Progress 10/01/03 to 09/30/06
Outputs
The goal of this project to modify Oncidium flower color thereby developing novel products to help the U.S. Pacific Basin orchid grower thrive in the expanding Oncidium floriculture industry. Objective 1. To express desired traits to change flower color in Oncidium Gower Ramsey. The yellow coloration of cut flower Oncidium is due to the presence of carotenoids. We have isolated a number of carotenoid biosynthesis genes or gene fragments including, the phytoene synthase, phytoene desaturase, carotenoid isomerase and 9-cis epoxycarotenoid dioxygenase from Oncidium. Also isolated was the anthocyanin biosynthesis gene, dihydroflavonol 4-reductase, responsible for the red splotches seen in the petals, sepals and lip crest. Interestingly northern analysis has demonstrated that not all genes were up-regulated in Oncidium flowers. Some of the genes isolated demonstrated multiple copies with variations caused by insertions or deletions. Suspecting that this is due to genetic
instability arising from the polyploid nature of the Oncidium Gower Ramsey cross, we have begun isolating the same genes from candidate parental plants. Although we have now isolated a number of these genes from the parental species, Oncidium sphacelatum, Oncidium flexuosum and Oncidium varicosum, we were unable to find an exact match with the genes from Oncidium Gower Ramsey. The observed minor differences are most likely due to geographic differences in source of species plants used. Objective 2. To develop a reliable and improved regeneration procedure for transformed orchids. Transformation experiments have been using both biolistic bombardment and Agrobacterium inoculation. Experiments had indicated that protocorm-like bodies (plbs) are sensitive to bombardment conditions causing sensitivity to antibiotic selection and an increased death. We have found that biolistic bombardment at lower pressures, with multiple shootings, followed by a longer recovery period better prepared the
plant material for selection. Previous experiments with Agrobacterium infection of etiolated internodes demonstrated successful transient expression beta-glucuronidase expression however, regeneration from these treated tissues still remains problematic. A similar approach, as used in biolistic bombardment, to let the etiolated internodes recover after treatment while on an Agrobacterium-specific antibiotic was also unsuccessful at achieving regeneration. The current focus is on generating more plant material to continue with biolistic bombardment and regeneration experiments.
Impacts
Oncidium orchids are a growing segment of orchid production in the US. Agricultural statistics in Hawaii show a steady increase in Oncidium cut-flower production and a booming up-and-coming market for potted Oncidium plants. The increase in potted plants is in part due to the excellent growth and propagation characteristics of Oncidiums. Although the cut-flower Oncidiums share these same growth characteristics their lack of variation in flower color limits their marketability and use. The mainstay of the Oncidium cut flower industry is the yellow flowered sprays of Oncidium Gower Ramsey and similar yellow variants. This project has defined the yellow floral color compounds and also the molecular basis behind them. This data is particular useful in further developing technologies to modify the flower color in Oncidium. The successful development of innovative new varieties is crucial to the overall growth of the U.S. Oncidium floriculture industry. New varieties will
directly benefit the U.S. Pacific Basin orchid grower by making them more competitive in the world orchid market.
Publications
- Hieber, AD., Kuehnle AR. Mudalige-Jayawickrama R.G. 2005. GenBank Accession Numbers AY953937-AY953939. Color genes in the orchid Oncidium Gower Ramsey:Dihydroflavonol 4-reductase
- Hieber, AD., Kuehnle AR. Mudalige-Jayawickrama R.G. 2005. GenBank Accession Number AY973631. Color genes in the orchid Oncidium Gower Ramsey:Phytoene synthase
- Hieber, AD., Kuehnle AR. Mudalige-Jayawickrama R.G. 2005. GenBank Accession Number AY973632. Color genes in the orchid Oncidium Gower Ramsey:Phytoene desaturase
- Hieber, AD., Kuehnle AR. Mudalige-Jayawickrama R.G. 2005. GenBank Accession Numbers AY973633- AY973634. Color genes in the orchid Oncidium Gower Ramsey:Carotenoid isomerase
- Hieber, AD., Kuehnle AR. Mudalige-Jayawickrama R.G. 2005. GenBank Accession Numbers AY974325-AY974327. Color genes in the orchid Oncidium Gower Ramsey:9-cis Epoxycarotenoid dioxygenase
- Obsuwan, K., Hieber A.D., Mudalige-Jayawickrama R.G., Kuehnle, A.R. 2006. Functional characterization of Dendrobium and Oncidium Dfr in Petunia hybrida model. 27th International Horticulture Congress, Seoul, Korea. Pp 335-336.
