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  • Author or Editor: Jude Grosser x
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Protoplast culture following polyethylene glycol (PEG)-induced fusion resulted in the regeneration of somatic hybrid plants from the following combinations: `Succari' sweet orange (C. sinensis L. Osbeck) + `Ponkan' mandarin (C. reticulata Blanco), `Succari' sweet orange + `Dancy' mandarin (C. reticulata), `Succari' sweet orange + `Page' tangelo [a sexual hybrid between `Minneola' tangelo (C. reticulata × C. paradisi Mcf.) × `Clementine' mandarin (C. reticulata)], `Valencia' sweet orange (C. sinensis) + `Page' tangelo. `Succari' and `Valencia' protoplasts were isolated -from ovule-derived embryogenic cell suspension cultures and from seedling leaves for the other parents. Somatic hybrid plants were Identified on the basis of leaf morphology and electrophoretic analysis of isozyme banding patterns. Root tip cell chromosome counting is being performed on all plants. Other putative somatic hybrids Include: `Succari' sweet orange + `Minneola' tangelo; `Succari' sweet orange + `Murcott' tangos (C. sinensis × C. reticulata); `Valencia' sweet orange + `Murcott' tangor; and `Valencia' sweet orange + `Dancy' mandarin. These plants may have direct cultivar potential, but there primary use will be for interploid hybridization with selected monoembryonic scions to produce improved seedless triploids.

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Protoplast culture following polyethylene glycol (PEG)-induced fusion resulted in the regeneration of somatic hybrid plants from the following combinations: `Succari' sweet orange (C. sinensis L. Osbeck) + Severinia disticha; `Hamlin' sweet orange (C. sinensisj + S. disticha: `Valencia' sweet orange (C. sinesis) + S. disticha; `Nova' tangelo (C. reticulata hybrid) + S. disticha; `Succari' sweet orange + S. buxifolia; `Nova' tangelo + Citropsis gilletiana; and `Succari' sweet orange + Atlantia ceylanica. `Succari', `Hamlin', `Valencia', and `Nova' protoplasts were Isolated from ovule-derived embryogenic callus and/or suspension cultures whereas protoplasts of S. disticha, S. buxifolia, C. gilletiana, and A. ceylanica were isolated from leaves of potted trees in a greenhouse. Plants were regenerated via somatic embryogenesis and somatic hybrids were identified on the basis of leaf morphology. Electrophoretic analysis of isozyme banding patterns and root tip chromosome counts are being performed. Somatic hybrids with S. disticha are apparently weak whereas the other somatic hybrid plants with S. buxifolia, C. gilletiana, and A. ceylanica exhibit adequate vigor. These are more examples that the the techique of protoplast fusion can be an important tool in overcoming barriers to hybridization of sexually incompatible species.

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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.

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Protoplasm culture following polyethylene glycol (PEG) -induced fusion resulted in the regeneration of somatic hybrid plants from the following six parental combinations: Citrus sinermis (L.) Osbeck cv. Hamlin + Severinia buxifolia (Poir.) Tenore (Chinese box-orange); C. reticulate Blanco cv. Cleopatra + Poncirus trifoliata (L.) Raf. cv. Flying Dragon; C. reticulate cv . Cleopatra + Swingle citrumelo (C. paradisi Macf. × P. trifoliata); C. sinensis cv . Hamlin + C. jambhiri cv . Rough lemon; C. sinensis cv . Valencia + C. jambhiri cv . Rough lemon; and C. paradisi cv . Thompson + `Murcott' tangor (purported hybrid of C. reticulate × C. sinensis). Diploid plants were regenerated from nonfused embryogenic culture-derived protoplasts of `Cleopatra' mandarin and `Hamlin' and `Valencia' sweet orange, and from nonfused leaf-derived protoplasts of Rough lemon and `Mnrcott'. Regenerated plants were classified according to leaf morphology, chromosome number, and isozyme analyses. All of the somatic hybrids reported herein are tetraploid (2n = 4x = 36), with the exception of the `Hamlin' + S. buxifolia hybrid, which was unexpectedly found to have a chromosome number of 2n = 27. These six new somatic hybrids have potential in citrus scion and rootstock improvement for commercial use.

