FOCUS ON: GENETIC ENGINEERING
TRANSGENETIC PAPAYA
Engineering a Virus Resistant Plant
Introduction
Papaya varieties grown today in Hawaii have bright names, like Rainbow, Sunrise, Kapoho Solo, Kamiya and Laie Gold, calling to mind warm, happy days on tropical islands, eating fruits bursting with flavor and sweetness. But these sunny names mask the desperation felt by Hawaiian papaya farmers as they watched their crops and livelihoods wither and die through the early 1990s as a terrible virus ravaged their crops. How were the farms able to rebound? Continue on to learn about how a transgenic organism saved the papaya farms of Hawaii.
How can plant breeders protect papaya against viruses?

About the Organism
The Papaya Plant
Carica papaya looks like a tree, but in fact it's just a 20-foot tall shrub. The main stem is green or purple and, unlike a tree's trunk, it is hollow. At the top of the plant are stems with large(1– 3.5 ft.) lobed leaves.

Flowers, which contain the plant's reproductive organs, develop near the leaves. Papaya flowers have five white petals. After pollen is transferred onto the stigma in the flower's center and travels down the style to the ovary where the ova are fertilized, a fruit begins to grow. Papaya plants can be male, female or hermaphroditic, depending upon which type of flower they have. Papaya growers in Hawaii tend to prefer hermaphroditic papaya and remove trees with only male or only female flowers.

What is papaya's scientific name?
Plant Reproduction
The female reproductive part is the pistil, with the sticky stigma opening into the tubular style that connects to the ovary. The male reproductive structure is the stamen, comprised of the thin filament on top of which sits the anther, which contains lots of pollen.
Male, Female, and Hermaphroditic Plants
Male flowers contain only male reproductive parts and won't grow fruits. Female flowers must be fertilized with pollen from a different tree in order for a fruit to develop. Hermaphroditic flowers, also called perfect flowers, contain both male and female parts. Those flowers can self-pollinate or receive pollen from different papaya trees.

The papaya plant (A), flower (1), developing fruit (2), mature fruit (3) and seed (4,5)
The papaya grown for grocery stores usually range from 1-5 pounds in weight. The skin is thin and waxy, green when it's still ripening and yellow when it's ripe. The flesh surrounds a middle cavity containing many black seeds. The flesh is red, orange or yellow, depending upon the variety.
Think and Apply
Plants rely on photosynthesis for energy. Photosynthesis, the process by which sunlight converts carbon dioxide and water into glucose, which the plant uses for energy, happens primarily in leaf cells. If the leaf cells become damaged in some way that results in decreased amounts of photosynthesis, what impact does that have on the rest of a fruit-producing plant?
Answer:
Interrupting photosynthesis can be devastating for fruit production. If the amount of glucose available to the plant decreases, the plant cannot put as much energy into producing seeds, fruits or other parts. Crop productivity decreases as photosynthesis decreases.
Sum of papaya production from 1994 – 2014.
Native to Central America, papaya trees have migrated - with human help - worldwide. In the 1600s, papaya seeds left Central America on trading ships bound for India, Malaysia and the Philippines. Papaya plants were first introduced to Hawaii in the early 1800s. Now papayas are grown in nearly 60 countries worldwide in tropical and subtropical regions of Asia, Africa, North America and South America. In the US, Hawaii is a major producer of papaya; papayas are also grown in California, Texas, Florida, Puerto Rico and the US Virgin Islands.
How Nutritious?

Papayas are a healthy food that contains lots of vitamins and fiber.
Papayas are delectable and a healthy food. Fat free and sodium free, these delicious fruits are low in calories and high in Vitamins A and C. And that 12% of your daily dietary fiber isn't a shabby number either. A serving of papaya also contains 360 mg, or 10% of the daily value, of potassium. All that goodness in just half a papaya!
Picture of organism

But wait! There's more! The nutrition label doesn't tell the whole story.

Papayas contain beta-carotene & lycopene. Both lycopene and beta-carotene are carotenoids, fat-soluble pigments found in many different organisms. Lycopene gives papayas a red hue, while beta-carotene gives the orange color. Both are thought to act as antioxidants, or substances that prevent cell damage by removing free radicals (oxidants) in our bodies. Beta-carotene is also a precursor for Vitamin A.

