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A new mandarin cultivar currently designated as selection LB8-9 [‘Clementine’ mandarin (Citrus reticulata) × ‘Minneola’ tangelo, Duncan grapefruit (Citrus paradisi) × Dancy tangerine (C. reticulata)], soon to be released by the University of Florida, Citrus Research and Education Center, Lake Alfred, has been evaluated for postharvest fruit quality and consumer acceptance. Comparisons were made with ‘Sunburst’ and ‘Minneola’ mandarins, which overlap the early and late LB8-9 maturity season, respectively. LB8-9 and ‘Sunburst’ fruit stored 2 weeks at 70 °F developed similar decay percentages (35% to 37%), while ‘Minneola’ showed only 16% decay. Postharvest pitting incidence was zero in LB8-9 and ‘Minneola’ and 3% in ‘Sunburst’ stored at 70 °F and 92% to 96% relative humidity (RH). At the same storage conditions, LB8-9 and ‘Sunburst’ developed better fruit peel color (hue = 60) in comparison with the ‘Minneola’ (hue = 65) mandarin. No differences were found in fruit external peel color (hue angle), chilling injury, or decay among three cultivars stored at 40 °F after 6 weeks. However, juice color was the best in ‘Sunburst’ as indicated by the highest color numbers (44), followed by LB8-9 (40) and ‘Minneola’ (38). Wax-formulation studies indicated that carnauba wax was the best formulation for coating LB8-9 because of low decay incidence, weight loss, and good color. No difference was found in fruit taste panels at day 6 after packing, while a better score of acceptance in the fruit taste panel was recorded for LB8-9 than ‘Minneola’ after 50 d of storage at 40 °F and 92% to 96% RH. LB8-9 fruit had a higher soluble solids concentration [SSC (14.0)] and acid (1.12) than ‘Minneola’ or ‘Sunburst’ mandarins, both having a SSC and acid lower than 11.5 and 0.86, respectively. Sucrose concentration was noticeably higher in the new cultivar (7.14 g/100 mL) than in ‘Minneola’ (5.27 g/100 mL) or ‘Sunburst’ (6.10 g/100 mL). Vitamin C concentration was 42.43 mg/100 mL for the new cultivar, which was considerably higher than ‘Minneola’ (23.27 mg/100 mL) or ‘Sunburst’ (26.25 mg/100 mL). Overall, LB8-9 has good potential as a new fresh fruit for the consumer, and no serious problems were noted with typical postharvest handling.

Although no longer as glamorous as it was a few decades past, the routine application of embryo rescue techniques, leading to plant recovery, is a valuable tool for citrus cultivar improvement. Embryo rescue approaches can be used to generate useful variation or to capture various kinds of spontaneous genetic variation. Embryo rescue, by in vitro culture of undeveloped, and presumably unfertilized, ovules in colchicine-supplemented media is a practical method of producing tetraploid clones, which are used then in crosses with diploids to produce seedless triploid hybrids. This same approach, i.e., in vitro culture of undeveloped ovules, is also used to recover plants from chimeric sectored fruit exhibiting economically important mutations for fruit characteristics, and for producing potentially variant somaclones. Seedlessness is an important objective for fresh citrus fruit cultivar improvement, and triploidy following 2x × 4x hybridizations is one approach being exploited for this objective. When monoembryonic diploid seed parents are crossed with tetraploid pollen parents, however, normal seed development is not usually possible. Embryos must be excised from abortive seeds fairly early in development and cultured appropriately to ensure the recovery of sufficient numbers of 3x offspring from these crosses, to increase the likelihood of identifying superior seedless hybrids. These applications will be described in some detail, and progress toward breeding objectives are highlighted.

Citrus tristiza virus (CTV) is the most-significant viral pathogen of citrus in the world. Rapid decline of trees on sour orange and stem pitting of grapefruit and sweet orange, two diseases induced by CTV, severely jeopardize citrus production worldwide. It is recognized that all future rootstocks should be resistant to this virus, and scion resistance to stem pitting stains is desirable. To facilitate introgression of the CTV resistance gene from Poncirus trifoliata and development of CTV-resistant varieties in citrus, gene mapping projects have been initiated and more than a dozen RAPD markers have been identified with tight linkage to the resistance gene. As part of our efforts to use marker-assisted selection with a large number of crosses, and ultimately to accomplish map-based cloning of the CTV resistance gene, we have been converting the most tightly linked RAPD markers into SCAR (sequence characterized amplified region) markers by cloning, sequencing the marker fragments, and designing locus-specific primers. One codominant and several dominant SCARs have been developed thus far. The updated progress and utilization of these SCARs in marker-assisted selection and possibly in characterization of a BAC library will be presented and discussed.

