Biotechnology Consultation Note to the File BNF No. 000101

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CFSAN/Office of Food Additive Safety
January 7, 2009
Biotechnology Consultation
Note to the File
BNF No. 000101
Date: January 7, 2009
Subject: Biotechnology Notification File (BNF) BNF 000101; Plum pox virus-resistant plum
line C5
Keywords: plum, Prunus domestica, plum pox virus, Sharka disease, Potyviridae, Potyvirus,
plant-incorporated protectant (PIP), Agrobacterium tumefaciens, neomycin phosphotransferase
type II, NPTII, β-glucuronidase, GUS
1. Introduction
In a submission dated January 12, 2007, Dr. Ralph Scorza of the United States Department of
Agriculture's Agricultural Research Service (USDA-ARS) submitted to the Food and Drug
Administration (FDA) a safety and nutritional assessment of the genetically engineered plum
pox virus-resistant plum, designated as plum line ARS-PLMC5-6 (C5). USDA-ARS submitted
additional information dated April 5, June 3, and June 12, 2007, and September 19, 2008. The
developer concluded that the C5 plum is as safe and nutritious as conventional plum varieties.
2. Intended Effect
The intended effect of the genetic engineering is to confer resistance to the plant pathogen plum
pox virus (PPV), the causative agent of Sharka disease in multiple Prunus species, including
plum. To accomplish this, the developer introduced the plum pox virus coat protein gene (ppv-
cp) as well as the neomycin phosphotransferase type II gene (nptII) from Tn5 and the β-
glucuronidase gene (uidA) from Escherichia coli. The nptII and uidA genes and resulting
expression products were used as selectable markers.
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
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regulates PIPs under the Federal Food, Drug, and Cosmetic Act and the Federal Insecticide,
Fungicide, and Rodenticide Act. Under EPA regulations, the ppv-cp gene in plum line C5 and
resulting expression products are considered pesticidal substances; and the nptII and uidA genes
and resulting expression products are considered inert ingredients. EPA considers the
recombinant DNA construct containing the ppv-cp, nptII, and uidA genes to be part of the PIP
in plum line C5, and therefore EPA is reviewing the recombinant DNA construct and resulting
expression products.
4. Genetic Modifications and Characterization
To generate the plum line C5, the developer used an Agrobacterium tumefaciens-mediated
embryo transformation method. The developer transformed embryos derived from the Prunus
domestica (L.) plum cultivar ‘Bluebyrd' using the pGA482GG/PPV-CP-33 transformation
vector. 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
pGA482GG/PPV-CP-33
Element
Description
RB
A restriction fragment from the pTiT37 plasmid containing the 24 bp
nopaline-type T-DNA right border used to initiate the T-DNA transfer from
Agrobacterium tumefaciens to the plant genome
nos 5'
Nopaline synthase promoter from A. tumefaciens
nptII
Neomycin phosphotransferase type II gene from Tn5
nos 3'
A 3' non-translated region of the nopaline synthase gene which functions to
terminate transcription and direct polyadenylation of the nptII mRNAs
nos 3'
A 3' non-translated region of the nopaline synthase gene which functions to
terminate transcription and direct polyadenylation of the ppv-cp mRNAs
ppv-cp
Insert containing the leader sequence of the coat protein mRNA of tobacco
mosaic virus and an ATG start codon fused in phase with the plum pox virus
coat protein gene from PPV-D strain
CaMV (35 S)
The cauliflower mosaic virus (CaMV) promoter to drive expression of the
ppv-cp gene
CaMV (35 S)
The CaMV promoter to drive expression of the GUS gene
uidA (GUS)
β-glucuronidase gene from Escherichia coli
nos 3'
A 3' non-translated region of the nopaline synthase gene which functions to
terminate transcription and direct polyadenylation of the GUS mRNAs
MUA10
Cosmid sequence
Origin of
replication
pBR322 sequence responsible for initiating plasmid replication
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The plasmid backbone of pGA482GG/PPV-CP-33 contains tetracycline and gentamycin
antibiotic resistance genes which are not intended for insertion into the plant genome. DNA
sequencing and PCR analyses of C5 did not reveal any evidence of genetic material from the
plasmid backbone.
Analysis of C5 suggests that the transgenic line contains multiple insertions including the nptII,