- Mudalige-Jayawickrama R.G., Kuehnle, A.R., Champagne M.M., Hieber A.D. 2006. Elucidating orchid flower colour and gene regulation of pigmentation and morphology. 4th International Workshop on Anthocyanins, Rotorua New Zealand, pg. 39.
- Hieber, A.D., R.G. Mudalige-Jayawickrama, A.R. Kuehnle. 2006. Color genes in the orchid Oncidium Gower Ramsey: identification, expression, and potential genetic instability in an interspecific cross. Planta 223: 521-531
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Progress 10/01/04 to 09/30/05
Outputs
This projects goal is to modify Oncidium flower color creating novel products to help the U.S. Pacific Basin orchid grower in the expanding Oncidium floriculture industry. Objective 1. To express desired traits to change flower color in Oncidium Gower Ramsey. Previously we had isolated the phytoene synthase (PSY) gene from Oncidium. Northern analysis has since demonstrated that this gene was not up-regulated in Oncidium flowers. This result was unexpected as it is up-regulated in a number of other flower models. To find a gene that is up-regulated for future promoter studies we isolated a number of other candidate genes. Other genes or gene fragments isolated include the carotenoid biosynthesis genes, phytoene desaturase (oPDS), carotenoid isomerase (ocrtISO) and 9-cis epoxycarotenoid dioxygenase (NCED). Also isolated was the anthocyanin biosynthesis gene, dihydroflavonol 4-reductase (DFR). Northern analyses demonstrated that some of these genes were up-regulated in
the floral tissue and as such are suitable for further promoter studies. Interestingly the isolation of these genes revealed in some cases multigene families with various insertions and deletions among the different members. We have hypothesized that this may be due to genetic instability arising from the polyploid nature of the Oncidium Gower Ramsey cross. This current research has culminated in a scientific paper that has recently been published in the journal Planta (Hieber et al. 2005). This work was presented to the Big Island Dendrobium Growers Association (BIDGA) in Fall 2005. Objective 2. To develop a reliable and improved regeneration procedure for transformed orchids. Previous experiments indicate that Oncidium protocorm-like bodies (plbs) are compromised by the biolistic pressure, causing sensitivity to antibiotic selection and an increase in wounding and death from certain pressures. To this end, experiments aimed at developing an Agrobacterium-based system have also been
pursued. We have been able to show Agrobacterium infection of etiolated internodes and plbs by following the transient expression of the beta-glucuronidase (GUS) expression. Although regeneration has been shown for untreated etiolated internodes, experiments attempting regeneration from the treated etiolated internodes are still ongoing. Similar regeneration experiments with antibiotic selection are currently planned for the plbs.
Impacts
Oncidium orchids are an important part of floriculture production to the US orchid grower. Hawaii agricultural statistics show that the value of cut-flower Oncidium sales has been steadily increasing across the last twelve years. Although cut flower Oncidium plants have excellent growth and propagation characteristics, the lack of variation in flower color limits their marketability and use as a cut flower. New colors would greatly benefit the U.S. orchid grower. This project is presently uncovering the molecular basis behind flower color in Oncidium and is in the process of developing technologies that are directly applicable to modifying the flower color in Oncidium. These technologies are of great value to the plant geneticist to be able to research orchid cut flower's biology and to facilitate production of even better varieties. The development of novel new colors is crucial to the U.S. Pacific Basin Oncidium grower to be competitive in the world orchid market.
