Volume 4 Special Issue 1 2010
TRANSGENIC PAPAYA

ISBN 978-4-903313-55-9

Guest Editor

Paula Tennant

The University of the West Indies, Jamaica

www.mona.uwi.edu/lifesciences

CONTENTS AND ABSTRACTS

Gustavo A. Fermin, Luz T. Castro (Venezuela), Paula F. Tennant (Jamaica) CP-Transgenic and non-Transgenic Approaches for the Control of Papaya Ringspot: Current Situation and Challenges (pp 1-15)

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ABSTRACT

Invited Review: In the last decade, successful cases of managing plant virus diseases using the transgenic approach have been reported, with the best known example being the Papaya ringspot virus (PRSV)-resistant transgenic papayas in Hawaii. Use of the coat protein (cp) gene has proven effective with not only papaya, but with various plant-virus systems such as squash. Although other viral sequences are equally effective in conferring resistance, few transgenic plants engineered with these sequences have made their way into the market. In addition, opposition to genetic manipulation of crop plants has prevented wide application of the technology, despite the fact that many countries (including Jamaica, Brazil and Venezuela) have produced and characterized several generations of resistant transgenic papayas. Using the papaya-PRSV system as a case study, we examine the transgenic cropping systems available, constraints to the adoption of transgenic papayas in various countries, as well as the impact the technology has made on world production of this fruit crop. Alternative non- cp and non- transgenic approaches of managing PRSV are also presented.

Maureen M. M. Fitch (USA) Papaya Ringspot Virus (PRSV) Coat Protein Gene Virus Resistance in Papaya: Update on Progress Worldwide (pp 16-28)

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ABSTRACT

Invited Review: Transgenic papayas have been marketed from Hawaii to the US mainland for 11 years and to Canada for 7 years. The resistance provided by a fragment of a Papaya ringspot virus (PRSV) coat protein (cp) gene enabled Hawaii’s papaya industry to recover from destruction. Commercialized in 1998, Hawaii papayas were joined in 2006 by China’s containing an untranslatable replicase gene. Worldwide, to our knowledge, these are the only commercialized transgenic papayas although Australia, Brazil, China, India, Jamaica, the Philippines, Taiwan, Thailand, Venezuela, and the US (Florida, Hawaii, US Virgin Islands) developed other transgenic virus resistant papayas. Taiwan’s dual cp genes protect against PRSV and Papaya leaf distortion mosaic virus and India seeks protection against PRSV and Papaya leaf curl virus. Florida has petitioned for deregulation of PRSV cp gene transformants. In other important papaya growing regions, either PRSV is not a major problem or alternative methods, for example, isolation, use of tolerant cultivars, or introgression of resistance genes from wild relatives, provide useful resistance. Brazil, India, Mexico, and Malaysia rely on broad expanses of buffer zones. In Thailand and the Philippines, tolerance was bred into local papayas and in Australia and the Philippines, resistance genes were introgressed from Vasconcellea quercifolia. The latter two examples show the importance of alternatives when hindrances impede adoption of new technologies, for example, conservative precautionary control policies adopted to provide time for government regulators to weigh benefits and detriments. Today, Hawaii supplies local, national, and international markets with high quality transgenic and/or nontransgenic fruit. Nontransgenic papayas are surrounded and protected by transgenic ones making it possible to produce both crops. Someday perhaps a similar scenario will be commonplace worldwide.

