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
Zhigang Ouyang, Huihui Duan, Lanfang Mi, Wei Hu, Jianmei Chen, Xingtao Li, and Balian Zhong
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
Hong Chen, Greg McCollum, Elizabeth Baldwin, and Jinhe Bai
identification of Candidatus Liberibacter species associated with citrus huanglongbing J. Microbiol. Methods 66 104 115 Liao, H.L. Burns, J.K. 2012 Gene expression in Citrus sinensis fruit tissues harvested from huanglongbing-infected trees: Comparison with
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.
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.
Lynn Jo Pillitteri, Carol J. Lovatt, and Linda L. Walling
Homologues of the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) were isolated from the hybrid perennial tree crop `Washington' navel orange (Citrus sinensis) and designated CsLFY and CsAP1, respectively. Citrus has an extended juvenile period unlike herbaceous plants and responds to different floral stimuli than herbaceous plants or deciduous tree species. Despite these differences, the deduced amino acid sequences of CsLFY and CsAP1 genes are at least 65% identical with their Arabidopsis thaliana L. Heynh counterparts and share even greater sequence similarity to LFY and AP1 from the deciduous woody perennials, Populus balsamifera Bradshaw and Populus tremuloides Michaux, respectively. Like A. thaliana LFY (AtLFY) and AP1 (AtAP1), CsLFY and CsAP1 expression was restricted almost exclusively to reproductive tissues, but observed expression of CsAP1 in the fourth whorl carpel tissue of mature flowers was distinct from other plant AP1 genes. Transgenic A. thaliana plants ectopically expressing CsLFY or CsAP1 showed early-flowering phenotypes similar to those described for overexpression of AtLFY and AtAP1. In addition, the 35S:CsLFY and 35S:CsAP1 transgenes partially complemented the lfy-10 and ap1-3 mutants, respectively. The severity of the overexpression phenotypes correlated with the accumulation of CsLFY or CsAP1 transcripts. LFY is a single-copy gene in flowering plants but consistent with its hybrid origin, the genome of C. sinensis `Washington' has two easily distinguishable CsLFY and CsAP1 alleles derived from it's parental genotypes, C. maxima L. Osbeck (pummelo) and C. reticulata Blanco (mandarin). Allelic polymorphism at both the CsLFY and CsAP1 loci was restricted to the 5′- and 3′-flanking regions.
Juan I. Valiente and L. Gene Albrigo
Citrus flowering is a complex phenological process influenced by a number of interacting factors. Low winter temperatures are recognized as an important factor, but the flowering response has not been quantified under Variable natural conditions. A study was conducted to monitor the flower bud induction response of `Valencia' and `Hamlin' sweet orange trees [Citrus sinensis (L.) Osbeck] to naturally occurring winter weather conditions during the 1999 and 2000 seasons. The flowering response was quantified and related to shoot age, bud position along the shoot, local weather information, and crop load status. Results indicate that buds on previous summer shoots developed 2.52 and 3.59 to 1 flower on spring shoots, for `Hamlin' and `Valencia', respectively. In addition, buds at apical positions produced more flowers than buds located far from the apex. These basal positions buds required higher induction levels. Under Florida conditions, greater accumulation of hours of temperatures 11 to 15 °C increased floral intensity by the combined effect on the number of sprouting buds with reproductive growth and the number of flowers per flowering bud. Some statistical analyses indicated that high winter temperatures reduced flowering in `Valencia' and `Hamlin' oranges. The presence of fruit consistently reduced reproductive response for both cultivars. Crop load reduced flowering by an average of 41.5% compared to no crop and varied by cultivar. A discussion on the different induction requirements as well as on the differential effect of crop load on flowering by cultivar is presented.
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.
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
Eduardo Esteves, Gabriel Maltais-Landry, Flavia Zambon, Rhuanito Soranz Ferrarezi, and Davie M. Kadyampakeni
The bacterial disease Huanglongbing (HLB) has drastically reduced citrus production in Florida. Nutrients play an important role in plant defense mechanisms and new approaches to manage the disease with balanced nutrition are emerging. Nutrients like nitrogen (N), calcium (Ca), and magnesium (Mg) could extend the productive life of affected trees, although interactions among these nutrients in HLB-affected citrus trees are still unclear. A 2-year study was established in Florida to determine the response of HLB-affected trees to applications of N, Ca, and Mg. The study was conducted with ‘Valencia’ trees (Citrus sinensis L. Osbeck) on Swingle citrumelo (Citrus paradisi Macf. × Poncirus trifoliata L. Raf.) rootstock on a Candler sand. Applications of N at 168, 224 (recommended rate), and 280 kg⋅ha−1 N were used as the main plots. Split-plots consisted of a grower standard treatment receiving only basal Ca (51 kg⋅ha−1) and Mg (56 kg⋅ha−1); supplemental Ca (total Ca inputs: 96 kg⋅ha−1) only; supplemental Mg (total Mg inputs: 101 kg⋅ha−1) only; and supplemental Ca (total Ca inputs: 73.5 kg⋅ha−1) and Mg (total Mg inputs: 78.5 kg⋅ha−1). The following variables were measured: tree size, fruit yield, and juice quality. Although some differences in tree growth among treatments were statistically significant (e.g., greater canopy volume with Mg fertilization at 168 kg⋅ha−1 N), there was no clear and consistent effect of plant nutrition on these variables. Fruit yield was higher with Ca and Mg relative to the grower standard at the lowest N rate in 2020, and there were no other statistically significant differences among treatments. Juice acidity was significantly higher with Mg fertilization relative to other treatments in 2019. As N rates had no significant effect in this study, unlike secondary macronutrients, N rates could potentially be reduced to 168 kg N⋅ha−1 in HLB-affected citrus without affecting vegetative growth, fruit yield, and juice quality. However, this will require optimizing the supply of secondary macronutrients and all other nutrients to develop a balanced nutritional program. Ultimately, the effects of N, Ca, and Mg obtained in this 2-year study should be confirmed with longer-term studies conducted at multiple sites.