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

DNA fingerprinting was applied for the molecular elucidation of taxonomic relationships within a genus of orchids which have previously been based on morphological characteristics. Phragmipediwn consists of 15-20 species native to Central and South America. This research project included two studies. In the first study DNA was isolated from 11 samples (including two unidentified ones). These individuals, which were mostly hybrids, were found in the Wheeler Orchid Collection and Species Bank at Ball State University. In order to position Phragmipediwn within the orchid family fingerprinting was also performed on individuals in the sister taxa, Cypripedium and Paphiopedium, which are members of the same subfamily, and on a member of the outgroup taxon Vanda. The polymerase chain reaction (PCR) was employed to yield fingerprints resulting from the use of random primers. Fifty nine random amplified polymorphic DNA (RAPD) bands were obtained using 5 different primers to yield 107 polymorphic bands. As many as 75% of genetic loci were found to be shared between hybrids that resulted from a cross of more than one individual in the same section. However the percentage dropped to 35-65 % when only one parent was shared in the cross. Furthermore, the sister group taxa Cypripedium and Paphilopedium shared from 12 % -35 % of their polymorphic loci with the members of the genus Phragmipedium. The outgroup taxon Vanda shared 17% of its polymorphic loci with the rest of the samples.In a second study DNA was isolated from one member of each of the five sections of the genus Phragmipedium, and RAPD-PCR fingerprinting was used to compare their genetic similarities to that of the two sister taxa and the outgroup taxon. It was found that individuals in different genera shared 25% or less of their polymorphic bands. Between sections of the same genus 20-50% of genetic loci were shared. Two sections, Platypetalwn and Phragmipedium showed the highest degree of genetic relatedness (41-53%). Again the outgoup taxon shared less than 20% Phragmipediwn samples on the phenograms produced but the percentage was again insignificant. However, genetic analyses of the members of the section Lorifolia gave conflicting results: 46% genetic identity was observed in the first trial and 20% in the second.In conclusion, RAPD-PCR fingerprinting results appeared to be effective in the positioning of sections within a genus indicating the degree of similarity of closely related taxa. Also RAPD-PCR was able to place an unknown individual within a specific section of the genus. However, it could not be employed to determine the identity of unknown species due to the high degree of genetic diversity observed between even closely related individuals. / Department of Biology

To study resistance (R) genes that are expressed when Sophrolaeliacattleya Ginny Champion 'Riverbend' orchid tissue was infected with the tobacco mosaic virus (TMV0), a subtraction library of cDNA clones was previously constructed using mRNA isolated before and after infection (Shuck, unpublished). From 200 clones collected, 5 clones were randomly selected, DNA was isolated, and the cDNA insert was sequenced. These sequences were imported into BLAST to search for homology to other R genes. This search revealed clone 4A to have an 84% homology to a 54 nucleotide region from the Arabidopsis thaliana oligouridylate binding protein which is highly expressed and known to bind RNA Polymerase III transcripts and adenovirus associated RNAs. Further bioinformatics analysis was performed utilizing databases and analysis packages available on the Internet, software such as Vector NTI (Informax, Bethesda, MD), and manual searches. However, no additional domains or motifs indicative of pathogen resistance genes were located in any of the 5 clones. Subsequently, total proteins expressed at various time points following infection were examined on denaturing 5-20% gradient polyacrylamide gels stained with the ProteoSilver Plus TM silver stain kit (Sigma, St. Louis, MO) in order to examine the timing and duration of expression of proteins involved in TMV-O resistance. One protein of-18 kDa was highly expressed at 4 hr after infection that was not seen in the negative control. By 8 hr the band was no longer expressed, it was expressed again from 30 - 48 hr, but was not seen again in later time points. Finally, total mRNA isolated from pooled time points and subjected to in vitro translation indicated a reduction in translation products after infection, providing evidence of posttranscriptional gene silencing (PTGS) following TMV-O infection. / Department of Biology

