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Assessment of Plant Pest Risk for International Flower Developments

Pty. Ltd. IFD-524Ø1-4 and IFD-529Ø1-9 Rosa x hybrida (rose) varieties

International Flower Developments Pty. Ltd. (IFD) (Victoria, Australia) has petitioned the 

Animal and Plant Health Inspection Service (APHIS) of the United States Department of 

Agriculture for a determination that IFD-524Ø1-4 and IFD-529Ø1-9 roses (Rosa x

hybrida) are unlikely to pose a plant pest risk and, therefore, should no longer be regulated 

articles under APHIS’ regulations at 7 CFR part 340. APHIS administers 7 CFR part 340 

under the authority of the plant pest provisions of the Plant Protection Act of 20001. This 

plant pest risk assessment was conducted to determine whether IFD-524Ø1-4 and IFD529Ø1-9 are unlikely to pose a plant pest risk. 

History of Development of IFD-524Ø1-4 and IFD-529Ø1-9 Rosa x hybrida varieties

Working with a rose grower in the U.S., Florigene and IFD have been researching and 

developing two lines of genetically engineered roses since 2004. Both of these rose lines 

include added genes for flavonoid 3’-5’ hydroxylase (from a black pansy, Viola tricolor) 

and anthocyanin 5-acyltransferase (from torenia, Torenia hybrida). These rose lines also 

contain the neomycin phosphotransferase gene (from the bacterium Escherichia coli)

which was used for selection in the laboratory. Both of these rose lines have been 

approved for commercial use, including environmental release, in Japan (IFD 2010). One 

line (IFD-524Ø1-4) has also been approved for commercial use/environmental release in 

Australia (IFD 2010) (costs of the regulatory request for the other rose line and the small 

size of the Australian market led the company to only request approval for one line there). 

Addition of the Viola and Torenia genes alter the anthocyanin biosynthesis pathways and 

shunt some of these biochemicals toward production of the delphinidin-based 

anthocyanins, resulting in production of blue pigments in these rose lines. Production of 

these blue pigments alters the flower color of these rose lines (IFD 2010). 

Description of added genes

Florigene’s IFD-524Ø1-4 and IFD-529Ø1-9 roses (Rosa x hybrida) were produced using 

disarmed Agrobacterium tumefaciens (IFD 2010) and contain 3 transgene fragments (IFD 

2010) from plasmid pSPB130 (IFD 2010): 

(1) A nopaline synthase promoter/ neomycin phosphotransferase gene/ nopaline synthase 

terminator (NOS/NPT II/ NOS) fragment:

• The NOS promoter is from Agrobacterium tumefaciens and drives production of 

the npt II gene. 

 1

Section 403 (14) of the Plant Protection Act (7USC Sec 7702(14)) defines plant pest as:

“Plant Pest - The term “plant pest” means any living stage of any of the following that can directly or indirectly injure, cause damage 

to, or cause disease in any plant or plant product: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic plant. (D) A bacterium. 

(E) A fungus. (F) A virus or viroid. (G) An infectious agent or other pathogen. (H) Any article similar to or allied with any of the 

articles specified in the preceding subparagraphs.” 
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• The npt II gene (from Escherichia coli) results in production of neomycin 

phosphotransferase and confers tolerance to the antibiotic kanamycin. Kanamycin 

is used to select for transgenic tissues under laboratory conditions. 

• The NOS terminator is from Agrobacterium tumefaciens. 

(2) A cauliflower mosaic virus 35S promoter/ flavonoid 3’, 5’- hydroxylase gene/ 

nopaline synthase fragment (CaMV35S/ F3’5’H/ NOS) fragment:

• The enhanced 35S promoter from cauliflower mosaic virus results in production of 

flavonoid 3’, 5’- hydroxylase.

• The flavonoid 3’, 5’-hydroxylase gene from Viola tricolor results in production of 

this biochemical. 

• The NOS terminator is from Agrobacterium tumefaciens. 

