Progress 10/01/03 to 09/30/06
Outputs
This research project successfully isolated and characterized regulatory genetic elements of Dendrobium orchid, namely two transcription factors and one promoter element, of the two most important floral traits of flower shape and color. For Objective 1, isolation of genes controlling pansy-lip and white flowers, a subtractive hybridization was performed between the cDNA of lip and the petal tissues from a normal-lip Den. Jaquelyn Thomas hybrid (UH232). This resulted in the isolation of cDNA fragment belongs to the TCP transcription factor family. This cDNA fragment was used to isolate the full clone from normal-lip and pansy-lip phenotypes of Den. Ethel Kamemoto lines. Nucleotide sequence of this clone, Den-TCP-1, was submitted to GenBank with a release date of December 31 2006 (accession no. DQ517495). Copy number and the restriction fragment pattern of Den-TCP-1 were determined in parents, F1 and F2 lines using Southern analyses. This showed a minimum of four copies
of TCP transcription factors in Dendrobium. Segregation of restriction patterns in the F2 generation of pansy-lip and normal lip closely resembled the parental phenotypes of pansy-lip in line D427-1 and normal lip in line D383, respectively, confirming the recessive nature of the pansy-lip trait, with semi-dominance for the intermediate F1. Flower color regulatory genes responsible for white phenotype was obtained using a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method with degenerate primers. It resulted in isolation of two partial clones of R2R3 Myb family of transcription factors. Full-length Myb transcription factor (Myb 9) from the flower buds of UH503 was isolated these partial sequences. A similar Myb transcription factor for Dendrobium has recently been described by others in GenBank. In addition to the regulatory genes, we isolated an anthocyanin biosynthetic gene, flavonoid 3', 5'-hydroxylase (F3'5'H) of the cytochrome p450 family (GenBank accession no.
DQ923127). A Northern analysis of flower bud, open flowers and leaves showed undetectable levels of expression at 20 microgram of total RNA loads. However, RT-PCR revealed expression of F3'5'H in buds as well as open flowers. Objective 2: Isolation and characterization of Dendrobium floral-specific promoters. We showed that the expression of the color gene Dihydroflavonol 4-reductase (Dfr) is floral specific and the temporal expression coincides with the color development of the flower buds. A 500 base pair fragment of the Dfr promoter region was cloned into an expression vector (pBI525) containing the reporter gene uidA for GUS expression. Bombardment conditions into petal tissues were optimized for the Particle Inflow Gun (PIG). Our transient expression studies revealed that this 500 bp fragment was sufficient to drive the expression of uidA gene in petal tissue.
Impacts
The main goal of this research was to isolate and characterize the genetic regulatory elements that control the flower shape and color. This was achieved through isolation of floral specific promoters and transcription factors that regulate color and shape. University of Hawaii is a leader in the classical and molecular breeding program for Dendrobium. Transcription factors and promoters isolated through this project will be an invaluable tool in strengthening the orchid molecular breeding program. The University helps the US Pacific basin orchid growers to remain globally competitive by providing new cultivars and hybrids for the cut flower and potted plants. This project underscores how imperative it is to use new molecular techniques in order to compete with the aggressive competition from the other orchid growing countries, which are already using molecular techniques to enhance their breeding programs. We have isolated transcription factors that regulate the two
most important features of Dendrobium flowers: their shape and color. We have also generated a set of binary vectors for transformation of orchids. The functionality of these genes and their efficacy in delivering the expected phenotype can now be tested in a fast flowering petunia model. With this newly developed set of molecular tools, manipulating flower color and shape will open a new door to an exciting variety of color and shape while minimizing any adverse effects on the plant growth and environment.
Publications
- Mudalige-Jayawickrama R.G., Champagne M.M., Hieber A.D., and A.R. Kuehnle. 2005. GenBank Accession Numbers AY741318-AY741319. Cloning and characterization of two anthocyanin biosynthetic genes from Dendrobium orchid (Dendrobium orchid dihyrdoflavonol 4-reductase and chalcone synthase).
