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

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

Pollen morphology of 8 species (TEM), 86 species (SEM), and leaf anatomy of 62 species (LM), were surveyed in the Oisinae, Coryclinae and, as outgroup taxa, the Orchideae and Satyriinae. Characters extracted from observations made of leaf anatomy and pollen were analysed using cladistic methods, and assessed in relation to the present phylogeny of the group. Leaf anatomy data gave little phylogenetic information. Sclerification associated with vascular bundles was systematically useful. Pollen data served to resolve taxa at the subtribal level. Both data sets provided evidence of relationship for taxonomically problematic taxa. The Coryciinae are palynologically defined by a suite of synapomorphies, including a secondarily tectate exine structure, fasciculate massulae, and elongated tetrads with linear microspore configuration. Bibliography: pages 107-123.

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

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Twenty five years after the last major morphological revision, phylogenetic relationships were inferred on the basis of a new DNA dataset for the African orchid subtribe Disinae, which includes the large genus Disa and the small genus Schizodium. One nuclear gene region (ITS) and two plastid gene regions (trnLF and matK) were sequenced for 136 ingroup, representing 70% of all known Disinae species, as well as for 7 outgroup taxa. The combined data matrix contained 4094 characters and was analysed using parsimony and Bayesian inference. The generic status of Schizodium can no longer be supported, as it is deeply embedded within the genus Disa. Furthermore, the currently recognised subgenera do not reflect the phylogenetic relationships. Several of the currently recognised sections are monophyletic, others contain misplaced elements, while some are polyphyletic. These results necessitate a re-classification of the Disinae. A monotypic subtribe Disinae and a subdvision of Disa into eighteen sections is formally proposed. These sections are monophyletic, well-supported, morphologically distinguishable and are delimited to maximize the congruence with the previous classification. All currently known species are enumerated and assigned to sections. Likelihood optimisation onto a dated molecular phylogeny is subsequently used to explore the historical biogeography of Disa, as well as of three other Cape lineages (Irideae p.p., the Pentaschistis clade and Restionaceae), to find out where these lineages originated and how they spread through the Afrotemperate region. Three hypotheses have been proposed: (i) a tropical origin with a southward migration towards the Cape; (ii) a Cape origin with a northward migration into tropical Africa and (iii) vicariance. None of these hypotheses, however, has been thoroughly tested. In all cases, tropical taxa are nested within a predominantly Cape clade and there is unidirectional migration from the Cape into the Drakensberg and from there northwards into tropical Africa. Dating estimates show that the migration into tropical East Africa has occurred in the last 17 million years, consistent with the Mio-Pliocene formation of the mountains in this area. The same technique is then utilised to reconstruct the temporal occurrence of ancestral ecological attributes of the genus Disa. The first appearance of species in