(3) A cauliflower mosaic virus 35S promoter/ anthocyanin 5-acyltransferase/ nopaline 

synthase fragment (CaMV35S/ 5AT/ NOS) fragment:

• The enhanced 35S promoter from cauliflower mosaic virus drives production of 

anthocyanin 5-acyltransferase.

• The anthocyanin 5-acyltransferase gene from Torenia hybrida results in 

production of this biochemical.

• The NOS terminator is from Agrobacterium tumefaciens. 

Production of F3’5’H and 5AT enzymes shunts production of anthocyanins in these rose 

lines away from the cyanidin based anthocyanins (pink) and toward production of 

delphinidin based anthocyanins (blue) (IFD 2010).

Southern blots were used to analyze gene insertion from plasmid pSPB130 into the rose 

genome and to examine the integrity and expression of the inserted DNA. APHIS

evaluated Florigene’s Southern blot data and came to several conclusions regarding 

insertion of the various genes into these 2 rose lines. Based on gel quantitation analysis of 

DNA, there are between 1 and 4 copies of each of the genes (npt II, F3’5’H, and 5AT) in 

both IFD-524Ø1-4 and IFD-529Ø1-9. Additionally, the Southern blots show the presence 

of multiple DNA bands (IFD 2010) indicating that it is also likely that some gene 

rearrangements have occurred. The DNA banding patterns does indicate that these genes 

were inserted differently in each of the two rose lines. 

Florigene also provided Northern analyses documenting production of RNA from the 

inserted genes (IFD 2010). Each gene was expressed in the transgenic plants and there 

was no expression noted in the control non-transformed rose line. 

Further, Florigene presented data on production of delphinidin and cyanidin in the petals 

of these roses. Both transgenic lines showed accumulation of delphinidin (a blue 

anthocyanin) and much decreased production of cyanidin (a red anthocyanin) (IFD 2010). 

The control non-transformed rose did not produce delphinidin but did produce cyanidin. 
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Plant Pest Risk Assessment

APHIS has prepared a Plant Pest Risk Assessment in response to a petition (APHIS No. 

08-315-01p) from International Flower Developments Pty Ltd. APHIS regulation 7 CFR 

340.6(c) (4) stipulates the information needed to be considered in a petition for 

nonregulated status. APHIS uses information submitted by the applicant related to plant 

pest risk characteristics, disease and pest susceptibilities, expression of the gene product, 

new enzymes, or changes to plant metabolism, weediness of the regulated article, any 

impacts on the weediness of any other plant with which it can interbreed, and the transfer 

of genetic information to organisms with which it cannot interbreed for IFD-524Ø1-4 and 

IFD-529Ø1-9 roses. Issues related to agricultural or cultivation practices and the effects of 

the regulated article on non-target organisms will be considered in the Environmental 

Assessment for these rose lines. 

Based on information on the biology of rose

(http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/riskassessments-1, accessed 

1/26/10), data presented in the Petition and scientific data relevant to a discussion of plant 

pest risk, APHIS has concluded the following regarding IFD-524Ø1-4 and IFD-529Ø1-9 

roses: 

Potential impacts of altered disease and pest susceptibilities 

USDA-APHIS assessed whether IFD-524Ø1-4 and IFD-529Ø1-9 roses are likely to have 

significantly altered disease and pest susceptibilities. The assessment encompasses a 

consideration of introduced traits and interactions with pests and diseases. 

Hybrid tea roses (Rosa x hybrida) are not considered plant pests in the United States. 

None of the gene sequences derived from the plant pests (Agrobacterium and cauliflower 

mosaic virus) that were incorporated into these rose lines result in the production of 

infectious agents or disease symptoms in plants, and so they are unlikely to pose a plant 

pest risk. The description of the genetic modifications, including genetic elements, 

expression of the gene products and their functions in these roses has been summarized 

above.

Cultivated hybrid tea roses are susceptible to numerous insect pests and diseases. 

