Papaya Ringspot Virus Resistance Research Articles

Differential expression of microRNAs in response to Papaya ringspot virus infection in differentially responding genotypes of papaya (Carica papaya L.) and its wild relative.

Papaya ringspot virus (PRSV) is one of the most devastating viruses of papaya that has significantly hampered papaya production across the globe. Although PRSV resistance is known in some of its wild relatives, such as Vasconcellea cauliflora and in some of the improved papaya genotypes, the molecular basis of this resistance mechanism has not been studied and understood. Plant microRNAs are an important class of small RNAs that regulate the gene expression in several plant species against the invading plant pathogens. These miRNAs are known to manifest the expression of genes involved in resistance against plant pathogens, through modulation of the plant's biochemistry and physiology. In this study we made an attempt to study the overall expression pattern of small RNAs and more specifically the miRNAs in different papaya genotypes from India, that exhibit varying levels of tolerance or resistance to PRSV. Our study found that the expression of some of the miRNAs was differentially regulated in these papaya genotypes and they had entirely different miRNA expression profile in healthy and PRSV infected symptomatic plants. This data may help in improvement of papaya cultivars for resistance against PRSV through new breeding initiatives or biotechnological approaches such as genome editing.

Mutagenesis of the melon Prv gene by CRISPR/Cas9 breaks papaya ringspot virus resistance and generates an autoimmune allele with constitutive defense responses.

The majority of plant disease resistance (R) genes encode nucleotide binding-leucine-rich repeat (NLR) proteins. In melon, two closely linked NLR genes, Fom-1 and Prv, were mapped and identified as candidate genes that control resistance to Fusarium oxysporum f.sp. melonis races 0 and 2, and to papaya ringspot virus (PRSV), respectively. In this study, we validated the function of Prv and showed that it is essential for providing resistance against PRSV infection. We generated CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] mutants using Agrobacterium-mediated transformation of a PRSV-resistant melon genotype, and the T1 progeny proved susceptible to PRSV, showing strong disease symptoms and viral spread upon infection. Three alleles having 144, 154, and ~3 kb deletions, respectively, were obtained, all of which caused loss of resistance. Interestingly, one of the Prv mutant alleles, prvΔ154, encoding a truncated product, caused an extreme dwarf phenotype, accompanied by leaf lesions, high salicylic acid levels, and defense gene expression. The autoimmune phenotype observed at 25 °C proved to be temperature dependent, being suppressed at 32 °C. This is a first report on the successful application of CRISPR/Cas9 to confirm R gene function in melon. Such validation opens up new opportunities for molecular breeding of disease resistance in this important vegetable crop.

Studies on molecular variability of coat protein gene of Papaya ringspot virus-P isolates from India

Papaya ringspot virus (PRSV) is the key pathogen responsible for damage of the papaya plant and significant loss of fruit yield globally. Its coat protein (CP) sequences are highly variable and provide adaptability to the virus in the papaya plant. Due to the variability, the major concern facing of papaya cultivation is the breakdown of CP-transgenic PRSV resistance. In this study, 16 PRSV isolates were collected from different locations in seven districts of Uttar Pradesh, India and the variability of the CP gene of the virus was analysed. Further, it was compared with the isolates from other geographical origins. High diversity in CP sequences of our isolates was observed when compared with other Indian isolates. Phylogenetic analysis also revealed that PRSV isolates were more divergent from isolates of other countries. This study of sequence variation in the CP could be further very useful in virus management and development of broad-spectrum virus-resistant transgenic papaya.

The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ring-spot virus resistance locus.

