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Gloria A. Moore

We have produced a number of transgenic citrus plants via Agrobacterium-mediated transformation of seedling stem segments with a vector plasmid containing a β-glucuronidase (GUS) gene. All regenerated green shoots produced in our experiments are assayed histochemically for expression of GUS by cutting a section from the base of the shoot. Many of the shoots express GUS only in sectors, which vary in size from shoot to shoot. Analyses suggest that sectored regenerated shoots are chimeric, consisting of nontransformed cells as well as transformed cells. However, plants derived from shoots with large GUS+ sectors in the original assays do not necessarily contain the GUS gene; conversely, some plants derived from shoots with small sectors appear solidly transformed. Plants that appear solidly transformed have maintained gene expression for up to 5 years. None of the transgenic plants have obviously altered morphologies. It has not been possible to analyze progeny plants because of the long juvenile periods and polyembryony of the primary transformants. However, because citrus is clonally propagated, long-term phenotypic stability of primary transformants is the most important factor in producing useful transgenic plants.

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Diane Luth and Gloria A. Moore

Many Citrus species accumulate large amounts of flavonoids, specifically flavanone glycosides, that impart an intensely bitter flavor to the fruit. In grapefruit, this bitterness decreases the acceptability of fresh fruit and juice; in other species, these compounds entirely prevent fruit consumption. No physiological purpose for the accumulation of these compounds has been determined; they do not function in color production or, as far as is known, in defense responses. As has been found in other plants, the accumulation of specific flavonoids in citrus appears to be under genetic control, but no definitive genetic analyses have been done. The long-term objective of this research is to determine whether the production of bitter-tasting flavanone glycosides (neohesperidosides) in citrus can be manipulated using molecular genetic techniques. As a first step, cDNAs for chalcone synthase and chalcone isomerase, the first two biosynthetic enzymes specific to the flavonoid pathway, were isolated from a grapefruit leaf cDNA library using heterologous probes. Southern analyses showed that both genes appear to be part of multigene families, as expected. Northern analyses are underway to determine steady state mRNA levels in various grapefruit tissues, and Western blots to characterize protein expression are also being attempted.

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Richard Durham, Gloria Moore and Charles Guy

Genetic linkage analysis was performed on an interspecific backcross of citrus [Citrus grandis (L.) Osbeck cv. Thong Dee X (Thong Dee X Poncirus trifoliata (L.) Raf. cv. Pomeroy)], using restriction fragment length polymorphism (RFLP) and isozyme analysis. Sixty-five progeny were analyzed for a total of 57 segregating markers including 9 isozymes and 48 RFLPs. Significant (p = 0.05) deviation from an expected 1:1 segregation ratio was observed for 21 (37%) of the 57 loci, but this did not exclude their use in the mapping study. Linkage analysis revealed that 50 loci mapped to 12 linkage groups while 7 loci segregated independently from all other markers. The total map distance included in the 12 linkage groups was 472 cM with the mean distance between markers being 12.8 cM. This does not represent a saturation of the genome with markers; however, this work demonstrates the potential for mapping traits of economic importance in citrus.

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Courtney A. Weber and Gloria A. Moore

A greater saturation of the previously constructed genetic linkage map of Citrus is important in the long term goal of mapping quantitative trait loci (QTL) such as those controlling cold and salt tolerance. Segregation for cold tolerance appears to be greatly enhanced in the intergeneric F1 population of Citrus grandis × Poncirus trifoliata as compared to the BC1 population previously used for mapping due to the higher percentage of P. trifoliata genes present. This is not unexpected since P. trifoliata is the source of cold tolerance in this cross and is a highly heterozygous species. An integration of the maps of the two populations using about 50 random amplified polymorphic DNA (RAPD) markers common to the two populations is possible using the JoinMap computer program. This will allow the placing of approximately 100 new polymorphic RAPD markers from the F1 population identified by screening from 42 random oligonucleotide primers onto the Citrus map. This saturated map will be used to locate QTL following bulk segregation analysis of cold tolerance in the F1 population.

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Ilhami Tozlu, Charles L. Guy, Ouinvin Cai and Gloria A. Moore

There is wide variation in Citrus and related genera in tolerance to cold and salt stress. While Poncirus trifoliata (L.) Raf. is an important rootstock for cold regions, it is salt sensitive. C. grandis (L.) Osb., on the other hand, is cold sensitive, but is relatively salt hardy. We are attempting to map genes (quantitative trait loci, QTLs) influencing salt and cold tolerance in Cirrus, using a BC1 population from [C. grandis × (C. grandis × P. trifoliata)]. As a first step, 2 year old containerized replicates of individual BC1 progeny plants have been salinized with 30 mM NaCl over a 9 month period under greenhouse conditions. Growth response under saline conditions, as evaluated by plant height and node number, varied significantly between individual progeny. Concentrations of 11 macro- and micro-elements, including Na and Cl, in leaf and root tissues were also determined. Ultimately, this data will be analyzed in conjunction with our current linkage map of this population, which consists of more than 200 marker genes, in order to map QTLs for salt tolerance.

