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- Author or Editor: Dennis Gonsalves x
Our laboratory has focused on the development of virus-resistant plants through the use of pathogen-derived resistance. We have analyzed transgenic plants, including tobacco, cucumbers, melons, squash, chrysanthemum, tomato, papaya, and lettuce, that contain the coat protein genes of viruses belonging to the potyvirus, tospovirus, nepovirus, cucumovirus, and comovirus groups. Field and greenhouse trials have generally shown that resistance is not correlated to the expression level of the coat protein gene—in a number of cases, plants that expressed low amounts of coat protein showed excellent resistance. However, we have found that transgenic progenies from a particular plant often vary in their resistance to virus infection. These observations will be discussed as they relate to gene stability.
Six summer squash (Cucurbita pepo L.) cultivars were regenerated via somatic embryogenesis using cotyledons excised from germinated or nongerminated seeds. Genotypes included were zucchini, commercial F1 hybrids, `President', `Seneca Zucchini', `Jade'; the noncommercial inbred line `Caserta Inbred 557311'; and two yellow squash hybrids `Dixie' and `Seneca Butterbar'. Somatic embryogenesis was initiated in induction medium containing 22.62 μm 2, 4-D, and embryos were germinated in maturation medium containing 0.27 μm NAA and 0.23 μm kinetin. Plants were elongated and rooted on basal medium without hormones. All media contained carbenicillin at 500 mg·liter–1. Sixty-one percent of the `Seneca Butterbar' cotyledons produced somatic embryos when kept on induction medium for 10 weeks. Overall, 7% of the initial explants produced plantlets, and regeneration efficiency was calculated as 0.3 plantlets per initial explant. The relative production of plants from cotyledons that were kept on induction medium for different time periods were determined for `Caserta Inbred 557311' and `Seneca Zucchini'. All cotyledons produced somatic embryos after 11 to 17 weeks on induction medium. However, plantlet production was optimal with explants kept on induction medium for 13 weeks for `Seneca Zucchini' and for 15 weeks for `Caserta Inbred 557311', producing an average of 4.5 and 9.3 plants per explant, respectively, from 90% to 70% of the explants. We recovered plants from all six cultivars; thus, our regeneration protocol may be applicable to other genotypes. The high percentage of regenerants obtained indicates that the regeneration method is efficient enough to be adapted successfully to squash transformation experiments. Chemical names used: α-carboxybenzylpenicillin (carbenicillin); 2,4-dichlorophenoxyacetic acid (2,4-D); 6-furfurylaminopurine (kinetin); α-napthaleneacetic acid (NAA).
Transgenic papaya line 55-1 with resistance to papaya ringspot virus (PRSV) originated in 1989 by particle bombardment of cultivar Sunset with the coat protein gene (cp) of mild mutant Hawaii PRSV strain HA 5-1. Hemizygous (+/cp) R0 clones of 55-1 displayed resistance to the virulent Hawaii HA strain in greenhouse tests in New York in 1991 and to local strains in a field trial in Hawaii from 1992 to 1994. In the R1 generation produced by crossing the pistillate R0 55-1 with `Sunset', up to 50% of the hemizygous transgenic segregants were susceptible to a local Oahu PRSV strain when inoculated as seedlings but not as mature plants. Similar inoculation experiments in New York showed that hemizygous R1 transgenics were susceptible in differing degrees to PRSV strains from regions other than Hawaii. Homozygous (cp/cp) R2, R3, and R4 populations planted in various locations in Hawaii since 1994 have consistently demonstrated high-level resistance to local strains at all stages of development. When inoculated in New York with eight non-Hawaii PRSV strains, homozygous R3 seedlings were resistant to all but a Thai strain. Transgenic resistance is the result of a complex interaction involving the stage of plant development, transgene dosage, the degree of homology between transgene and challenge virus, and environmental variables. Papaya plants transformed with nontranslatable versions of various cp genes are also highly resistant to PRSV, indicating that the resistance mechanism operates at the RNA level. No loss of resistance due to the appearance of resistance-breaking virus strains or to transgene inactivation has been noted thus far.
Tomato ringspot virus (TmRSV) was detected by ELISA indexing in leaf tissue from 23 of 59 clones of Malus domestica Borkh., 1 to 6 years after inoculation with TmRSV-infected buds. However, TmRSV was repeatedly detected from only 10 of the 23 clones: Mailing (M) 26, MM 106, MAC-30, MAC-39, and P-2 and the fruiting cultivars ‘Northern Spy’, ‘Spigold’, ‘Spijon’, ‘Stayman’, and ‘Wayne’. ‘Northern Spy’ and most of its derivatives appeared to be highly susceptible to TmRSV infection, in that the virus was consistently recovered from inoculated plants.
Tomato ringspot virus (TmRSV) was consistently detected in myrobalan (Prunus cerasifera) and in 8 other plum rootstocks that exhibited prune brownline (PBL) symptoms at the graft unions. The infected rootstocks included Prunus domestica cvs. St. Julien A, Brompton, Ackermanpflaume, and Pershore and 6 seedling lines of P. cerasifera. Own-rooted ‘Stanley’ prune trees remained free of natural infection by TmRSV for 14 years on a site where 36% of the ‘Stanley’ trees on other rootstocks became infected. Mortality among TmRSV-infected trees on myrobalan stocks was higher than among infected trees on P. domestica roots. Own-rooted ‘Stanley’ trees are suggested for extensive commercial trial in areas infected with TmRSV.
