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  • Author or Editor: Frederick G. Gmitter Jr x
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Seeds of Coreopsis leavenworthii Torr. & Gray (Asteraceae) are being commercially produced but the lack of genetic diversity information has hindered growers and end users from addressing several critical issues affecting wild collection, commercial production, distribution, and the use of seeds. In this study, the genetic diversity and differentiation among natural, production, and introduced populations were analyzed at the molecular level using 320 amplified fragment length polymorphism (AFLP) markers. A high level of diversity [68.6% average polymorphism; total genetic diversity (H t ) = 0.309] and a moderate level of genetic differentiation [total genetic diversity residing among populations (G st ) = 0.226; Φ st = 0.244; Bayesian analog of Nei's G st (G st -B) = 0.197] was detected among six natural populations—two each from northern, central, and southern Florida. Two distance-based clustering analyses, based on an individual's AFLP phenotypes or a population's allele frequencies, grouped natural populations into three clusters, concordant with our previous results from a common garden study of phenotypic variation. Clustering of populations was mostly according to their respective geographical origin within Florida. The correlation between geographical distances and pairwise F st values between populations was very significant (r = 0.855, P < 0.0001). Two central Florida natural populations were divergent and grouped into separate clusters, indicating that the existence of factors other than physical distance alone were contributing to genetic isolation. Three production populations maintained a level of genetic diversity comparable to that in the natural populations and were grouped with the natural populations from which the production populations were derived, suggesting that the genetic identity of the seed origin was maintained under production practices. The genetic diversity of the introduced population was comparable to that of the source populations (central Florida natural populations), but genetic shift seems to have occurred, causing the introduced population to cluster with local (northern Florida) populations where planted. The observed genetic differentiation among natural populations may indicate a need to develop appropriate zones within Florida for preservation of genetic diversity during seed collection, increase, and distribution. This high level of population differentiation also suggests a need to collect and analyze more natural populations across Florida and from Alabama for a better understanding of the species' genetic diversity and population structure across its distribution range.

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Protoplasm culture following polyethylene glycol-induced fusion resulted in the regeneration of tetraploid somatic hybrid plants from the following attempted parental combinations: Cleopatra mandarin (Citrus reticulata Blanco) + Argentine trifoliate orange [Poncirus trifoliata (L.) Raf.]; `Succari' sweet orange [C. sinensis (L.) Osb.] + Argentine trifoliate orange; sour orange (C. aurantium L.) + Flying Dragon trifoliate orange (P. trifolita); sour orange + Rangpur (C. limonia Osb.); and Milam lemon (purported sexual hybrid of C. jambhiri Lush × C. sinensis) + Sun Chu Sha mandarin (C. reticulate Blanco). Protoplasm isolation, fusion, and culture were conducted according to previously published methods. Regenerated plants were classified according to leaf morphology, chromosome number, and peroxidase, phosphoglucomutase, and phosphoglucose isomerase leaf isozyme profiles. All of the somatic hybrid plants were tetraploid, as expected (2n = 4x = 36), and all five selections have been propagated and entered into commercial citrus rootstock trials.

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Allotetraploid somatic hybrid plants of `Nova' tangelo [a sexual hybrid of `Clementine mandarin (C. reticulata Blanco) × `Orlando' tangelo (C. reticulata × C. paradisi Macf.)] + `Succari' sweet orange (C. sinensis L. Osbeck), and `Hamlin' sweet orange (C. sinensis L. Osbeck) + `Dancy' tangerine (C. reticulata) were regenerated following protoplast fusion. `Nova' and `Hamlin' protoplasts were isolated from ovule-derived embryogenic callus and suspension cultures, respectively, and fused using a polyethylene glycol method with seedling leaf-derived protoplasts of `Succari' and `Dancy', respectively. Plants were regenerated via somatic embryogenesis, and somatic hybrids were identified on the basis of leaf morphology, root-tip cell chromosome number, and electrophoretic analysis of peroxidase and phosphoglucose mutase isozyme banding patterns. Diploid plants were regenerated from unfused protoplasts of `Hamlin', `Nova', and `Succari'. Tetraploid plants of `Hamlin' and `Succari' were also recovered, apparently resulting from homokaryotic fusions. No `Dancy' plants were recovered. The somatic hybrid and autotetraploid plants can be used for interploid hybridization with selected monoembryonic scions to generate improved seedless triploid tangor/tangelo cultivars. The lack of suitable tetraploid breeding parents has previously inhibited the development of quality seedless cultivars by this method.