Citrus genetic studies and cultivar improvement have been difficult with conventional techniques. Alternative approaches are needed to enhance efficiency of such studies. Our objectives were to characterize the Citrus genome and to initiate development of a linkage map using RFLP and isozyme analysis. Methods of Citrus DNA extraction were developed to allow the isolation of chromosomal DNA of acceptable quality for recombinant' DNA manipulations. A PstI Citrus genomic library was constructed to create DNA clones for the RFLP survey. A rapid, reliable procedure was developed to facilitate screening of the library for useful clones. The methods used and strategy followed minimized contamination with organelle DNA, increased the frequency of single copy clones, and allowed rapid screening of the newly–constructed library. Linkage relationships of 49. markers, including 36 RFLP and 6 isozyme loci, were analyzed and a map comprised of 8 linkage groups was constructed. Insertions or deletions were responsible for at least 30% of the RFLPs identified. A hypothesis of transposon activity in Citrus was proposed based on our observations.

A family of 63 citrus intergenemic backcross hybrids was used for this study. The parents and hybrids were multiplied by rooted cuttings, with 6 uniform replicates selected per hybrid, and each plant was inoculated with citrus nematodes (Tylenchulus semipenetrans) 5 times over 2 mo. The number of nematode female larvae per gram of fine fresh root was determind 2 mo after the last inoculation. The phenotypic variation of the hybrids was continuous and wide-ranged, from 8.0 females· g^-1 of root tissue (resistant parent Swingle citrumelo=15.6) to 620.0 females· g^-1 of root tissue (susceptible parent LB 6-2=540.5). Bulked segregant analysis (BSA), using RAPD fragments, was conducted with 2 DNA bulks of individuals from the extremes of the phenotypic distribution. Three hundred twenty primers were screened and 5 were found to generate repeatedly single RAPD fragments specific to the resistant bulk. The segregation of resistance-associated fragments among the individuals was examined, and the linkage between these markers and potential nematode resistance loci was estimated.

Mapping quantitative trait loci (QTL) associated with freeze tolerance was accomplished using a Citrus grandis (L.) Osb. × Poncirus trifoliata (L.) Raf. F₁ pseudo-testcross population. A progeny population of 442 plants was acclimated and exposed to temperatures of -9 °C and -15 °C in two separate freeze tests. A subpopulation of 99 progeny was genotyped for random amplified polymorphic DNA (RAPD), cleaved amplified polymorphic sequence (CAPS), sequence characterized amplified region (SCAR), and sequence tagged site (STS) markers to produce a linkage map for each parent. Potential QTL were identified by interval mapping, and their validity was corroborated with results from means comparison (t test), one-way analysis of variance (F test), and bulked segregant analysis (BSA). Multiple analytical methods provided evidence supporting putative QTL and decreased the probability of missing significant QTL associated with freeze tolerance. QTL with a large effect on freeze tolerance were located on both the Citrus and Poncirus linkage maps. In addition, clusters of markers with significantly different means between marker present and absent classes indicating minor QTL that contribute smaller effects on the level of tolerance were found on the linkage maps of both species.

Most of the commercially important citrus scion cultivars are susceptible to Huanglongbing (HLB), which is the most devastating disease the citrus industry has ever faced. Because the rootstock can influence the performance of the scion in various ways, including disease and pest tolerance, use of superior rootstocks can assist citrus growers with minimizing the negative effects of HLB. The objective of this study was to assess rootstock effects on the horticultural performance and early production potential of ‘Hamlin’ sweet orange (Citrus sinensis) trees in commercial field settings under HLB-endemic conditions. Two field trials were conducted in different locations in Central and Southeast Florida. The trials were established in 2015 and included 32 diverse diploid and tetraploid rootstock cultivars and advanced selections. One trial was performed in Highlands County, FL, on a poorly drained flatwoods-type site. Another trial was performed in Polk County, FL, on a well-drained sandy Central Florida Ridge site. Horticultural traits including tree height, canopy volume, trunk diameter, canopy health, leaf nutrient content, yield, and fruit quality were assessed during the 2018–19 and 2019–20 production years. Significant differences were found among trees on different rootstocks for most of the measured traits, particularly tree vigor and productivity, but rootstock effects also varied by location. Rootstocks that induced large tree sizes, such as the diploid mandarin × trifoliate orange hybrids ‘X-639’, ‘C-54’, ‘C-57’, and ‘C-146’, also induced higher yield, but with lower yield efficiency. Most of the tetraploid rootstocks significantly reduced tree size, among which ‘Changsha+Benton’, ‘Green-3’, ‘Amb+Czo’, ‘UFR-3’, and ‘UFR-5’ induced high yield efficiency. Therefore, these rootstocks have the potential to be used in high-density plantings. However, trees on some of these small size-inducing rootstocks had a higher mortality rate and were more vulnerable to tropical force winds. This study provides important information for the selection of rootstocks with the greatest production potential in an HLB-endemic environment, especially during the early years of production.