This research was an attempt to identify and provide a phylogeny of Vanilla orchids using PCR sequencing/ fingerprinting.Traditional classification methods haved relied heavily on morphological traits and are often contradictory, subjective and incomplete. This research attempted to discover an objective and reliable method for identification and classification of selected tropical orchids species using molecular techniques. Vanilla orchids were chosen for their commercial importance in the scent and flavoring industry.Reconstruction of phylogenetic data is possible using relatively new molecular techniques: Polymerase Chain Reaction (PCR) fingerprinting and sequencing. PCR fingerprinting reveals restriction fragment length polymorphisms (RFLPs) within an organism's genome. Thesevariations can be used to construct genetic maps in a variety of species. PCR sequencing reveals genetic variation at an even greater level: nucleotide sequence.The literature suggests that these methods are fairly quick and simple; however, the crucial step is obtaining high molecular weight DNA digestable with restriction enzymes or amplifiable by the PCR. DNA isolation has proven to be difficult with Vanilla orchids due to their numerous phenolic compounds, tough fibrous tissue and high number of polysaccharides. This research developed a method of isolating high molecular weight DNA from orchids. This DNA was digestible with restriction enzymes. The DNA was subjected to DNA fingerprinting with primers specific to the 18S-26S ribosomal RNA gene and with RAPD primers. The DNA was also sequenced using the PCR technique.Variation between Vanilla species genomes was discovered and compared to traditional phylogenetic information. It was found that banding patterns and nucleotide sequences were almost identical for Vanilla planifolia and planifolia varegata. The banding patterns for Vanilla barbellata were similar to those of the two unknown Vanilla orchids. Vanilla humboltii exhibited a banding pattern far different from either barbellata or planifolia. These results confirm the hypothesis that PCR fingerprinting/ sequencing is a useful technique for the identification and phylogenetic analyses of Vanilla orchids. / Department of Biology

Thesis (Ph.D.)--University of Western Australia, 2008.

Resumo: 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 / Abstract: 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 / Orientador: Maria das Graças Sajo / Coorientador: Fábio de Barros / Banca: Neuza Maria de Castro / Banca: Tatiane Maria Rodrigues / Banca: Adelita Aparecida Sartori Paoli / Banca: Sandra Maria Carmello Guerreiro / Doutor

Orientador: Renato de Mello Prado / Banca: Paulo Hercilio Viegas Rodrigues / Banca: Renato Fernandes Galdiano Júnior / Resumo: A produção comercial de orquídeas é uma atividade de destaque na floricultura mundial, entretanto há falta de informações sobre a adubação nitrogenada quanto às proporções de nitrato e amônio como fonte de nitrogênio e aplicação de elementos benéficos como o silício. O silício via foliar pode beneficiar e em excesso pode prejudicar o crescimento de orquídeas dependendo da fonte e da concentração do elemento na solução. O crescimento adequado das orquídeas epífitas depende da proporção entre amônio e nitrato utilizada como fonte de nitrogênio, entretanto, não há informações sobre o efeito a longo prazo. Portanto, foram desenvolvidos dois experimentos com duas orquídeas epífitas em cada experimento: Phalaenopsis Golden Peoker e Dendrobium Valentine. As plantas foram cultivadas em bandejas plásticas com Sphagnum seco e mantidas em casa de vegetação recebendo solução nutritiva nos primeiros seis meses, depois foram transplantadas para vasos plásticos (0,9L) individuais e aplicados os tratamentos. O experimento 1 foi em esquema fatorial 5x3, com cinco concentrações de Si (controle; 14,3; 28,6; 42,9 e 57,2 mmol L-1) e três fontes (ácido monossilícico, silicato de potássio e silicato de potássio e sódio) com cinco repetições e em delineamento inteiramente casualizado. Após 18 meses de aplicação de Si foram avaliados nos dois híbridos comerciais o acúmulo de: Si, C, N, P, K, Ca, Mg e S na parte aérea; teor de lignina e proteína e variáveis biométricas. No experiment... (Resumo completo, clicar acesso eletrônico abaixo) / Abstracts: The commercial production of orchids is a prominent activity in the world floriculture, however there is a lack of information regarding the management and recommendation of the fertilization regarding the proportions of nitrate and ammonium as a source of nitrogen and application of beneficial elements such as silicon. Foliar silicon may benefit and in excess may impair the growth of orchids depending on the source and the concentration of the element in the solution. The adequate growth of epiphytic orchids depends on the ratio of ammonium and nitrate used as a source of nitrogen, however, there is no information on the long-term effect. Therefore, two experiments were carried out with two epiphytic orchids in each experiment: Phalaenopsis Golden Peoker and Dendrobium Valentine. The plants were grown in plastic trays with dry Sphagnum and kept in a greenhouse receiving nutrient solution in the first six months, then transplanted to individual plastic vessels (0.9 L) and applied treatments. Experiment 1 was a 5x3 factorial scheme with five concentrations of Si (control: 14.3, 28.6, 42.9 and 57.2 mmol L-1) and three sources (monosilicic acid, potassium silicate and sodium potassium silicate) with five replicates and in a completely randomized design. After 18 months of application of Si, the accumulation of: Si, C, N, P, K, Ca, Mg and S in aerial part was evaluated in the two species; lignin and protein content and biometric variables. In experiment 2, the treatments were five proportions of nitrate / ammonium (0/100, 25/75, 50/50, 75/25, 100/0) with five replicates arranged in a completely randomized design. The sources of nitrate and ammonium were calcium nitrate and ammonium sulfate, respectively. After 12 months of the beginning of the treatments, when the plants were able to flowering, the accumulation of: N, P, K, Ca and Mg in... (Complete abstract click electronic access below) / Mestre