Bottle gourd (Lagenaria siceraria) is an important vegetable crop as well as a rootstock for other cucurbit crops. In this study, we report a high-quality 313.4-Mb genome sequence of a bottle gourd inbred line, USVL1VR-Ls, with a scaffold N50 of 8.7Mb and the longest of 19.0Mb. About 98.3% of the assembled scaffolds are anchored to the 11 pseudomolecules. Our comparative genomic analysis identifies chromosome-level syntenic relationships between bottle gourd and other cucurbits, as well as lineage-specific gene family expansions in bottle gourd. We reconstructed the genome of the most recent common ancestor of Cucurbitaceae, which revealed that the ancestral Cucurbitaceae karyotypes consisted of 12 protochromosomes with 18534 protogenes. The 12 protochromosomes are largely retained in the modern melon genome, while have undergone different degrees of shuffling events in other investigated cucurbit genomes. The 11 bottle gourd chromosomes derive from the ancestral Cucurbitaceae karyotypes followed by 19 chromosomal fissions and 20 fusions. The bottle gourd genome sequence has facilitated the mapping of a dominant monogenic locus, Prs, conferring Papaya ring-spot virus (PRSV) resistance in bottle gourd, to a 317.8-kb region on chromosome 1. We have developed a cleaved amplified polymorphic sequence (CAPS) marker tightly linked to the Prs locus and demonstrated its potential application in marker-assisted selection of PRSV resistance in bottle gourd. This study provides insights into the paleohistory of Cucurbitaceae genome evolution, and the high-quality genome sequence of bottle gourd provides a useful resource for plant comparative genomics studies and cucurbit improvement.

Transcriptome Profiling Revealed Stress-Induced and Disease Resistance Genes Up-Regulated in PRSV Resistant Transgenic Papaya.

Papaya is a productive and nutritious tropical fruit. Papaya Ringspot Virus (PRSV) is the most devastating pathogen threatening papaya production worldwide. Development of transgenic resistant varieties is the most effective strategy to control this disease. However, little is known about the genome-wide functional changes induced by particle bombardment transformation. We conducted transcriptome sequencing of PRSV resistant transgenic papaya SunUp and its PRSV susceptible progenitor Sunset to compare the transcriptional changes in young healthy leaves prior to infection with PRSV. In total, 20,700 transcripts were identified, and 842 differentially expressed genes (DEGs) randomly distributed among papaya chromosomes. Gene ontology (GO) category analysis revealed that microtubule-related categories were highly enriched among these DEGs. Numerous DEGs related to various transcription factors, transporters and hormone biosynthesis showed clear differences between the two cultivars, and most were up-regulated in transgenic papaya. Many known and novel stress-induced and disease-resistance genes were most highly expressed in SunUp, including MYB, WRKY, ERF, NAC, nitrate and zinc transporters, and genes involved in the abscisic acid, salicylic acid, and ethylene signaling pathways. We also identified 67,686 alternative splicing (AS) events in Sunset and 68,455 AS events in SunUp, mapping to 10,994 and 10,995 papaya annotated genes, respectively. GO enrichment for the genes displaying AS events exclusively in Sunset was significantly different from those in SunUp. Transcriptomes in Sunset and transgenic SunUp are very similar with noteworthy differences, which increased PRSV-resistance in transgenic papaya. No detrimental pathways and allergenic or toxic proteins were induced on a genome-wide scale in transgenic SunUp. Our results provide a foundation for unraveling the mechanism of PRSV resistance in transgenic papaya.

Update on gene transfer biotechnology of papaya

Papaya ringspot virus (PRSV) is the most devastating disease of papayas worldwide. For 15 years, since 1998, genetically engineered, PRSV resistant Hawaii papayas, 'Rainbow', 'SunUp', and 'Laie Gold' have been sold locally and 'Rainbow' has been exported to the western US. Following deregulation, it has been exported for eight years to Canada and for three years, on a very small scale, to Japan. Markets like Hong Kong that do not differentiate between non-transgenic products and genetically engineered organisms (GMOs) also import Hawaii GMO papayas. At least fifteen research groups attempted to develop GMO papayas, mostly for PRSV resistance. A majority of the papayas were resistant to their specific PRSV strains; however at least initially China is the only other country to have commercialized transgenic virus resistant papaya. Activity in GMO projects is reduced because of negative publicity and lack of funding, but several projects continue: delayed ripening in Malaysia and the Philippines (field testing); PRSV and Papaya leaf distortion mosaic virus (PLDMV) broad resistance with single, double, and super-constructs in Taiwan; PRSV resistance construct development in Pune, India; multiplication of transgenic plants for PRSV resistance testing in Hainan, China; transformation of papaya for PRSV resistance with a gene from a papaya relative, Vasconcellea pubescens; and the final stages of deregulation of a second PRSV resistant papaya for Florida in the US.

Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance.

Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana.

Genetic analysis and gene mapping of papaya ring spot virus resistance in cucumber

The papaya ring spot virus (PRSV) causes significant fruit yield loss in cucurbit crops. Understanding of the inheritance and molecular mapping of PRSV resistance will facilitate development of resistant varieties to control this disease. In the present study, an F2 population was developed from the cross between susceptible ‘65G’ and resistant ‘02245’ cucumber inbred lines. Genetic analysis of PRSV resistance in 144 F2:3-derived F3 families showed that resistance is controlled by a single recessive gene which was designated as prsv02245. Simple sequence repeat (SSR) markers were employed in polymorphism screening between PRSV-susceptible and resistant DNA pools. The PRSV resistance gene, prsv02245, was mapped on chromosome 6 that was flanked by two SSR markers, SSR11-177 and SSR11-1, which was 1.1 and 2.9 cM away from the prsv02245 locus, respectively. The physical distance between the two markers was approximately 600 kb. The accuracy rate of marker-assisted selection of PRSV resistance among 35 cucumber lines using the marker, SSR11-177 was more than 80 %. Results from this study provide a valuable tool for fine mapping, gene cloning, and marker-assisted breeding for PRSV resistance in cucumber.

The Application of Irap Markers in the Breeding of Papaya (Carica Papaya L.)

Papaya is an important medicinal crop which is used in industries and other healthcare products. This study was conducted to facilitate the selection of Papaya Ring Spot Virus (PRSV) resistant papayas in breeding programs by DNA fingerprinting method. By using Inter-Retrotransposons Amplified Polymorphism molecular markers, 5 out of 45 possible primer combinations (28 IRAP primers combination and RT primers) showed significant polymorphism in DNA banding patterns that determined parental-progeny relations and classified progenies based on their resistance towards PRSV. Primers combination of LTR 6149 and LTR 6149 had the highest degree of polymorphism which was 75% of polymorphic bands. It is observed that only 1 out of 5 combinations of primers demonstrated DNA banding patterns similar to the previous study by Malaysian Agriculture and Research Development Institute (MARDI). Phylogenetic trees were constructed to detect the relation of papaya samples, and those which based on primers combination of LTR 6150 and Nikita showed resistant progenies with resistant parent. Furthermore, progenies from L90 were found to be closely related to the parent Tainung (T5) whilst progenies from L13 and L33 were closely related to another parent, Eksotika (E6). L90 with symptom-free fruits was the most tolerant followed by L33 and L13. So, the best primers in terms of inheritance pattern based on the PRSV resistance trait (LTR 6150 and Nikita primers) were suggested to be further applied in Molecular Assisted Breeding (MAB) or Molecular Assisted Selection (MAS) for papaya breeding programs.

