by Brian Dick, PhD

Pathogen-derived Resistance (PDR)

In the mid-1980s, plant geneticists developed a new approach to controlling plant viruses known as parasite- or pathogen-derived resistance (PDR). The strategy was to develop transgenic plants that incorporated gene products from the pathogen into the host plant, which would then confer resistance. Plant geneticists John Sanford at Cornell and Stephen Johnston at Duke proposed the idea in a 1985 article in the Journal of Theoretical Biology. They noted that “there are certain parasite-encoded cellular functions which are essential to the parasite but not to the host. These functions represent the ‘Achilles’ heel’ of the parasite. If one of these functions is disrupted, the parasitic process should be stopped.” This line of reasoning was validated in 1986 when it was reported that tobacco expressing the coat protein (CP) gene of the tobacco mosaic virus (TMV) could confer resistance to the virus, which would then be passed on to subsequent generations. In 1994, a transgenic squash was developed on the basis of PDR, followed four years later by the transgenic papaya.

How to do Plant Biotechnology

Transgenesis is a method for enhancing genetic variation. When foreign genes are incorporated into a plant, it is said to have undergone transformation. Transformation is accomplished via several different vectors or transport systems. The most widely employed is Agrobacterium tumefaciens, a soil-dwelling pathogen that infects plant cells, resulting in the formation of tumorous growths. It naturally incorporates foreign DNA into plant genomes and can be modified to deliver specific genes of interest. Other techniques include vector-free chemical and electrical systems, microinjection, and microprojectile bombardment using a ‘gene gun.’ Researchers aim to confer resistance to herbicides, viruses, insects, and other biotic and abiotic stresses, or to enhance or modulate color, ripening pathways, pollen production, or nutritional value.

John Sanford's Gene Gun

The gene gun was invented by John Sanford and collaborators at Cornell between 1983 and 1987. The ingenious device transforms plant cells through brute force. DNA is coated onto gold or tungsten particles, propelled at high speed into plant material, and incorporated into plant genomes. Transformed cells can then be selected using antibiotic resistance markers. The gene gun was an important innovation because Agrobacterium tumefacians cannot be used as a vector for all plants. The use of the gun to transform plants has come to be known as ‘biolistics.’

Puna District Papaya Production

LSF Magazine: Summer 2013

The Fate of a Fruit

Papaya is one of Hawaii’s leading agricultural products, for both export and consumption. In the mid-1950s, a papaya ringspot virus (PRSV) epidemic broke out on the island of Oahu and caused massive crop losses. Orchards were relocated to the Big Island, but in 1992 another outbreak forced growers to cut down thousands of infected trees. Three years later, the virus had nearly wiped out the state’s papaya industry. A recovery began in 1998 with the introduction of transgenic, virus-resistant papaya varieties called SunUp and Rainbow. Growers were rescued; an economic disaster was averted. The transgenic papaya was widely hailed by scientists, farmers, food suppliers, and civic leaders as a biotech success story. Environmental activists and organic growers protested the deregulation of the (genetically modified) GM plant, but did not manage to intervene in the policymaking process.

In papaya-growing regions in other parts of the world, however, environmental groups mounted vigorous campaigns to prevent transfers of the technology. In order to sway public opinion and influence the actions of farmers and governments, they portrayed SunUp and Rainbow as commercial failures and environmental calamities. The story of the transgenic papaya illustrates the power of molecular biology to reshape agriculture, and simultaneously to generate controversy.


 The Big Island’s Puna District

A PAPAYA PREDICAMENT

When the papaya ringspot virus (PRSV) appeared in the Puna district of Hawaii’s Big Island in 1992, there was no treatment or cure. The Hawaiian Department of Agriculture cut down thousands of infected trees, but failed to stem the spread. Ken Kamiya, a papaya farmer with property in the Waikane Valley north of Puna, commented on growers’ bleak prospects: “When you see disease coming, you salvage what you can and go out of business, or you cut down your trees and go out of business.”

