Paula Tennant

Papaya Ringspot Virus Resistance of Transgenic Rainbow and SunUp is Affected by Gene Dosage, Plant Development, and Coat Protein Homology

R1 plants of the transgenic papaya line 55-1, which expresses a single coat protein (CP) gene of the mild strain of the papaya ringspot virus (PRSV) HA from Hawaii, were previously shown to be resistant only to PRSV isolates from Hawaii. Two transgenic papaya cultivars were subsequently derived from line 55-1. UH SunUp (SunUp) is homozygous for the CP gene insertion and UH Rainbow (Rainbow) is hemizygous for the CP gene because it is a F1 hybrid of a cross between SunUp and the nontransgenic papaya cultivar Kapoho. To determine the various parameters that affect the resistance of SunUp and Rainbow, plants at different developmental stages (younger and older) were inoculated with PRSV isolates from Hawaii, Brazil, Jamaica, and Thailand. Hawaiian isolates shared nucleotide sequence identities of 96.7–99.8% to the CP transgene, and the other isolates shared sequence identities of 89.5–92.5%. Resistance was affected by CP gene dosage, plant developmental stage, and CP sequence identity of the challenge isolate. Young and older hemizygous Rainbow plants were resistant to the homologous PRSV HA (99.8% homology to CP transgene), while only older Rainbow plants were resistant to the other Hawaiian isolates (96.7% homology). However, all inoculated Rainbow plants were susceptible to PRSV isolates collected from Jamaica, Brazil, and Thailand. In contrast, SunUp was resistant to all PRSV isolates, except the one from Thailand, regardless of the plant developmental stage. Resistance to the Thailand isolate, which shares 89.5% homology to the transgene, was observed only with SunUp plants inoculated at an older stage. Steady state RNA analysis and nuclear run-on experiments suggested that resistance of the transgenic papaya is RNA-mediated via post-transcriptional gene silencing.

A protocol for efficient transformation and regeneration of Carica papaya L.

SummaryA reproducible and effective biolistic method for transforming papaya (Carica papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors in this protocol included: 1) spreading of young somatic embryo tissue that arose directly from excised immature zygotic embryos, followed by another spreading of the actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of kanamycin selection from all subsequent steps after kanamycin-resistant clusters were first isolated from induction media containing kanamycin; 3) transfer of embryos with finger-like extensions to maturation medium; and 4) transferring explants from germination to the root development medium only after the explants had elongating root initials, had at least two green true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 transgenic papaya lines expressing the nontranslatable coat protein gene of papaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. This also represents an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated this procedure in our laboratory by visiting researchers who did four independent projects to transform seven papaya cultivars with coat protein gene constructs of PRSV strains from four different countries. The method is described in detail and should be useful for the routine transformation and regeneration of papaya.

Phylogeography and molecular epidemiology of Papaya ringspot virus

Papaya ringspot virus (PRSV) is the most important virus affecting papaya and cucurbit plants in tropical and subtropical areas. PRSV isolates are divided into biotypes P and W: both the P and W types naturally infect plants in the family Cucurbitaceae, whereas the P type naturally infects papaya (Carica papaya). Understanding the origin and nature of the PRSV genetic diversity and evolution is critical for the implementation of control strategies based on cross-protection and the deployment of transgenic plants that show resistance to virus isolates highly similar to the transgene. The molecular epidemiology of PRSV was evaluated by analyzing the nucleotide sequence of the capsid protein (CP) and helper component-proteinase (HC-Pro) genes of isolates from around the world, including newly characterized ones from Colombia and Venezuela, using a relaxed molecular clock-based approach and a phylogeographic study. Our results confirm previous estimates on the origin of PRSV around 400 years ago and suggest distinct dispersion events from the Indian Peninsula to the rest of Asia, via Thailand, and subsequently to the Americas. A historical reconstruction of the P- and W-type characters in the phylogenetic study supports the need to revise the hypothesis that PRSV-P derives from PRSV-W since our results suggest that the ancestral state could be either of the two biotypes. Moreover, estimates of epidemic growth predict an increasing genetic diversity of the virus over time that has direct implications for control strategies of PRSV based on cross-protection and the use of transgenic plants.

