Database Product Description

55-1/63-1 (CUH-CP551-8)

Host Organism

Carica papaya (Papaya)

Trait

Resistance to viral infection, papaya ringspot virus (PRSV).

Trait Introduction

Microparticle bombardment of plant cells or tissue

Proposed Use

Production for human consumption.

Product Developer

Cornell University

Summary of Regulatory Approvals

Introduction Expand

Papaya lines 55-1 and 63-1 were developed using recombinant DNA techniques to resist infection by papaya ringspot virus (PRSV), a major limiting factor in papaya production. These papaya lines were developed by inserting virus-derived sequences that encode the PRSV coat protein (CP). The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology. PRSV belongs to the potyvirus group and is an aphid-transmissible RNA virus that commonly infects papaya, causing serious disease and economic loss.

These transgenic papayas exhibit “pathogen-derived resistance” to infection and subsequent disease caused by PRSV through a process that is related to viral cross-protection. Although the exact mechanism by which the viral protection occurs is unknown, most evidence suggests that expression of viral CP by a plant interferes with one of the first steps in viral replication, uncoating (removal of CP) from the incoming virus (Register & Nelson, 1992). Other modes of action of cross protection have also been suggested (Matthews, 1991).

An antibiotic resistance marker gene (neo) encoding the enzyme neomycin phosphotransferase II (NPTII), which inactivates aminoglycoside antibiotics such as kanamycin and neomycin, was also introduced into the genome of these varieties. This gene was derived from a bacterial transposon (Tn5 transposable element from Escherichia coli) and was included as a selectable marker to identify transformed plants during tissue culture regeneration and multiplication. The expression of the neo gene in these plants has no agronomic significance and the safety of the NPTII enzyme as a food additive was evaluated by the United States Food and Drug Administration in 1994 (US FDA, 1994).

Line 55-1 expresses a reporter gene (uidA) encoding the enzyme beta-D-glucuronidase (GUS) from E. coli. The expression of GUS activity was used during the development phase to identify transgenic plants containing the introduced gene sequences.

Summary of Introduced Genetic Elements Expand

Code	Name	Type	Promoter, other	Terminator	Copies	Form
CP	papaya ringspot virus (PRSV)	VR	CaMV 35S	CaMV 35S		Native
nptII	neomycin phosphotransferase II	SM	nopaline synthase (nos) from A. tumefaciens	A. tumefaciens nopaline synthase (nos) 3'-untranslated region		Native
gus	beta-D-glucuronidase	SM	CaMV 35S	A. tumefaciens nopaline synthase (nos) 3'-untranslated region		Native; not present in line 63-1

Characteristics of Carica papaya (Papaya) Expand

Center of Origin	Reproduction	Toxins	Allergenicity
Believed to be native to tropical America, possibly southern Mexico or northwestern South America.	Commercial papaya lines are mostly gynodioecious as the hermaphroditic plants are generally self-pollinating. A low rate of out-crossing still occurs since the plants produce copious pollen during most of the year.	Green tissues contain benzyl isothiocyanate (BITC), which has been linked to incidents of spontaneous abortions in pregnant women and with a higher incidence of prostate cancer in Japanese men over age 70. Ripe fruit has no latex and virtually no BITC.	The latex is either hyperallergenic or an irritant.

Modification Method Expand

The transgenic papaya lines 55-1 and 63-1 were produced by biolistic (particle bombardment) transformation of embryogenic cultures of the papaya cultivar ‘Sunset’ with DNA-coated tungsten particles. The Agrobacterium tumefaciens binary plasmid pGA482GG/cpPRSV-4 used for the transformation contained three plant-expressible genes, the PRSV CP, neo, and uidA genes. The plasmid also had two genes encoding resistance to tetracycline and gentamycin antibiotics, respectively, but their associated DNA regulatory sequences enabled expression only in bacteria. The plasmid included the right- and left-border regions derived from the A. tumefaciens T-DNA.

Expression of the PRSV CP gene was controlled by including promoter and transcription termination and polyadenylation signal sequences derived from the 35S transcript of cauliflower mosaic virus (CaMV). In addition, the CP gene sequences were fused to the 5' untranslated sequence and the first 39 nucleotides from the cucumber mosaic virus (CMV) CP to enhance translation of the transgene mRNA. The inclusion of these additional sequences was necessary because PRSV naturally encodes its CP as part of a polyprotein and, therefore, the CP coding region normally lacks a translation initiation ATG codon.