Resumo: A realização do trabalho objetivou desenvolver uma técnica comercial de aplicação do ácido giberélico via pulverização foliar para o controle da floração de orquídeas Phalaenopsis, Cattleya e Brassocattleya, além da redução do tempo para a primeira florada em Phalaenopsis, fatores limitantes no cultivo. Os experimentos foram realizados no Setor de Biotecnologia e Orquidicultura da Fundação Shunji Nishimura de Tecnologia, Pompéia-SP. Foram conduzidos três experimentos. Como híbridos foram utilizados Cattleya Ireni Holguim, Brassocattleya Marcela Koss, Phalaenopsis FSNT "Snow F1" e Phalaenopsis FSNT "Dai-Itigô". Para C. Ireni Holguim e Bc. Marcela Koss testaram-se 4 concentrações de GA3 (125, 250, 500 e 1.000 mg L-1) em 4 aplicações com intervalos de 7 dias via pulverização foliar, nos meses de Outubro/Novembro para Bc. Marcela Koss e Janeiro/Fevereiro para C. Ireni Holguim, em plantas adultas, além de 2 condições hídricas (sem e com estresse hídrico). Não foi possível induzir a floração em Cattleya Ireni Holguim com o uso de GA3. Para Bc. Marcela Koss, a associação da aplicação de 250 mg L-1 de GA3 e a colocação das plantas em condições de estresse hídrico induziu cerca de 83% das plantas a florescerem fora da época normal de florescimento. Na mesma concentração de GA3, porém em condições normais de irrigação, apenas 17% das plantas foram induzidas a florescer. O número e o tamanho das flores aumentaram com o aumento das concentrações de GA3 utilizadas no experimento. Também se testou a utilização do ácido giberélico (GA3) na indução floral fora de época e qualidade do florescimento de orquídeas adultas de Phalaenopsis FSNT "Snow F1", híbrido de coloração branca. Foram realizadas duas e 4 aplicações de GA3, a intervalos de 7 dias nos meses de Novembro/Dezembro. O melhor resultado ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this works was to develop a commercial technique to control blooming in orchids, via the use of foliar sprays with gibberellic acid in Phalaenopsis, Cattleya, and Brassocattleya orchids, in addition to reducing the time required for the first blooming in Phalaenopsis, which are factors that restrict growth. It was conducted three works. The influence of gibberellic acid and water stress on the induction and quality of flowering was tested in two hybrid genera of orchids, Cattleya and Brassocattleya. The experiment was conducted at the Biotechnology and Orchidculture Sector of the Shunji Nishimura Technology Foundation, in Pompéia, SP. As hybrids was use Cattleya Ireni Holguim, Brassocattleya Marcela Koss, Phalaenopsis FSNT "Snow F1" and Phalaenopsis FSNT "Dai-Itigô". In C. Ireni Holguim and Bc. Marcela Koss, four GA3 concentrations (125, 250, 500, and 1,000 mg L-1) were tested in four applications at 7 day- intervals by foliar sprays on adult plants, in addition to two humidity conditions (with or without water stress). Applications were made in October/November in Bc. Marcela Koss and January/February in C. Ireni Holguim. It was not possible to induce flowering in Cattleya Ireni Holguim with gibberellic acid. In Brassocattleya Marcela Koss, the association between 250 mg L-1 GA3 and plants grown under water stress conditions induced blooming in 83% of plants treated under adverse conditions. At the same GA3 concentration, but without water stress, only 17% of the treated plants bloomed. The number and size of flowers increased as GA3 concentration increased. Another objective of this work was to test the use of GA3 to induce off-season flowering and improve blooming quality in adult Phalaenopsis FSNT "Snow F1". Four GA3 concentrations (125, 250, 500, and 1,000 mg L-1) in 2 and 4 applications (at 7-day intervals) were tested. Spray applications were ...(Complete abstract click electronic access below) / Orientador: João Domingos Rodrigues / Coorientador: Elizabeth Orika Ono / Banca: Armando Tavares / Banca: Carmen S. F. Boaro / Mestre