Florigene has noted some of the major ones (IFD 2010). A variety of insecticides, 

miticides and other pesticides are commonly used on roses 

(http://urbanext.illinois.edu/roses/disease.cfm, accessed 1/25/10; 

http://www.ipm.ucdavis.edu/PMG/PESTNOTES/pn7466.html, accessed 1/25/10). 

Florigene has contracted for growing these rose lines in greenhouses in California for over

4 years. The grower (Jackson and Perkins Wholesale, Inc.) has noted that these rose lines

grow normally in all respects (IFD 2010). They further make note of the major pests of 

roses in California: two spotted spider mites, powdery mildew and downy mildew. They 

also indicate that all of these pests are controlled using common treatments and 

conventional protocols (IFD 2010). These rose lines have also been grown extensively in 
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Columbia under typical rose cultivation conditions there. The grower there indicates that 

both the transgenic and non-transgenic parental lines are susceptible to mildew but that 

there was no difference between the transgenic and non-transgenic plants (IFD 2010). 

Finally, this information submitted by Florigene indicates that there are no significant 

differences in terms of pest or disease susceptibilities between these rose lines and their 

non-transgenic counterparts.

Potential impacts from new gene products, changes to plant metabolism or 

composition

As a point of reference, Figure 4 in the Petition (p. 22) presents the basic biochemical 

pathways for production of anthocyanins in plants. 

New proteins produced in both IFD-524Ø1-4 and IFD-529Ø1-9 include the following:

1. Neomycin phosphotransferase (NPT II)

2. Flavonoid 3’, 5’-hydroxylase (F3’5’H)

3. Anthocyanin 5- acyltransferase (5AT)

NPT II is a common protein found in a number of genetically engineered plants that have 

been widely planted across the U.S. and in other parts of the world. In every case, no 

issues related to health or environmental safety have been noted (APHIS petitions 04-317-

01p, 04-264-01p, 01-137-01p, 01-206-02p, 01-206-01p, 95-352-01p, 96-051-01p, 95-045-

01p, 94-308-01p) (USDA-APHIS 2011). NPT II confers tolerance to the antibiotic 

kanamycin and is used in a laboratory setting to select tissues transformed with the genes 

of interest. 

Flavonoid 3’, 5’-hydroxylase (F3’5’H) is an enzyme that is widely found in nature in 

plants producing anthocyanins, most often blue colors. This enzyme can be found in 

grapes (Bogs 2006), petunia (Toguri 1993), eggplant (Solanum melongena) (Chapple 

1998), gentian, torenia, campanula and many other plants (Tanaka 2006). F3’5’H is in the 

cytochrome P450 family of enzymes (designated in the CYP75A subfamily) (Nelson 

2009, http://drnelson.utmem.edu/CytochromeP450.html, accessed 1/26/10). Plant species 

lacking flavonoid 3’-5’-hydroxylase, such as non-engineered roses, do not make the blue 

delphinidin-based anthocyanins (Deng 2001).

Anthocyanin 5-acyltransferase (5AT) is also an enzyme that is widely found in nature in 

plants producing anthocyanins. In the anthocyanin biosynthesis pathways, this enzyme, as 

well as related anthocyanin acyltransferases, act to alter the biochemical structure of 

anthocyanin glucosides (AGS) (such as pelargonidin GS, cyanidin GS, and delphinidin 

GS) and make the resulting pigment (anthocyanin) more chemically stable in the plant cell 

(Nakayama et al 2003). 

As described above, compositional assessment data supplied in the Petition regarding 

production of DNA, RNA and delphinidin supports the conclusion that IFD-524Ø1-4 and 
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IFD-529Ø1-9 roses contain introduced f3’5’h and 5at genes from Florigene plasmid 

pSPB130 (IFD 2010). 