Paula F. Tennant, Simone E. Pinnock, Melissa Powell, Andrew O. Wheatley, Donna A. Minott (Jamaica) An Overview of the Safety Assessment of Transgenic Papaya for the Management of Papaya ringspot virus in Jamaica (pp 29-36)

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ABSTRACT

Invited Review: Papaya (Carica papaya L.) production in Jamaica suffered severe losses due to the type p Papaya ringspot virus (PRSV), causing devastation to over 40% of local papaya orchards in the past 15 years. Absence of genetic resistance in commercial cultivars has directed disease management efforts towards examination of transgenic resistance. Recently a number of transgenic lines were engineered with the coat protein gene (cp_T) of a Jamaican PRSV strain via microprojectile bombardment. Under field tests, R₁ transformants exhibited virus-resistant (70-75%) phenotypes. Trees were monoecious, producing red-flesh fruits similar in weight to the non-transgenic controls although some yellow-flesh fruits were obtained from two lines. Nutritional (e.g. vitamins, sugars) and anti-nutritional components (benzyl isothiocyanate, oxalate and cyanide) were assessed. With the exception of one cp_T transgenic line, no significant differences to the control were observed in the levels of nutrients and anti-nutrients at various stages of maturity, although a few random variations were noted. A sub-chronic feeding study in rats revealed markers of general health, body weight, food intake, and activities of plasma, liver and kidney function enzymes, to be comparable for animals fed diets of cp_T transgenic papaya and those of the control group. No effects were observed with the liver or kidney, in organ weights or histopathology. Although not statistically relevant, variations were recorded in some parameters. Thus, the cp_T transgenic papaya lines possess nutritional attributes and resistance to PRSV that can be manipulated in subsequent generations for development of products with acceptable commercial performance. Factors affecting the deregulation and commercialization of the transgenic product are discussed.

Shyi-Dong Yeh, Yi-Jung Kung, Huey-Jiunn Bau, Tsong-Ann Yu, Joseph A. J. Raja (Taiwan) Generation of a Papaya Hybrid Variety with Broad-Spectrum Transgenic Resistance to Papaya ringspot virus and Papaya leaf-distortion mosaic virus (pp 37-44)

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ABSTRACT

Invited Mini-Review: The aphid-borne potyvirus, Papaya ringspot virus (PRSV), is the major limiting factor for papaya production worldwide. Transgenic resistance conferred by the PRSV-coat protein (CP) gene and based on the mechanism of post-transcriptional gene silencing (PTGS), has become the most effective method to protecting papaya from infection by the noxious virus. The PRSV-CP transgenic lines of cultivars ‘Rainbow’ and ‘SunUp’ have been commercialized in Hawaii since 1998, and to date, they have retained their resistance against the virus. However, another aphid-borne potyvirus, Papaya leaf-distortion mosaic virus (PLDMV), which occurs in Okinawa and Taiwan, has emerged as a serious threat for PRSV-CP transgenic papaya. To deal with the new emerging problem, double resistance in transgenic papaya carrying a chimeric construct containing partial CP genes of PRSV and PLDMV was generated. In addition, a super strain of PRSV was recently identified, that contains a stronger gene silencing suppressor capable of effectively shutting off PTGS and single or double CP-transgenic resistance in a homology-independent way. To solve this problem, transgenic resistance generated by an untrans-latable construct targeting the PRSV HC-Pro gene was developed and the transgenic papaya lines provided broad-spectrum resistance against the PRSV super strain and PRSV isolates from different geographical locations. The event-specific molecular markers, derived from the flanking sequences of the transgene integration, in combination with the sex-linked markers, significantly fastened the molecular breeding process for pyramiding of single, double and super transgenic resistance into a commercial hybrid papaya cultivar. The super commercial hybrid cultivar of papaya with broad-spectrum resistance to different strains of PRSV and PLDMV has a great potential for application in different geographic regions of the world.

Laura Silva-Rosales, Diego González-de-León, Salvador Guzmán-González, Michelle Chauvet (Mexico) Why there is no Transgenic Papaya in Mexico (pp 45-51)

