• CORRECTION: This story was updated on April 9, 2013, to correct the location of the photo of Neal Carter of Okanagan Specialty Fruits. He is pictured in Washington state, not British Columbia.
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Volume 91 Issue 14 | pp. 31-33
Issue Date: April 8, 2013

Engineered Apples Near Approval

Fruit with nonbrowning genes may get green light in U.S.
Department: Government & Policy
News Channels: Environmental SCENE
Keywords: genetic engineering, apples, organic, GMO
Okanagan hopes that by inhibiting enzymatic browning, it can cut down on food waste.
Credit: Okanagan Specialty Fruits

Any parent will tell you, it’s hard to get a kid to eat an apple that’s turned brown. So one Canadian firm is using genetic engineering to turn the browning reaction off in Granny Smith and Golden Delicious apples, and the resulting fruit is close to clearing the last regulatory hurdles before it can be sold in U.S. grocery stores.

Okanagan Specialty Fruits of British Columbia is using well-known laboratory methods to insert genes designed to prevent the chemical reaction responsible for browning into their Arctic apples. The modified apples are moving through a complex and time-consuming regulatory process, which must be completed before the apples can be sold alongside conventionally bred varieties.

The genetically engineered (GE) traits most commonly inserted into commercial crops are tolerance to herbicides such as Monsanto’s glyphosate weed killer, Roundup, and pest resistance, often via internal production of pesticides. These traits, harvested from bacteria, are controversial. And a tenacious community of opponents of GE crops, who prefer the phrase “genetically modified organism” or GMO, has emerged. Nonetheless, in the U.S., 88% of corn, 93% of soybeans, and 94% of cotton is genetically engineered, according to 2012 data from the U.S. Department of Agriculture.

No evidence that Arctic apples are unsafe has come to light nor has any reasonable mechanism by which they could be. Okanagan triggers a selective gene-silencing pathway and inserts a selection gene that is broadly recognized as harmless to humans. Even skeptical experts usually don’t assert that any of the GE foods currently on the market are dangerous.

The real concern is that existing regulations wouldn’t be able to catch a truly dangerous product, or one that could contaminate other plants through cross-pollination.

Three federal agencies have some form of oversight on GE crops. USDA’s Animal & Plant Health Inspection Service (APHIS) evaluates whether GE crops are significantly weaker against plant pests and could therefore endanger other nearby crops. The Food & Drug Administration looks at the GE crops to see whether they could create or spread a food-borne illness among people. And the Environmental Protection Agency analyzes any plant engineered to produce its own pesticides; Arctic apples don’t, so EPA isn’t involved in their approval.

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ORCHARD
Carter inspects genetically engineered apple trees in a test plot in Washington state.
Credit: Okanagan Specialty Fruits
Photo of Neal Carter reaching in to pick an apple.
 
ORCHARD
Carter inspects genetically engineered apple trees in a test plot in Washington state.
Credit: Okanagan Specialty Fruits

Some GE crop producers say the regulatory process is too burdensome. Critics, on the other hand, say it lacks scientific rigor and adequate enforcement authority. “We have a very broken regulatory process,” says Michael K. Hansen, a senior scientist at Consumers Union, the advocacy arm of Consumer Reports.

The main browning reaction in apples starts when a dicopper enzyme known as polyphenol oxidase, or PPO, oxidizes certain phenols to quinones. Subsequent reactions polymerize the quinones to form melanins and other dark-colored polyphenols. In intact apples, PPO is kept away from its phenolic substrates, but damage to the cells from cutting or bruising brings them together, which results in browning at the site of the wound.

PPO’s substrate phenols are antioxidants, so keeping them from converting to quinones actually improves the nutritional profile of the apple, Okanagan notes.

The evolutionary advantage offered by PPO to plants is under debate. One theory holds that it protects the plant from insects by reducing the available nutrition at the damaged surface, another that insects get trapped in the brown melanin polymer. But studies of plants with naturally or artificially low levels of PPO do not show consistent correlations with changes to pest resistance, so the advantage offered to a plant by the enzyme remains unclear.

