native DFR enzyme in petunia
favours the conversion of the anthocyanin precursor dihydrokaempferol to the pigments
cyanidin (red) and delphinidin (blue). The maize DFR enzyme can also drive conversion of
dihydrokaempferol to the pigment pelargonidin (orange), and thus alter flower colour
(Meyer et al. 1987; Griesbach 1993). The total anthocynanin content of GM petunias
containing an introduced
A1 gene was measured and is similar to non-GM petunias
(Griesbach 1993). Humans are naturally exposed to anthocyanins through ingestion of fruit
and vegetables (Wu et al. 2006), and anthocyanins are authorised food additives used for
colouring in Australia (FSANZ 2014).
2. Potential for toxicity or allergenicity
Petunias are not grown for human food or animal feed in Australia. Non-GM petunias are
not considered toxic if accidental y ingested by children or pets (Safe and Poisonous Garden
Plants, ASPCA Toxic and Non-Toxic Plants List), and are recommended plants for low-
allergen gardens (Asthma Foundation Victoria 2013). This suggests that petunias do not
produce any native toxins or allergens that cause problems when petunias are grown in
gardens and not intentional y consumed. Based on experience to date with a range of GM
plants, it is unlikely that the genetic modifications in the GM petunia would alter levels of
native toxins or allergens, and, even if this were to occur it is not expected to lead to harm.
The introduced
A1 gene is derived from maize and is naturally expressed in all maize tissues
(Bernhardt et al. 1998). Maize kernels have a long history of safe consumption by people and
animals, whole maize plants are used as grazing for livestock, and maize flowers are
regularly visited by insect pollinators without ill effects. In addition, the encoded maize DFR
protein is homologous to the geranium and petunia DFR proteins, which have been
previously assessed by the Regulator as being of negligible risk to people and the
environment. GM torenias containing geranium DFR were approved for field trials (DIR
068/2006). GM carnations containing petunia DFR were approved for commercial releases
and also placed on the GMO Register for uncontained release without a licence (DIR
030/2002, DIR 134, GMO Register). Thus, the maize DFR protein is not expected to be toxic
or allergenic to people or other organisms, including insect pollinators.
The introduced
nptI gene from
E. coli is used extensively as a selectable marker in the
production of GM plants and is present in many GMOs approved for commercial release in
Australia and other countries. 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. Regulatory agencies in Australia and other countries have found no
evidence that the NPTII protein is toxic or allergenic to people or other organisms, including
insect pollinators.
3. Potential for weediness
Petunias are typically planted in gardens as annuals; they are killed by frost but may survive
mild winters (Small 2014). Petunias can also self-seed in humid subtropical climates such as
Florida and South Carolina (Burch & Demmy 1995; Russ 2007). However, most petunias will
die if not watered for 14 days (Estrada-Melo et al. 2015). Thus, unwanted petunias could be
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minor weeds in gardens, but probably only if the gardens are in a warm climate and
frequently watered.
Petunias may spread outside gardens by several mechanisms, including wind transport of
their tiny seeds, which weigh around 0.1 mg each (Jauron 2013). Seeds are initially dormant
and require light (cannot be buried), warm temperatures and moisture in order to germinate
(Jauron 2013; Small 2014; Petruzzelli et al. 2003).
Petunia populations (often classified as
P. axillaris, which is one parent of the hybrid
P. hybrida) are occasionally naturalised in Australia, primarily in northern NSW and southern
Queensland (Harden 1992)(Atlas of Living Australia). Naturalised
P. axillaris plants are
considered a minor problem warranting control in NSW and are present but not considered
to warrant control in Queensland (Groves et al. 2003). Petunias are not classified as noxious
weeds or weeds of national interest (National weeds lists).
Neither the altered flower colour trait conferred by the
A1 gene, nor the antibiotic
resistance conferred by the
nptI gene are expected to increase the weediness potential of
the GM petunias.
4. Potential for gene transfer
Petunias are insect pollinated. They are sexually compatible with cultivated petunias, other
species from the genus
Petunia and possibly species from the closely related genus
Calibrachoa (Smal 2014; Vandenbussche et al. 2016). No plants from
Petunia or
Calibrachoa are native to Australia. Cultivated petunias and
Calibrachoa species are grown as ornamental
garden plants in Australia. Petunias are occasionally naturalised as discussed in section 3.
The species
C. parviflora is sparsely naturalised but not considered to warrant control in
northern NSW (Groves et al. 2003; Harden 1992)(Atlas of Living Australia).
As discussed in sections 2 and 3 of this document, the introduced
A1 and
nptI genes are not
expected to increase toxicity, al ergenicity or weediness in GM petunia. For the same
reasons, if gene transfer to related species occurred, these genes would not be expected to
increase the toxicity, al ergenicity or weediness of the related species.