Field Resistance of Coat Protein Transgenic Papaya to Papaya ringspot virus in Jamaica

Transgenic papayas (Carica papaya) containing translatable coat protein (CPT) or nontranslatable coat protein (CPNT) gene constructs were evaluated over two generations for field resistance to Papaya ringspot virus in a commercial papaya growing area in Jamaica. Reactions of R0 CPT transgenic lines included no symptoms and mild or severe leaf and fruit symptoms. All three reactions were observed in one line and among different lines. Trees of most CPNT lines exhibited severe symptoms of infection, and some also showed mild symptoms. R1 offspring showed reactions previously observed with parental R0 trees; however, reactions not previously observed or a lower incidence of the reaction were also obtained. The transgenic lines appear to possess virus disease resistance that can be manipulated in subsequent generations for the development of a product with acceptable commercial performance.

Comparative development and impact of transgenic papayas in Hawaii, Jamaica, and Venezuela.

We present data concerning the creation of transgenic papayas resistant to Papaya ringspot virus (PRSV) and their adoption by three different countries: the United States (e.g., Hawaii), Jamaica, and Venezuela. Although the three sets of transgenic papayas showed effective resistance to PRSV, the adoption rate in each country has varied from full utilization in Hawaii to aggressive testing but delay in deregulating of the product in Jamaica to rejection at an early stage in Venezuela. Factors that contributed to the rapid adoption in Hawaii include a timely development of the transgenic product, PRSV causing severe damage to the papaya industry, close collaboration between researchers and the industry, and the existence of procedures for deregulating a transgenic product. In Jamaica, the technology for developing the initial field-testing of the product progressed rather rapidly, but the process of deregulation has been slowed down owing to the lack of sustained governmental efforts to complete the regulatory procedures for transgenic crops. In Venezuela, the technology to develop and greenhouse test the transgenic papaya has moved abreast with the Jamaica project, but the field testing of the transgenic papaya within the country was stopped very early on by actions by people opposed to transgenic products. The three cases are discussed in an effort to provide information on factors, other than technology, that can influence the adoption of a transgenic product.

Momordica charantia is a Weed Host Reservoir for Papaya ringspot virus Type P in Jamaica

Papaya rinsgpot virus type P (PRSV), a member of the genus Potyvirus in the family Potyviridae, is primarily transmitted by aphids in a nonpersistent manner (2). The virus is geographically widespread but has a narrow host range within the plant families Caricaceae, Chenopodiaceae, and Cucurbitaceae (2). The first reported epidemic of PRSV in Jamaica was during the late 1980s (1). Since then, the virus has spread across the island and is recognized as a potential problem for continued production of papaya (Carica papaya L.). In the summers of 1999 and 2000, prominent vein clearing symptoms were observed on leaves of a common weed, cerasee (Momordica charantia L.), in papaya orchards of western Jamaica. This weed, a climbing annual in the Cucurbitaceae family used in a variety of local herbal preparations, was found to be growing on fences or the ground along the periphery of the orchards. Leaf samples were collected and tested for PRSV by double-antibody sandwich (DAS)-ELISA with polyclonal antibodies (Agdia Inc, Elkhart, IN). In addition, crude sap extracts from 12 cerasee leaf samples that were diluted 1:20 were mechanically inoculated onto six plants each of cerasee and papaya. Within 2 weeks, vein clearing symptoms were observed on cerasee and symptoms (vein clearing followed by mosaic development and leaf distortions) typical of PRSV infection were obtained on papaya (2). All original leaf samples and inoculated plants tested positive in DAS-ELISA. In subsequent vector transmission tests, 10 healthy cerasee or papaya seedlings were inoculated with aphids (Aphis gossypii) that were previously permitted to feed on PRSV-infected papaya or cerasee. High rates of virus transmission were achieved in three tests from cerasee to papaya (77 to 83%), papaya to cerasee (90 to 93%), and cerasee to cerasee (60 to 70%). Total RNA from papaya samples was subjected to reverse transcriptase-PCR using primers to the capsid protein gene (3). A single fragment of the expected size (approximately 996 bp) was amplified and sequenced and showed high nucleotide identity (90.3 to 91.4%) with previously reported PRSV type P from Jamaica (GenBank Accession No. DQ104823), Cuba (GenBank Accession No. DQ089482), Florida (GenBank Accession No. AF196839), Brazil (GenBank Accession No. AF344650), and Hawaii (GenBank Accession No. S46722). To our knowledge, this is the first report of the natural occurrence of PRSV on a weed host in Jamaica. Because of its widespread distribution and potential of serving as a reservoir of PRSV, cerasee may play a role in the epidemiology of PRSV. References: (1) M. Chin et al. Jam. J. Sci. Technol. 14:58, 2003. (2) D. Purcifull et al. No 292 in: Descriptions of Plant Viruses. CMI/AAB, Surrey, England, 1984. (3) J. Slightom. Gene 100:251, 1991.