CURRENT STATUS OF PAPAYA PRODUCTION IN THAILAND

Papaya (Carica papaya) is an important economic fruit crop in the tropical and subtropics. The world production of papaya in 2008 was over 9.1 million metric tons in 20 countries. Thailand ranged eighth, while India was first in the production of papaya followed by Brazil, Indonesia, Nigeria, Mexico, Ethiopia and others. In Thailand papaya is one of the most important fruit crops. The total cultivation area in Thailand covered about 17,430 ha. The eastern and the southern regions have the largest papaya area of 44 and 16%, respectively, in 2008, with an overall production volume of just over 201,099 t. The overall quantity of product exported in 2008 was about 3,458 t worth around Baht 111.89 mil. One of the main problems of papaya cultivation and the most ubiquitous and widespread is the Papaya ring spot virus (PRSV). The occurrence of PRSV disease was first observed in 1975 in the northeastern region has spread all over the country today. The Department of Agriculture (DOA), Ministry of Agriculture and Cooperatives have conducted papaya breeding since 1984. These programs were divided into two types, conventional breeding and genetic engineering. Conventional breeding included collection, selection and crossing of papayas. Papaya cultivars were collected from various sources, planted and selected for agricultural characteristics. They were self-pollinated to increase the purity of the lines. The DOA accepted and approved ‘Khaek Dam Si Sa Ket’ as a new cultivar in 1994. With regard to cross breeding, ‘Khaek Dam’ was crossed with ‘Coimbatire No. 2’ (CO2) cultivar. The cross breeding can be selected from the F1 hybrid papaya. The DOA approved this cultivar ‘Phichit’ in 1997. There is also a cross between the ‘Khaek Dam’ cultivar and the ‘Florida Tolerant’ cultivar. There were large amounts of F1 hybrid seeds which were grown, selected and self-pollinated for an inbred line. From this program, new cultivars, ‘Khaek Dam Tha Phra’ and ‘Khon Kaen 80’ were selected based on good agricultural traits and then they were approved in 1997 and 2010, respectively. Genetic engineering improvement under the DOA was in collaboration with Cornell University. The coat protein gene (CP gene) of PRSV was transferred into Thai papaya, ‘Khaek Dam’ and ‘Khaek Nuan’ cultivars. Potential resistant cultivars were selected for the R0 PRSV resistance, including one ‘Khaek Dam’ and four ‘Khaek Nuan’. Currently, one cultivar ‘Khaek Dam’ (R3 300KD) and two ‘Khaek Nuan’ (R3 319-1KN-180 and R3 319-1KN-181) cultivars were selected for the R3 PRSV resistance.

Differential gene expression in response to Papaya ringspot virus infection in Cucumis metuliferus using cDNA-amplified fragment length polymorphism analysis.

A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment length polymorphism (cDNA-AFLP) method. Transcript derived fragments (TDFs) identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future.

Genomics of Helper Component Proteinase Reveals Effective Strategy for Papaya Ringspot Virus Resistance

Papaya ringspot virus (PRSV) causes severe economic losses in both cucurbits and papaya throughout the tropics and subtropics. Development of PRSV-resistant transgenic plants faces a major hurdle in achieving resistance against geographically distinct isolates. One of the major reasons of failing to achieve the broad-spectrum PRSV resistance is the involvement of silencing suppressor proteins of viral origin. Here, based on sequence profile of silencing suppressor protein, HcPro, we show that PRSV-HcPro, acts as a suppressor of RNA silencing through micro RNA binding in a dose- dependent manner. In planta expression of PRSV-HcPro affects developmental biology of plants, suggesting the interference of suppressor protein in micro RNA-directed regulatory pathways of plants. Besides facilitating the establishment of PRSV, it showed strong positive synergism with other heterologous viruses as well. This study provides a strategy to develop effective and stable PRSV-resistant transgenic plants.

COMMERCIALIZATION OF GENETICALLY ENGINEERED PAPAYA: BREEDING PERSPECTIVES

Papaya is a breeder-friendly plant well suited to improvement by conventional methods. However, breeding failed to produce resistance to papaya ringspot virus (PRSV) due to inadequate genetic variation within the crop and sterility in interspecific hybrids. The transgenic approach succeeded because it provides effective resistance, is genetically simple to transfer (single dominant gene), and requires no change in farming practices. Success was aided by deploying transgenic resistance in a worthy Fl hybrid cultivar ('Rainbow') having excellent flavor, high yield, and broad environmental adaptation. An on-going program to improve 'Rainbow' for resistance to Phytophthora aerial blight and other highly heritable traits is based on phenotypic recurrent selection. Virus resistance results from post-transcriptional gene silencing of the PRSV coat protein gene and is most effective against PRSV strains with high homology to Hawaiian strains. Resistance in Hawaii has held up very well since the first field tests in 1992, but dependence on a single transgene raises concern about durability. Field tests with transgenic plants suggest that resistance-breaking PRSV strains are less likely to arise by viral recombination with coat protein transgenes than by accidental introduction of heterologous PRSV strains. In the future, complex quantitative traits will continue to be enhanced through conventional breeding, aided by marker-assisted selection. Genetic engineering will be most useful for contributing new single-gene traits when natural variation is lacking within papaya germplasm. Objectives of various on-going transformation projects include extending postharvest shelf life, developing durable PRSV resistance, and creating a phenotypic marker for early sexing of seedlings.