Papayas are also rich in an enzyme called papain. Remember that an enzyme is a protein that acts as a catalyst in chemical reactions. Some people use papain to tenderize meat; it breaks down some of the proteins. In our bodies, papain can aid digestion and it's been used to treat ulcers. Interestingly, papain is also helpful on the outside of the body: studies have demonstrated that it has anti-microbial properties and can be used to reduce certain scar tissues after surgery.
Vitamins A & C
Vitamin A is a fat-soluble vitamin that plays an important role in vision, bone growth, reproduction, cell division, and cell differentiation. It helps regulate the immune system and may help fight infections. Vitamin A promotes healthy surface linings of the eyes and the respiratory, urinary, and intestinal tracts, which helps prevent infection. Vitamin C is a water soluble vitamin that acts as an antioxidant in our bodies.
Plant Dietary Fibers
Dietary fiber is the parts of plants that your body cannot digest. Although it passes undigested and intact out of your body, it helps maintain bowel health and healthy cholesterol and blood sugar levels.
Potassium
Potassium is a mineral that acts as an electrolyte, helping our bodies maintain the right amount of fluid and promoting proper muscle and nerve function.
Think and Apply
US nutritional guidelines recommend that female teenagers eat 25 grams of fiber per day and male teenagers eat 31 grams of fiber per day. The chart below lists the dietary fiber found in different fruits and vegetables. Evaluate papaya as a source of dietary fiber compared to other fruits and vegetables.
Answer:
A serving of papaya provides 3 g of dietary fiber. This is similar to many fruits and vegetables, like dates, guava, orange and winter squash. Other fruits and vegetables, such as apples, blackberries, raspberries, pears, baked potatoes (with skin), collards and even mixed vegetables, provide a little bit more dietary fiber per serving. Many beans provide much more dietary fiber; small white beans, for example, provide 9.3 grams of fiber per serving.
The Challenge:
Stop the Ringspot Virus!
Papaya trees grow beautifully in the rich volcanic soil of the Hawaiian Islands. However, a deadly virus was creeping across the Hawaiian Islands, threatening to destroy the papaya industry. The virus causes, a slow death—as one farmer described it—taking out one infected plant after another until few remain. As the virus started spreading more rapidly, it was clear that it was a significant threat to papaya farms in Hawaii, as well as elsewhere in the world.
Healthy papaya, symptoms of ringspot virus (a) affected papaya tree and (b) ringspot on papaya fruit
Papaya Ringspot Virus, sometimes called PRSV, had been in Hawaii since the 1940s, but a new, more severe strain was described in the 1950s. Papaya Ringspot Virus is devastating to the papaya plants. Symptoms of infection include an upper trunk with oily streaks, leaves with a mosaic pattern and a lighter yellow color due to loss of chlorophyll, as well as fruits that have 'ringspots'. If young plants develop the disease, they don't make fruit.

The virus is spread by aphids, a small insect. The virus replicates in the plant and causes the devastating symptoms, then aphids acquire the virus as they feed on infected papayas. The aphids then transfer virus to uninfected plants as they feed on new plants. In a papaya plantation, where thousands of papaya plants grow in close proximity, aphids can quickly spread the virus as they travel from plant to plant.

A papaya field with early symptoms of PRSV (left) and a grove with all trees infected with PRSV (right)
Farmers unsuccessfully tried many strategies to stop the virus. Some farmers tried managing the virus' vector– the aphids – by spraying, but aphids are plentiful, move quickly, and are difficult to manage. Scientists would have liked to have used plant breeding via artificial selection to develop a strain of disease-resistant plants, but none of the papaya varieties grown commercially showed any natural resistance to the virus. Scientists even tried infecting plants with a milder form of the virus (known as cross-protection), but that technique didn't prevent the disease. Cutting down PRSV-infected trees kept the virus somewhat contained in some areas, but government officials, farmers and scientists knew it was a matter of time before the virus did significant damage.
The papaya ringspot virus decimated Hawaiian farms in the mid 1990s.
In 1992, the virus situation grew serious, quickly. PRSV was detected on the Big Island of Hawaii, near the Puna district where most of the papayas were grown. During the first week of May that year, a months-old infection was discovered in a grove 1-3 miles from major papaya plantations. The virus was also found in abandoned orchards and in young orchards nearby. The Hawaii Department of Agriculture cut down all the infected plants and even went so far as to suggest to growers that they cut down all the papaya plants in the area—infected or not. The growers did not approve that idea, but growers continued to try to contain the infection. They did not succeed. As growers experienced high infection rates, they abandoned their orchards rather than destroying them and the aphids continued to spread the virus. By late 1994, less than 5 years from the virus's onset, nearly all the area's papaya was infected.

