Citrus Tristeza Closterovirus (CTV) induces mild and/or severe symptoms on Citrus species. It may cause death of trees if the rootstock-scion combination is susceptible. It has been found in other plant/virus combinations that transformation with partial or complete viral genes (e.g., coat protein genes) can confer resistance to the resulting transgenic plants. We previously reported A. tumefaciens mediated transformation and production of two sour orange (C. aurantium L.) plants expressing the coat protein gene of CTV, which was the first report of production of transgenic Citrus using a viral gene. However, in order to properly evaluate resistance, it is necessary to obtain as many transgenic Citrus plants from single transformation events as possible. Therefore, we are currently transforming grapefruit (Citrus paradisi) `Marsh' and `Star Ruby' and sweet orange (C. sinensis) `Valencia' with CTV coat protein genes. These species are susceptible to CTV and more amenable to transformation than sour orange. Epicotyl segments of etiolated seedlings were inoculated with A. tumefaciens strain EHA101 harboring binary plasmid pGA482GG containing the coat protein gene of mild Florida CTV strain T30 (CP-T30) or severe Florida strain T36 (CP-T36). Putatively transformed shoots were regenerated on selection medium containing kanamycin. Regenerated shoots were evaluated with GUS assays; those shoots positively identified by GUS were then evaluated with PCR. We have currently identified 17 `Marsh' grapefruit, 20 `Star Ruby' grapefruit, and seven sweet orange putatively transformed plants.
Elvia C. Palacios-Torres, M. Alejandra Gutièrrez-Espinosa, Gloria A. Moore, Gustavo Mora-Aguilera, Daniel L. Ochoa-Martínez, and Angel Villegas-Monter
Diane Luth and Gloria A. Moore
Many Citrus species accumulate large amounts of flavonoids, specifically flavanone glycosides, that impart an intensely bitter flavor to the fruit. In grapefruit, this bitterness decreases the acceptability of fresh fruit and juice; in other species, these compounds entirely prevent fruit consumption. No physiological purpose for the accumulation of these compounds has been determined; they do not function in color production or, as far as is known, in defense responses. As has been found in other plants, the accumulation of specific flavonoids in citrus appears to be under genetic control, but no definitive genetic analyses have been done. The long-term objective of this research is to determine whether the production of bitter-tasting flavanone glycosides (neohesperidosides) in citrus can be manipulated using molecular genetic techniques. As a first step, cDNAs for chalcone synthase and chalcone isomerase, the first two biosynthetic enzymes specific to the flavonoid pathway, were isolated from a grapefruit leaf cDNA library using heterologous probes. Southern analyses showed that both genes appear to be part of multigene families, as expected. Northern analyses are underway to determine steady state mRNA levels in various grapefruit tissues, and Western blots to characterize protein expression are also being attempted.
N.G. Beck, M.L. Arpaia, J.S. Reints Jr., and E.M. Lord
Deformations consisting of longitudinal ridges in the rind of Citrus fruits have recently been found in Southern California Citrus groves. Here, we report the correlation between ridge formation and applications of chlorpyrifos (Lorsban, Dow Chemical Company, Midland, MI) during the feather-growth stage of bud break. All chlorpyrifos formulations resulted in significant ridging. Addition of agricultural oil and 2,4-D (2,4-dichlorophenoxyacetic acid (2,4-D) to chlorpyrifos resulted in the greatest ridging damage and widened the window of susceptibility by 2 weeks in 1988. In 1989, no significant difference was seen between treatments of chlorpyrifos, although all were significantly greater than the control. The susceptible stages of bud growth are described, as are the non-susceptible stages which precede and follow it. Floral buds in which carpels are initiating are susceptible to fruit ridging upon application with chlorpyrifos. These ridges are the result of an increase in cell size of the flavedo tissue which may be the result of a polyploid chimera.
Catalina M. Anderson, William S. Castle, and Gloria A. Moore
Isozyme analysis was the basis for determining the frequency of occurrence and the characteristics of zygotic plants in Swingle citrumelo seedling populations from various sources of open-pollinated seeds, in a commercial nursery of Swingle citrumelo before and after roguing, and in commercial orchards and rootstock trials where this rootstock was used. Most zygotic seedlings identified by isozyme analysis could be distinguished by careful examination of morphological characteristics. Frequencies of zygotic seedlings varied among seedling populations, but were in the range (≈5% to 10%) found in previous studies. Roguing based primarily on size and growth habit of seedlings was effective in removing some, but not all, zygotic seedlings. Most of the remaining zygotic plants in the rogued population were found among the smaller seedlings. Trees budded on zygotic rootstock seedlings were found in two of the three groves studied, and in some instances an apparent incompatibility was developing in young trees.