Somatic hybridization is a powerful tool for the genetic improvement of citrus rootstocks, and it is part of an efficient in vitro-based breeding system described here. An essential component of the system is the requirement of confirming tetraploidy and the combination of the two donor genomes. Expressed sequence tag–simple sequence repeat (EST-SSR) markers provide a means to accomplish both of these objectives, and their application to a population of pummelo [Citrus grandis (L.) Osbeck] + mandarin (C. reticulata Blanco) somatic hybrids developed for the specific purpose of providing alternative rootstocks for sour orange (Citrus aurantium L.) is detailed. Nineteen new somatic hybrids were produced from various mandarin and pummelo parents, and their ploidy level and the complementation of their nuclear genomes were confirmed using four EST-SSR markers. These markers were selected from markers previously mapped in sweet orange [C. sinensis (L.) Osbeck] and trifoliate orange [Poncirus trifoliata (L.) Raf.] and prescreened for suitable allelic polymorphism within the mandarin and pummelo lines used. After polymerase chain reaction amplification of sequences from the parents and putative hybrids, the products were separated on a genetic sequencer and visualized electronically. Additionally, EST-SSR markers identified the unexpected zygotic origin of a presumed nucellar embryogenic callus line. Integration of EST-SSR techniques for high-throughput genotyping with previously developed approaches to somatic hybrid creation increases substantially the effectiveness and efficiency of this in vitro-based breeding system for citrus rootstock improvement.

Completely enclosed screen houses can physically exclude contact between the asian citrus psyllid [ACP (Diaphorina citri)] and young, healthy citrus (Citrus sp.) trees and prevent huanglongbing (HLB) disease development. The current study investigated the use of antipsyllid screen houses on plant growth and physiological parameters of young ‘Ray Ruby’ grapefruit (Citrus ×paradisi) trees. We tested two coverings [enclosed screen house and open-air (control)] and two planting systems (in-ground and container-grown), with four replications arranged in a split-plot experimental design. Trees grown inside screen houses developed larger canopy surface area, canopy surface area water use efficiency (CWUE), leaf area index (LAI) and LAI water use efficiency (LAIWUE) relative to trees grown in open-air plots (P < 0.01). Leaf water transpiration increased and leaf vapor pressure deficit (VPD) decreased in trees grown inside screen houses compared with trees grown in the open-air plots. CWUE was negatively related to leaf VPD (P < 0.01). Monthly leaf nitrogen concentration was consistently greater in container-grown trees in the open-air compared with trees grown in-ground and inside the screen houses. However, trees grown in-ground and inside the screen houses did not experience any severe leaf N deficiencies and were the largest trees, presenting the highest canopy surface area and LAI at the end of the study. The screen houses described here provided a better growing environment for in-ground grapefruit because the protective structures accelerated young tree growth compared with open-air plantings while protecting trees from HLB infection.

Huanglongbing (HLB) disease is a threat to most citrus (Citrus sp.) producing areas and is associated with the bacterium Candidatus Liberibacter asiaticus. The disease is transmitted by the vector asian citrus psyllid [ACP (Diaphoria citri)]. Antipsyllid screen houses can potentially reduce and eliminate HLB development in young citrus plantings by excluding the insect vector. These structures are also anticipated to represent a new environmental platform to cultivate high-valued fresh citrus. The purpose of this investigation was to evaluate the effect of screen houses on excluding infective ACP from inoculating grapefruit (Citrus ×paradisi) trees and determine changes on environmental conditions caused by the screen cloth. We tested two coverings [enclosed screen house and open-air (control)] and two planting systems (in-ground and container-grown), with four replications arranged in a split-plot experimental design. Psyllid counting and HLB diagnosis were performed monthly, and the antipsyllid screen excluded the HLB vector from the houses. ACP and HLB-positive trees were found only at the open-air plots. Weather monitoring was performed every 30 minutes from 22 Feb. to 31 July 2014. Solar radiation accumulation averaged 6.7 W·m⁻²·minute⁻¹ inside the screen houses and 8.6 W·m⁻²·minute⁻¹ in the open-air. Air temperature was greater inside the screen houses whereas wind gusts were higher in the open-air. Reference evapotranspiration accumulation averaged 3.2 mm·day⁻¹ inside the screen houses and 4.2 mm·day⁻¹ in the open-air. There was no difference in cumulative rainfall between screen houses and open-air. The antipsyllid screen houses reduced solar radiation, maximum wind gust, and reference evapotranspiration (ET_o). The environmental conditions inside the protective screen houses are suitable for grapefruit production.