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Six mandarin cultivars, Ponkan (Citrus reticulata), Willowleaf (Citrus deliciosa), Kinnow (Citrus nobilis × C. deliciosa), Murcott (purported C. reticulata × Citrus sinensis), W. Murcott [purported (C. reticulata × C. sinensis) × C. reticulata)], and Snack (purported C. reticulata hybrid), were used in protoplast fusion with different parental combinations to generate somatic hybrids. Sixty-five somatic regenerants were obtained using optimized formulation of enzymes and molecular weight of polyethylene glycol for improved protoplast yield and heterokaryon fusion rate, respectively. Flow cytometry was used to determine the ploidy level of somatic regenerants, and nuclear expressed sequence tag–simple sequence repeat (EST-SSR) markers to determine their parental source. Of the 65 somatic regenerants, 46 were identified as autotetraploids, 18 allotetraploids, and one undefined. The EST-SSR markers also revealed that some ‘W. Murcott’ embryogenic callus lines that were presumed to be of nucellar origin were actually derived unexpectedly from individual ovules of zygotic origin. These mandarin-derived tetraploids are valuable as potential breeding parents for interploid crosses with an aim at seedlessness and easy-peeling traits.

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The primary objective of this experiment was to determine if the selection of rootstock (Citrus and hybrids) could enhance the development of huanglongbing (HLB)-related symptoms associated with the pathogen Candidatus Liberibacter asiaticus (CLas) in sweet orange (Citrus sinensis). If so, then it may permit more rapid identification of HLB-susceptible compared to HLB-resistant scion types. The secondary objective was to assess the impact of different rootstocks on plant growth parameters and health to determine if trees on any rootstocks displayed reduced sensitivity to HLB-influenced growth restriction. ‘Valencia’ sweet orange was budded on each of the following eight genotypes: Carrizo (C. sinensis × Poncirus trifoliata); Cleopatra (C. reshni); Green-7 {a complex allotetraploid from somatic hybrids [C. clementina × (C. paradisi × C. reticulata) + C. grandis] × [(C. aurantium + (C. sinensis × P. trifoliata)]}; UFR-2 (a complex allotetraploid from somatic hybrids {[C. clementina × (C. paradisi × C. reticulata)] + C. grandis} × (C. reticulata + P. trifoliata)); UFR-4 (same pedigree as UFR-2); rough lemon (C. jambhiri); sour orange (C. aurantium); and US-897 (C. reticulata × P. trifoliata). Half of the trees on each rootstock were bud-inoculated with CLas and half were inoculated with the asian citrus psyllid [ACP (Diaphorina citri)], which is the CLas vector. During both experiments, no rootstock conferred significantly greater HLB symptom severity compared to trees on Carrizo; however, trees on several rootstocks had reduced HLB severity compared to those on Carrizo. Regarding the bud-inoculated trees after 3 years, trees on UFR-4 displayed greater overall health than trees on Carrizo, Green-7, sour orange, and US897, and trees on UFR-4 had a higher percentage of plants with leaf cycle threshold (Ct) values >36 compared with trees on Cleopatra and rough lemon (62 vs. 26-29 respectively). Regarding the ACP-inoculated trees after 3 years, trees on UFR-4 had better overall health than trees on Carrizo, rough lemon, and US-897, and trees on sour orange had a higher percentage of plants with leaf Ct values greater than 36 only compared to Cleopatra and US-897. The percentage increase in the trunk diameter per month over the course of each entire experiment was significantly greater for UFR-2 in both trials than all rootstocks except UFR-4. Only root CLas titers were sometimes significantly higher for trees on other rootstocks compared to those on Carrizo. Although no rootstock provided acceleration of HLB symptom development compared with Carrizo, some rootstocks conferred significantly greater health compared to Carrizo. However, it is uncertain whether the modest differences in health and growth observed in these greenhouse trials would translate to economic benefits in the field.