Expression of the neo gene was under control of the promoter and terminator sequences from the nopaline synthase (nos) gene of A. tumefaciens. The second marker gene, uidA, was modified for plant expression by the addition of 35S promoter region from CaMV and the nos 3'-termination region.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analyses of genomic DNA from line 55-1 verified that it contained the PRSV CP gene, intact copies of two plant-expressible marker genes encoding NPTII and GUS, respectively, and a partial copy of the tetracycline resistance marker gene. Genomic DNA did not hybridize with probes to the gentamycin marker genes or to the origin of bacterial replication (Ori V/Tet) region. The partial tetracycline resistance gene was not expressed in plants, due both to the fact that the gene was incomplete and under the regulatory control of a bacterial promoter.

Southern blot analyses indicated that line 63-1 contained intact, functional genes encoding the PRSV CP and NPTII, and did not contain the GUS encoding gene. Genomic hybridization with probes to the gentamycin resistance gene and the Ori T/Tet region indicated that either all or part of the genes for gentamycin and tetracycline resistance had integrated into the papaya genome. However, these genes were not functional since their bacterial promoters cannot drive expression of these genes in plants.

Environmental Safety Considerations Expand

Field Testing

The transgenic papaya lines 55-1 and 63-1 were field tested in the United States (1991 through 1996). Extensive testing of papaya lines 55-1 and 63-1 in laboratory, greenhouse, and field experiments, determined that the plants exhibited the typical agronomic characteristics of the parent papaya variety ‘Sunset’, with the addition of resistance to PRSV infection. The variation in agronomic characteristics did not differ significantly from that seen in commercial cultivars of papaya. Field trial reports demonstrated that the transformed papaya lines 55-1 and 63-1 had no effect on nontarget organisms or the general environment.

Outcrossing

Papaya (Carica papaya L.) plants have different mating systems and may be either dioecious (staminate and pistillate plants) or gynodioecious (hermaphrodite and pistillate plants). Commercial papaya lines are mostly gynodioecious lines and are preferred because of their potential for inbreeding and consequent uniformity. The hermaphroditic plants are generally self-pollinating due to the position of the anthers relative to the stigma such that in commercial production self-pollination occurs without manual assistance or bagging. A low rate of out-crossing still occurs since hermaphrodites tend to produce copious pollen from staminate flowers during most of the year. Additionally, the pistillate plants never produce anthers and are consequently obligate out-crossers. In nature, pollination of papaya occurs through bees, butterflies, and wind.

Papaya is usually described as sexually incompatible with other member of the Carica genus. Initial steps have been taken to develop methods for somatic hybridization of C. papaya with C. stipulata and with C. pubescens, but no hybrid plants have been regenerated to date.

Papaya is the best known member of the Caricaceae, a small dicotyledonous family consisting of four genera. Carica is the largest genus with 23 described species with overlapping distributions in the foothills of the Andes Mountains in northwestern South America, although members of the genus range from southern Brazil, Argentina and Chile to southern Mexico. Most likely, the center of origin for papaya is the Caribbean coast of Central America. United States commercial production of papaya is generally in Hawaii with secondary production in Puerto Rico and southern Florida.

Weediness

No Carica species is considered a weed, and there is no evidence in the scientific literature to suggest that susceptibility to PRSV is the factor that prevents these plants from being weed pests. Therefore, it seems likely that even if the PRSV-resistance trait could be transferred from line 55-1 or 63-1 to another Carica species, the resultant offspring would not be weed pests.

Secondary and Non-Target Adverse Effects

It was concluded that the genes inserted into the transgenic papaya lines 55-1 and 63-1 would not result in any deleterious effects or significant impacts on nontarget organisms, including threatened and endangered species or beneficial organisms. The PRSV coat protein expressed in these papaya lines is found in all PRSV-infected plants, and there are no reports of this protein having any toxic effects. In fact, this viral coat protein is routinely ingested by virtually all animals, including humans, when papaya is consumed. Naturally occurring infections of susceptible papaya varieties result in concentrations of coat proteins far higher than those that occur in the tissues of the transgenic papaya lines 55-1 and 63-1. No direct pathogenic properties, nor any hypothetical mechanisms for pathogenesis toward beneficial organisms, such as bees and earthworms, were identified for papaya lines 55-1 and 63-1.

Food and/or Feed Safety Considerations Expand

Nutritional Data

Papaya is a tropical fruit crop that is normally consumed fresh and is valued as a rich source of vitamins A and C. In assessing the nutritional composition, samples of fruit from transgenic and non-transgenic control plants were analyzed for vitamins A and C, and total soluble solids (TSS), which is a measure of sugar content. In some cases, the levels of TSS were compared between PRSV-infected fruit and non-infected fruit.