Six years later, in the face of the papaya industry’s imminent collapse, a small group of public-sector scientists introduced two transgenic varieties of virus-resistant fruit, the yellow-fleshed Rainbow and red-fleshed SunUp. Farmers enthusiastically embraced the new cultivars. Expressing a sentiment that was shared broadly among Hawaiian agriculturists, Kamiya called genetic engineering “our hope for the future.”

Human beings have been manipulating plant genomes for thousands of years through the breeding, selection, and cultivation of crops. Recombinant DNA (rDNA) technology is the latest refinement, a means of transferring genes and phenotypic characteristics with high precision. Rather than selecting from genetic recombinations produced by nature or conventional breeding methods, or from random mutations induced by chemicals or radiation, rDNA technology enables scientists to direct the propagation of specific genes and traits.

The cultivation and consumption of genetically modified (GM) crops is widespread. In 2012, 17.3 million farmers in twenty-eight countries cultivated GM crops on 425 million acres. In the United States, an estimated 60 to 70 percent of processed foods contain GM ingredients, primarily in the form of high-fructose corn syrup derived from GM corn.

Still, GM crops remain controversial. General understandings of agricultural biotechnologies are impoverished and often confused. From a scientific point of view, the ratio of fact to fabrication in public discourse and media reporting is disturbingly low.

PAPAYA CULTIVATION AND PRSV

The papaya (Carica papaya) is an herbaceous tropical fruit tree that originated in Southern Mexico and Central America. It was imported from Barbados to Hawaii in 1911. Papaya trees grow rapidly, producing mature fruit in about eleven months. Fruit is picked for two years; productivity then declines and trees become too tall for efficient harvesting.

Papaya is an important crop in Hawaii. Sales of the vitamin-rich fruit are second only to the pineapple, and an enzyme derived from the plant, papain, has many industrial and biomedical applications. Three varieties were grown in Hawaii before the outbreak of the papaya ringspot virus: the yellow fleshed ‘Kapoho,’ the dominant cultivar accounting for 90 percent of production; the red-fleshed ‘Sunrise;’ and the pink-fleshed ‘Sunset’ hybrid.

PRSV posed a dire and sustained threat to this economic resource throughout the latter half of the twentieth century. The ringspot virus is a member of the genus Potyvirus, and is transmitted by various species of aphids. The name refers to symptomatic rings that appear on the leaves. The virus interferes with photosynthesis, and results in stunted growth, loss of vigor, and fruit unfit for market. The papaya has no natural resistance to PRSV.

The virus was first discovered in Hawaii by D. D. Jensen in 1945, on the island of Oahu. By the mid-1950s it had caused severe damage, and the transfer of orchards to Puna began. By the 1970s, over 90 percent of the state’s papaya crop was grown on the Big Island. Respite from the virus was temporary. The 1992 epidemic in Puna devastated the industry—Hawaiian fruit yields declined from 53 million pounds in 1992 to 27 million pounds five years later.

CREATING THE TRANSGENIC PAPAYA

Development of the transgenic papaya was led by plant pathologist Dennis Gonsalves, a native Hawaiian. Gonsalves earned his PhD in plant pathology at the University of California, Davis in 1972. He then accepted a faculty position at the University of Florida where he worked on citrus fruit viruses. In 1977, he moved to Cornell. That same year, he took a trip to Honolulu and met with Richard Hamilton, a papaya breeder and horticulturist.

In 1978, Hamilton and Gonsalves went to see William Furtick, Dean of the Department of Agriculture at the University of Hawaii (UH), to discuss Gonsalves’s citrus virus work. The Dean mentioned that PRSV had appeared in Hilo, and surmised that it was only a matter of time before it reached Puna. Gonsalves was interested in the subject, and when Furtick returned with a modest sum of money to fund PRSV research, he decided to pursue it.

Between October 1982 and April 1983, researchers in Gonsalves’s lab explored the possibility of ‘cross protection,’ a method similar to inoculation, in which plants are purposefully infected with a mild viral strain to confer resistance to a severe one. Shyi-Dong Yeh, a graduate student who had been sent to Cornell by the Taiwanese government to investigate PRSV, created mild strains of the virus (HA5-1 and HA6-1) by subjecting the severe strain (HA) to nitrous acid mutagenesis.

