Currently the breeding of desirable traits in orchid flowers is a lengthy and unpredictable process. A shortened breeding time and a more direct method of introducing specific genetic characteristics could be achieved if more information were available on the specific genes responsible for flower characteristics. In order to identify some of these genes, the genetic relationships between a hybrid, Dendrobium Gatton 'Sunray', and the parent species bred to produce it, D. chrysotoxum Lindley and D. pu/che//um Lindley were examined.Ball State UniversityMuncie, IN 47306These results were supported by Restriction Fragment Length Polymorphisms (RFLPs) observed following amplification of the Internal Transcribed Spacer (ITS) regions of the rDNAs.In order to clone genes responsible for specific flower characteristics, mRNA differential display was performed using total RNA isolated from the leaves, immature flowers, and mature flowers of the hybrid orchid and its two parents. Bands unique to D. Gatton 'Sunray' flower tissue, which were common to the hybrid and a single parent, were excised from a denaturing acrylamide gel. Four of the bands, which represented expressed genes determining inherited flower characteristics, were re-amplified, cloned, and three were sequenced. Partial sequence information obtained for two of the clones was used to search the GenBank database for homologous genes. One of the clones had sequence homology to plant 26S ribosomal genes and the other clone was homologous to sequences encoding regulatory proteins active during development (for example, the human retinoblastoma susceptibility gene or the Caenorhabditis e/egans cosmid R06F6 containing a serine/threonine protein kinase gene). / Department of Biology

Genes responsible for differences in gene regulation and expression in normal cells and freezing tolerant cells were identified using two related wintergreen orchid species, Aplectrum hyemale and Tipularia discolor. Changes in gene expression observed in field-collected tissues obtained from different seasons were compared as were changes observed in plants subjected to cold shock in a laboratory environmental chamber. In order to clone these differentially expressed genes which may confer photosynthesis cold tolerance, the recently developed technique, mRNA differential display was employed. Using this process, mRNA was isolated from the tissue and reverse transcribed to cDNAs, which were amplified using specific anchored 3' primers and various random 5' primers. The 50-100 bands resulting from specific primers were compared on denaturing polyacrylamide gels. Bands differently expressed were excised from the gel and purified. In the future, if partial sequence analysis indicates they may code important regulatory proteins, they will be used as probes to obtain full-length genes from a cDNA library for further characterization. This study provides an opportunity not only to obtain important regulatory genes in plants, but also to understand more about temperature regulated gene expression in orchids. / 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

This research was an attempt to use a dicot transformation vector to transform a monocot. The initial purpose of this thesis was to transform orchids with the sense and antisense coat protein genes from the Odontoglossum ringspot virus (ORSV) in an effort to mitigate viral symptoms in transgenic plants using the transformation vector, Agrobacterium tumefaciens. However, it soon became apparent that much time would be needed to develop a transformation protocol. The transformation vectors used included the Agrobacterium tumefaciens disarmed strain LBA4404 with the binary plasmid pB1121, the disarmed strain At699 with the binary plasmid pCNL65, and the wild-type strain Chry5. The marker gene on the binary plasmids of both disarmed strains was p-glucuronidase (GUS).Several transformation protocols were used in an effort to determine if this transformation system would work on orchids. Transformation was not achieved even though a number of experimental conditions were varied. These included using two different types of orchid tissue, callus and protocorms; using two different species of orchids, Cattleya Chocolate Drop x Cattleytonia Kieth Roth and Cymbidium maudidum; varying the time the plant tissue was exposed to the bacteria from 1 hour to 96 hours; performing experiments with and without the wound signal molecule acetosyringone; and exposing the tissue to the virulent strains of A. tumefaciens mentioned previously.This research also developed GUS assay conditions necessary to decrease the number of false positives due to bacterial contamination. These conditions included chloramphenicol in the GUS assay buffer. / Department of Biology