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Robert C. Ebel, Said Hamido, and Kelly T. Morgan

impact Citrus sinensis cv. Valencia affected by HLB. Because it is impossible to conduct studies with trees not infected with C Lasiaticus under commercial conditions, this study was conducted under greenhouse conditions where psyllid vectors could be

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Zhigang Ouyang, Huihui Duan, Lanfang Mi, Wei Hu, Jianmei Chen, Xingtao Li, and Balian Zhong

at the functional characterization of these YTH genes and their utilization for genetic improvement. Materials and Methods Plant materials. Citrus sinensis (L.) Osbeck cv. Gannan Zao was used for expression assays. ‘Gannan Zao’ navel orange seedlings

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Hong Chen, Greg McCollum, Elizabeth Baldwin, and Jinhe Bai

-year-old ‘Hamlin’ and ‘Valencia’ orange trees [ C. sinensis (L.) Osbeck] on ‘Swingle’ citrumelo [ Citrus paradisi Macf. × Poncirus trifoliata (L.) Raf.] rootstock, in a commercial grove located in Vero Beach, FL, were selected for this experiment. The trees

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Ahmad A. Omar, Wen-Yuan Song, James H. Graham, and Jude W. Grosser

Citrus canker disease caused by the bacterial pathogen Xanthomonas axonopodis pv. citri is becoming a worldwide problem. Xa21 gene is a member of the Xa21 gene family of rice, which provides broad spectrum Xanthomonas resistance in rice. `Hamlin' sweet orange [Citrus sinensis (L.) Osbeck) is one of the leading commercial cultivars in Florida because of its high yield potential and early maturity. `Hamlin' also has a high regeneration capacity from protoplasts and is often used in transformation experiments. Since the citrus canker pathogen is in the same genus, this gene may have potential to function against canker in citrus. The wild-type Xa21 gene contains an intron, and there are some questions whether dicot plants can process genes containing monocot introns (the cDNA is intron-free). Plasmids DNA, encoding the non-destructive selectable marker EGFP (Enhanced Green Fluorescent Protein) gene and the cDNA of the Xa21 gene were transformed or co-transformed into `Hamlin' orange protoplasts using polyethylene glycol. More than 200 transgenic embryoids were recovered. More than 400 transgenic plants were developed from 75 independent transgenic events. PCR analysis revealed the presence of the cDNA of the Xa21 and the GFP genes in the transgenic plants. Some of the plants have the GFP only. Southern analysis is showing integration of the cDNA into different sites ranges from one to five sites. Western analysis is showing the expression of the cDNA of the Xa21 gene in the transgenic citrus plants. This is the first time that a gene from rice has been stably integrated and expressed in citrus plants. Canker challenge assay is in progress.

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Lynn Jo Pillitteri, Carol J. Lovatt, and Linda L. Walling

and provided the C. reticulata `Fairchild' DNA and template for the production of the prenyltransferase-stimulating protein probe. We also thank Dr. Tracy Kahn (Citrus Variety Collection, UC Riverside) for donating fruit from the “seedy” navel

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Mohammad R. Karim, Glenn C. Wright, and Kathryn C. Taylor

A field trial conducted at Yuma, Ariz., examined the effect of foliar boron application on fruit yield and quality of Citrus sinensis cv. Hamlin. Boron was applied to 5-year-old trees at five treatment levels (0, 500, 1000, 2000, and 3000 ppm) before or after flowering in a split plot design. At harvest, fruit number, size and quality were determined. Yield (P = 0.01) and average fruit number per tree (P = 0.02) were different among treatments. The highest yield was obtained with the 500 ppm treatment. In this first year of the trial there was no difference in average fruit weight, fruit pH, titratable acidity, peel thickness, juice volume, or soluble solid content of fruit between the treatments. Previous studies indicate that boron influenced in vivo and in vitro pollen germination in many crops. Increased fruit yield may have occurred because boron was transported to the flowers where it exerted its influence on increased fruit set through an effect on pollen viability or pollen tube growth. Further investigation of this hypothesis is underway.

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Elvia C. Palacios-Torres, M. Alejandra Gutièrrez-Espinosa, Gloria A. Moore, Gustavo Mora-Aguilera, Daniel L. Ochoa-Martínez, and Angel Villegas-Monter

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.

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Raquel L. Boscariol, Mariza Monteiro, Elizabete K. Takahashi, Sabrina M. Chabregas, Maria Lucia C. Vieira, Luiz G.E. Vieira, Luiz F.P. Pereira, Francisco de A.A. Mourão Filho, Suane C. Cardoso, Rock S.C. Christiano, Armando Bergamin Filho, Janaynna M. Barbosa, Fernando A. Azevedo, and Beatriz M.J. Mendes

Citrus canker, caused by Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al., is one of the main problems affecting citrus production. In order to obtain resistance to phytopathogenic bacteria, insect genes, coding for antimicrobial proteins, have been used in plant genetic transformation. In this study, transgenic Citrus sinensis (L.) Osb. `Hamlin' plants expressing the antimicrobial insect-derived attacin A gene (attA) were obtained by Agrobacterium tumefaciens (Smith and Towns.) Conn-mediated transformation. Initially, the cDNA clone was used to construct a binary plasmid vector (pCattA 2300). The construction included the native signal peptide (SP) responsible for directing the insect protein to the extracellular space where bacteria is supposed to accumulate in vivo. In order to investigate the native SP effectiveness in a plant model system, onion (Allium cepa L.) epidermal cells were transformed, via biobalistics, using plasmids containing the attA gene with or without SP, fused with the green fluorescent protein gene (pattA 1303 and pSPattA 1303). Fluorescence accumulation surrounding the cells was observed only in tissues transformed with the plasmid containing the gene with SP, indicating the protein secretion to the apoplast. Citrus transformation was confirmed by PCR and Southern blot hybridization analysis in 12 regenerated plants. Transcription of attA gene was detected by Northern blot analysis in all transgenic plants. Eight selected transgenic lines were propagated and inoculated with a 106 cfu/mL suspension of the pathogen X. axonopodis pv. citri. Compared to control (non-transformed plant), seven transgenic lines showed a significant reduction in susceptibility to citrus canker. The results obtained here indicate the potential use of antibacterial proteins to protect citrus from bacterial diseases.

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Juan I. Valiente and L. Gene Albrigo

by the Florida Agricultural Experiment Station and was partially funded by a grant from the Florida Citrus Production Research Advisory Council (FCPRAC) and reports part of the PhD dissertation work of the senior author and was approved for

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Naveen Kumar and Robert C. Ebel

abscission in tomato ( Lycopersicon esculentum Mill.) fruit ( Djanaguiraman et al., 2004 ). The objective of this study was to explore the role of CMNP-induced oxidative metabolism in AZ of ‘Valencia’ sweet orange [ Citrus sinensis (L.) Osbeck] and to