It turned out that cross protection was not economical in Taiwan. It was more cost-effective in Hawaii, but wasn’t adopted due to the adverse effects of the attenuated virus, the requirement of sustained cultural management, and the hesitancy of farmers to infect their orchards.

In the mid-1980s, Gonsalves was contacted by the Upjohn Company, which was conducting research on vegetable viruses. He met one of the company’s molecular biologists, Jerry Slightom, who was skilled at DNA sequencing and had established the company’s plant antiviral program. Gonsalves and Slightom decided to collaborate on cloning and sequencing PRSV.

Over the next few years, their efforts were aided by two important developments: the discovery of pathogen-derived resistance (PDR) and the invention of the ‘gene gun.’ PDR demonstrated that disease resistance could be conferred to plants by incorporating a partial gene sequence from the pathogen (such as the protein coat of a virus), while the invention of the ‘gene gun’ furnished a method for transporting this genetic sequence into a plant genome.

In 1986, UH horticulturalist Richard Manshardt and his graduate student, Maureen Fitch, joined the team. Gonsalves had been traveling back and forth between Cornell and the University of Hawaii when he met Manshardt and found that they shared similar interests. The pair sought funds from the US Department of Agriculture’s Tropical and Subtropical Agricultural Research (TSTAR) Program. Fitch, a technician with the US Department of Agriculture (USDA), was recruited to carry out the tissue culture work.

They established a division of labor in which Gonsalves and Slightom cloned and sequenced the PRSV HA 5-1, Fitch transformed and regenerated the papayas, and Manshardt bred them in the field.

In 1987, Fitch began transforming the Sunrise, Sunset, and Kapoho varieties with the gene gun. She bombarded embryogenic plant tissues with tungsten particles coated with the viral gene. “It was crazy,” Fitch recalls. “We used a .22 caliber blank to propel the DNA. Cultures were flying all over the place. Stuff would fall in the gun. We’d scoop it up and stick it back in the plate. There were holes in some of the plates—it was wild.”

Inoculation tests showed that the first fruit line developed by microprojectile bombardment, line 55-1, was resistant to the virus. Experiments were conducted on 55-1 using PRSV isolates from Hawaii and other countries. Resistance was limited to the Hawaiian strains, but conscious of the practical import of the work, Gonsalves was eager to move on to field trials: “I said, ‘Forget this stuff, man. Let’s clone this and go into the field.’”

Field tests required permission from the Animal and Plant Health Inspection Service (APHIS) of the USDA. The federal agency had previously approved field trials of the transgenic squash in 1990. Using the squash application as a template, Gonsalves and Manshardt wrote up a petition. Approval was granted in April 1992, and Manshardt commenced the field trials in June at the University of Hawaii’s experimental farm at Waimanalo on Oahu.

The results were dramatic. While 95 percent of the non-transgenic plants showed PRSV symptoms after seventy-seven days, no symptoms were observed in the line 55-1 plants. (A few exceptions were explained by the timing of the introduction of the virus.) This was the first experimental evidence demonstrating that coat protein-mediated protection (CPMP) could be extended from annuals, such as squash, to trees, such as papayas.

The red-fleshed SunUp transgenic cultivar was derived from plants bred for this initial trial. Since consumers favored yellow-fleshed papayas, Manshardt also produced the “Rainbow” by crossing the SunUp with the yellow-fleshed Kapoho.

The field trial results were timely, because the PRSV epidemic had by then reached Puna. The virus appeared in early May. When the removal of plants was ineffective and farmers rejected a proposed one-year moratorium on papaya cultivation, the urgency of the research intensified. Although cross-pollination of transgenic and non-transgenic plants was a concern, Gonsalves weighed the arguments for a pivotal large-scale field test that would determine the fate of the fruit:

1) Line 55-1 had performed well in field trials on Oahu.
2) The risk of PRSV-resistant papaya becoming a weed was virtually non-existent because wild relatives of C. papaya are not grown in Hawaii.
3) Drastic action was needed—it might not be possible to eradicate PRSV in Puna.