Pest resistance, however, is an important parameter because APHIS’s authority over biotech crops is currently derived from the Plant Protection Act (PPA), a law passed in 2000 that consolidates regulation related to plant pests and noxious weeds. A plant pest is defined as any living thing that can directly or indirectly injure, cause damage to, or cause disease in any plant or plant product.

“APHIS’s regulatory determinations are based on the best available science,” an APHIS spokesman says. “If an organism does not meet the definition of a plant pest as defined in PPA, APHIS has no authority to regulate that organism.”

Petitioners such as Okanagan, who want to have a GE food approved for market, submit materials to APHIS that they hope show their engineered crop is not a plant pest risk. The goal is to have their crop earn a “deregulated” status. This status allows the GE crop to be sold as if the inserted traits were achieved through conventional breeding. Okanagan began the APHIS process for its two apple varieties in May of 2010.

The Arctic Granny Smith showed an increased incidence of tentiform leafminer, a leaf-eating bug found in the Northwest. But in all other cases the two Arctic varieties performed the same as or better than their conventional counterparts against the 13 pests or diseases studied in Okanagan’s field trials. On the basis of those results, the apples are likely to qualify for deregulation by APHIS, observers note.

The trials, however, were done under standard orchard conditions, says Doug Gurian-Sherman of the Union of Concerned Scientists. He raises the concern that those conditions might mask any increased vulnerability because they include heavy use of fungicides and pesticides.

To gather the input of laypeople as well as experts such as Gurian-Sherman, the regulatory process includes public comment periods. APHIS combines information from trials with feedback from the public to make its decision.

The first 60-day public comment period on the deregulation of Arctic apples ended on Sept. 11, 2012. Nearly 2,000 comments, mostly opposed, were submitted by the closing day. Many of these comments expressed a philosophical opposition to GE crops but had sparse scientific objections specific to Arctic apples.

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VECTOR
Agrobacterium tumefaciens injects this plasmid, modified by Okanagan scientists, into infected cells. Only the genes between the border sequences of this plasmid are incorporated into the apple genome. The rest of the plasmid contains genes used by the bacterium (yellow).
Credit: Adapted from APHIS
Scheme shows plasmid modified by Okanagan scientists to help create nonbrowning apples.
 
VECTOR
Agrobacterium tumefaciens injects this plasmid, modified by Okanagan scientists, into infected cells. Only the genes between the border sequences of this plasmid are incorporated into the apple genome. The rest of the plasmid contains genes used by the bacterium (yellow).
Credit: Adapted from APHIS

“One of the big challenges is to educate people about what we’re doing and how we’re doing it, and how it’s really not that scary,” says Neal Carter, founder of Okanagan. “I feel it’s time for opponents to stop referring to biotechnology as a science experiment.”

A large portion of the opposing comments were copies of fill-in-the-blanks-style form letters. A common feature of public comment periods, such campaigns are not an effective way to sway this process. Federal law requires APHIS to consider every comment submitted, but “it is the substance of comments rather than the quantity that impacts our decisions,” the APHIS spokesman says.

During a second APHIS public comment period, expected to start this month, Okanagan’s supporting documents will be available again, alongside draft assessments by APHIS on the plant pest risk and environmental impact of Arctic apples. No GE crop has ever been denied deregulation, but companies have pulled a handful of products midway through the process rather than risk the black mark of an APHIS rejection.

One key opponent of Arctic apples is the U.S. Apple Association, an industry trade group. In its comment to APHIS, the group argues that the commercial benefit of a nonbrowning apple isn’t worth the “costs to the industry in the form of labeling and marketing efforts that would be required to differentiate conventional apples from the GE apples.” U.S. Apple was unable to provide an estimate for those costs.

