Resistance (R) genes, a key factor in determining the resistance of plants, have been shown often to be highly allelic entities existing in duplicated regions of the genome. This characteristic suggests that R-gene acquisition may have arisen through frequent genetic rearrangements as a result of transient, reduced genome stability. Tabacco plants transgenic for a recombination construct exhibited reduced genome stability upon infection with a virulent pathogen (tobacco mosaic virus). The reduced genome stability manifested as an increase in recombination events in the transgene. Such increases were observed following a virulent pathogen attack. This increase in recombination was shown to be systemic and was observed prior to systemic viral movement suggesting the presence of a systemic recombination signal. Further molecular analyses revealed that specific R-gene loci experience a large frequency of rearrangements following a virulent pathogen encounter. The possible targeting of instability to R-gene regions may be controlled through epigenetic processes, in particular, DNA methylation. / xiii, 119 leaves ; 29 cm.

In this study, we have demonstrated that inoculation of bean seeds with non-pathogenic binucleate Rhizoctonia (np-BNR) at sowing protected bean seedlings from infection of R. solani. Using quantitative real-time RT-PCR (QRT-PCR), transcript levels of defense genes encoding 1,3-beta-glucanase (GLUC), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) in one-week old bean seedlings was monitored during np-BNR and R. solani interaction. The results revealed that protection effect of np-BNR correspond to a systemic suppression of these three defense genes' expression from significant higher level elicited by R. solani to the level of non-infected plants. This indicates that bio-protection by np-BNR isolates is not correlated to activation of these three defense genes' expression. Similar suppression was achieved for pre-colonization of bean seedlings with arbuscular mycorrhizal (AM) Glomus introradices on GLUC gene expression, although the AM fungus did not significantly reduce rot symptoms. Possible mechanisms implicated in down-regulation during plant-pathogen and np-BNR or AM interaction are discussed.

Resistances to bean common mosaic virus, halo, common and Ascochyta blight, angular leaf spot, anthracnose and rust pathogens of beans in South Africa were combined by reverse dichotomous crossing. Full resistance to Uromyces appendiculatus from Carioca 80 was conditioned by a single dominant gene. Partially dominant resistance to Phaeoisariopsis griseola was conditioned by a single gene in Carioca 80 and two genes in PAl 127. Differences in aggressiveness of isolates of Phoma exigua var. exigua were found. Different levels of Ascochyta blight resistance were found in the glasshouse, but field testing showed little difference after flowering. Inoculations of differential cultivars indicated the presence of at least eight races of U. appendiculatus and the a-Brazil race of Colletotrichum lindemuthianum . Inoculations of the old set of halo blight differential cultivars identified races 1 and 2. Forty-five lines with partial resistance to rust were obtained by recurrent selection. Very highly significant differences were noted between ratings of percentage leaf area affected by rust and yield of 23 cultivars planted in field trials. Significant genotype x environment interaction was noted for rust ratings. Ratings at different dates within a trial were correlated with one another, showing few ratings are required per trial, and a correlation of -0.678 between yield and rust rating was found. Inheritance of partial resistance and improved yield of eight cultivars crossed in a full diallel was mostly due to additive effects but non-additive effects were also very highly significant. Reciprocal effects were not significant for yield and rust ratings. Genotype x environment interactions were significant for rust ratings and yield. High estimates of narrow-sense heritability for rust resistance were obtained. No relationship between resistance and time to flowering, pustule size, leaf hairs and stomata was found. Latent periods in unifoliate leaves did not correlate with resistance but a closer match was found in the fourth trifoliate leaves. Inoculations with three additional single-pustule isolates of the 23 parent cultivars indicated the cultivars had similar levels of resistance. Ring necrosis was found in nine cultivars or crosses with them. The ring reaction was conditioned by a single dominant gene and possibly by the epistatic interaction of two dominant genes in Carioca 80. Differences in symptom severity in plants derived from Epicure indicated the possibility of additional gene interaction. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1994.

The Wheeler Orchid Collection is home to some of the most endangered species of orchids in the world. This fantastic reservoir of endangered species has been enhanced and broadened by its function as a plant rescue station for the U.S. customs service. Unfortunately, this responsibility increases the risk of bringing orchids, which harbor contageous diseases, into the greenhouse where sap transmitted diseases such as the Tobacco Mosaic Virus (TMV), can run rampant. Although manipulation of orchid characteristics is typically done by classical plant breeding techniques, genetic engineering is emerging as a useful technique for the introduction of desirable traits into the orchid genome. Through the use of genetic engineering techniques it may be possible to mitigate the symptoms associated with this destructive virus. Virus resistance may be achieved through the expression of either the sense or antisense viral coat protein gene in orchid tissues if an efficient means of orchid transformation is developed. In this research two transformation protocols were examined for their ability to efficiently transform orchid tissue. The first transformation protocol explored utilized the native ability of Aq bacterium tumefaciens to incorporate DNA into host plants to achieve transformation. The second mechanism explored was particle bombardment transformation.Many strains of A. tumefaciens were employed using direct exposure of Cattleya_ orchid protocorm and callus tissue. Particle bombardment using DNA coated 0.5 um diameter tungsten particles and high pressure helium tank acceleration was employed. The particle bombardment procedure employed the pG35barB plasmid which confers herbicide resistance to the herbicide basta when integrated and expressed in plant tissues.GUS fluorescence assays and PCR analysis indicate that T-DNA is present in orchid tissues, while Southern blot analysis was unable to display that integration had occurred. Particle bombardment yielded herbicide resistant orchid tissues which have yet to be analyzed by Southern blot analysis to confirm integration due to limited tissue quantities. / Department of Biology