Orchids are an endangered group of plants, protected both in the Czech Republic and in the whole world. Questions of their protection are therefore lively discussed, but not all factors, affecting their presence, are known so far. The purpose of this work was to find out, which environmental factors influence the existence of certain orchid species at their localities in the selected area. This is important for better protection of orchids, because only by knowing these factors we can find new sites, or improve management plans of the existing ones. Another purpose of this work was to find out what is the main reason for extinction of orchids at their historical localities and whether or not there is a possibility of finding other, yet unknown localities of these species. This thesis is based on data from databases, which were also updated during the data collection. The data were processed by computer software MaxEnt, which produces species distribution models and allows to predict potential occurrence of orchids even at yet unknown localities. This software also analyses the environmental factors affecting species presence. I found that the main reason of extinction of orchids at their historical localities was overgrowing. Main environmental factors affecting orchid occurrence were analysed for...

The morphology, anatomy and pollination biology of Mystacidium Lindl., a small, epiphytic genus of orchids, was investigated within a phylogenetic context. Morphological and anatomical studies were carried out in order to obtain characters for a cladistic analysis of the genus using Cyrtorchis arcuata (Lindl.) Schltr. as an outgroup. The phylogenetic analysis indicated that the genus may not be monophyletic. Two species of the closely related genus Diaphananthe Schltr., D. caffra (H.Bol.) Linder and D. millarii (H.Bol.) Linder, appear to be nested within Mystacidium. Mystacidium species grow in habitats varying from mistbelt forest to dry savanna. Analysis of stable isotope composition (Ȣ¹³C values) of leaves and roots showed that all Mystacidium species, as well as D. caffra and the outgroup C. arcuata, employ CAM photosynthesis. The Ȣ¹³C values were significantly negatively correlated with mean annual rainfall at the collection sites. Breeding system experiments revealed that Mystacidium is dependent on pollinators for fruit set, and that self-pollination results in substantially reduced seed set due to either inbreeding depression or partial self-incompatibility. Field observations revealed that M. venosum Harv. ex Rolfe and M. capense (L.f.) Schltr. are hawkmoth-pollinated, and that M. gracile Harv. and M. pusillum Harv. are pollinated by settling moths. The spurs of the flowers contain dilute, sucrose-dominant nectar. Mystacidium venosum and M. capense showed evidence of nectar reabsorption. Nocturnal emission of scent occurred in all species except M. aliceae H. Bolus and M. brayboniae Summerh., which are unscented, and was composed largely of a combination of monoterpenes and benzoids. Despite substantial variation in spur length (1 - 4.7 cm) among species, no evidence for directional selection on spur length was found in M. venosum, M. capense or M. gracile. Hand pollinations significantly increased fruit set in M. capense in two consecutive seasons at different sites, indicating pollen limitation. Although pollen removal was greater than pollen receipt in M. venosum, M. capense and M. gracile, suggesting transport loss or insufficient visitation, a remarkably high percentage of removed pqllen reached stigmas (35 - 50%). Experiments on M. venosum revealed that flower longevity is reduced by pollination, and that pollinia removed from flowers remained viable for up to 20 days under field conditions. The phylogeny indicated that long-spurred, hawkmoth-pollinated species are basal within the genus, and that shorter-spurred species pollinated by noctuid moths are derived. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2002.