Varying genetic diversity of Papaya ringspot virus isolates from two time-separated outbreaks in Jamaica and Venezuela

SummaryCoat protein sequences of 22 Papaya ringspot virus isolates collected from different locations in Jamaica and Venezuela in 1999 and 2004, respectively, were determined and compared with sequences of isolates from earlier epidemics in 1990 and 1993. Jamaican isolates collected in 1999 exhibited nucleotide sequence identities between 98 and 100% but shared lower identities of 92.2% with an isolate collected in 1990. Isolates from the 2004 epidemic in Venezuela exhibited more heterogeneity, with identities between 88.7 and 98.8%. However, isolates collected in 1993 were more closely related (97.7%). The viral populations of the two countries are genetically different and appear to be changing at different rates; presumably driven by introductions, movement of plant materials, geographical isolation, and disease management practices.

Genetic Transformation in Carica papaya L. (Papaya)

The genus Carica (family Caricaceae) comprises about 21 species (Purseglove 1968), but only C. papaya L. is of economic importance. Papayas are grown extensively in the tropics and subtropics where dooryard gardens and plantations contribute to an annual production rate of 4.43 million metric tons (MT) (FAO 1990). Nearly all of the fruit is grown in countries with developing market economies, for example, Latin America produces more than half of the crop in extensive plantings in Brazil (1.65 MT) and Mexico (0.65 MT). About one-fourth of the crop is grown in Asia, and Africa and the USA (Hawaii and Florida) make up the small remaining portion of total world production. The major product is fresh fruit which is usually consumed locally for breakfast or dessert, while papain, the basic component of meat tenderizer, is produced from the sap of immature fruit in Africa, Sri Lanka, and India (Poulter and Caygill 1985). The flesh of ripe fruit is rich in vitamins A and C (Arriola et al. 1980).

Opportunities and constraints to biotechnological applications in the Caribbean: transgenic papayas in Jamaica and Venezuela

In this opinion article, we briefly review the status of crop biotechnology research—with emphasis on the development of GM crops—in Jamaica and Venezuela. We focus on the transgenic papayas developed for both countries, and examine the factors hindering not only the development and application of this biotechnological commodity for the improvement of agricultural productivity, but also on the challenges influencing societal acceptance of the technology.

Physicochemical and biochemical characterization of transgenic papaya modified for protection against Papaya ringspot virus

BACKGROUND Papaya, a nutritious tropical fruit, is consumed both in its fresh form and as a processed product worldwide. Major quality indices which include firmness, acidity, pH, colour and size, are cultivar dependent. Transgenic papayas engineered for resistance to Papaya ringspot virus were evaluated over the ripening period to address physicochemical quality attributes and food safety concerns. RESULTS With the exception of one transgenic line, no significant differences (P > 0.05) were observed in firmness, acidity and pH. Lightness (L*) and redness (a*) of the pulps of non-transgenic and transgenic papaya were similar but varied over the ripening period (P < 0.05). Fruit mass, though non-uniform (P < 0.05) for some lines, was within the range reported for similar papaya cultivars, as were shape indices of female fruits. Transgene proteins, CP and NPTII, were not detected in fruit pulp at the table-ready stage. CONCLUSION The findings suggest that transformation did not produce any major unintended alterations in the physicochemical attributes of the transgenic papayas. Transgene proteins in the edible fruit pulp were low or undetectable.