Gonsalves concluded that potential benefits far outweighed potential risks. He received a permit from APHIS, and a large-scale trial in Puna began in October 1995 under the supervision of plant and environmental protection scientist Steve Ferreira of the University of Hawaii. Using a papaya farm in the Kapoho section of Puna, Ferreira compared the rates of PRSV infection of transgenic varieties with conventional papayas. While nearly all of the non-transgenic plants became infected, the transgenic papayas remained unscathed, generating the yields and fruit quality required for commercialization.

DEREGULATION AND FARMER ADOPTION

Introducing the seeds to US markets required deregulation—the waiver of special government oversight. Three agencies had to give assent. APHIS investigated potential environmental harm; the Environmental Protection Agency (EPA) considered whether the papaya ought to be classified as a pesticide (due to the virus coat protein); and the Food and Drug Administration (FDA) evaluated whether the fruit was safe for consumption.

The respective permits were issued in September 1996, August 1997, and September 1997. There was no sign of public opposition. The APHIS application, for example, was made available for public review for sixty days. The agency received eighteen comments. The Federal Register noted that “all were favorable to the petition.”

Intellectual property rights to elements of the technology were held by Monsanto, Asgrow Seed Company, Cambria Biosystems, and the Massachusetts Institute of Technology. Michael Goldman, a lawyer hired by the industry’s trade association, the Papaya Administrative Committee (PAC), successfully negotiated use licenses with the companies in April 1998.

The licenses included a number of limitations-of-use and compliance provisions: planting was restricted to Hawaii and the fruit could be exported only to countries that accepted GMOs, growers had to attend training workshops and sign compliance agreements, and the PAC was to be the sole organization permitted to distribute seeds. In May of 1998, seeds were supplied to farmers free of charge through a lottery system.

In the face of the devastation wrought by PRSV, growers were desperate to obtain and apply the new technology. The New York Times reported that some had broken into Department of Agriculture experimental stations to acquire seeds prior to the scheduled release.

After the distribution, Gonsalves’s wife Carol conducted adoption and opinion surveys with farmers (for a master’s thesis project at the University of Hawaii). She found that farmers rapidly adopted the transgenic papaya. Seventy-six percent planted transgenic seeds. Farmers cited lower production risks, chances for higher profits, and opportunities to experiment with transgenic fruit as reasons for adopting, but 96 percent identified the virus threat as the principal rationale.

Reasons for not adopting the transgenic papaya were generally economic—concerns, for example, over market demand since the transgenic fruit could not be exported to important markets in Japan and Canada. These countries, and others, did not accept modified food products. Interestingly, Carol Gonsalves found that “none of the farmers were personally against the use of genetically modified plants.”

THE PAPAYA PARADOX

The popularity of the transgenic fruit among Hawaiian growers contrasts starkly with responses elsewhere. As news of Gonsalves’s work spread internationally, developing countries began sending researchers to Cornell to develop papaya varieties suitable for use in other geographic regions. However, despite the efforts of Gonsalves and international collaborators to promote the technology, the transgenic papaya was not adopted in developing countries facing similar threats from PRSV. China was the sole exception.

Virus-resistant papaya have been produced for cultivation in Brazil, the Philippines, Thailand, Venezuela, and Jamaica. Researchers assumed that the transgenic fruit would be readily embraced, but growers in these countries have rejected it largely due to misinformation concerning environmental, economic, and public health consequences spread by anti-GMO activists.

In 2004, Greenpeace protesters clad in full protective gear scaled a barbed wire fence and entered an experimental orchard in Thailand. They uprooted transgenic papaya trees and discarded them in biohazard waste bins. Media coverage of the event fueled public fears. The activists told farmers that the transgenic product had been disastrous for Hawaiian growers. Patwajee Srisuwan, one of many Greenpeace members arrested for trespassing and destruction of property, justified the group’s action: “A single papaya could give thousands of seeds and the spread [through cross-pollination] would become exponential, totally out of control. It’s like a time-bomb waiting to explode.”