Breeding for Papaya ringspot virus resistance in Carica papaya via hybridisation with Vasconcellea quercifolia

Papaya ringspot virus type P (PRSV-P) is a major threat to the papaya industry worldwide. F1 hybrids have been produced when Carica papaya L. female flowers have been pollinated with pollen of the PRSV-P resistant species Vasconcellea quercifolia. F1 plant production required embryo rescue 90 days post-pollination, and plantlet regeneration in vitro. Three hundred F1 hybrids were grown to maturity in the field and had morphological characteristics that were identical to 1 or both parents, were intermediate between those of the parents, or were greater than either parent. They had a sex ratio of 2 (male) : 49 (hermaphrodite) : 49(female). Eighteen plants (7 male and 11 hermaphrodite) produced some viable pollen. Pollen viability of these plants as measured by germination on agar medium varied between 1.1 and 6.1% (mean of 3.37 ± 0.18) compared with >90% for C. papaya. Cytogenetic studies showed limited homology between C. papaya and V. quercifolia genomes. The intergeneric hybrids varied in their reaction to strains of PRSV-P in Australia and the Philippines. Of the hybrids, 75% were resistant to PRSV-P while 25% produced virus symptoms.

Molecular markers linked to papaya ring spot virus resistance and Fusarium race 2 resistance in melon

In melon, the Fom-1 gene confers monogenic resistance against the soil-borne fungus Fusarium oxysporum f. sp. melonis, races 0 and 2, while the closely linked Prv gene specifies resistance against the papaya ring spot virus. Markers linked to these resistance (R) genes were identified using two recombinant inbred line populations, derived from crosses between Cucumis melo Vedrantais and C. melo PI 161375, and between C. melo Vedrantais and C. melo PI 414723, respectively. Using bulked segregant analysis, as well as systematic scoring of the mapping populations, we developed two amplified fragment length polymorphism markers, two random amplified polymorphic DNA markers and five restriction fragment length polymorphism (RFLP) markers linked to this locus. Four of the RFLP sequences bear homology to nucleotide-binding site-leucine-rich repeat R genes, indicating the presence of a significant R-gene cluster in this locus. Our study provides the most closely linked markers published so far for these important traits. It also improves the resolution of the whole linkage group IX, which was difficult to order in our previous studies. Two of the markers were converted to cleaved amplified polymorphic sequence markers to facilitate their application in marker-assisted selection. Testing these two markers in several melon lines revealed different marker haplotypes in the melon germplasm and supported multiple, independent origin of the Fusarium races 0 and 2 resistance trait.

Cloning of the papaya ringspot virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gene

Papaya ringspot virus (PRSV) can cause a destructive disease in papaya (Carica papaya L.). Based on observations that viral replicase (RP) gene confers resistance to virus in other plants, we designed a pair of primers and cloned the RP gene from PRSV by RT-PCR. The 3′-truncated and 5′-extended RP gene fragment was then oriented under the control of the CaMV35 S promoter and nos termination sequence in the mini Ti plasmid vector pRok to construct a plant expression vector, designated pRPTW. Papaya (C. papaya L.) cv. Tai-nong-2 embryogenic calli were transformed by Agrobacterium tumefaciens LBA4404 harboring the pRPTW vector. After selection on 100 mg/ml kanamycin, 20 putative transgenic papayas were regenerated and confirmed by PCR-Southern blot and Southern blot analyses. PRSV inoculation tests showed that the RP gene conferred resistance to PRSV in transgenic papayas and those offspring carrying the RP gene. The consistency of the presence of the RP gene and PRSV resistance indicates that replicase-mediated resistance against PRSV was attained in papaya. Possible mechanisms include RNA-mediated resistance and protein-mediated resistance, as well as others, although further studies are required.