The agricultural support system had tried everything to stop the spread of Papaya Ringspot Virus—pest control, eliminating infected plants, finding resistant varieties. Nothing worked. Was the virus the end of Hawaiian papaya farming?
Think and Apply
The Puna area is Hawaii's most important papaya-growing region. In 1992, when PRSV was first detected in Puna, how much of Hawaii's papaya did the area produce? By 1997, what had happened to papaya production in Puna and Hawaii as a whole?
Answer:
In 1992, Puna produced 95% of Hawaii's 55,800 million pounds of papaya. By 1997, Puna's production shrank dramatically to about half (52%) of 1992 levels.The region was producing less (78%) of Hawai's papaya and overall Hawaiian production had shrunk to 64% of 1992 levels. Even though the infection blanketed the area, farmers were still able to deliver some papaya to market.
The Solution
In the 1980s, while papaya ringspot virus was beginning to wreak havoc on Hawaiian papaya plantations, scientists from the University of Hawaii, the USDA, and Cornell University collaborated to try creating a virus-resistant papaya. Scientists were entertaining wild ideas. Could they somehow"vaccinate"plants against a virus? Vaccination works in animals, but plants don't have immune systems, so the mechanism would have to be different. What if the plant contained and produced one of the elements of the virus? Would the virus still be able to infect it?

The scientists decided to try to create a transgenic papaya. A transgenic organism – also sometimes called a genetically engineered (GE) or genetically modified (GM) organism - is an organism whose genome has been altered to include genetic material from another species. In the case of papaya, it would contain part of the virus, which hopefully would act as a"vaccine."Creating a transgenic organism is no simple process. Scientists knew the process would take three main steps: understanding the virus, creating a virus-resistant transgenic papaya, and then gaining approval for farmers to grow it in the field.
> Step 1: Understanding the Virus

The papaya ringspot virus (PRSV), like all viruses, is made of RNA surrounded by a protein coat or capsid. Once PRSV attaches to a papaya cell, the virus releases its RNA into the host cell where the RNA encodes one large protein. That protein is then broken into smaller proteins, all of which have different functions. Inside the plant cell, the virus interrupts the plant's metabolic processes as it uses the plants resources to copy more viral RNA and build more virus particles.

Researchers focused on the virus' outermost covering, the protein coat. That protein coat determines how the virus enters cells, how the genetic material is released into the cell, and how new virus particles are made. Could scientists use the virus protein coat in the papaya to interfere with the virus' function?

PRSV is a long, thin filamentous virus made of RNA surrounded by a protein coat.
> Step 2: Creating a virus-resistant transgenic papaya

Inspired by virus resistance in tobacco and tomatoes after a virus protein coat was transferred into the genomes of those plants, plant researchers were curious whether a similar solution would work for papayas. The researchers thought that if they could introduce the virus' protein coat gene into the papaya genome, the papaya would express the protein coat gene, which would act somewhat like a vaccine and make the papaya resistant to ringspot virus.
How did scientists create a virus-resistant papaya variety?
Scientists successfully created two virus resistant varieties of papaya. SunUp has reddish-flesh fruit and has 2 copies of the coat protein (cp) gene. Rainbow is a hybrid of SunUp and a popular non-transgenic variety. It has a yellow-flesh fruit, and only one copy of the coat protein (cp) gene. In what turned out to be serendipitous timing, PRSV resistant transgenic papaya was being field tested just as the Puna region papaya plantations were being decimated by PRSV in the early 1990s. Despite the positive results in the early field trials of resistant papaya, some farmers initially were pessimistic about the potential of the transgenic fruit. This is understandable, of course, given that their livelihoods were at stake.

> Step 3: Getting Approval to Grow

"What we had done through the field trial would only be academic unless the papaya got through
the red zone of translational biotechnology… getting the papaya deregulated and commercialized."
~ Dennis Gonsalves

Although the experiments indicated that the transgenic papaya were resistant to PRSV, the plants could not be used commercially until they were approved by regulatory agencies. The virus protein coat gene inserted into the papaya genome is considered a pesticide because it acts against the virus—a papaya pest. So the Environmental Protection Agency (EPA) and US Department of Agriculture (USDA) had to approve the transgenic organism's environmental and agricultural safety. The Food and Drug Administration (FDA) considers food safety; it also granted approval and deemed the transgenic papaya safe for consumption since PRSV breaks down quickly in the human stomach. All three agencies provided approval. After getting the licenses to commercialize the transgenic papaya, in 1998 the seeds for the transgenic papayas were distributed free to growers.