Soon after the incident in Thailand, activist groups in Hawaii led by the Hawaii Organic Farmers Association began protesting the deregulation of the fruit. Melani Bondera, an organic farmer and head of the Hawaii Genetic Engineering Action Network (HIGEAN), complained in 2004 that transgenic papayas were contaminating organic papayas through cross-pollination, making them ineligible for export to countries that ban GMOs. A group of organic farmers on the Big Island financed a study purportedly showing major contamination of organic crops (although crop scientists found serious faults in its methodology).

Representations of the transgenic papaya make for a study of contrasts—of hope, on the one hand, and catastrophe on the other. In a 2008 article in Plant Physiology entitled, “Forbidden Fruit: The Transgenic Papaya in Thailand,” plant biologist Sarah Nell Davidson outlined the reasons behind the rejection of virus- resistant plants:

There is a lack of farmer engagement in the debate, and to the extent that networking with farmers does occur, it is often dominated by anti-GE [genetic engineering] nongovernmental organization (NGO) networks and less by government or university extension agents. Many developing countries still lack biosafety laws and too often countries lack sufficient infrastructure and training to carry out the regulatory testing needed prior to commercialization. Fear of biopiracy by foreign entities is directly tied to concerns over intellectual property because most of the intellectual property has been developed and previously implemented in wealthier nations. Finally, many countries’ markets are dependent on the political and consumer demands of importing countries.

In the introduction of transgenic crops, economic and political considerations intermingle with scientific assessments of health and safety. Over the past twenty-five years, a consensus has emerged in the scientific community (and among governmental regulatory agencies in the United States) that transgenic crops pose no hazards distinct from crops modified by conventional means. This does not mean that there are no risks, or that GM crops can be considered 100 percent safe, but it undermines arguments for special regulation and restriction.

In both traditional crop breeding and transgenesis there is the possibility of unwanted mutations increasing allergenicity or toxicity. Many non-GM crops cause severe allergic reactions, such as peanuts, soybeans, wheat, and tree nuts. Other non-GM crops naturally produce toxins that can be accidentally enhanced through traditional breeding practices. Potatoes, for example, contain the glycoalkaloid solanine, which can be toxic at high levels. Consumers therefore, are advised to avoid eating uncooked or greening potatoes, and conventional breeders must carefully select new varieties.

In contrast to conventionally bred crops, transgenic crops are subject to tests designed to identify changes that may inadvertently lead to allergies or higher toxicity levels. For example, when researchers in the early 1990s attempted to engineer a high-protein soy bean by inserting a gene from the Brazil nut, testing indicated that the nut allergy was present in the new soybeans. The project was halted and the soybeans were never marketed.

Such testing is not conducted on conventionally bred crops, and adverse effects have occasionally resulted. For example, when the Kiwi fruit was introduced in the United States in the 1960s, it was found that the fruit caused allergic reactions, some of which were fatal. Nevertheless, activists continue to perpetuate unfounded myths about the special dangers of GMOs with support from organic farmers, New Age healers, and promoters of nutritional supplements and alternative medicines.

Another objection frequently raised by anti-GMO activists is the possibility of transgenes moving to non-GM plants through pollen flow. This is a legitimate concern for farmers who wish to grow only organic foods. More research is required, but the problem of gene flow—whether from conventional or GM crops—must be addressed through the cultural management of agricultural practices, and, at least in the case of the Hawaiian transgenic papaya, the best available evidence indicates that GM and organic papayas can coexist.

In a 2007 article published in the Annual Review of Phytopathology, Gonsalves and plant pathologist Marc Fuchs addressed a number of concerns about the transgenic papaya raised by activists, including the issue of transgene flow. They pointed to the success of the identity preservation protocol (IPP) adopted by the Hawaiian Department of Agriculture, which ensured that papayas exported to Japan were GM free. (Japan lifted its ban in 2011.) A preliminary study of neighboring transgenic and nontransgenic papaya orchards showed that “transgene flow is minimal in nontransgenic papaya orchards growing in close proximity to transgenic papaya under commercial conditions in Puna. Coexistence is routinely and successfully practiced in Hawaii.”