5. Consideration of proposed dealings
The dealings with GMOs proposed in the licence are:
(a) disposing of the GMOs;
(b) conducting experiments with the GMOs for purposes relating to disposing of the
GMOs;
(c) growing the GMOs for purposes relating to disposing of the GMOs;
(d) transporting the GMOs for purposes relating to disposing of the GMOs; and
(e) possession or supply of the GMOs for purposes relating to disposing of the GMOs.
The GM petunia cultivars are already present in the Australian environment, as they have
been imported and grown since 2013. The effect of the proposed dealings would be to
reduce the number of GM petunias present in the environment, thus reducing exposure of
people and animals to the GMOs and reducing the potential for spread and persistence of
the GMOs. The proposed dealings are not expected to pose any new risks that do not
already exist due to the presence of the GMOs in the environment.
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Methods of disposal
Methods of disposal for GM petunia plants listed in the licence include herbicide application,
uprooting, desiccation and incineration. Methods such as mowing or shredding are not listed
due to some uncertainty regarding their effect, as petunias can regrow from cuttings under
favourable conditions (Ahkami et al. 2009). Listed methods of disposal for GM petunia seeds
include various types of heat treatment, which are expected to denature proteins and
render the seeds non-viable, or deep burial. Rationales for the recommended methods can
be found in Appendix A to this document.
6. Conclusions of risk analysis document
Based on the information currently available, the GM petunias are considered to pose
negligible risks to human health and safety or the environment. Should new and/or different
information become available, then further risk analysis may be required.
7. References
Ahkami, A.H., Lischewski, S., Haensch, K.T., Porfirova, S., Hofmann, J., Rolletschek, H. et al.
(2009) Molecular physiology of adventitious root formation in
Petunia hybrida cuttings:
involvement of wound response and primary metabolism.
New Phytol 181: 613-625.
Asthma Foundation Victoria (2013) The Low Allergen Garden.
Bashandy, H., Teeri, T.H. (2017). Genetical y engineered orange petunias on the market.
http://dx.doi.org/10.1101/142810
Bernhardt, J., Stich, K., Schwarz-Sommer, Z., Saedler, H., Wienand, U. (1998) Molecular
analysis of a second functional
A1 gene (dihydroflavonol 4-reductase) in
Zea mays.
Plant J
14: 483-488.
Burch, D., Demmy, E.W. (1995) Last year's garden this year.
Proc Fla State Hort Soc 108: 404-
405.
Estrada-Melo, A.C., Chao, Reid, M.S., Jiang, C.Z. (2015) Overexpression of an ABA
biosynthesis gene using a stress-inducible promoter enhances drought resistance in petunia.
Hortic Res 2: 15013.
FSANZ (2014) Food additive names and code numbers. Document prepared by Food
Standards Australia New Zealand. Available online.
Griesbach, R.J. (1993) Characterization of the flavonoids from
Petunia x
Hybrida flowers
expressing the
A1 gene of
Zea mays.
HortScience 28: 659-660.
Groves, R.H., Hosking, J.R., Batianoff, G.N., Cooke, D.A., Cowie, I.D., Johnson, R.W. et al.
(2003)
Weed categories for natural and agricultural ecosystem management. Bureau of
Rural Sciences, Canberra.
Harden, G.J. (1992)
Flora of New South Wales Volume 3. Harden, G.J., ed. University of New
South Wales Press.
Jauron, R. (2013) Growing Petunias. Iowa State University of Science and Technology,
https://store.extension.iastate.edu/Product/Growing-Petunias-PDF.
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Meyer, P., Heidmann, I., Forkmann, G., Saedler, H. (1987) A new petunia flower colour
generated by transformation of a mutant with a maize gene.
Nature 330: 677-678.
Petruzzelli, L., Muller, K., Hermann, K., Leubner-Metzger, G. (2003) Distinct expression
patterns of beta-1,3-glucanases and chitinases during the germination of Solanaceous seeds.
Seed Science Research 13: 139-153.
Russ, K. (2007) Petunia. Clemson University, South Carolina,.
Small, E. (2014) Top Canadian Ornamental Plants. 7. Petunia.
CBA/ABC Bulletin 47: 49-55.
Vandenbussche, M., Chambrier, P., Rodrigues, B.S., Morel, P. (2016) Petunia, your next
supermodel?
Frontiers in Plant Science 7: 72.
Wu, X., Beecher, G.R., Holden, J.M., Haytowitz, D.B., Gebhardt, S.E., Prior, R.L. (2006)
Concentrations of anthocyanins in common foods in the United States and estimation of
normal consumption.