Florigene also provided an assessment of delphinidins found in common foods and 

common flowers (IFD 2010). As noted in the tables, many foods, which are consumed 

widely, and ornamental flowers, which are grown widely, contain measurable quantities 

of delphinidin. None of the foods or ornamental plants noted are known to pose unique 

environmental risks because of the presence of delphinidin, its precursor biochemicals or 

catalytic enzymes (i.e., F3’5’H or 5AT) in the anthocyanin pathways. Specific data on 

toxicity and potential environmental effects of F3’5’H and 5AT is sparse but information 

on the chemistry of delphinidins and other anthocyanins is noted (Beheshti 2008; 

Vilanova 2009; Yu 2006). None of the documents identified or noted raise environmental 

concerns related to new gene products, changes in plant metabolism or plant composition. 

Based on all the noted considerations, APHIS concludes that IFD-524Ø1-4 and IFD529Ø1-9 roses pose no more of a plant pest risk from new gene products, changes to plant 

metabolism or composition than conventional hybrid tea roses.

Potential impacts from outcrossing of IFD-524Ø1-4 and IFD-529Ø1-9 roses 

to wild relatives 

Florigene has described the biology of roses, including hybrid tea roses, in its Petition 

(IFD 2010). They note that the first hybrid tea roses were produced in France in 1867 and 

that since then, hundreds of new cultivars of hybrid tea roses have been introduced. They 

also note that cultivated rose is the most widely produced cut flower crop in the world and 

that over 60 million rose plants are planted each year just to meet demand for cut flower 

production. The Australian Office of Gene Technology Regulator (OGTR) has also 

produced an extensive document on the biology of Rosa x hybrida

(http://www.health.gov.au/internet/ogtr/publishing.nsf/Content/rose3/$FILE/biologyrose09.pdf, accessed 1/25/10) and completed a risk assessment of one of 

these rose lines (IFD-524Ø1-4) in 2009 

(http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/Content/dir090-

4/$FILE/dir090rarmp.doc, accessed 1/25/10). They describe the taxonomy of roses, rose 

species, centers of diversity and domestication, development and commercialization of 

modern rose cultivars, cultivation practices, breeding work (including use of “sports” as 

well as irradiation induced mutations), plant morphology, plant development and 

reproduction, biochemistry, biotic and abiotic interactions, disease and insect pests and 

weediness. Most of the information in those documents is directly relevant to this 

application and plant pest risk assessment and is incorporated here by reference. 

Florigene’s description and analysis of IFD-524Ø1-4 and IFD-529Ø1-9 roses indicates 

that they are both L1 periclinal chimeras2. The significance of this is that the f3’5’h and 

5at genes introduced are only found and expressed in the epidermal tissues and cannot be 

 2 An L-I periclinal chimera is a type of plant chimera in which the L-I cell layer has a different genetic 

make-up than the L-II and L-III cell layers. The L-I cell layer gives rise to epidermal tissues of a plant while 

the L-II and L-III layers give rise to reproductive tissues, vascular tissues and other internal plant tissues. 
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passed on to progeny through cross pollination (i.e., outcrossing). Florigene demonstrated 

this in the various pollination and in situ hybridization experiments they conducted during 

development of these lines (IFD 2010). Florigene analyzed over 100 seeds of hybrid 

pollinations with grandiflora and floribunda roses and found none that contained the IFD524Ø1-4 and IFD-529Ø1-9 transgenes. They also analyzed 19 seeds from pollinations 

with wild rose (Rosa multiflora) and found none that contained the introduced genes. 

They also conducted specific RNA hybridization experiments using petal tissues of 

control and transgenic lines and identified stained cells only in the epidermis of the 

transgenic rose lines (IFD 2010). 

In assessing the risk of gene introgression from IFD-524Ø1-4 and IFD-529Ø1-9 roses into 

its sexually compatible relatives, APHIS considers two primary issues: 1) the potential for 

gene flow and introgression and, 2) the potential impact of introgression. 

Given the demonstrated L-I chimeric nature of these rose lines, the potential for gene flow 

and introgression into sexually compatible relatives of rose is essentially zero. Since gene 

flow cannot occur, any potential impact of introgression, therefore, is also zero. 