T.G. McCollum and R.E. McDonald
Storage of `Marsh' white seedless grapefruit (Citrus paradisi Macf.) for 2 weeks at 5C resulted in the development of chilling injury (CI). Electrolyte leakage from chilled fruit did not increase significantly until CI had become severe, and was therefore considered to be of limited value as an early indicator of CI. In contrast to electrolyte leakage, respiration and ethylene evolution were significantly higher in chilled than in nonchilled fruit, even before the onset of visual symptoms of CI. Respiration rates ranged from ≈8 to 11 and 5 to 7 ml CO2/kg per hour in chilled and nonchilled fruit, respectively. Ethylene evolution was not detected from nonchilled fruit, whereas chilled fruit produced from 45 to 250 nl ethylene/kg per hour. Results of this study indicate that electrolyte leakage does not increase until visible pitting of the flavedo has occurred, whereas stimulation of respiration and ethylene evolution occur early in the development of CI.
E. Echeverria, J.K. Burns, and W.M. Miller
The effect of fruit temperature and fruit maturity on the development of blossom end clearing (BEC) in Florida grapefruit (Citrus paradisi Macf. vars. Ruby Red and Marsh) was investigated. Field and storage temperature studies indicated that development of BEC was directly associated with temperature; BEC increased when fruit temperature rose above 21 °C. Cooling fruit prior to packingline operations reduced BEC significantly. Older fruit were more susceptible to BEC than were younger fruit.
Peter D. Petracek, Wilfred F. Wardowski, and G. Eldon Brown
A postharvest peel disorder, morphologically similar to chilling injury (CI), was detected on nonchilled `Marsh' white grapefruit (Citrus paradisi Macf.). Like CI, the disorder was characterized by pitting of the peel caused by the collapse of oil gland clusters. This disorder is distinguished from CI in that pitting developed within the first 10 days of postharvest storage on fruit held at high (21.0C), but not low (4.5C), temperatures and on waxed fruit, but not unwaxed fruit. Pathogens isolated from pitted fruit were similar to those of nonpitted fruit. No preharvest pitting or visual clues of fruit susceptibility were observed.
Michael A. Maurer and Frederick S. Davies
Two field studies conducted from 1990 to 1991 evaluated the effects of reclaimed water on growth and development of 1- and 2-year-old `Redblush' grapefruit (Citrus paradisi Macf.) trees on Swingle citrumelo [Citrus paradisi (L.) Osb. ×Poncirus trifoliata (L.) Raf.] rootstock. Treatments were arranged as a3 (water sources) x 3 (irrigation levels) factorial at two locations on an Arredondo (well drained) and Kanapaha (poorly drained) fine sand near Gainesville, Fla. Irrigation treatments included 1) reclaimed water, 2) reclaimed water plus fertigation, and 3) well water plus fertigation. The reclaimed water was formulated to simulate that of a sewage treatment plant at Vero Beach, Fla. Irrigation was applied at 20% soil moisture depletion, or at 19 or 25 mm·week regardless of rainfall. In both experiments, visual ratings of tree vigor, and measured tree height and trunk diameter, were significantly lower for trees watered with reclaimed water without fertilizer than for the others in both years. Moreover, there was no fourth leaf flush in 1991 with reclaimed water. There was a significant increase in leaf Na, Cl, and B concentrations for the reclaimed water and reclaimed water plus fertigation treatments in 1990; however, in 1991 only leaf B concentrations showed a similar trend. In 1991, there were no significant differences in leaf Cl concentrations. Visual symptoms of N deficiency were observed by the end of the first season in trees grown with reclaimed water. Irrigation levels generallv did not affect tree growth.
Yong-Soo Hwang, D.J. Huber, and L.G. Albrigo
Cell wall composition and structure were examined in visually normal (N), granulated (G), and collapsed (VC) juice vesicles of `Marsh Seedless' grapefruit (Citrus paradisi) Macf.). According to gel-filtration data, VC appeared to be associated with a modification of water-soluble (WSP) and chelate-soluble (CSP) pectin molecular weight (Mr); small-Mr pectins increased, whereas large-J4. pectins decreased. The difference in M = of pectins did not appear to be mediated by polygalacturonases. Molecular weight of hemicelluloses did not differ. Granulated vesicles contained about two times more structural polysaccharides (pectins, hemicelhdose, and cellulose) than N vesicles, although hemicellulose and pectin M = modification were absent. Ion-exchange profiles of WSP, CSP, and hemicelhrlose fractions of VC and G vesicles were not different from those of N vesicles. Individual cells in vesicles with G and these vesicles themselves were much larger than those of N vesicles, whereas cells in VC were partially or completely collapsed.
Roy E. McDonald, William R. Miller, T.G. McCollum, and G. Eldon Brown
The fungicides thiabendazole (TBZ) or imazalil were applied at 1 g·liter-1 at 24 or 53C to `Marsh' and `Redblush' grapefruit (Citrus paradisi Macf.) to reduce fruit susceptibility to chilling injury (CI) and decay. Generally, there was more CI and decay on `Marsh' grapefruit than on `Redblush'. Severity of CI was lower in grapefruit that had been dipped at 53C than at 24C. Fruit dipped in fungicides had less CI than fruit dipped in water alone. Imazalil was more effective in reducing CI than TBZ. Fungicides reduced decay at both temperatures, and imazalil was better than TBZ. Chemical names used: 2-(4-thiazolyl)benzimidazole (thiabendazole, TBZ); 1-[2-(2,4-dichlorophenyl)-2-(2-propenyloxy)ethyl] -1H -imidazole (imazalil).