Okanagan’s Carter, a member of U.S. Apple, doesn’t agree: “We don’t see any other trait that would be better” for the first GE apple. “It has something for everybody. It reduces losses for the grower and packer, reduces shrinkage for the retailer,” and the consumer ends up wasting less of what they buy. Carter also says that relative to the 466,000 acres devoted to apple production in the U.S. and Canada in 2011, Arctic apples “will be an insignificant component of the apple industry for many years to come,” with only a few hundred acres planted over the next five years.

Another concern about Arctic apples is that GE pollen from apple trees may contaminate nearby conventional or organic fields. Although possible, this scenario is unlikely, Carter argues. Analysis summarized in Okanagan’s documents suggests that because the flowering trees provide a glut of available pollen, a professional beehive under normal conditions has no need to wander far for food. But, to address this concern, Okanagan’s APHIS materials mention measures such as buffer rows that their model indicates bring the risk of inadvertent cross-pollination nearly to zero.

U.S. Apple is skeptical that cross-pollination can be mitigated, saying that “in the growing practices common in the Pacific Northwest, there is reasonable concern about genetic flow.” This could, in theory, affect the ability of nearby orchards to export to the European Union and their ability to earn organic certification. Many GE crops are banned in the EU, and its policies have a strong effect on the global export market.

Also, GE crops are not considered organic, so cross-pollination from such crops could affect the ability of contaminated organic crops to be certified as such. But, according to spokespeople from USDA and from the European Commission, inadvertent pollination of an organic tree by GE pollen would not make fruit of the organic tree ineligible for organic certification in the U.S. or the EU or interfere with export to the EU.

New biotech crops, such as Arctic apples, are also reviewed by FDA in a voluntary process. Although it isn’t required, “I wouldn’t expect anybody to try to commer­cialize any of these crops without going through FDA review,” says Gurian-Sherman.

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SLICED
Arctic apples (right) have been genetically engineered to inhibit browning.
Credit: Okanagan Specialty Fruits
Photos show, on the left, a conventional granny smith apple with slices cut out; the slices have significant browning. On the right, a genetically engineered apple sliced the same way shows little to no browning.
 
SLICED
Arctic apples (right) have been genetically engineered to inhibit browning.
Credit: Okanagan Specialty Fruits

The review generally starts with the GE crop producer submitting data that it thinks show the new crop is safe for human consumption. After evaluating these documents and conducting a public comment period, FDA regulators correspond with the crop producer about any questions that remain and ask for any additional data needed to assess the safety of the GE crop. If and when the regulators are satisfied, they send a letter to the GE crop producer stating that they have no more questions and post the same “completed consultation” letter on the FDA website.

Gurian-Sherman stresses that a completed consultation is not a safety approval by FDA. In the final paragraph of each one is a reminder that it is the producer’s “continuing responsibility to ensure that foods marketed by the firm are safe, wholesome, and in compliance with all applicable legal and regulatory requirements.”

The trouble, according to critics, is that the FDA process is optional and has no regulatory teeth. If GE crop producers don’t feel like supplying additional data, they can just decline to do so. If a GE crop fails to earn a completed consultation—which has never happened—that doesn’t prevent the GE crop from being marketed and sold. Gurian-Sherman says that the letter “gives a fig leaf to the companies that something has been done; that’s really all it is.”

An FDA spokesperson says that if a GE crop were found to be unsafe, before or after release to the market, “FDA would have available all the enforcement tools it has for nongenetically engineered foods,” including ordering a recall of the crop from grocery store shelves. Critics counter that such mandatory recall authority would be cold comfort if an unsafe food made it into the food supply.

Okanagan started FDA’s process in June of 2011. After a few rounds of correspondence, Carter says, FDA told them in October 2012 that it has no more questions, though Okanagan is still waiting for the completed consultation letter. “We were hoping for before Christmas; it would have been a very nice present,” Carter says.

