Richard M. Manshardt

Stable transformation of papaya via microprojectile bombardment.

SummaryStable transformation of papaya (Carica papaya L.) has been achieved following DNA delivery via high velocity microprojectiles. Three types of embryogenic tissues, including immature zygotic embryos, freshly explanted hypocotyl sections, and somatic embryos derived from both, were bombarded with tungsten particles carrying chimeric NPTII and GUS genes. All tissue types were cultured prior to and following bombardment on half-strength MS medium supplemented with 10 mg 1−1 2,4-D, 400 mg 1−1 glutamine, and 6% sucrose. Upon transfer to 2,4-D-free medium containing 150 mg 1−1 kanamycin sulfate, ten putative transgenic isolates produced somatic embryos and five regenerated leafy shoots. Leafy shoots were produced six to nine months following bombardment. Tissues from 13 of these isolates were assayed for NPTII activity, and 10 were positive. Six out of 15 isolates assayed for GUS expression were positive. Three isolates were positive for both NPTII and GUS,

Pathogen-derived resistance provides papaya with effective protection against papaya ringspot virus

Transgenic Carica papaya plants (cv. Sunset, R0 clone 55-1) carrying the coat protein gene of papaya ringspot virus (strain HA 5-1) remained symptomless and ELISA-negative for 24 months after inoculation with Hawaiian strains of papaya ringspot virus under field conditions. Non-transgenic and transgenic control plants lacking the coat protein gene developed disease symptoms within one month after manual inoculation or within four months when natural aphid populations were the inoculum vectors. Mean trunk diameter was significantly greater in cloned 55-1 plants compared with virus-infected controls (14.7 cm versus 9.3 cm after 18 months). Fruit brix, plant morphology, and fertility of 55-1 plants were all normal, and no pleiotropic effects of the coat protein gene were observed. These results indicate that pathogen-derived resistance can provide effective protection against a viral disease over a significant portion of the crop cycle of a perennial species.

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

SummaryImmature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l−1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l−1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos.

SummaryTransgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 μg·ml−1 kanamycin. Four putative transgenic plant lines were obtained from the cp gene− vector and two from the cp gene+ vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene+ plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.

A phylogenetic analysis of the genus Carica L. (Caricaceae) based on restriction fragment length variation in a cpDNA intergenic spacer region

The phylogenetic relationships among twelve wild and cultivated species of Carica (Caricaceae) were analyzed using restriction fragment length variation in a 3.2-kb PCR amplified intergenic spacer region of the chloroplast DNA. A total of 138 fragments representing 137 restriction sites accounting for 5.8% of the amplified region were examined. Both parsimony and neighbor joining cluster analyses confirmed the close association among South American wild Carica species. However, cpDNA data did not support the traditional monophyly hypothesis for the evolution of Carica. Further, cpDNA analyses showed two basic evolutionary lineages within the genus Carica, one defined by cultivated C. papaya and another consisting of the remaining wild species from South America in a well resolved but poorly supported monophyletic assemblage. This evolutionary split in Carica strongly suggests that C. papaya diverged from the rest of the species early in the evolution of the genus and evolved in isolation, probably in Central America.

Tissue differential expression of lycopene β-cyclase gene in papaya

Carotene pigments in flowers and fruits are distinct features related to fitness advantages such as attracting insects for pollination and birds for seed dispersal. In papaya, the flesh color of the fruit is considered a quality trait that correlates with nutritional value and is linked to shelf-life of the fruit. To elucidate the carotenoid biosynthesis pathway in papaya, we took a candidate gene approach to clone the lycopene β-cyclase gene, LCY-B. A papaya LCY-B ortholog, cpLCY-B, was successfully identified from both cDNA and bacterial artificial chromosome (BAC) libraries and complete genomic sequence was obtained from the positive BAC including the promoter region. This cpLCY-B shared 80% amino acid identity with citrus LCY-B. However, full genomic sequences from both yellow- and red-fleshed papaya were identical. Quantitative real-time PCR (qPCR) revealed similar levels of expression at six different maturing stages of fruits for both yellow- and red-fleshed genotypes. Further expression analyses of cpLCY-B showed that its expression levels were seven- and three-fold higher in leaves and, respectively, flowers than in fruits, suggesting that cpLCY-B is down-regulated during the fruit ripening process.

