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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.
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.
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 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.
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.
| 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 |
| MUA10 | Cosmid sequence |
| β-lactamase | A gene for resistance to ampicillin contained in pBR322 |
| cos1 | 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 |
| 1The cos site interrupts the β-lactamase gene, rendering the β-lactamase gene non-functional | |
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.
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.
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 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.
| 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 (B1) riboflavin (B2) niacin (B3) |
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