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In cybridization, new combinations of nuclear and cytoplasmic genes result in a unique genotype that may bring cellular, physical, physiological, and biochemical changes to the plant. This has been demonstrated in the unexpected cybrids generated from the fusion of citrus (Citrus sp.) protoplasts in two independent experiments. The first experiment was conducted to generate potentially seedless triploids by fusing diploid protoplasts of embryogenic ‘Dancy’ mandarin (Citrus reticulata) suspension culture cells with haploid ‘Ruby Red’ grapefruit (C. paradisi) protoplasts derived from tetrad-stage microspores. After multiple attempts, only one triploid was recovered, but several diploid plants with typical grapefruit morphology were also regenerated. In the second experiment, protoplasts derived from embryogenic ‘Dancy’ mandarin suspension culture were fused with nonembryogenic protoplasts from ‘Duncan’ grapefruit leaves in an effort to produce an allotetraploid somatic hybrid. The fruit from the resulting trees resembled grapefruit in morphology and type, and maintained excellent quality throughout the summer, when commercial grapefruit rapidly loses quality. Fruit on these trees remained firm with exceptional sweetness and good flavor into August, and without seed germination. The regenerants obtained in the protoplast fusion experiments were confirmed as cybrids by genetic marker analyses. The test grapefruit were identical to commercial ‘Ruby Red’ grapefruit at six nuclear simple sequence repeat (SSR) marker loci, but identical to ‘Dancy’ with respect to a mitochondrial intron marker. The plastid genomes of individual trees originated from either fusion partner. In the first experiment, haploid ‘Ruby Red’ protoplast preparations must have also contained contaminant diploid protoplasts. Apart from the value of altered fruit quality attributes in the marketplace, these plants provide an opportunity to understand the contributions of cytoplasmic organelle genetics to important citrus fruit-breeding objectives.

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Two adjacent rootstock trials were conducted in the east coast Indian River region of Florida with ‘Marsh’ grapefruit (Citrus paradisi Macf.) scion. The objective was to find rootstocks to replace sour orange (C. aurantium L.) because of losses to citrus tristeza virus, and to replace Swingle citrumelo [C. paradisi × Poncirus trifoliata (L.) Raf.] because of its limited usefulness in certain poorly drained coastal sites. The trials were conducted in randomized complete blocks with 12 single-tree replicates spaced 4.6 × 6.9 m. The soils were of the Wabasso and Riviera series. The first trial consisted largely of trees on citrange [C. sinensis (L.) Osb. × P. trifoliata] and citrumelo rootstocks, ‘Cipo’ sweet orange (C. sinensis), and various hybrid rootstocks. The second trial involved mandarin rootstocks (C. reticulata Blanco) and sour orange and related rootstocks. Trees were grown for 7 years and yield and juice quality data were collected for the last 4 years of that period. Those rootstocks identified as the most promising, based on combinations of smaller tree size and high productivity and juice quality, were two Sunki mandarin × Swingle trifoliate orange (TF) hybrids (C-54, C-146), a Sunki mandarin × Flying Dragon TF hybrid, C-35 citrange, and a Cleopatra mandarin × Rubidoux TF hybrid (×639). The trees on these five rootstocks cropped well leading to soluble solids (SS) values of 3000 to 4000 kg/ha when they were 7-years old. The trees on C-54 and C-146 were relatively large, somewhat taller than trees on sour orange, whereas those on C-35 and the Sunki × Flying Dragon hybrid were smaller and similar to sour orange in tree height. Fruit quality among the trees on C-35 and the Sunki × Flying Dragon hybrid had relatively high SS concentration (better than sour orange), and the other three rootstocks had relatively lower solids concentration (poorer than sour orange). The trees on C-35 and the Sunki × Flying Dragon hybrid would be good candidates for higher density orchards.

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Somatic hybridization through protoplast fusion has proven to be a valuable technique in citrus for producing unique allotetraploid breeding parents that combine elite diploid selections. Many citrus somatic hybrids are now flowering and being used in interploid crosses to generate triploid hybrids that produce seedless fruit, a primary objective of citrus breeding programs. Most of the early somatic hybrids produced for mandarin improvement combined sweet oranges with mandarins, because the performance of sweet oranges in tissue/protoplast culture generally exceeds that of most mandarin selections. However, a high percentage of triploid progeny from interploid crosses using sweet orange + mandarin somatic hybrids as the tetraploid parent produce fruit that are difficult to peel. We report nine new allotetraploid somatic hybrids and five new autotetraploids from somatic fusion experiments involving easy-peel mandarin parents. These tetraploids can be used in interploid crosses to increase the percentage of seedless triploid progeny producing easy-to-peel fruit. Ploidy level of the new tetraploids was determined by flow cytometry and their genetic origin by expressed sequence tag–simple sequence repeat marker analysis.

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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.

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