Line 63-1 is a `Sunset'-derived transgenic papaya expressing the coat protein (CP) gene from a mild mutant of a Hawaiian isolate of Papaya ringspot virus (PRSV). Previous work showed that line 63-1 R1 plants exhibited a range of resistance to severe PRSV isolates from Hawaii (HA), Jamaica (JA), Thailand (TH), and Brazil (BR). Genetic and molecular data obtained in this study confirm that line 63-1 has two CP transgene insertion sites; segregation analysis shows that the CP and the npt II genes are present at both loci. To study the potential effect of gene dosage on resistance, various populations of R1, R2, and R3 seedlings were challenged by PRSV HA, BR, and TH. A R1 population obtained by self-pollination of line 63-1 hermaphrodite R0 plant exhibited resistance to all three isolates. The percentage of plants resistant to all three PRSV isolates increased in 63-1-derived populations as a result of recurrent selection. Additional genetic studies demonstrate that the number of resistant plants in a 63-1-derived population is directly correlated with the number of plants with multiple transgene copies. We conclude that transgene dosage plays a major role in affecting the resistance of 63-1 to PRSV isolates from various geographical locations.
A single regeneration procedure using cotyledon explants effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens (using `Burpee Hybrid' and `Hales Best Jumbo') or microprojectile bombardment (using `Topmark') methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), β-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 μm 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg·liter-1 and carbenicillin (Cb) at 500 mg·liter-1. Our comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R0 plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes. This analysis showed that plants transformed by A. tumefaciens contained all three genes, although co-transferring the genes into bombarded plants was not always successful. R1 plants were challenge inoculated with CMV-FNY, a destructive strain of CMV found in New York. Resistance levels varied according to the different transformed genotypes. Somaclonal variation was observed in a significant number of R0 transgenic plants. Flow cytometry analysis of leaf tissue revealed that a significant number of transgenic plants were tetraploid or mixoploid, whereas the commercial nontransformed cultivars were diploid. In a study of young, germinated cotyledons, however, a mixture of diploid, tetraploid, and octoploid cells were found at the shoot regeneration sites.
Transgenic plum plants expressing the papaya ringspot virus (PRV) coat protein (CP) were produced by Agrobacterium-mediated transformation of hypocotyl slices. Hypocotyl slices were cocultivated with Agrobacterium tumefaciens strain C58/Z707 containing the plasmid pGA482GG/CPPRV-4. This plasmid carries the PRVCP gene construct and chimeric NPTII and GUS genes. Shoots were regenerated on Murashige and Skoog salts, vitamins, 2% sucrose, 2.5 μm indolebutyric acid, 7.5 μm thidiazuron, and appropriate antibiotics for selection. Integration of the foreign genes was verified through kanamycin resistance, GUS assays, polymerase chain reaction (PCR), and Southern blot analyses. Four transgenic clones were identified. Three were vegetatively propagated and graft-inoculated with plum pox virus (PPV)-infected budwood in a quarantine, containment greenhouse. PPV infection was evaluated over a 2- to 4-year period through visual symptoms, enzyme-linked immunosorbent assay, and reverse transcriptase PCR assays. While most plants showed signs of infection and systemic spread of PPV within l-6 months, one plant appeared to delay the spread of virus and the appearance of disease symptoms. Virus spread was limited to basal portions of this plant up to 19 months postinoculation, but, after 32 months symptoms were evident and virus was detected throughout the plant. Our results suggest that heterologous protection with PRVCP, while having the potential to delay PPV symptoms and spread throughout plum plants, may not provide an adequate level of long-term resistance.
The disease resistance of a transgenic line expressing the coat protein (CP) gene of the mild strain of the papaya ringspot virus (PRSV) from Hawaii was further analyzed against PRSV isolates from Hawaii and other geographical regions. Line 63-1 originated from the same transformation experiment that resulted in line 55-1 from which the transgenic commercial cultivars, `Rainbow' and `SunUp', were derived. Plants of line 63-1 used in this study consisted of a population from a self pollinated R0 bisexual plant. ELISA and PCR tests provided evidence that there are at least two segregating CP loci. To allow for comparison with reactions of the previously reported line 55-1, virus isolates from Hawaii, Brazil, Thailand, and Jamaica were used to challenge seedlings of 63-1. Unlike line 55-1, a significant percentage of inoculated transgenic plants were susceptible to isolates from Hawaii. However, a proportion of plants were resistant to the non-Hawaiian isolates. In contrast, previous work showed that all plants of the hemizygous line 55-1 were susceptible to PRSV isolates from Brazil, Thailand, and Jamaica. Line 63-1, therefore, presents Hawaii with PRSV-resistant transgenic germplasm that could be used as a source of transgenes for resistance to PRSV isolates within and outside of Hawaii.