Once the regulatory hurdles were cleared, farmers in Puna quickly accepted and started growing the transgenic papaya. They were delighted to have virus resistant plants that produced good yields. As of 2014, transgenic papaya made up 85% of the Hawaiian papaya crop. US consumers bought the transgenic papaya; Hawaiian transgenic papayas are also sold in Canada, Hong Kong and Japan.
Eating Infected Papayas
For many years, people have eaten papayas infected with the virus, with no ill effects. The coat protein (cp) gene inserted into the transgenic papaya is from the virus that has been safely eaten.
Researchers have studied the of allergenicity of transgenic papaya and found no allergy issues.
Think and Apply
What happened to papaya production in Hawaii and the Puna region after transgenic seeds were released in 1998?
Answer:
In 2001, the Puna region was recovering and producing 55% more papaya than its low point in 1999. However, the area had not fully recovered and only was producing 76% of the 1992 peak amount.
Continued Monitoring
The success – both in resisting infection and in the marketplace – of the transgenic papaya varieties does not mean that scientists' work is over. Resistance to any pathogen, whether papaya virus or influenza, requires continued monitoring. A virus-resistant plant creates a strong selection pressure on the virus population. How long will it be until the virus evolves resistance to the virus-resistant plant? Additional studies suggested that plants resistant to Hawaiian PRSV were less resistant to PRSV in other places. Therefore, introduction of different strains of the virus from other locations could threaten the fragile new balance. Furthermore, seedlings of the Rainbow variety, which contains only one copy of the transgene, has a window of vulnerability to Hawaiian PRSV, even though they were resistant later in their lives. Scientists continue to monitor the relationships between resistant plants and their viral pathogens.

It's important to note that although the transgenic papaya have been approved and successfully sold, non-transgenic papayas are also still grown in Hawaii. In 2015 transgenic papaya totaled approximately 85% of the papaya crop in Hawaii. The remaining 15% was non-transgenic papaya. Researchers at the University of Hawaii have evidence the non-transgenetic papaya benefit from nearby plantations growing PRSV resistant papaya because the transgenic papayas provide a buffer to disease spread by the aphids.

Genetically engineered crops do face opposition, however, and not all Hawaiian farmers and consumers accept transgenic papaya. Some people are very concerned about keeping the coat protein gene that was inserted into the transgenic papaya out of non-transgenetic papaya. Although papaya plants usually self-pollinate, they can also cross-pollinate. So DNA from the transgenic papaya could potentially mix with non-transgenic papaya planted nearby. Scientists monitor this potential movement of genes.


Think and Apply
Read the quote below from Dr. Dennis Gonsalves. What do you think accounts for the rate of gene flow the researchers found?

"We recently completed a very thorough gene flow study under commercial conditions where the GE field was adjacent to non-GE field. In non-GE papaya fields separated from GE field by a 12-foot road, the rate of gene flow to the non-GE papaya row that bordered the road was about one percent. Further in the field we did not detect any gene flow."

*Recall that "GE", or genetically engineered, is another way to describe transgenic crops.
Answer:
The very low rate was probably due to the fact that commercial papaya in Hawaii are hermaphroditic (flowers have male and female parts). The flowers usually self-pollinate, therefore there isn͛t much movement of genes, via cross-pollination, from transgenic to non-transgenic plants.

Conclusion
To create a transgenic papaya, scientists built on ideas pioneered with tomato and tobacco. They used their knowledge of papaya ring spot virus to engineer a papaya plant that produces a PRSV coat protein. They tested to see if this "long-shot" approach worked, and it did. The transgenic papaya varieties underwent field tests and were evaluated for safety by the FDA, USDA and EPA regulatory agencies. Eighty-five percent of farmers in Hawaii are now growing transgenic papaya. Scientists continue to monitor resistance of the plants to the virus.

PRSV-resistant papaya has been called the "poor man's transgenic." While most transgenic crops are developed by the private sector businesses, the papaya was the first public-sector developed transgenic crop in the US. The team of researchers set out to develop a solution to help Hawaiian papaya farmers survive after all other methods of controlling the spread of the virus had failed. They succeeded. As a result, papaya plantations in Hawaii are growing strong today.
Commercial papaya trees are grown in large groves
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