Journal of Agricultural and Food Chemistry 54: 4069-4075.
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Appendix A GM petunia disposal guidelines
Seedless petunia plants may be destroyed by any of the fol owing methods:
• Herbicide treatment. Tips on safe herbicide use can be found in the web page of
Australian Pesticides and Veterinary Medicines Authority
• Burning. Burn only in accordance with all federal, state, and local laws and
ordinances and permits. Monitor weather conditions prior to ignition to avoid
hazardous fires (Rowe 2013)
• Composting. Plants can be pul ed and left with roots exposed to dry out. This
material can be then composted. Composting at 55°C for three continuous days kil s
most plant propagules, but note that this is not 100% effective if seeds or flowers
are present (Rowe 2013)
• Burial. Dig a hole of at least 1 meter deep and bury the plants. Such a pit is best
located in an out-of-the-way spot in the yard where it will not be disturbed (Timber
Press 2013).
• Desiccation. Stop watering the petunia plants. Water-deprived plants usual y die
after 14 days in greenhouse conditions (Estrada-Melo et al. 2015)
• Plants can be placed into plastic bags and stored in a lidded waste bin for at least a
month. Non-viable plant material can then be disposed of via standard methods (eg
landfill). This method has been previously used to destroy GM-Torenia plants,
another herbaceous ornamental, after a field trial (OGTR 2008).
• Plants may also be bagged in a black plastic bag, sealed and then 'baked' in the sun
until destroyed (Australian Department of Environment).
If petunia plants bear seeds the preferred method of disposal is burning. If this is not
possible, seeds need to be harvested prior applying any of the other methods to kil the
plants. Harvested seeds and seeds in packets may be destroyed by:
• Boiling in water for 20 min. Petunia seeds do not have hard seed coats (Sink 1984),
therefore they are sensitive to hot water. Even plants with hard seed coats that
require scarification treatments to germinate can be killed by boiling them in water
for prolonged periods (Fulbright & Flenniken 1987; Kimura & Islam 2012)
• Heating in the oven at 150°C for 30 minutes. Petunia seeds are not known for their
resistance to heat (Sink 1984). Seeds of plant species that live in fire prone
environments and are built to withstand high temperatures, have their germination
completely inhibited by exposure to 150°C for 5 minutes (Gashaw & Michelsen
2002)
• Microwaving at 800 watt or more for 10 minutes. Microwaving annual rye grass
seeds at 800 watt for 240 seconds was sufficient to stop completely the germination
of seeds (GRDC 2015)
• Deep burial at a biosecurity waste class 8.2 site approved by the Department of
Agriculture and Water Resources. This approved arrangement for deep burial
requirements ensures that the burial site wil be undisturbed. Petunia seeds require
light (cannot be buried) in order to germinate (Jauron 2013; Small 2014; Petruzzelli
et al. 2003).
References
Estrada-Melo, A.C., Chao, Reid, M.S., Jiang, C.Z. (2015) Overexpression of an ABA
biosynthesis gene using a stress-inducible promoter enhances drought resistance in petunia.
Hortic Res 2: 15013.
6
Fulbright, T., Flenniken, K. (1987) Temperature and scarification effects on germination of
prostate bundleflower seeds.
Journal of Range Management 40: 170-173.
Gashaw, M., Michelsen, A. (2002) Influence of heat shock on seed germination of plants
from regularly burnt savanna woodlands and grasslands in Ethiopia.
Plant Ecology 159: 83-
93.
GRDC (2015) Using microwaves to kill weed seeds and snails.
Jauron, R. (2013) Growing Petunias. Iowa State University of Science and Technology,
https://store.extension.iastate.edu/Product/Growing-Petunias-PDF.
Kimura, E., Islam, M.A. (2012) Seed scarification methods and their use in forage legumes.
Res J Seed Sci 5: 38-50.
OGTR (2008) Risk Assessment and Risk Management Plan for DIR 084/2008. Limited and
control ed relase of torenia genetical y modified for enhanced phosphate uptake.
Petruzzel i, L., Mul er, K., Hermann, K., Leubner-Metzger, G. (2003) Distinct expression
patterns of beta-1,3-glucanases and chitinases during the germination of Solanaceous seeds.
Seed Science Research 13: 139-153.
Rowe, R (2013) Disposal of terrestrial invasive plants. Florida Exotic Pest Plant Council.
Sink, K. (1984)
Petunia. Sink, K.C., ed. Monographs on theoretical and applied genetics ; 9
Monographs on theoretical and applied genetics ; 9 Springer-Verlag, Berlin ; New York.
Small, E. (2014) Top Canadian Ornamental Plants. 7. Petunia.
CBA/ABC Bulletin 47: 49-55.
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