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ABSTRACT

Invited Mini-Review: Mexico is the world’s second largest papaya producer and its top exporter. Papaya ringspot virus (PRSV) may cause commercial crop losses of up to 100% in some regions, especially affecting small, low-income, and medium sized papaya producers. The introduction of genetically engineered (GE) PRSV-resistant papaya could therefore help alleviate this problem. Scientific expertise for in-country development of GE PRSV-resistant papaya is already in place and the regulatory, intellectual property rights and public perception settings are encouraging. The introduction of GE papaya would be greatly facilitated by the fact that a single variety, ‘Maradol’, would need to be modified for adoption since it completely dominates local and export markets. However, part of Mexico is within the proposed center of origin of Carica papaya. Risk assessment of GE PRSV-resistant papaya should consider transgene flow to cultivated, wild or weedy relatives. The role of farmers’ practices and social values as well as the structure of the papaya seed system in shaping the nature of gene flow needs to be investigated. What then has hampered, over the last 20 years, any serious efforts to develop, introduce and evaluate this technology? An ex ante assessment of the socioeconomic impact of GE papaya is being conducted to foresee possible impacts of transgenic papaya, taking into consideration the prevailing agricultural production systems. Current data indicate that resource poor farmers would benefit most from this technology but that large producers are averse to its introduction. Moreover, two foundations whose main objective is to facilitate the development and transfer of innovative technologies to farmers, have dropped their support of two public projects for the development of GE papaya.

Sunee Kertbundit (Thailand/Czech Republic), Miloslav Juříček (Czech Republic) Application of Transgenic Technologies to Papaya: Developments and Biosafety Assessments in Thailand (pp 52-57)

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ABSTRACT

Invited Mini-Review: Papaya (Carica papaya) is one of the staple foods in Thailand. Since 1975, papaya production in Thailand has been severely limited by Papaya ringspot virus (PRSV) which is now endemic. The great success of transgenic papaya resistant to PRSV from Hawaii in the 1990s signaled a new strategy to combat PRSV. However, transgenic papaya resistant to PRSV isolated from Hawaii is not resistant to PRSV isolated from Thailand and other countries. Consequently, three independent Thai research groups: the Department of Agriculture (DOA), Plant Genetic Engineering Unit (BIOTEC) and Mahidol University, set out to use transgene technology to develop papaya resistant to Thai isolates of PRSV. All obtained resistant papaya plants, but testing the levels of resistance to PRSV in the field has been thwarted because of the moratorium on field trials launched by Thai government in 2001. Only small experimental fields for research purposes are permitted. During 2004-2007, the entire experimental field test of transgenic papaya was banned on account of the argument of contamination of the environment by transgenic papaya materials from the DOA station. This ban was repealed in 2007 and currently a National Biosafety Law awaits ratification by the Thai parliament. If approved, this law will support the expansion of biotechnology research and commercialization of transgenic crops in Thailand.

Changming Ye, Huaping Li (China) 20 Years of Transgenic Research in China for Resistance to Papaya ringspot virus (pp 58-63)

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ABSTRACT

Invited Mini-Review: Papaya is a favorite fruit and important industrial raw material in China. It is traditionally planted in southern China. Due to its wide uses and mystical health-related effects, papaya has been listed in fruits of high priority. Papaya ringspot virus (PRSV) is the main limitation of papaya production. PRSV infection rate reaches 100% in field in the late season of planting, which considerably reduces productivity and quality of papaya. Several research groups in China started around 1990 to develop transgenic papaya lines for resistance against PRSV, adopting pathogen-derived resistance strategy. The evaluation of resistance against PRSV and biosafety assessments with genetically modified (GM) papaya lines have been conducted in greenhouse and field. GM papaya with replicase gene from PRSV has been approved for commercial production in Guangdong province by Chinese government since 2006. In this review, we discuss 20 years research and regulatory management on transgenic papaya in China.