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An efficient in vitro regeneration system through direct shoot organogenesis was established for Murraya paniculata (L.) Jack (Orange Jessamine). Epicotyls, leaves, roots, and cotyledons from in vitro-germinated seedlings and several plant growth regulators (PGRs) were evaluated for their effects on plant regeneration. Longitudinally cut epicotyl segments were observed to be the optimal explants followed by uncut epicotyls (not longitudinally cut). Roots, leaves, and cotyledons were not suitable as explants as a result of little or no shoot induction. Adventitious shoot induction was enhanced by the addition of 6-benzyladenine (BA). The highest percentage of shoot induction (87%) and the greatest number of shoots per explant (12.7) occurred on Murashige and Skoog (MS) medium supplemented with 15 μM BA from longitudinally cut epicotyls followed by 5.2 shoots per explant from uncut epicotyls. Optimal concentration of gibberellic acid (GA3) for shoot elongation was observed to be 15 μM. Eighty-five percent of the regenerated shoots produced roots with an average of three roots per shoot on MS medium supplemented with 5 μM indole-3-butyric acid (IBA). Our protocol for direct shoot organogenesis can potentially lead to the development of a robust method for production of transgenic plants of M. paniculata through Agrobacterium-mediated genetic transformation.

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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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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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Citrus fruit with sector chimeras were collected in commercial packinghouses and from the field. Chimeric fruit from eight cultivars of sweet oranges [Citrus sinensis (L.) Osbeck], grapefruit (C. paradisi Macf.), tangelo (C. paradisi × C. reticulate Blanco), and tangors (C. reticulate × c. sinensis) were found at a frequency of 0.009% to 0.271%. Tetraploid plants obtained from one type of sector mutant (termed gigas) and albino plants obtained from another type of sector mutant confirmed that some genetic mutations observed in fruit rind can be recovered in nucellar seedlings. The gigas chimeras were identified as a source of citrus tetraploids. Several types of potentially useful sector mutants with altered rind color were observed, and plants were produced from some mutant sectors by developed seed or culture of aborted ovules. HPLC analysis of rind tissues from sectors of one chimeric fruit revealed substantial quantitative and qualitative differences in pigment composition. Propagation of plants from mutant sectors may yield cultivars with improved fruit color, altered maturation date, and reduced disease or mite susceptibility and may eventually lead to breeding of seedless triploid hybrids.

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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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As one of the Florida's state wildflowers, Coreopsis leavenworthii is highly desirable for roadside plantings in all parts of the state. Seeds of this species are being produced by growers. Where should seed be produced for different ecotypes? Where can the seed be used? These are among questions that have arisen in commercial seed production and distribution. To address these questions, it was necessary to assess the levels of genetic diversity. Eleven populations (242 total individuals) were collected from different parts of Florida, grown at one location in central Florida, and observed for morphological variations. North Florida natural populations had more complex leaves, while south Florida natural populations had smaller flowers. Principal component analyses revealed that two of the seven characteristics studied accounted for as much as 88% of the morphological variations observed. Molecular diversity was analyzed by using the fluorescent amplified fragment length polymorphism (AFLP) technique and the capillary sequencing system. Four primer combinations detected 320 AFLP fragments, of which 90.6% were polymorphic. The overall genetic diversity in the species was 0.2206 (estimated using AMOVA), of which 77.9% was within populations and 22.1% was among populations. The genetic distance among populations seemed to be loosely correlated with geographical distances. A high level of gene flow was found in several populations. Based on the results, a model has been developed to describe the genetic relationship of Coreopsis leavenworthii populations.

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