ppv-cp, and uidA genes and parts thereof.
The developer states that the stability of the locus has been repeatedly assessed by restriction
analysis over a period of fifteen years. These assessments indicate that the transformation event
is stable in both vegetatively propagated C5 trees and in C5 progeny carrying the event.
5. Food/Feed Use
Plums (primarily P. domestica) are consumed as human food in a variety of forms including
fresh, dried, canned, as juice, as concentrate, and frozen. Plums are also used as a production
source for alcoholic beverages. Dried plums may be sold intact or pitted. Fresh and dried plums
are used in baked goods, sauces and marinades, snack foods, and energy bars. Plums are not
ordinarily used for animal feed.
6. Compositional Analysis
Fruits from plum line C5 and five nontransgenic comparator lines were produced under similar
growing conditions at the USDA-ARS Appalachian Fruit Research Station in Kearneysville,
WV and subjected to compositional analysis. The comparator lines included ‘Bluebyrd,' a
relatively new cultivar released by USDA-ARS that is the female parent of C5, as well as other
major plum cultivars (‘Early Italian,' ‘Green Gage,' ‘Reine Claude,' and ‘Stanley'). Several
factors introduced additional variation into the compositional analysis. Although all C5 trees
were grown on their own roots, other cultivars were grown on one of several rootstocks
including peach (Prunus persica), myrobalan plum (Prunus cerasifera), and European plum (P.
domestica). Rootstock can affect fruit composition. In addition, the determination of fruit
ripeness is relatively subjective. The developer attempted to collect fruit at the same stage of
ripeness based on fruit size, color and firmness, but the maturity of each fruit is another source
MUA10
Cosmid sequence
β-lactamase
A gene for resistance to ampicillin contained in pBR322
cos
1
A bacteriophage lambda cos site that leads efficient packaging of lambda
DNA into virus particles
β-lactamase
A gene for resistance to ampicillin contained in pBR322
MUA10
Cosmid sequence
LB
Restriction fragment from the octopine Ti plasmid, pTi15955, containing the
24 bp T-DNA left border used to terminate the transfer of the T-DNA from A.
tumefaciens to the plant genome
1
The cos site interrupts the β-lactamase gene, rendering the β-lactamase gene non-functional
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of variation. Finally, the developer notes that abiotic factors such as soil quality and sunlight are
capable of affecting fruit composition and can vary significantly even between trees in the same
plot.
For all compositional analyses except antioxidant capacity and phenolic content, approximately
500 grams of accumulated frozen, pitted fruit of each variety harvested from a single tree were
analyzed as a single sample per variety for each analyte. For antioxidant activity and phenolic
content, two to five subsamples from the sample collection described above were analyzed. The
developer also provided literature values for analytes when the units presented in the literature
were compatible with those used in the analyses. The developer notes that in some cases the
method of analysis was different or was not specified, and in other cases the species of plum
was not defined. A list of all measured components is shown in Table 2. The developer
concluded that there were no substantial nutrient differences between C5 plums and
conventional counterparts.
7. Conclusions
USDA-ARS has concluded that plum line C5 is not materially different in composition, safety,
wholesomeness, or any relevant parameter from plums now grown, marketed, and consumed.
At this time, based on USDA-ARS's data and information, the agency considers USDA-ARS's
consultation on plum line C5 to be complete.

Jeremiah Fasano


Biotechnology
| Products: Completed Consultations

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FDA/Center for Food Safety & Applied Nutrition
Hypertext updated by jmf
February 5, 2009
Table 2: Components measured
Proximates
Minerals
Carbohydrates
Secondary Metabolites
Vitamins
ash
fat
moisture
total dietary fiber
carbohydrates
protein
calcium
magnesium
sodium
potassium
iron
total sugar
glucose
sucrose
lactose
maltose
fructose
starch
phenolics
antioxidant capacity
thiamine (B
1
)
riboflavin (B
2
)
niacin (B
3
)
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