Marisela Hernández González, Jacquelynne Cervantes Torres, José Luis Cabrera Ponce, Luis Herrera Estrella, Gabriela Rosas Salgado, Maria Luisa Villarreal, Gladis Fragoso González, Edda Sciutto Conde (Mexico) Development of an Oral Anti-Cysticercosis Vaccine Delivered in Genetically Modified Embryogenic Callus of Papaya (pp 64-70)

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ABSTRACT

Invited Mini-Review: Vaccination of pigs may interrupt Taenia solium transmission by reducing the number of cysticerci, the precursors of adult intestinal tapeworms in humans. A novel preventive cost/effective polyvalent anti-cysticercosis vaccine expressed in papaya (S3Pvac-papaya) has been developed with the aim to increase its effectiveness at a reduced cost and offer an alternative oral route to increase the feasibility of its administration. Transgenic papaya plants have been recently developed with very few applications. This chapter reviews the advances of one application that is the development of an anti-cysticercosis vaccine expressed in papaya (Carica papaya L.). Sequences that codify for the three peptides included in the S3Pvac anti-cysticercosis vaccine named KETc7, KETc12 and KETc1 were used to transform papaya embryogenic callus. The presence integration of the transgene (KETc1.6His, KETc12.6His and KETc7) and its expression were confirmed in 19 clones by PCR, RT-PCR and real time RT-PCR. The protective capacity against the experimental murine cysticercosis of 19 transgenic lines 15 lines corresponding to KETc1.6His, two to KETc12.6His and two to KETc7 was determined after subcutaneous immunization. The clones that induced the highest protective capacity were selected and used for oral immunization. A high level of protection, similar to that induced by subcutaneous immunization was observed. Interestingly, non-transformed papaya embryogenic callus also induced protection when subcutaneously administered albeit at a lower level, a finding that merits additional studies which are in progress. Some initial findings were obtained in respect to the immunity elicited by S3Pvac-papaya: increased levels of specific IgG antibodies were detected in sera of immunized mice and an increased CD4+ and CD8+ proliferative response in spleen cells and CD8+ in Peyer patches. The potential of this new oral version of the anti-cysticercosis vaccine was demonstrated. Papaya cells are an advantageous delivery system that can be used for the design of an effective, sustainable and affordable oral subunit vaccine against different pathogens. Thus, the scale-up of embryogenic callus and cell suspension cultures of the S3Pvac-papaya vaccine is presently being developed for its mass production.

Rodolfo López-Gómez, Plinio Guzmán (Mexico) Toward the Control of Ethylene Production in Papaya Fruit: A Model for Tropical Fruits (pp 71-76)

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ABSTRACT

Invited Mini-Review: Papaya (Carica papaya) is one of the healthiest and nutritious fruits in the world. This tropical plant contains diverse biologically active compounds of industrial and nutraceutical interest. As a climacteric fleshy fruit, papaya is highly vulnerable to the effects of the growth regulator ethylene, reducing its nutritional value and causing significant postharvest losses. To extend the shelf life of fruits several strategies are available to control the production of this gas during fruit ripening. Knowledge at the molecular level of the ethylene biosynthesis and action pathways permits to devise strategies to control ethylene metabolism in transgenic plants. The control of ethylene production is well documented in transgenic tomato plants and has been initiated in papaya. This paper reviews the molecular basis of ethylene metabolism in fruits and centers on papaya as an example of a climacteric fruit.

Chi-Chu Lo, Huey-Jiunn Bau, Shu-Chuan Chen, Chien-Ju Lin, Yi-Chien Huang, Shyi-Dong Yeh (Republic of China) Persistence and Bioavailability of Transgenic Genes Released from Genetically Modified Papaya and the Influence on Soil Bacterial Communities in Taiwan (pp 77-89)