Genetic variability in Macadamia

A genetic variability analysis involving 45 accessions of Macadamia including four species, M. integrifolia, M. tetraphylla, M. ternifolia, and M. hildebrandii and a wild relative, Hicksbeachia pinnatifolia was performed using eight enzyme systems encoded by 16 loci (Gpi-1 and 2, Idh-1 and 2, Lap, Mdh-1, 2, and 3, 6Pgd-2, Pgm-2 and 3, Tpi-1 and 2, Ugpp-1, 2, and 3). Forty-three accessions possessed distinct isozyme fingerprints indicating a high level of genetic variation. Examination of multivariate relationships among accessions using a cluster analysis resulted in the identification of four clusters, two of which contained one accession each representing H. pinnatifolia and M. hildebrandii. All Hawaiian cultivars were included in two sub-clusters within the largest cluster, which encompassed all the M. integrifolia and three M. ternifolia accessions, suggesting that: (1) the Hawaiian cultivars must have originated from at least two genetically diverse ancestral populations and (2) M. ternifolia may be a conspecific variant of M. integrifolia. Macadamia tetraphylla has diverged marginally from the M. integrifolia and M. ternifolia complex suggesting that these taxa represent a species complex. The different measures of genetic variability such as mean number of alleles per locus, polymorphic index, and observed and expected levels of heterozygosity, indicated significant levels of genetic variation in the Macadamia collection.

Allergenicity assessment of the papaya ringspot virus coat protein expressed in transgenic rainbow papaya.

The virus-resistant, transgenic commercial papaya Rainbow and SunUp (Carica papaya L.) have been consumed locally in Hawaii and elsewhere in the mainland United States and Canada since their release to planters in Hawaii in 1998. These papaya are derived from transgenic papaya line 55-1 and carry the coat protein (CP) gene of papaya ringspot virus (PRSV). The PRSV CP was evaluated for potential allergenicity, an important component in assessing the safety of food derived from transgenic plants. The transgene PRSV CP sequence of Rainbow papaya did not exhibit greater than 35% amino acid sequence homology to known allergens, nor did it have a stretch of eight amino acids found in known allergens which are known common bioinformatic methods used for assessing similarity to allergen proteins. PRSV CP was also tested for stability in simulated gastric fluid and simulated intestinal fluid and under various heat treatments. The results showed that PRSV CP was degraded under conditions for which allergenic proteins relative to nonallergens are purported to be stable. The potential human intake of transgene-derived PRSV CP was assessed by measuring CP levels in Rainbow and SunUp along with estimating the fruit consumption rates and was compared to potential intake estimates of PRSV CP from naturally infected nontransgenic papaya. Following accepted allergenicity assessment criteria, our results show that the transgene-derived PRSV CP does not pose a risk of food allergy.

Isozyme variation in cultivated and wild pineapple

SummaryIsozyme variation was studied in 161 accessions of pineapple including four species of Ananas and one of Pseudananas. Six enzyme systems (ADH, GPI, PGM, SKDH, TPI, UGPP) involving seven putative loci revealed 35 electromorphs. Considerable variation exists within and between species of Ananas. Sixty-six distinct zymotypes were identified. Multivariate analyses of isozyme variation indicated that A. comosus contains five genetically diverse groups that do not match perfectly with the traditional varietal groups. Isozyme evidence also suggests that A. erectifolius is a conspecific variant of A. comosus, and that among other wild species, A. ananassoides is more closely related to A. comosus than A. bracteatus. Pseudananas is genetically distinct from all species of Ananas. It is evident from our study that differentiation among the species of Ananas may be due to ecological isolation rather than genetic divergence with breeding barriers and therefore may represent a species complex.

Isozyme variation in lychee (Litchi chinensis Sonn.)

Abstract A genetic diversity analysis involving 49 lychee ( Litchi chinensis Sonn.) accessions using eight enzyme systems encoding 12 loci ( Idh-1, Idh-2, Mdh-2, Per-1, Pgi-2, Pgm-1, Pgm-2, Skdh, Tpi-1, Tpi-2, Ugpp-1 , and Ugpp-2 ) revealed moderate to high levels of genetic variability. Cluster analysis of the isozyme data from 40 genetically different accessions of the total 49 identified three groups at the 50% level of genetic similarity, the largest of which contained 32 of the 40 accessions distributed in three sub-groups. The groups including the three sub-groups differed markedly both in frequency and composition of alleles at different loci. On average, 77% of the loci were polymorphic with an overall mean of 2.2 alleles per locus and an observed heterozygosity of 0.387. The unbiased genetic identities ( I ) between groups ranged from 0.809 between Groups 1b and 2 to 0.937 between Groups 1b and 1c. Group 2 has diverged from the other groups with an average genetic identity of 0.821. Summing over all 11 polymorphic loci, 16% of gene diversity was due to differentiation between groups and 84% to that within groups. Comparison of isozyme fingerprints revealed that some accessions identically named, such as ‘No mai tsz’, ‘Kwai mi’, and ‘Hak ip’, possessed different isozyme genotypes, whereas some others with different names displayed identical isozyme genotypes.