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Pan-chi Liou, Fred G. Gmitter Jr. and Gloria A. Moore

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.

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Catalina M. Anderson, William S. Castle and Gloria A. Moore

Isozyme analysis was the basis for determining the frequency of occurrence and the characteristics of zygotic plants in Swingle citrumelo seedling populations from various sources of open-pollinated seeds, in a commercial nursery of Swingle citrumelo before and after roguing, and in commercial orchards and rootstock trials where this rootstock was used. Most zygotic seedlings identified by isozyme analysis could be distinguished by careful examination of morphological characteristics. Frequencies of zygotic seedlings varied among seedling populations, but were in the range (≈5% to 10%) found in previous studies. Roguing based primarily on size and growth habit of seedlings was effective in removing some, but not all, zygotic seedlings. Most of the remaining zygotic plants in the rogued population were found among the smaller seedlings. Trees budded on zygotic rootstock seedlings were found in two of the three groves studied, and in some instances an apparent incompatibility was developing in young trees.

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Ed Stover, David G. Hall, Jude Grosser, Barrett Gruber and Gloria A. Moore

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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Ed Stover, Robert G. Shatters Jr., Barrett Gruber, Prem Kumar and Gloria A. Moore

Plants inoculated with the huanglongbing (HLB)-associated bacterium, Candidatus Liberibacter asiaticus (CLas) typically must be monitored for 8–10 months to identify differences in susceptibility between genotypes. Continuous light is reported to accelerate development of HLB symptoms and field observations suggest that trees girdled by tags or tree ties showed greater symptoms. Therefore, an experiment was conducted assessing HLB susceptibility as influenced by light/dark periods of 12 hours: 12 hours and 24 hours: 0 hours, in combination with scoring tree trunks to disrupt phloem. Sixty trees of each of three citrus genotypes (‘Kuharske’, previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and ‘Valencia’, highly HLB susceptible) were bud grafted using two CLas-infected buds (rough lemon and citron) per tree on 26 Mar. 2012, and were placed in controlled growth rooms (one 12 hour light: 12 hour dark and one constant light) on 4 June 2012. Ten trees of each genotype in each growth room were scored 10 cm above the soil (cutting through the bark but not the wood) with a knife on 18 July 2012 and the scoring was repeated at the same scoring wounds on 30 Aug. 2012. Trees were removed from growth rooms on 12 Dec. 2012 and subsequently maintained in a greenhouse. At two to three month intervals between June 2012 and May 2013, HLB symptoms and stem diameter at 5 cm above the soil were assessed, and three leaves per tree were collected for quantitative polymerase chain reaction (qPCR) determination of CLas titer. Six months after inoculation and 3 months following imposition of treatments, the ‘Valencia’ scored in the 12 hour light: 12 hour dark regime, the ‘Valencia’ non scored trees in 24 hours of light and the ‘Kuharske’ scored trees in 24 hours of light displayed higher CLas titers than most other trees. After an additional two months, both scored and non-scored trees of all three genotypes in 24 hours of light had significantly elevated CLas titers compared with trees in 12 hour light: 12 hour dark regime, but within most treatments all three genotypes had titers which were not statistically different from each other. Growth of ‘Kuharske’ and rough lemon was enhanced; whereas ‘Valencia’ growth was reduced when graft-inoculated plants were maintained in continuous light. Scoring enhanced early CLas development in ‘Kuharske’ when combined with continuous light, had no effect in rough lemon, and showed inconsistent effects in ‘Valencia’. Although continuous lighting enhanced disease progression, it did not reveal differences in HLB susceptibility.

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Courtney A. Weber, Gloria A. Moore, Z. Deng, F. Gmitter and Courtney A. Weber

Specific primers were designed for 61 cloned RAPD fragments and from 10 Citrus EST sequences for the production of SCAR, CAPS, and STS markers for a Citrus grandis `DPI 6-4' × Poncirus trifoliata `Rubideaux' F1 pseudo-testcross population. Fifteen SCAR, three CAPS, and one EST/STS markers were developed. An additional 17 SCAR and CAPS primer pairs developed at the Citrus Research and Education Center for a Citrus grandis `Thong Dee' × (Citrus grandis `Thong Dee' × Poncirus trifoliata `Pomeroy') BC1 population were screened in the pseudo-testcross population. A total of 27 markers were identified and scored in the pseudo-testcross population in which 24 were mapped; 13 in the Citrus parental linkage map on seven linkage groups and 11 in the Poncirus parental map on five linkage groups. In the BC1 population, 20 of 27 markers tested were found to be polymorphic and 13 mapped to seven of nine linkage groups. Of these, 11 were mapped in both populations and could be used for aligning presumed homologous regions on the three linkage maps.