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E.S. Louzada, H.S. del Rio, D. Xia, and J.M. Moran-Mirabal

Large-scale production of microprotoplasts from `Ruby Red' grapefruit (Citrus paradisi Macf.) and from the Citrus L. sp. relative Swinglea glutinosa (Blanco) Merr., was performed after treatment of suspension cells with APM. An average of 75.2% of the microprotoplasts contained a single chromosome, followed by 17.1% with two, 4.6% with three, and 2.0% with four. Only 1.1% had more than five chromosomes. Maximum chromosome number observed was eight and the average yield was 2 × 106 of total microprotoplasts per gram of suspension cells. Flow cytometry analysis confirmed low DNA content. The polyethylene glycol fusion method was used to fuse microprotoplasts from `Ruby Red' grapefruit with protoplasts of `Succari' sweet orange [Citrus sinensis (L.) Osbeck], and microprotoplasts from S. glutinosa with protoplasts from sour orange (C. aurantium L.). Embryos or suspension cells from the recipient species with a few additional chromosomes were obtained; however, embryogenesis of the fusion products was reduced or inhibited. Chemical name used: amiprophos-methyl (APM).

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John L. Jifon, James P. Syvertsen, and Eric Whaley

Correlations between extractable leaf chlorophyll (Chl) concentration and portable, nondestructive leaf “greenness” meter readings imply that such estimates can be used as surrogate measurements of leaf nitrogen (N) status. However, few studies have actually found a direct relationship between Chl meter readings and leaf N. We evaluated the utility of two handheld transmittance-based Chl content meters (SPAD-502, Minolta Corp. and CCM-200, Opti-Sciences) and one reflectance-based meter (Observer, Spectrum Technologies), in estimating Chl and N concentrations in intact leaves of several citrus cultivars. Total Chl determined analytically, correlated well with nondestructive Chl meter readings (r 2: 0.72 to 0.97; P < 0.0001), but regression models differed among cultivars using the same meter and also among meters for a given cultivar. The relationships were generally more linear and stronger at low Chl concentrations (<0.5 mmol·m-2) than at higher Chl concentrations, reflecting increased variability in Chl meter readings with increasing leaf Chl. Significant relationships between Chl meter readings and measured leaf N concentrations were also found in all the cultivars tested (r 2: 0.23 to 0.69; P < 0.01), but the data were more variable than those for Chl. Field-grown leaves were significantly thicker and had higher Chl meter readings than greenhouse-grown leaves of similar Chl or N concentrations. The results suggest that nondestructive Chl content meters can overestimate Chl and N in thicker leaves and/or leaves with high Chl concentrations. A single prediction equation derived from a wide range of Chl or N concentrations could be applicable across the range of citrus cultivars when grown in the same environment. Potential limitations associated with leaf thickness as influenced by environmental factors may necessitate the development of more specific calibration equations.

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Kim D. Bowman and Frederick G. Gmitter Jr.

A diverse population of grapefruit-like Citrus growing in Saint Lucia (West Indies), called forbidden fruit, was examined as a potential germplasm source for Citrus genetic improvement. Four clones from this population were studied by leaf isozyme analysis, and a distinct resemblance between forbidden fruit and grapefruit (C. × paradisi Macfady.) was observed at several loci, including identical banding patterns for peroxidase, phosphoglucose mutase, phosphohexose isomerase, and shikimic acid dehydrogenase. These results support morphological and historical indications of a close taxonomic relationship between modern grapefruit cultivars and Caribbean forbidden fruit. Comparison of isozyme allele segregation among seedlings of several forbidden fruit clones and grapefruit cultivars demonstrated a much higher degree of zygotic embryony in the former. Morphological diversity and zygotic embryony in the Caribbean forbidden fruit population may make it a useful genetic resource for breeding grapefruit and other Citrus species.

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Milton E. Tignor Jr. and Courtney A. Weber

Current efforts in the study of citrus freeze hardiness including gene mapping and elucidating early induction processes require large populations of uniform seedlings. Related genera and intergeneric hybrids are often used in these studies and little is known about factors effecting their seedling emergence. We tested a total of 8 genotypes including Poncirus trifoliata `Rubidoux', Citrus grandis, C. sinensis `Pineapple', C. jambhiri `Schaub', C. paradisi `Duncan', C. aurantium (Brazilian), Carrizo citrange (P. trifoliata × C. sinensis), and Troyer citrange. A total of seven pre-planting treatments were used to evaluate seedling emergence rates. Expanding on the work of previous researchers, treatments were seed coat removal, hydrating in water (96 hours) at either 4, 25, or 35°C, acid scarification, or boiling. Generally, seed coat removal resulted in the most uniform emergence as compared to untreated controls. Presoaking at each temperature enhanced emergence in most varieties tested and 25°C was the best hydrating temperature. Acid scarification greatly delayed emergence in all genotypes tested except Troyer citrange and `Pineapple' orange which had enhanced emergence rates as compared to controls. Preplanting treatment with 100°C water was lethal in all varieties. Pretreatment of citrus seeds can enhance uniformity of germination, although optimum treatments for individual genotypes vary.