For plants grown in PRSV-infested locations, there was a slightly higher observed level of TSS in transgenic fruit than in PRSV-infected non-transgenic fruit. This difference was not observed when comparisons to non-infected non-transgenic fruit were made.

The vitamin A content of transgenic line 55-1 was comparable to other red-pigmented varieties of papaya and was within the range normally reported for several commercial papaya varieties. Vitamin C content was observed to by slightly lower in transgenic line 55-1 compared to non-transgenic papayas, but was within the range reported for papaya in the literature. Because a number of factors, including ripeness of fruits and growing conditions, can affect vitamin content, this variation was not considered a significant concern. The amount of vitamin C in line 55-1 represents a high level of vitamin C for a food product (49.4-53.6 mg/100g).

Endogenous Toxicants

The presence of benzyl isothiocyanate (BITC) in the latex of green papaya tissues has been linked to incidents of spontaneous abortions in pregnant women and with a higher incidence of prostate cancer in Japanese men over the age of 70. Because ripe papaya fruits have no latex they also have virtually no BITC content, and, therefore, the consumption of ripe fruit would not be expected to be linked to any abortifactant effects. The levels of BITC were measured by gas chromatography in extracts from immature and ripe fruits obtained from transgenic papaya and three varieties of non-transgenic control papayas, and there were no significant differences observed between samples derived from transgenic or non-transgenic plants, respectively. It was noted that the isothiocyanate concentrations in ripe papaya fruits are 10 to 100 times lower than similar values reported for other foods, such as broccoli, Brussels sprouts, and cabbage.

Toxicity and Allergenicity

There is a history of safe consumption of PRSV CP derived from virus-infected papaya fruit, and there is no indication that the form of the CP expressed in transgenic papaya is materially different in any way that would affect its potential for toxic or allergenic effect. Additionally, the PRSV CP does not possess any of the physiochemical properties normally associated with protein allergens or toxins, such as heat stability and resistance to digestion by simulated gastric fluids. Also, there were no regions of homology when the deduced amino acid sequence of PRSV CP was compared to the amino acid sequences of known protein allergens or toxins.

Abstract Collapse

The major producers of papaya are Brazil, Mexico, Nigeria, India, Indonesia, Ethiopia and the Democratic Republic of Congo. Papaya is grown primarily for the fresh market and processed food industry, and also has industrial applications.

Papaya is a tropical fruit crop that is normally consumed fresh and is valued as a health food due to the high content of vitamins C and A. The pulp is eaten fresh, made into juice, preserves, and desserts. Immature pulp is used as a vegetable and in some regions young leaves are consumed like spinach. Papaya is used in traditional medicine with seed used as a vermifuge and leaves boiled for treatment against malaria, as examples. When the fruit is green and hard it is rich in white latex. As it ripens, it becomes light- or deep-yellow externally and the thick wall of succulent flesh becomes aromatic, yellow, orange or various shades of salmon or red. The dried latex, or milk, of immature fruit yields papain, a proteolytic enzyme similar in action to pepsin, which is used as a meat tenderizer, in breweries (to clarify beer), and in pharmacy. Papain is also used in bathing hides, degumming silk and softening wool.

Hawaii, partly because of its distance from other papaya-growing areas, is less afflicted with disease problems than Florida and Puerto Rico, but still has to combat a number of major and minor maladies. Most serious of all is the papaya ringspot potyvirus (PRSV), which is common in Florida, Cuba, Puerto Rico, Trinidad, and first seen in Hawaii in 1959. It is transmitted mechanically or by the green peach aphid, Myzus persicae, and other aphids including the green citrus aphid, Aphis spiraecola, in Puerto Rico. Papaya plants infected by PRSV lose their photosynthetic capacity and as a result display stunted growth, deformed and inedible fruit, and eventually, plant mortality. When plants are infected at the early stages they will not produce mature fruit. If they are infected at a later stage, fruit production is reduced and of poor quality because of ringspots on the fruit and a decrease in sugar concentration. PRSV is very difficult to control and once a plant has become infected there is no remedy, but measures to avoid spread include the destruction of affected plants, control of aphids by pesticides, and elimination of all members of the Cucurbitaceae (alternate hosts for the virus) from the vicinity.

Papaya lines 55-1 and 63-1 were developed using recombinant DNA techniques to resist infection by PRSV by inserting virus-derived sequences that encode the PRSV coat protein (CP). The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology.