Potential impacts based on the relative weediness of IFD-524Ø1-4 and IFD529Ø1-9 rose.

APHIS assessed whether Florigene IFD-524Ø1-4 and IFD-529Ø1-9 roses are any more 

likely to become weeds than the non-transgenic recipient rose line, or other rose currently 

cultivated. The assessment encompasses a thorough consideration of the basic biology of 

rose and an evaluation of unique characteristics of these rose lines.

The biology and cultivation practices of hybrid tea rose have been well described 

(http://www.health.gov.au/internet/ogtr/publishing.nsf/Content/rose3/$FILE/biologyrose09.pdf) and Florigene has also provided information on Rosa x 

hybrida cultivation, taxonomy, pollination, weediness and potential modes of gene flow 

(IFD 2010). Cultivated hybrid tea roses are all complex hybrids (IFD 2010) derived from 

breeding work done over centuries from more than 5 different rose species and numerous 

rounds of selection and cross hybridization at each step. Cut flower rose varieties are the 

most widely cultivated cut flower crop worldwide with sales of over 6 billion stems per 

year (IFD 2010). Florigene has noted that USDA lists over 100 species of Rosa in its 

Plants database (http://plants.usda.gov/). Only 3 of these Rosa species are common weeds 

in the U.S. (Rosa arkansana, Rosa multiflora, and Rosa rugosa) 

(http://plants.usda.gov/java/noxiousDriver#state) and Rosa arkansana is native to the U.S. 

None of the Rosa species are listed as Federal noxious weeds. 

In the U.S., Rosa x hybrida is not listed as a weed in several major weed references 

(Crockett 1977; Holm et al. 1979; Muenscher 1980) nor is it listed as a noxious weed 

species by the U.S. Federal Government 

(http://www.aphis.usda.gov/plant_health/permits/organism/federal_noxious_weeds.shtml,

accessed 12/22/09). As noted by Florigene, these roses also do not readily propagate by 

vegetative means (IFD 2010). Because of the L-I chimeric nature of these rose lines, they 
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also will not disperse the genes for f3’5’h and 5at by pollen. As is typical of hybrid tea 

roses grown for cut flowers, the likelihood of moving genes around by seed is very small 

as these roses are cut and harvested long before hips or seeds would mature. Compared to 

the non-transgenic progenitor roses, these attributes are no different. 

Weediness for the purposes of this part of the plant pest risk assessment is an attribute, 

which causes a crop to act as a weed due to the addition of genes, in comparison to the 

non-transgenic comparator. If the fitness of these rose lines improves in natural or 

agricultural ecosystems due to the inserted DNA, the potential for weediness could 

increase. The following analysis of the inserted DNA is intended to document that IFD524Ø1-4 and IFD-529Ø1-9 roses have a negligible likelihood of increased weediness. As 

described previously, these rose lines differ only in the expression of the F3’5’H and 5AT 

enzymes which ultimately result in production of blue delphinidin-based anthocyanins. 

Florigene collected data on numerous phenotypical features of these rose lines. Data 

collected related to plant height, flower stem length, flower height, flower diameter, petal 

length and width, number of pistils, number of stamens, and others (IFD 2010). While 

some of these characteristics showed statistical differences between the non-transgenic 

and transgenic lines, none of these characteristics stand out as ones that would provide a 

fitness advantage to these lines in an unmanaged situation. Florigene also collected data 

on pollen viability, pollen grain germination, and pollen diameter (IFD 2010). None of 

the data on pollen characteristics showed any statistical significance between the nontransgenic and transgenic lines. 

None of the characteristics that APHIS considers in its assessment of weediness point to 

the potential for these rose lines to become more weedy or invasive than the nontransgenic comparator lines. APHIS concludes that introduction of these rose lines do no 

result in increased plant pest risk related to weediness. 