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S. Verdejo-Lucas, F.J. Sorribas, J.B. Forner, and A. Alcaide

The response of 52 citrus hybrid rootstocks to a Mediterranean biotype of Tylenchulus semipenetrans Cobb was determined in greenhouse tests. Seven selections of the cross `Cleopatra' mandarin [Citrus reshni Hort. × Poncirus trifoliata (L.) Raf], and one of Citrus volkameriana Pasq. × P. trifoliata did not support nematode reproduction and were considered as highly resistant to the citrus nematode. The nematode showed very low infectivity and reproductive potential on seven additional selections of `Cleopatra' mandarin × P. trifoliata, one of `King' mandarin × P. trifoliata, and two C. volkameriana × P. trifoliata. These selections were considered as nematode resistant. All the selections with `Troyer' citrange [Citrus sinensis (L). Osbeck × P. trifoliata (L.) Raf] in their parentage supported nematode reproduction but showed different levels of susceptibility.

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Frederick S. Davies and Glenn R. Zalman

Several studies suggest that optimum N rate and application frequency differ among citrus rootstocks. `Rhode Red' valencia orange trees [Citrus sinensis (L.) Osb.] on three rootstocks, C. volkameriana Ten. and Pasq., `Carrizo' citrange [C. sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.], and `Swingle' citrumelo [C. paradisi Macf. × P. trifoliata (L.) Raf.], were used to determine if N rate and application frequency should be adjusted, based on rootstock, during the first 3 years in the field. Treatments were arranged in a 3×3×3 (rootstock, N rates, N application frequency, respectively) factorial experiment. Annual N application rates ranged from 68 to 272 g/tree depending on tree age, and N was applied biweekly, weekly or monthly. Application frequency had no effect on trunk diameter or leaf N concentration in any year. Rootstock had a significant effect on growth in all 3 years, with trees on C. volkameriana being largest and having the greatest yields, followed by those on `Carrizo' and `Swingle', respectively. Trees on C. volkameriana were larger than those on the other rootstocks because they were larger at planting, grew over a longer period during the year, and often grew at a faster rate. Nitrogen rate had no effect on growth during the first 2 years in the field, but the highest N rate increased yields in year 3 for trees on C. volkameriana and `Swingle' rootstocks. Interaction between rootstock and N rate was nonsignificant for trunk diameter, but it was significant for yield, suggesting that trees on C. volkameriana responded more to increased N than did those on the other rootstocks.

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H.K. Wutscher and K.D. Bowman

Twenty-one selections consisting of 13 numbered hybrids, one ornamental, and seven named cultivars were tested as rootstocks for `Valencia' orange, Citrus sinensis L. Osbeck. The test included six, four-tree replications in randomized complete blocks on sandy soil typical of the center of the Florida peninsula. Trees propagated on Vangasay lemon, HRS 812 (Sunki × Benecke trifoliate orange), and HRS 942 (Sunki × Flying Dragon trifoliate orange) produced more fruit than trees on the other 18 rootstocks in the test. Trees on 10 rootstocks, including the widely used commercial rootstocks, Swingle citrumelo and Carrizo citrange, were intermediate in cumulative fruit production. Trees on five rootstocks, including Sun Chu Sha, Gou Tou #1, and Tachibana, had low yields and trees on HRS 939 (Flying Dragon trifoliate orange × Nakorn pummelo) and sour orange #2 were extremely dwarfed and were minimally productive because of tristeza virus disease. Fouryear cumulative fruit production ranged from 52 to 317 kg per tree. Fruit from trees on HRS 954 and HRS 952 (Pearl tangelo × Flying Dragon trifoliate orange) had the highest, and fruit from trees on Vangasay and Gou Tou #1 had the lowest total soluble solids concentration.

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Richard Bourgault, J. Derek Bewley, Aurelia Alberici, and Delphine Decker

High amounts of endo-β-mannanase (EC activity were extracted from tomato (Lycopersicon esculentum Mill.) fruits when a high-salt-containing buffer was used. Two pI forms of the fruit enzyme were identified, one being much more basic than the many seed isoforms. The number of isoforms increased if a protease inhibitor was not used during extraction. The enzyme was found in the ripe fruits of many other species, and was particularly active in those of muskmelon (Cucumis melo L. Cantalupensis Group) and watermelon [Citrullus lanatus (Thunb.) Matsum. and Nak.]. In most fruits, enzyme activity was localized in the skin and the epidermal and subepidermal regions. The enzyme in several fruits had a molecular weight of ≈40,000 and reacted immunologically with the tomato seed endo-β-mannanase antibody.

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Paul J.R. Cronje, Graham H. Barry, and Marius Huysamer

., Rockville, MD El-Otmani, M. Ait-Oubahou, A. Zacarías, L. 2011 Citrus sp.: Orange, mandarin, tangerine, clementine, grapefruit, pomelo, lemon and lime, p. 467–474. In: Yahia, E.M. (ed.). Postharvest biology and technology of tropical and subtropical fruits

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Antonios E. Tsagkarakis, Michael E. Rogers, and Timothy M. Spann

applications to Citrus sp. on the reproductive biology of the asian citrus psyllid. The second experiment was conducted from Jan. to Feb. 2010 and was carried out in a custom-built walk-in growth chamber (Mechanical Refrigeration, Winter Haven, FL) to provide