UNRAVELING THE GMO CONTROVERSY

Perceptions of new technologies—as threats or opportunities—depend on how the technologies are framed by different audiences. In assessing the risks of GMOs, US regulators addressed health, safety, and, to a lesser extent, environmental impacts. Socioeconomic debates on corporate control were not on the agenda. In the aftermath of the anti-GMO movement that swelled in Europe during the late 1990s, the rhetoric circulating in global activist networks cited the profit motive in corporate capitalism as a sound basis for assuming the subversion of science, the distortion of risk assessments, and the likelihood of environmental contamination and invisible health hazards.

Protests against the cultivation of GM crops intensified after Monsanto introduced herbicide-resistant soybeans into Europe in 1996. Consumers viewed Monsanto’s refusal to segregate or label the product as arrogant. Environmental groups such as Greenpeace and Friends of the Earth initiated sustained campaigns against ‘Frankenfoods.’ Although opposition to GM products was based on hypothetical risks, activists effectively persuaded large segments of populations around the world that progress in agricultural biotechnology served the interests of large multinational corporations at the expense of human health, consumer welfare, and environmental safety.

Critics of the commercialization of science insist that the general public no longer views scientists as disinterested stewards of truth. They maintain that faith in scientific authorities and regulatory agencies has eroded because technical experts and inspectors negotiate standards with industrial concerns and trade associations. Even academic institutions that accept industry funding are subject to growing suspicion.

Social scientist Les Levidow has noted:

In both environmental and food safety controversies, public-sector research institutes have had a complex role, partly due to their dependence on industry funding. Whenever their risk-assessment research has revealed unexpected results, NGOs have called for greater precaution or bans. Whenever institute staff have downplayed uncertainties or emphasized the manageability of risk, however, NGOs have suggested commercial pressures were involved.

The creators of the transgenic papaya encountered similar resistance. Gonsalves recalls attending a conference sponsored by an organic farming association: “It was a hostile environment. Everybody was against us. Many people said, ‘We don’t trust you. You’re in Monsanto’s back pocket.’”

The entanglement of scientific and socioeconomic concerns in the GMO controversy has politicized scientific disagreements. Studies indicating GMO-associated risks to human health and the environment—e.g., damaging effects of GM potatoes on rats, harm to Monarch butterflies caused by Bt corn pollen, and the cross-pollination of GM maize with local varieties in Mexico—have been subjected to intense scientific scrutiny. All have been discredited, but perhaps too late.

Prior to consensus formation in the scientific community, uncertainties and differences of opinion among experts on appropriate degrees of risk acceptance provided anti-GMO activists with opportunities to promote unfounded claims, which were then amplified in the media. Subsequent scientific investigations that failed to confirm negative health or environmental effects were dismissed as industry propaganda.

The anti-GMO movement equates transgenic crops with the corporate plunder of nature and the pursuit of profit at the expense of public interests, even when, as in the Hawaiian case, biotechnologies are developed through public and private cooperation, and are dedicated to the protection and enhancement of specialty crops. The transgenic papaya, Gonsalves points out, “helps farmers that don’t have much money. A lot of the big companies are not going to pick up projects like this because there’s not enough money in it. If you give it away, it has to come from the public sector.”

The development of the transgenic papaya is an instructive case. It had immediate benefits both for farmers, who could survive to grow another day, and consumers, who would have otherwise paid much higher prices for diminished supplies of inferior fruit. But the value of the project was not universally appreciated. Despite the scientific evidence mustered to support it, the fruit was widely perceived as a risk and a threat.

The full promise of agricultural biotechnologies will not be realized without greater public trust in scientific expertise and regulatory processes. Some have suggested that the situation could be ameliorated through the revival of public breeding programs and seed banks. In an era of austerity, however, this may prove unfeasible. Can public trust in private, for-profit science be restored? The answer may determine the fates of agricultural biotechnologies in coming decades.

 

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