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ABSTRACT

Invited Review: Papaya ringspot virus (PRSV) is the most destructive disease of papaya (Carica papaya L.) in Taiwan, and transgenic papaya resistant to PRSV and PLDMV (papaya leaf distortion mosaic virus) were developed in Taiwan. Horizontal gene transformation (HGT) of kanamycin resistant gene nptII from transgenic papaya, the persistence of transgenic genes in soil samples, and the influence of transgenic papaya on the soil diversity of microorganisms were reported. Two strains of Acinetobacter sp. BD413 and BD413 (pFG4ΔnptII) were used as recipient cells, and no transformation was detected in both bacteria when transgenic papaya genomic DNA were added. However, transformations were detected in Acinetobacter sp. BD413 (pFG4ΔnptII) on filter and in soil microcosms when PCR products of different lengths (1396, 786, and 604 bp, contained nptII) amplified from transgenic papaya DNA were added. The persistence of transgenic gene of 398 bp (located between plasmid pBI121 and NOS terminator, pBI121/NOS-T) in soil was 0.06 μg g^-1 soil, whereas the residues of 769 bp (located between 35S promoter and coat protein, 35S-P/PRSV-CP) and 200 bp (located between NOS promoter and nptII gene, NOS-P/nptII) were less than 30 pg g^-1 soil (detection limit). The influence of transgenic papaya on the soil diversity of microorganisms were conducted by DGGE (Denaturing Gradient Gel Electrophoresis) method, and the results showed that some differences appeared on the bacterial DGGE patterns at the beginning of planting, but the difference reduced after six months.

Namthip Phironrit, Bencharong Phuangrat, Parichart Burns, Wichai Kositratana (Thailand) Resistance of Coat Protein Transgenic Papaya and Development of Homozygous Transgenic Papaya Line 116/5 Resistant to Papaya ringspot virus (PRSV) under Screenhouse Conditions in Thailand (pp 90-93)

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ABSTRACT

Original Research Paper: Transgenic papaya, cv. ‘Khak Nual’, line 116/5 R₀ was regenerated via particle bombardment-mediated transformation with coat protein (CP) gene of Papaya ringspot virus (PRSV), Chiang Mai isolate (PRSV-CM). This line showed high resistance to PRSV-CM and contained 4 copies of the transgene. Homozygous lines of transgenic papaya line 116/5 were developed by self-pollination. Progenies were tested for resistance to PRSV by mechanical inoculation under screenhouse conditions. PRSV-CP transgene was detected by PCR, dot blot and Southern blot analysis. The results showed that R₁ progenies contained 2-4 copies and 20% were resistant to PRSV-CM. We selected resistant lines that contained only 2 copies of transgene for self-pollination to produce R₂, R₃ and R₄ progenies. The level of resistance in the R₂, R₃ and R₄ were 34, 71 and 95-100%, respectively. All 426 plants of the R₄ generation contained the PRSV-CP transgene. Sixty R₄ resistant plants were randomly selected for the determination of CP transgene copy. The results showed that all plants contained 2 copies of the transgene. Stability of R₅ homozygosity and biosafety assessment are being investigated.

Katherine Noorda-Nguyen, Heather McCafferty, Ayumi Aoki, Wayne Nishijima, Yun J. Zhu (USA) Agrobacterium rhizogenes Generates Transgenic Hairy Roots in Carica papaya L.: A New Approach for Studying and Improving Resistance to the Root-rot Pathogen, Phytophthora palmivora (pp 94-96)

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Short Communication: Phytophthora palmivora is an oomycete pathogen of Carica papaya L. that has a wide host range and causes especially severe root- and fruit-rot diseases. P. palmivora flourishes on papaya during the rainy season when fungicide treatments are often washed away. There are no varieties of C. papaya L. with resistance to P. palmivora. Genetic engineering with significant resistance genes has the potential to improve C. papaya’s tolerance to P. palmivora and to better understand the interactions between the host plant and this pathogen. However, there must first be an efficient Agrobacterium rhizogenes-mediated transformation protocol to produce rapid regeneration of transformed roots to evaluate the functional roles of the defense-related genes and proteins in papaya. In this study, we report the production of in vivo composite C. papaya L. plants by direct injection of cultures of A. rhizogenes transformed with the binary vector, pCNL65, into the cotyledonary nodes of papaya seedlings. Hairy roots were also produced by inoculating in vitro hypocotyl and cotyledon pieces of tissue culture plants with A. rhizogenes. Both procedures produced hairy roots within 1-3 weeks. Approximately 20% of the roots derived from inoculated seedlings tested positive for beta-glucuronidase (GUS) activity. This method of papaya transformation has the potential for the rapid evaluation of candidate genes involved in plant – pathogen interactions, particularly those involving roots.