the grassland and savanna biomes, as well as in the subalpine habitat, are in agreement with the existing, reliable geological and paleontological information. This suggests that phylogenies can be used to date events for which other information is lacking or inconclusive, such as the age of the fynbos biome and the start of the winter rainfall regime in southern Africa. The results indicate that these are much older than what is currently accepted and date back to at least the Oligocene. / AFRIKAANSE OPSOMMING: Vyf-en-twintig jaar na die laaste groot morfologiese hersiening, is die filogenetiese verwantskappe van die Afrika orgideë subtribus Disinae, wat die groot genus Disa en die klein genus Schizodium insluit, in hierdie studie op grond van ‘n nuwe DNA datastel afgelei. Daar is van 136 binnegroep, wat 70% van alle bekende Disinae spesies verteenwoordig, sowel as sewe buitegroep taksa geenopeenvolgings van een nukleêre geen streek (ITS) en twee plastiedgeen streke (trnLF en matK) bepaal. Die gekombineerde data matriks het 4094 karakters bevat en is met die parsimonie en Bayesian metodes ontleed. Die generiese status van Schizodium kan nie hieruit ondersteun word nie, en is diep ingebed binne die genus Disa. Die huidiglik aanvaarde subgenera word ook nie deur hierdie filogenie ondersteun nie. Verskeie van die huidiglik herkende seksies is bevind om monofileties te wees, ander bevat verkeerd geplaasde spesies, terwyl ander polifileties blyk te wees. ’n Monotipiese subtribus Disinae en ’n onderverdeling van Disa in agtien seksies word formeel voorgestel. Dié seksies is monofilities, goed ondersteun, morfologies onderskeibaar en omskryf om maksimaal ooreen te stem met die vorige klassifikasie. Alle huidiglik bekende spesies word gelys en toegewys aan seksies. Waarskynlikheidsoptimalisering op ’n gedateerde molekulêre filogenie is dan gebruik om die historiese biogeografie van Disa te ondersoek, tesame met drie ander Kaapse groepe (Irideae p.p., die Pentaschistis klade en Restionaceae), om te bepaal waar hierdie groepe hulle oorsprong gevind het en hoe hulle na die “Afrotemperate“ streek versprei het. Drie hipoteses word voorgestel: (i) ’n tropiese oorsprong met ’n suidwaartse migrasie na die Kaap; (ii) ’n Kaapse oorsprong met ’n noordwaartse migrasie na tropiese Afrika, en (iii) vikariansie. Geen van hierdie hipoteses is egter vantevore deeglik getoets nie. In alle gevalle is bevind dat die tropiese taksa oorwegend binne ’n Kaapse klade gesetel is, en dat daar ’n eenrigting migrasie is van die Kaap na die Drakensberge en van daar noordwaarts na tropiese Afrika. Dateringsskattings toon dat die migrasie na tropiese Oos-Afrika in die laaste 17 miljoen jaar plaasgevind het, ooreenstemmend met die Mio-Plioseen vorming van die berge in die area. Dieselfde tegniek is daarna aangewend om die temporale voorkoms van voorvaderlike ekologiese eienskappe van die genus Disa te rekonstrueer. Die eerste voorkoms van die spesies in die grasveld en savanna biome, sowel as die subalpiene habitat, is in ooreenstemming met bestaande, betroubare geologiese en paleontologiese informasie. Dit suggereer dat filogenieë gebruik kan word om gebeurtenisse te dateer waarvoor daar informasie ontbreek of nie beslissend is nie, soos die ouderdom van die Fynbos bioom en die begin van die winterreënval stelsel in suider-Afrika. Die resultate dui daarop dat dit heelwat ouer is as wat tans aanvaar word en terugdateer na ten minste die Oligoseen.

