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Frederick G. Gmitter Jr.

Traditional genetic manipulation methods have proven ineffective or irrelevant for many citrus breeding objectives. Alternative approaches to Citrus genetic improvement are now available as a result of technological developments in genetics and tissue culture. For example, mapping DNA marker polymorphisms should lead to identifying markers closely linked to important loci, thereby facilitating early selection and minimizing costs associated with plant size and juvenility. Genetic transformation methods allow trait-specific modification of commercial cultivars. By selecting beneficial variants from sectored fruit chimeras and the recovering plants via somatic embryogenesis, the problems of nucellar embryony and the hybrid nature of commercial cultivar groups can be avoided. Induced mutagenesis from mature vegetative buds may overcome these problems, as well as juvenility. Ploidy level manipulation in vitro can increase the number and diversity of tetraploid breeding parents, leading to the development of seedless Citrus triploids and mitigating sterility, incompatibility, and nucellar embryony.

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Frederick G. Gmitter Jr

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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Kim D. Bowman and Frederick G. Gmitter Jr.

Chinotto is a selection of sour orange (Citrus aurantium L.) with short internodes and small leaves and fruit. Mature fruiting trees of Chinotto grafted on standard rootstocks produce healthy, but dwarf, trees. Most seedlings recovered from Chinotto fruit are of nucellar (maternal) origin and will faithfully grow to reproduce the Chinotto genotype and phenotype. Vigorous greenhouse-grown nucellar seedlings of Chinotto have internodes 5 to 10 mm in length and leaves 30 to 40 mm in length, about 30% the dimensions of the corresponding organs on standard sour orange nucellar seedlings. Sexual hybrids with Chinotto have been produced by controlled crosses with several other parents. Some hybrids with shortened internodes and small leaves were recovered among all hybrid progenies, regardless of whether Chinotto was used as seed or pollen parent. In some cases, segregation among Chinotto hybrids was about 1 normal: 1 dwarf. In other progenies, some intermediate forms were recovered along with normal and dwarf plants.

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Kim D. Bowman and Frederick G. Gmitter Jr.

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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Frederick G. Gmitter Jr. and Xubai Ling

A method was developed to produce nonchimeric, autotetraploid Citrus plants via in vitro somatic embryogenesis in the presence of colchicine. Undeveloped ovules from immature fruit of `Valencia' sweet orange (Citrus sinensis [L.] Osb.) and `Orlando' and `Minneola' tangelos (Citrus reticulata Blanco × Citrus × paradisi Macf.) were held on Murashige and Tucker medium with 500 mg malt extract/liter and 0.0090, 0.01%, or 0.10% colchicine for 21 days. Embryogenesis from tangelo ovules was suppressed by 0.10% colchicine, but no such effect was observed among sweet orange ovules. Colchicine treatments had no subsequent effect on embryo germination. The numbers of chromosomes in root tip cells showed that both tetraploid and diploid `Valencia' and `Orlando' plants were recovered from colchicine treatments. `Minneola' cultures produced only diploid plants. Tetraploid plant morphology was typical for Citrus tetraploids. Examination of chromosome numbers in root tip, shoot, and leaf meristems indicated that the regenerants were nonchimeric. Such nonchimeric tetraploids will be useful parents for interploid hybridization directed toward development of seedless triploid Citrus scion cultivars.

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Xiuli Shen, William S. Castle and Frederick G. Gmitter Jr

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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Jacob B. Bade, Frederick G. Gmitter Jr. and Kim D. Bowman

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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Xiuli Shen, William S. Castle and Frederick G. Gmitter Jr

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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Zeynel Dalkilic, L.W. Timmer and Frederick G. Gmitter Jr.

The inheritance of resistance to a host-specific isolate (Shinn) of Alternaria alternata (Fr.:Fr.) Keissl. from `Minneola' tangelo (a cross between Citrus paradisi Macf. `Duncan' and C. reticulata Blanco `Dancy') was shown to be controlled by a single recessive allele, aaM1, within the citrus genome. A backcross between resistant `Clementine' mandarin (C. reticulata) and susceptible LB#8-10 (a hybrid of `Clementine' mandarin and `Minneola' tangelo) resulted in 61 resistant (R) and 58 susceptible (S) plants (χ2 = 0.0756, P ≥ 0.05), but the reciprocal cross deviated from the expected 1R:1S ratio (87 R and 36 S plants (χ2 = 21.1463, P ≥ 0.05). A dominant allele, AaM1, of this resistance gene was found in a loose coupling phase linkage with two RAPD markers, P12850 (15.3 cM) and AL31250 (36.7 cM), after JOINMAP computer analysis.