These transgenic papayas exhibit “pathogen-derived resistance” to infection and subsequent disease caused by PRSV through a process that is related to viral cross-protection. Although the exact mechanism by which the viral protection occurs is unknown, most evidence suggests that expression of viral CP by a plant interferes with one of the first steps in viral replication, uncoating (removal of CP) from the incoming virus (Register & Nelson, 1992). Other modes of action of cross-protection have also been suggested (Matthews, 1991).

Papaya lines 55-1 and 63-1 were tested in field trials in the United States (1991–1996). These tests demonstrated that 55-1 and 63-1 plants exhibited the typical agronomic characteristics of conventional papaya varieties, with the addition of resistance to PRSV infection. Papaya lines 55-1 and 63-1 were comparable to conventional papaya varieties and did not exhibit weedy characteristics, and had no effect on nontarget organisms or the general environment.

Papaya plants have different mating systems and may be either dioecious (male and female flowers are on separate plants) or gynodioecious (plants with perfect flowers/ hermaphrodite and plants with only female flowers). Commercial papaya lines are mostly gynodioecious as the hermaphroditic plants are generally self-pollinating. A low rate of out-crossing still occurs since the plants produce copious pollen during most of the year. The degree of cross-pollination among papaya varieties is dependent on the mating system and type of commercial production. In nature, pollination of papaya occurs through bees, butterflies, and wind.

Papaya is usually described as sexually incompatible with other members of the Carica genus of which there are 23 described species. Initial steps have been taken to develop methods for somatic hybridization of C. papaya with C. stipulata and with C. pubescens, but no hybrid plants have been regenerated to date. The exact origin of papaya is unknown but thought to be native to tropical America, possibly southern Mexico or northwestern South America. Distribution now extends throughout tropical America, South-East Asia, India, Oceania, and Africa. Commercial production of papaya in the United States is primarily in Hawaii with secondary production in Puerto Rico and southern Florida.

The food and livestock feed safety of papaya lines 55-1 and 63-1 was established based on several standard criteria. As part of the safety assessment, the nutritional composition of papaya fruit was found to be equivalent to conventional varieties by the analyses of vitamins A and C, and total soluble solids, which is a measure of sugar content. The latex of green papaya tissues contains a toxin called benzyl isothiocyanate (BITC) that has been linked to incidents of spontaneous abortions in pregnant women and with a higher incidence of prostate cancer in Japanese men over the age of 70. Because ripe papaya fruits have no latex they also have virtually no BITC content. The levels of BITC in transgenic papaya and conventional papaya varieties were measured and found to be much lower in ripe fruit compared to immature fruit. There were no differences between transgenic papaya and conventional papaya from either immature or ripe fruit.

The PRSV coat protein does not possess characteristics typical of known protein allergens or toxins such as heat stability and resistance to digestion by simulated gastric fluids. Comparisons of the deduced amino acid sequence of the plant-expressed PRSV CP did not reveal any homology to known protein allergens and toxins. Furthermore, PRSV-infected papaya fruit naturally contains higher levels of the viral CP than expressed in these transgenic papayas, and there has been no evidence of adverse effects linked to the consumption of virus-infected fruit.

Links to Further Information Expand

• Japan Mnistry of Health, Labor and Welfare: Biological Diversity Risk Assessment Report Papaya resistant to papaya ringspot virus - PRSV (Modified PRSV CP, uidA, nptII, Carica papaya L.) (55-1, OECD UI: CUH-CP551-8)
  [PDF Size: 387.00K bytes]

International Committee on Taxonomy of Viruses (ICTV) Universal Virus Database

• 57.0.1.0.045 Papaya ringspot virus
  [PDF Size: 135.25K bytes]

Office of Food Biotechnology, Health Canada

• NOVEL FOOD INFORMATION– FOOD BIOTECHNOLOGY HUMAN FOOD USE OF VIRUS RESISTANT PAPAYA LINE 55-1
  [PDF Size: 131.97K bytes]

US Food and Drug Administration

• Memorandum to file concerning virus-resistant papaya line 55-1.
  [PDF Size: 376.38K bytes]

USDA-APHIS Botechnology Regulatory Service

• Petition for determination of non-regulated status for transgenic papaya lines 55-1 and 63-1 and their derivatives.
  [PDF Size: 673.81K bytes]

USDA-APHIS Environmental Assessment

• USDA/APHIS Petition 96-051-01P for the Determination of Nonregulated Status for Transgenic Sunset' Papaya Lines 55-1 and 63-1
  [PDF Size: 48.68K bytes]

This record was last modified on Tuesday, September 15, 2015