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

The orchids known today make up one of the largest Angiospermous families in the world. Recent estimates place the number of genera at about 450 which estimates between 10,000 and 15,000 species (some authorities go as high as 20,000). They are without doubt one of the most highly specialized groups of green plants. Botanically the flowers are of more than passing interest for they deviate so distinctly from the norm of the Monocots. They are the possessors of a number of unique structures that are found in no other family of flowering plants.

Made available in DSpace on 2014-06-11T19:30:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-15Bitstream added on 2014-06-13T21:01:39Z : No. of bitstreams: 1 moraes_cp_dr_rcla.pdf: 1437388 bytes, checksum: e066c411adea82f23bca10e7e585f5c6 (MD5) / As Orchidaceae, facilmente reconhecíveis pelo aspecto modificado de suas flores, compreendem mais de 20.000 espécies cosmopolitas distribuídas em cinco sub-famílias. A subtribo Zygopetalinae é um grupo tipicamente neotropical incluído na subfamília Epidendroideae e compreende cerca de 30 gêneros e 418 espécies de hábito vegetativo variado. Visando complementar as informações sobre a estrutura dos órgãos vegetativos dos representantes dessa subtribo, estudou-se a anatomia das raízes, caules e folhas de alguns táxons brasileiros, muitos deles de posição taxonômica incerta e pouco conhecidos, sob o ponto de vista anatômico. Comparando os resultados obtidos com os descritos para outros representantes da mesma subtribo, observou-se uma uniformidade anatômica entre os órgãos das espécies brasileiras e das anteriormente estudadas, corroborando a inclusão de Zygopetalinae na tribo monofilética Cymbidieae. Observou-se, também, uma uniformidade nas características anatômicas, dentro dos gêneros, indicando que muitos aspectos podem ser úteis na análise sistemática da subtribo / The Orchidaceae, easily recognized by the modified aspect of their flowers, comprises more than 20,000 cosmopolitan species distributed in five subfamilies. The Zygopetalinae subtribe is a typically neotropical group included in the Epidendroideae subfamily and comprises around 30 genera and 418 species of varied vegetative habit. Aiming to complement the information about the structure of the vegetative organs of the representatives of this subtribe, the anatomy of roots, stems and leaves of some Brazilian taxa, may of them of uncertain and little known taxonomic position under the anatomical point of view, were studied. Comparing the results obtained with the ones described for the other representatives of the same subtribe, an anatomical uniformity among the organs of the Brazilian species and the ones previously studied was observed corroborating the inclusion of Zygopetalinae in the Cymbidieae monofiletic tribe. It was also observed an uniformity in the anatomical characteristics, within the genera, indicating that many aspects can be useful in the systematic analysis of the subtribe

Orchid growing is a hobby that includes not only acquiring and caring for orchids, but also learning about the diverse care requirements of various orchids, attending meetings of orchid groups, having one’s orchids evaluated by American Orchid Society judges or being a judge, or even creating hybrids. In this way, orchid hobbyists compose a distinctive subculture (Hansen 2000). Yet the activity of orchid growing also forms a nexus between the non-human and the human, two categories that are often constructed as an opposing binary. This thesis focuses on how orchid growing represents both the embedded, institutionalized characteristic of the binary between the non-human and the human and how this binary is actively deconstructed; that is, orchid growers often reinforce this binary through positioning their orchids as a part of “nature” and also blur it by participating in the activity of orchid growing. Through observations of monthly meetings of two local orchid groups and affiliated events and walking tours of individual participants’ orchid growing spaces and semi-structured interviews with them, I show how orchid growing represents such a “hybrid” form of nature (Whatmore 2002). Specifically, the ways in which orchid growers appreciate the novelty of their orchids, care for them, and establish authenticity in orchid growing demonstrates the nuanced ways orchid growing forms a relationship with “nature.”

Background and Aims Sexual deception is a species-specific pollination strategy commonly found in Orchidaceae. Sexually deceptive orchids lure male insect pollinators by mimicking the sex pheromones and/or appearance of female insects, which elicit copulatory behaviour with the flower by the male insects. This specialised pollination strategy has recently been found in a Pterostylis species in Australia. Pterostylis orchids also occur in New Zealand, although very few studies have been done on this genus, and no such specialised insect pollination strategy has been documented in New Zealand. Methods I investigated the breeding system and pollinators of three Pterostylis spp. to determine whether sexual deception may be operating in P. oliveri, P. irsoniana and P. venosa growing in native beech forests in Arthur's Pass. We also investigated the floral headspace volatiles of P. oliveri to determine which compounds are present, and which may be responsible for pollinator attraction. Key Results Breeding system experiments suggest that P. oliveri and P. irsoniana are self compatible, but exclusively dependent on insects for pollination. Only male fungus gnats (Diptera: Mycetophilidae) were found carrying pollinia attached to their thoraxes in traps set up over the flowers. Insect identification and ITS DNA analysis of the pollinia showed that each orchid species was pollinated by a specific fungus gnat species; Mycetophila latifascia males found with pollen of P. oliveri; Morganiella fusca males found with pollen of P. irsoniana; and Tetragoneura sp. males found with pollen of P. venosa. Field tests of an unidentified compound found in headspace volatiles of P. oliveri did not attract any Mycetophila latifascia males. Conclusions These results indicate that pollination via sexual deception may be operating in these three Pterostylis spp. However, further floral volatile analyses are required to confirm whether the flowers emit volatile compounds that resemble the sex pheromones of the specific pollinators.