Potential Impacts on Target and Non-target Organisms, Including Beneficial 

Organisms

Based on the data provided by the applicant and existing literature, APHIS evaluated the 

potential for deleterious effects or significant impacts of these rose lines on non-target or

beneficial organisms.

The genes introduced into these rose lines result directly in production of the F3’5’H and 

5AT proteins and indirectly in production of delphinidin, a blue pigment. Florigene notes 

that delphinidin and delphinidin derivatives are contained in many common foods in 

relatively large amounts (IFD 2010). Anyone or anything consuming these foods, 

therefore, consumes delphinidin as well as the F3’5’H and 5AT proteins required for its 

production. The 5AT protein is also found in foods containing other related anthocyanin 

pigments (IFD 2010). As noted by Florigene, the amount of delphinidin found in these 

rose lines is approximately 100 times less than that found in fresh blueberries (IFD 2010). 

Florigene also notes that, as a group, anthocyanins have a very low toxicity (IFD 2010; 

http://www.inchem.org/, accessed 1/25/10). 
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In addition to this lack of toxicity associated with these rose lines, Florigene notes that 

these roses will be grown in a limited number of locations under highly controlled 

conditions by experienced rose growers (IFD 2010). As such, exposure to organisms 

outside these conditions will be extremely limited. 

This data submitted by the applicant indicates that the interactions between these rose 

lines and other roses, including the control lines, are similar. Considering all this, APHIS 

concludes that Florigene rose lines IFD-524Ø1-4 and IFD-529Ø1-9 are unlikely to pose 

safety risks to non-target or beneficial organisms. 

Potential impacts from transferring genetic information from IFD-524Ø1-4 

and IFD-529Ø1-9 rose to organisms with which it cannot interbreed.

APHIS examined the potential for the new genetic material inserted into these rose lines 

to be horizontally transferred to other organisms without sexual reproduction and whether 

such an event could lead directly or indirectly to disease, damage, injury or harm to plants, 

including the creation of more virulent pathogens. First, many genomes (or parts thereof) 

have been sequenced from bacteria that are closely associated with plants including 

Agrobacterium and Rhizobium (Kaneko et al. 2000; Wood et al. 2001; Kaneko et al. 

2002). There is no evidence that these organisms contain genes derived from plants. 

Second, in cases where review of sequence data implied that horizontal gene transfer 

occurred, these events are believed to occur on an evolutionary time scale on the order of 

millions of years (Koonin et al. 2001; Brown 2003). Third, transgene DNA promoters and 

coding sequences are optimized for plant expression, not prokaryotic bacterial expression. 

Thus even if horizontal gene transfer occurred, proteins corresponding to the transgenes 

are not likely to be produced. Fourth, the FDA has evaluated horizontal gene transfer from 

the use of antibiotic resistance marker genes, and concluded that the likelihood of transfer 

of antibiotic resistance genes from plant genomes to microorganisms in the 

gastrointestinal tract of humans or animals, or in the environment, is remote (Council for 

Biotechnology Information, 2001; http://vm.cfsan.fda.gov/~dms/opa-armg.html, accessed 

1/26/10). Finally, a recent review of issues related to horizontal gene transfer concluded 

that this type of gene transfer is unlikely to occur and poses negligible risks to human 

health or the environment (Keese 2008). Therefore APHIS concludes that horizontal gene 

transfer is unlikely to occur and thus poses no significant environmental or plant pest risk. 

Conclusion 

APHIS has reviewed and conducted a plant pest risk assessment on Florigene’s IFD524Ø1-4 and IFD-529Ø1-9 roses. Due to the lack of plant pest risk from the inserted 

genetic material, the lack of weediness characteristics of these rose lines, the lack of 

atypical responses to disease or plant pests, the lack of deleterious effects on non-targets 

or beneficial organisms in the agro-ecosystem, and the lack of horizontal gene transfer, 

APHIS concludes that IFD-524Ø1-4 and IFD-529Ø1-9 roses are unlikely to pose a plant 

pest risk.
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