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

Apple scab is one of the greatest apple management problems throughout the world Much work has been done on cultivars resistant to Venturia inaequalis (Cke.) Wint., but few have been a commercial success. This frequently leaves fungicides as the only control method used. As Quebec growers select new cultivars for planting, more information is needed on their relative susceptibility for efficient scab control. In this light, 21 cultivars common to central and eastern Canada, were examined for their relative susceptibility using several components of partial resistance; disease severity, incubation period, latent period, lesion size, and conidial production. The cultivars used were Cortland, Early Geneva, Empire, Golden Delicious, Golden Russet, Idared, Jersey Mac, Jonagold, Jonamac, Lobo, Lodi, Summerland McIntosh, Mutsu (Crispin), Northern Spy, Paulared, Red Cortland, Red Delicious, Royal Gala, Spartan, Sunrise, and Vista Bella. A final ranking of the cultivars and selection of partial resistance components was done using the principal components analysis. (Abstract shortened by UMI.)

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.

An interaction was recently detected between the potyviral protein, genome-linked (VPg) and the Arabidopsis thaliana translation initiation factor eIF(iso)4E (Wittmann et al., 1997). / Here, experiments were undertaken to address biological aspects of the VPg-eIF4E interaction. First, coimmunoprecipitation experiments performed with purified recombinant proteins have shown that VPg not only associates with eIF4E, as was previously published, but also with the larger eIF4F complex, of which eIF4E is a subunit. These results were confirmed by ELISA-type binding assays. It was also shown that there is no direct interaction between VPg and the other subunit of eIF4F, namely eIF4G. Finally, with the same experimental system, it was shown that the presence of eIF4G does not influence the binding affinity of VPg and eIF4E. / The interaction of VPg with the plant translation apparatus suggests that potyviral infection may alter the host protein expression profile. This hypothesis was investigated with the use of a protoplast system. We have shown that the global rates of protein synthesis in protoplasts transfected with an infectious TuMV cDNA clone dropped shortly after transfection, by as much as an estimated 70%. Recovery to normal levels occurred within 48 hours. / Evidence was obtained that the interaction between VPg and eIF4E is instrumental in this transient down-regulation of protein expression: protoplasts transfected with a mutant TuMV cDNA clone, the VPg of which has no affinity for eIF4E, failed to exhibit the drop in protein synthesis observed with the wild-type clone.

No description available.

Turnip mosaic potyvirus (TuMV) is a single-stranded RNA plant virus. One of its proteins, the cylindrical inclusion (CI) protein, was hypothesized to interfere with host transcription via interaction with histone H3 protein. Interaction between CI and histone H3 was previously observed in Dr. Fortin's laboratory. Based on previous studies that demonstrated the importance of the H3 tail domain in gene regulation and chromosome arrangement, it was hypothesized that CI would interact with the tail rather than the globular domain. The objective of this project was to identify which histone H3 domains CI protein interacts with. The full-length, globular, and tail domains of histone H3 DNA were expressed in E. coli and purified. Based on in vitro interaction experiments, the CI protein was observed to interact with the globular domain of histone H3.

No description available.

The effect of the arbuscular mycorrhizal symbiosis at reducing the incidence of root diseases has received considerable attention. However, information on the role of mycorrhizae in reducing disease incidence of Fusarium root rot of beans (Phaseolus vulgaris), caused by the root pathogen Fusarium solani f. sp. phaseoli, is scarce. A study was undertaken to investigate how the arbuscular mycorrhizal fungus (AMF) Glomus intraradices affects disease development and population number of F. solani f. sp. phaseoli in the mycorhizosphere of bean plants growing in an experimental microcosm unit. This newly designed unit facilitated the spatial monitoring and quantification of both the symbiont and pathogen in different ecological soil regions of the mycorrhizosphere using compartmentation based on a physical segregation of roots, colonized or not by AMF (rhizosphere), AMF mycelium alone (mycosphere), or none (bulk soil). To study the interaction between both organisms, the experimental set-up consisted of a randomized complete block design using bean seedlings pre-colonized or not for 28 days by G. intraradices and infected or not for 6 days with F. solani f. sp. phaseoli. Monitoring of population number of the symbiont and the pathogen in bean plants and in the different mycorrhizosphere soil compartments was achieved with quantitative real-time PCR using specific molecular probes for each fungus, and with cultivation-dependant or morphological based methods. The results of this study indicated that non-mycorrhizal bean plants infected with the pathogen had typical root rot symptoms while infected plants that were pre-colonized by G. intraradices remained free of disease. The population number of F. solani f. sp. phaseoli was significantly reduced in the root system and in each of the mycorrhizosphere soil compartments of mycorrhizal infected plants. The mycorrhizosphere population of G. intraradices was not significantly modified, although the p

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