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.

Yield and Virus Resistance of Summer Squash Cultivars and Breeding Lines in North Carolina

Yellow and zucchini squash (Cucurbita pepo L.) cultigens (breeding lines and cultivars) were evaluated over a 2-year (1995 and 1996) period in North Carolina. Yellow squash cultigens that performed well (based on total marketable yields) were `Destiny III', `Freedom III', `Multipik', XPHT 1815, and `Liberator III' in Fall 1995 and HMX 4716, `Superpik', PSX 391, `Monet', `Dixie', XPH 1780, and `Picasso' in Spring 1996. Some of the yellow squash cultigens evaluated had superior viral resistance: XPHT 1815, XPHT 1817, `Freedom III', `Destiny III', `Freedom II', TW 941121, `Prelude II', and `Liberator III' in Fall 1995 and XPHT 1815, `Liberator III', `Prelude II', and `Destiny III' in Fall 1996; all these cultigens were transgenic. The yellow squash cultigens that performed well (based on total marketable yields) in the Fall 1995 test had transgenic virus resistance (`Destiny III', `Freedom III', XPHT 1815, and `Liberator III') or had the Py gene present in its genetic background (`Multipik'). Based on total marketable yields, the best zucchini cultigens were XPHT 1800, `Tigress', XPHT 1814, `Dividend' (ZS 19), `Elite', and `Noblesse' in Fall 1995; and `Leonardo', `Tigress', `Hurricane', `Elite', and `Noblesse' in Spring 1996. The zucchini cultigens with virus resistance were TW 940966, XPHT 1814, and XPHT 1800 in Fall 1995 and XPHT 1800, XPHT 1776, XPHT 1777, XPHT 1814, and XPHT 1784 in Fall 1996. Even though TW 940966 had a high level of resistance in the Fall 1995 test, it was not as high yielding as some of the more susceptible lines. Viruses detected in the field were papaya ringspot virus (PRSV) and watermelon mosaic virus (WMV) for Fall 1995; while PRSV, zucchini yellow mosaic virus (ZYMV), and WMV were detected for Fall 1996. Summer squash cultigens transgenic for WMV and ZYMV have potential to improve yield, especially during the fall when viruses are more prevalent. Most transgenic cultigens do not possess resistance to PRSV, except XPHT 1815 and XPHT 1817. Papaya ringspot virus was present in the squash tests during the fall of both years. Thus, PRSV resistance must be transferred to the transgenic cultigens before summer squash can be grown during the fall season without the risk of yield loss due to viruses.

364 FIELD TEST OF VIRUS RESISTANCE IN TRANSGENIC PAPAYAS

Twenty transgenic Carica papaya plants ('Sunset', Roclone 55-l) carrying the coat protein gene (cp) of papaya ringspot virus (PRV) strain HA 5-l have remained symptomless and ELISA-negative for 18 mo. after inoculation with Hawaiian PRV under field conditions. Control plants showed disease symptoms within 1 mo. after manual inoculation or within 4 mo. when aphid populations were the inoculum vectors. Trunk diameter was significantly greater in cp + plants (14.3 cm) than in PRV-infected controls (9.3 cm). Fruit brix, plant morphology, and fertility of cp + plants were all norm al. Segregation analysis in R1 seedlings indicated that 55-1 contains a single transgenic insertion site. PRV resistance in R1 plants was linked with the cp gene, although in some progenies, up to 50% of cp + plants developed mild PRV symptoms more than 3 mo. after inoculation. Preliminary tests suggest that this is not due to genesis of virulent mutant strains of PRV.