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  • Author or Editor: Frederick G. Gmitter Jr x
  • HortScience x
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Traditional methods of genetic manipulation have proven ineffective or irrelevant for many citrus breeding objectives. Alternative approaches to genetic improvement of citrus are now available as a result of technological developments in genetics and tissue culture. Mapping DNA markers on the Citrus genome should lead to identification of markers closely linked to important loci, thereby facilitating early selection and minimizing costs associated with plant size and juvenility. Genetic transformation methods provide opportunities for trait-specific modification of commercial cultivars. The selection of beneficial variants from sectored fruit chimeras, and the recovery of plants via somatic embryogenesis, can overcome the problems of nucellar embryony and the hybrid nature of commercial cultivar groups. Induced mutagenesis, using mature vegetative buds, may overcome size and juvenility, as well as nucellar embryony and hybridity. Ploidy level manipulation in vitro provides methods to overcome sterility, incompatibility, and nucellar embryony, and it can increase the number and diversity of tetraploid breeding parents available for development of seedless citrus triploids.

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A diverse population of grapefruit-like Citrus growing in Saint Lucia (West Indies), called forbidden fruit, was examined as a potential germplasm source for Citrus genetic improvement. Four clones from this population were studied by leaf isozyme analysis, and a distinct resemblance between forbidden fruit and grapefruit (C. × paradisi Macfady.) was observed at several loci, including identical banding patterns for peroxidase, phosphoglucose mutase, phosphohexose isomerase, and shikimic acid dehydrogenase. These results support morphological and historical indications of a close taxonomic relationship between modern grapefruit cultivars and Caribbean forbidden fruit. Comparison of isozyme allele segregation among seedlings of several forbidden fruit clones and grapefruit cultivars demonstrated a much higher degree of zygotic embryony in the former. Morphological diversity and zygotic embryony in the Caribbean forbidden fruit population may make it a useful genetic resource for breeding grapefruit and other Citrus species.

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Volatile oils were extracted from aqueous leaf suspensions of sweet orange [Citrus sinensis (L.) Osb.] cultivars Hamlin, Navel, and Valencia and grapefruit (Citrus paradisi Macf.) cultivars Marsh and Ray Ruby. Pressurized air was used as the sparging gas, and volatile oils were collected in a C-18 cartridge. Gas-liquid chromatography was used to separate and quantify 17 volatile components. Significant quantitative differences for individual components made it possible to distinguish sweet orange from grapefruit (four components), `Marsh' from `Ray Ruby' grapefruit (two components), `Hamlin' from `Valencia' or `Navel' orange (six components), and `Valencia' from `Navel' (three components). The simplicity and sensitivity of the procedure suggest potential use for Citrus taxonomic, genetic, and breeding research.

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We examined the relationship between seed size and shape in Citrus and the number and type of seedlings produced by individual seeds for each of three citrus cultivars. Seed size and shape were related to the number of seedlings produced and the likelihood of recovering a zygotic seedling. The relationship between seed size and shape and the likelihood of recovering a zygotic seedling most often was connected with weight and thickness of a seed. This relationship might be of sufficient strength to use in some aspects of cultivar development. However, the relationship did not appear strong enough to be of practical value for application in commercial production of purely nucellar rootstock seedlings.

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Casuarina cunninghamiana Miq. is an introduced species to Florida that has potential as a windbreak plant to help manage canker in citrus groves; however, only Florida sources can be used for that purpose. Local sources of Casuarina are generally adequate seed producers, but germination percentages are frequently poor. Thus, the causes of low seed germination and methods to improve germination were investigated using C. cunninghamiana and a local hybrid (C. equisetifolia L. × C. glauca Sieb. ex Spreng.). Seeds of the hybrid were larger and heavier (88 mg/100 seeds) than those of C. cunninghamiana (mean wt. 67 mg/100 seeds). Shrunken, insect-damaged, and empty seeds, present in all unsorted seed lots, were responsible for poor seed germination of the four seed sources studied. Petroleum ether separation improved germination by dividing seeds into floaters and sinkers. The floater fraction consisted of 47.5% to 93% insect-damaged seeds compared with 9.0% to 43.5% among sinkers. More than 50% of the sinkers were filled seeds and less than 21% in floaters. No empty seeds were sinkers except for one source of C. cunninghamiana. In sorted hybrid seeds, petroleum ether separation eliminated a large proportion of ungerminable seeds (floaters) and seed germination among sinkers was faster with a higher germination percentage than floaters. Cumulative germination of hybrid seeds in a trial involving two temperatures was 23.0% for sunken seeds at 30 °C at the end of 8 weeks compared with 1% of unsorted seeds. Temperature had no significant effect on seed germination. The germination percentage of hybrid seeds with seedcoats removed was 91.0% in the first week of culture compared with only 1.2% in the first week and 12.6% seed germination at the end of 8 weeks' culture of intact seeds.

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This study examined the effects of plant growth regulators, explant types, and their orientations on in vitro shoot proliferation of Casuarina cunninghamiana Miq. and also the subsequent rooting ability of shoots. Results showed that shoot proliferation occurred only in shoot tip explants cultured vertically on Murashige and Skoog (MS) medium supplemented with 2 or 4 μM thidiazuron (TDZ). Neither 6-benzylaminopurine alone nor in a combination with 1-naphthalene acetic acid (NAA) or gibberellic acid had any effect on shoot proliferation. TDZ at 4 μM resulted in the greatest percentage of axillary bud sprouting (70%) and mean number of sprouts per explant (2.3). Additionally, no shoot proliferation was observed from detipped or single-node explants or from horizontally placed shoot tip explants when cultured on the same TDZ-containing medium. The induced shoots produced adventitious roots on MS medium supplemented with 2.5, 5, or 10 μM indole-3-butyric acid (IBA), not with indole-3-acetic acid and NAA. Although the mean number of roots per explant was not significantly different between 2.5 and 5 μM IBA, the highest rooting percentage (68%) and mean length of roots per explant (0.7 cm) was achieved at 5 μM IBA. The current study provided preliminary information toward commercial in vitro propagation of Casuarina cunninghamiana male plants.

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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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