Publications
- Hieber, A.D., Mudalige-Jayawickrama, R.G. and Kuehnle A.R. 2005. Color genes in the orchid Oncidium Gower Ramsey: identification, expression, and potential genetic instability in an interspecific cross. Planta. Sep 6:1-11 [Epub]
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Progress 10/01/03 to 09/30/04
Outputs
The goal of this project to modify Oncidium flower color thereby supporting creating novel product development to help the U.S. Pacific Basin orchid grower thrive in an expanding Oncidium floriculture industry. Objective 1. To express desired traits to change flower color in Oncidium Gower Ramsey. We have already demonstrated that the yellow coloration is due to the presence of carotenoids. The first step specific of the carotenoid pathway is catalyzed by phytoene synthase (PSY). Using PCR technology we have been able to isolate a full-length phytoene synthase gene from Oncidium flower buds. Analysis of the DNA sequence shows 82% homology to daffodil and 80% homology to marigold, both of whose flower color is due to carotenoids. Tissue section analysis was performed on the Oncidium flowers to establish the site and nature of the coloration. Sections across the lip showed that the majority yellow color is localized in the upper layers of the lip in the conical
papillate cells that comprise the top layer and the underlining epidermal cells. On the underside of the lip some yellow coloration is seen in a thin layer of underlying epidermal cells but not in the papillate cells on the lower surface. In contrast to the lip, sectioning of the petal and sepals showed the surface cells are flat epidermal cells containing yellow coloration. Although the yellow is also localized in the upper layers, a greenish color, presumably from chloroplasts, can also be seen spread throughout the various layers of the sepals and petals. Interestingly, the red coloration appears to be localized in only the top layer of conical papillate cells in the lip crest and also only in the top layer of epidermal cell of the sepals and petals. The red coloration was not extractable in organic solvents but extracted in acid. This result together with preliminary tissue cross-sections, suggested that the red coloration is due to an anthocyanin-based pigmentation. Thin layer
chromatography (TLC) has confirmed the red coloration of the petals, sepals and lip crest to be anthocyanins. Objective 2: To develop a reliable and improved regeneration procedure for transformed orchids. Experiments aimed at developing an Agrobacterium based system using etiolated internodes has been started. Initial experiments indicate that although the etiolated internodes are susceptible to Agrobacterium infection they are vulnerable to immersion in the Agrobacterium solution. Agrobacterium treated internodes have also been shown to be more sensitive to antibiotic inhibition. Similar results have been obtained from the gene gun shooting experiments. Although we have been able to demonstrate transient gene expression in protocorm-like bodies (plbs), use of the same bombardment conditions causes cellular death before antibiotic selection pressure can be applied. This suggests a serious or fatal wounding resulting from the biolistic pressure. The conditions for bombardment may have
been suitable for transient expression, however due to these findings experiments using decreased biolistic pressures are now being investigated for creation of stable nuclear transformants.
Impacts
Orchids are one of the fastest growing segments of floriculture in the Pacific. The development of methodologies to genetically engineer the Oncidium orchid will be of great value to the plant geneticist who will be able to research this cut flower's biology and facilitate the development of better varieties for the orchid grower. These developed technologies will be directly applied to modifying the color of Oncidium orchids. The mainstay of the Oncidium cut flower industry is the yellow flowered sprays of Gower Ramsey and similar yellow variants. Although these orchids have excellent growth and propagation characteristics the lack of variation in flower color limits their use as a cut flower. Success in the development of innovative new varieties is crucial to the overall growth of the U.S. Oncidium floriculture industry and will directly benefit the U.S. Pacific Basin orchid grower by making them more competitive in the world orchid market.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
No progress to report. This project was initiated on October 1, 2003.
Impacts
The development of methodologies to genetically transform the Oncidium orchid, Gower Ramsey and similar yellow variants, will be of great value to the plant geneticist who will be able to research this cut flower's biology and facilitate the development of better varieties for the orchid grower. The developed technologies will be directly applied to modifying the color of Oncidium orchids. The establishment of new colored varieties is crucial to the overall growth of the U.S. Oncidium floriculture industry. The development of innovative new varieties will benefit the U.S. Pacific Basin orchid grower by making them more competitive in the world orchid market.
Publications
- No publications reported this period
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