Barring any surprises from FDA or USDA, orchards in the U.S. will likely be able to purchase young Arctic apple trees from Okanagan by the end of the year. The fruit, which Okanagan plans to require growers to label as an Arctic variety, could start appearing on grocery store shelves a few years after that. If everything goes well, more GE projects are likely to follow.

The fight over regulation will continue as well. A provision in the Consolidated & Further Continuing Appropriations Act of 2013, signed into law on March 26 by President Barack Obama, weakens judicial oversight of APHIS’s deregulation determinations—including the one Okanagan is seeking. Though the provision will expire at the end of September along with the rest of the bill, GE opponents are incensed and fear a dangerous precedent has been set.

“It is highly likely that the biotech industry influenced the introduction and passage of this rider,” Gurian-Sherman wrote in a blog post about the provision. “Once a country throws open its doors to the biotech industry, it can expect a similar effort to weaken regulations for food safety and environmental protection.”

SILENCE
Without polyphenol oxidase (PPO), chlorogenic acid, and other phenols cannot be converted into melanin precursors.

Genetic Engineering: Making A Nonbrowning Apple

A key part of the process that turns an apple brown is polyphenol oxidase (PPO) expression. To silence it, Okanagan Specialty Fruits uses a cellular mechanism called RNA interference. RNAi is one of the ways that eukaryotic cells regulate development and resist the expression of viral DNA, explains John Armstrong, research manager at Okanagan. “The cell naturally uses this pathway for turning on and off genes.”

In the 1990s, geneticists working on petunias inserted an additional copy of a gene involved in pigment production into the flower in hopes of getting deeper colors. Instead, the resulting flowers were mottled or white. That fluke led to the discovery of co­suppression, the RNAi-based technique used at Okanagan.

In cosuppression, additional copies of the genes targeted for silencing are inserted into the host genome. After these transgene segments are transcribed by the cell into single-stranded RNA, they are recognized as “aberrant” and converted to a double-stranded form with a stem-loop feature, Armstrong says. That stem-loop, a common feature of viral DNA, triggers the RNAi pathway and becomes a target for a “dicer” enzyme, which chops the aberrant double-stranded RNA into 20–25 base-pair fragments called small interfering RNAs (siRNAs).

One strand of each siRNA is taken up by an RNA-induced silencing complex (RISC) and used as a targeting tool. The siRNA-wielding RISCs destroy any RNA strands they encounter that contain a matching sequence. Because the genes inserted in Okanagan’s process are 300–1,800 base pairs long and are near-exact matches for the native PPO genes, each insertion produces a host of RISCs dead set on preventing PPO expression.

Four genes encode for PPO in the apple genome, so four insertions were required to turn off PPO expression entirely. The counterintuitive result of inserting an additional copy of each PPO gene is an apple tree that expresses no PPO whatsoever.

Okanagan accomplishes this feat through a relatively standard set of laboratory tools and the bacterium Agrobacterium tumefaciens. The microbe infects plants’ cells and injects a short circular DNA segment called a plasmid, a portion of which inserts itself into the genome of the host. In wild type A. tumefaciens, the expression of genes on the plasmid causes tumor formation.

Genetic engineers replace the harmful plasmid with a custom-designed one. The insertion segment includes the four PPO genes as well as a gene imparting tolerance to the antibiotic kanamycin.

At Okanagan, tiny cuttings from a standard apple tree are infected with the modified A. tumefaciens. The cuttings are then exposed to the antibiotic, which would normally kill them. But those that successfully incorporate the transgene—with its PPO-silencing cargo—survive.

NptII, which is the name of both the gene and product protein that impart kanamycin tolerance, is a common tool and earned “generally regarded as safe” status from the Food & Drug Administration in 1993, meaning it can be used in food without specific approval from the agency.

Out of 2,000 cuttings, only around 10 are transformed, and one of those transformations results in satisfactory PPO silencing, says Neal Carter, Okanagan’s president. All in all, he says, it takes around 10 years from the time a plant strain is brought into the program to get it onto a grocery store shelf.

 
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