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U.S. Department of Health and Human Services


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Biotechnology Consultation Note to the File BNF No. 000100

Return to inventory: Completed Consultations on Foods from Genetically Engineered Plant Varieties

See also Biotechnology: Genetically Engineered Plants for Food and Feed and about Submissions on Bioengineered New Plant Varieties

Date: December 18, 2008

Subject: Biotechnology Notification File (BNF) BNF 000100; Papaya ringspot virus-resistant papaya line X17-2

Keywords: papaya, Carica papaya L., papaya ringspot virus (PRSV) coat protein, plant-incorporated protectant (PIP), Agrobacterium tumefaciens, neomycin phosphotransferase type II, NPTII, kanamycin resistance gene (kanr)

1. Introduction

In a submission dated September 19, 2007, Dr. Michael J. Davis of the University of Florida's Citrus Research and Education Center (University of Florida) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of the genetically engineered papaya ringspot virus-resistant papaya designated as papaya line X17-2. The University of Florida submitted additional information dated February 15, and September 19, 2008. The University of Florida concluded that papaya line X17-2 is as safe and nutritious as conventional papaya varieties.

2. Intended Effect

The intended effect of the genetic engineering is to confer resistance to the plant pathogen papaya ringspot virus (PRSV), the causative agent of papaya ringspot disease in papaya. To accomplish this, the developer introduced a copy of the PRSV coat protein gene (prsv-cp) from PRSV isolate H1K with a thymidine inserted after the initiation codon to yield a frameshift. The developer also introduced the neomycin phosphotransferase type II (nptII) gene from Escherichia coli which confers resistance to kanamycin and was used as a selectable marker.

3. Regulatory Considerations

The Environmental Protection Agency (EPA) defines a plant-incorporated protectant (PIP) as “a pesticidal substance that is intended to be produced and used in a living plant, or the produce thereof, and the genetic material necessary for the production of such a pesticidal substance,” including “any inert ingredient contained in the plant, or produce thereof” (40 CFR 174.3). EPA regulates PIPs under the Federal Food, Drug, and Cosmetic Act and the Federal Insecticide, Fungicide, and Rodenticide Act. Under EPA regulations, the prsv-cp gene in papaya line X17-2 and resulting expression products are considered pesticidal substances; and the nptII gene and resulting expression products are considered inert ingredients. EPA considers the recombinant DNA construct containing the prsv-cp and nptII genes to be part of the PIP in papaya line X17-2, and therefore EPA is reviewing the recombinant DNA construct and resulting expression products.

4. Genetic Modifications and Characterization

To generate the papaya line X17-2, the developer used an Agrobacterium tumefaciens-mediated embryo transformation method. The developer transformed papaya embryos using the pBI121fs transformation vector, a T-DNA vector containing the prsv-cp and nptII genes. The T-DNA region of the vector which is intended for insertion into the plant genome contains the genetic elements listed in Table 1.

Table 1: Genetic elements contained in the T-DNA region of the plasmid vector pBI121fs
Element Source Description
RB A. tumefaciens T37 (nopaline strain) Right border of the plasmid pTiT37, used to initiate T-DNA transfer from A. tumefaciens to the plant genome.
NOS-pro A. tumefaciens T37 Promoter for transcription of the nptII gene from plasmid pTiT37
nptII(Kanr) E. coli Neomycin phosphotransferase type II (NPTII) enzyme conferring resistance to kanamycin and thus allowing for selection of transformed plant cells
NOS-ter A. tumefaciens A 3' nontranslated region of the nopaline synthase gene from plasmid pTiT37 involved in transcription termination and polyadenylation
CaMV 35S Cauliflower mosaic virus Promoter that directs transcription of the coat protein gene
uidA leader E. coli Enhances expression of PRSV CP in papaya
PRSV-CP Papaya ringspot virus Coat protein gene of PRSV H1K with a thymidine inserted after the initiation codon yielding a frameshift
NOS-ter A. tumefaciens A 3' nontranslated region of the nopaline synthase gene from plasmid pTiT37 involved in transcription termination and polyadenylation
LB A. tumefaciens Left border of the plasmid pTiT37, used to delineate the DNA transferred from A. tumefaciens to the plant genome during transformation.

The developer reports that the insert in line X17-2 has persisted as a single copy through six generations of progeny following the initial transformation and that no vector backbone sequence is present, as determined by Southern blot. The developer also notes that PCR analysis of the 5th generation progeny of the original transformant reveals the elimination of the frame shift in the prsv-cp gene, and that low level expression of PRSV-CP is detectable by Western blot.

5. Food/Feed Use

Papayas are grown primarily for ripe unprocessed fruit, but can be used in the green stage if first boiled. The ripe fruit may also be processed in various ways, including baking, pickling, as a base for sauces, jams, candy, purees, and in juices and nectars. The developer is not aware of any significant feed uses for papaya fruit.

6. Compositional Analysis

Mature fruits from four different specimens of line X17-2 were subjected to compositional analysis. Published information on other transgenic papaya lines (Hawaiian cultivars 55-1 and 63-1) and nontransgenic varieties was used for comparison. A list of all measured components is shown in Table 2.

Table 2: Components measured in ripe fruit
Proximates Minerals Carbohydrates Fats Secondary Metabolites Vitamins and Vitamin Precursors
total dietary fiber
total fat
total sugar saturated fatty acids
monounsaturated fatty acids
polyunsaturated fatty acids
benzyl isothiocyanate1 beta carotene
Vitamin A
Vitamin C

The developer concluded that compositional values of the X17-2 line were not meaningfully different from those given for the other varieties. Vitamin C values were comparable to the literature values of other varieties cited by the developer. Although Vitamin A values were somewhat higher on average, the range overlapped with the value reported in the United States Department of Agriculture's National Nutrient Database. The developer notes that benzyl isothiocyanate (BITC) was assayed in green fruits of seven different X17-2 breeding lines with concentrations ranging from 0.2 milligrams (mg) to 4.8 mg per 100 mg of green fruit. Similar or greater ranges were found with the same assay in three conventional cultivars.

7. Conclusions

The developer has concluded that papaya line X17-2 is not materially different in composition, safety, wholesomeness, or any relevant parameter from papaya now grown, marketed, and consumed. At this time, based on the developer's data and information, the agency considers the developer's consultation on papaya line X17-2 to be complete.


Jeremiah Fasano



1Benzyl isothiocyanate was measured in green not ripe fruit.