The work reported here was done at the Wheeler Orchid Collection and Species Bank and the Department of Biology at Ball State University. We have developed a research teaching program with two applied research goals: genetically transforming and DNA fingerprinting orchid tissue. As part of their molecular biology education, students have investigated the genetic transformation of orchids for mitigating viral symptoms and the identification of unknown orchids by DNA fingerprinting. In a second application of the technology, DNA fingerprinting has been used to determine evolutionary relationships and to quantify genetic diversity among orchids.This dissertation details the background and need for this project and the research that was done to start it. As the early work has, developed and students have added their contributions, the data have developed into two papers formatted for submission to scientific journals. They are included as results.The first is a project designed to insert exognenous DNA into orchid tissue. The soil microbe Agrobacterium tumefaciens causes crown-gall tumors to develop in its plant hosts by inserting DNA into their cells which then controls the biosynthesis of development-controlling hormones. A. tumefaciens which has been disarmed has been routinely used to bioengineer dicotyledonous plants but its use has been rare on monocotyledons. In this paper, we report that A. tumefaciens transformed embryonic orchid tissue and caused alteration in its normal developmental course.The second paper details the DNA fingerprinting of tissue from Aplectrum hymale, a terrestrial orchid native to this climate. Three populations of A. hymale have been sampled and DNA extracted from the tissue samples. RAPD primers were used to prime PCR amplifications of random sequences of the DNA and the amplified DNA was visualized by gel electrophoresis. Loci of the resulting bands were treated as potentially multiallelic gene loci and heterozygosity between and within subpopulations was calculated. We report that the three populations could be partially differentiated by this procedure and that the two populations located nearest to each other yielded the least between -ubpopulation heterozygosity. We report very high levels of genetic diversity between individuals within small subpopulations in spite of the fact that these subpopulations are considered to be primarily clonal in reproductive nature. / Department of Biology

A major goal of our laboratory is to confer resistence specifically to the Odontoglossum ringspot virus [ORSV; sometimes referred to as tobacco mosaic virus strain O (TMV-O)] in orchids. The chosen strategy may also provide cross-protection to other pathogens. The experimental design for the entire project is presented here along with the results obtained in several preliminary experiments performed in this research. Our approach involved RT-PCR amplification of the viral coat protein gene with gene-specific primers and digestion of the cDNAs into oligonucleotides. These fragments were cloned into the selectable vector pG35barB (which confers herbicide resistence) in both sense and antisense orientations. The cloned DNA was coated with tungsten beads and shot into orchid callus tissue using a makeshift biolistic gun. Tranformant callus cells were selected for by herbicide resistance. Unfortunately the potential transformants became contaminated with fungus and could nto be analyzed to determine which oligonucleotide was received and the effect each oligonucleotide had on pathogen resistance. Due to the uncertainty of the relatedness between ORSV and TMV-O, we also sequenced the coat protein gene of TMV-O and compared the amino acid sequence with those of several strains of ORSV: the Japanese strain had the highest percent amino acid similarity (99.4%), the Type strain the second highest (98.7%), and the Korean strain the lowest (96.9%). It was concluded that TMV-O is most likely one strain of ORSV, the Japanese strain. / Department of Biology

A time course study was conducted to monitor disease development and expression of the defense-related genes phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and hydroxyproline-rich glycoprotein (HRGP) in bean (Phaseolus vulgaris L.) plants colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices , and post-infected with the soil-borne pathogen Rhizoctonia solani. Pre-colonization of bean plants by the AM fungus did not significantly reduce the severity of rot symptoms. RNA blot analysis revealed a systemic increase in transcript levels of the four defense-related genes in response to R. solani infection. On the other hand, pre-colonization of bean plants with G. intraradices elicited no change in PAL, CHS and CHI transcripts, but an increase of HRGP transcripts in leaves was detected. A differential and systemic alteration in the expression of all four defense genes was observed in AM beans post-infected with R. solani. Depending on the time after infection with R. solani and the tissue examined, varying responses from stimulation, suppression, to no change in transcript levels were detected.

No description available.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 136-152). / Microfiche. / xiv, 152 leaves, bound ill. 29 cm

Bibliography: leaves 173-200 / iv, 200 leaves, 7 leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1986