- Kuehnle A. 2005. Adventures in orchid bioengineering for improved color and disease resistance. 16th biennial meeting of the New Zealand branch of the IAPTC&B, pg. 9
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Progress 10/01/04 to 09/30/05
Outputs
This research project is directed towards isolation and characterization of regulatory genetic elements (transcription factors and promoter elements) of the two most important floral traits: flower shape and color. Objective 1: Isolation of genes controlling pansy-lip and white flowers of Dendrobium A subtractive hybridization between F1 and F2 hybrid lines of Dendrobium Ethel Kamemoto (K1337=normal lip and K1449=pansy lip) was performed in order to isolate the gene that regulates the lip formation. This method did not yield the expected due to viral infection of K1449 lines. A second subtractive hybridization was performed between the cDNA of lip and the petal tissues from a normal-lip Dendrobium Jaqelyn Thomas hybrid. This resulted in the isolation of two cDNA fragments that are transcription factors. Their role in lip formation and expression patterns in floral tissue is currently being investigated. A similar subtractive hybridization is being done between the
colored lips and white petals of a Dendrobium in order to isolate the genes that regulate flower color. Objective 2: Isolation and characterization of Dendrobium floral-specific promoters. Two full-length cDNA clones, Den-CHS-4 and Den-DFR-1, encoding chalcone synthase (CHS) and dihydro-flavonol 4-reductase (DFR) were obtained from flower bud RNA of a lavender cyanidin- accumulating Dendrobium Sw. hybrid using reverse transcription-polymerase chain reaction PCR (RT-PCR). Northern analyses indicated that both genes are expressed in all developmental stages of buds, with highest expression in the medium size. RT-PCR analyses showed that DFR expression was confined to floral tissue while CHS was expressed in floral and vegetative tissues but not in pseudobulbs. The nucleotide sequence of a DFR clone isolated from a pale orange pelargonidin-accumulating Dendrobium hybrid was exactly the same as Den-DFR-1, ruling out the substrate specificity of DFR as a possible cause of the color
difference. We have used a Genome Walker kit to isolate the 5-prime promoter region of the DFR. A 387 base pair fragment was cloned into a expression vector containing the reporter gene uidA for GUS expression. The bombardment conditions into petal tissues has been optimized for the Particle Inflow Gun. This vector is currently being tested for efficacy in different Dendrobium petals for transient GUS expression.
Impacts
The main goal of this research is to isolate and characterize the genetic regulatory elements that control the flower shape and color. This will be achieved through isolation of floral specific promoters and transcription factors that regulate color and shape. University of Hawaii is a leader in the classical and molecular breeding program for Dendrobium. Transcription factors and promoters isolated through this project will be an invaluable tool in strengthening the orchid molecular breeding program. The University helps the US Pacific basin orchid growers to remain globally competitive by providing new cultivars and hybrids for the cut flower and potted plants. It is imperative to use new molecular techniques in order to compete with the aggressive competition from the other orchid growing countries who are already using molecular techniques to enhance their breeding programs. Manipulating flower color and shape with newly isolated transcription factors that are
targeted to the flower by tissue specific expression will open a new door to an exciting variety of colors and shapes while minimizing any adverse effects on the plant growth and environment.
Publications
- Mudalige-Jayawickrama, R.G., Champagne, M.M., Hieber, A.D. and Kuehnle, A.R. 2005. Cloning and characterization of two anthocyanin biosynthetic genes from Dendrobium orchid. J. Amer. Soc. Hort. Sci. 130:611-618.
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Progress 10/01/03 to 09/30/04
Outputs
The main goal of this research project is to equip the Dendrobium molecular breeding program with genetic tools to understand and control flower phenotypes. This will be achieved through isolation and characterization of regulatory genetic elements (transcription factors and promoter elements) of the two most important floral traits: flower shape and color. Objective 1: Isolation of genes controlling pansy-lip and white flowers of Dendrobium Total RNA from normal and pansy-lip phenotypes were extracted from dissected floral buds of the F1 and F2 hybrid lines of Dendrobium Ethel Kamemoto (K1337 and K1449). Flower buds of these plants were dissected into petals and sepals before extraction. RNA samples from each tissue of the normal and pansy lip siblings were separated on agarose gels and blotted on to membranes for hybridization with the potential heterologous probes of floral symmetry genes isolated from other plants. In the model flower snapdragon, floral symmetry
is controlled by two genes, CYCLOIDEA and DICHOTOMA, which belong to the TCP gene family of transcription factors. Recently, CYCLOIDEA related genes from other plants related to Snapdragon were isolated using PCR technique. Total RNA extracted from snapdragon flower buds was used to isolate the CYCLOIDEA gene through RT-PCR. The resultant PCR product has been cloned and verified by sequence similarity. We will use this as a heterologous probe to identify any potential CYCLOIDEA related genes in Dendrobium through northern hybridization of RNA blots from petals sepals and lips of flower buds. If there is any hybridization to Dendrobium lip tissues, this probe will be used to screen the subtractive library made from normal lip and pansy-lip types for isolation of peloria genes. Objective 2: Isolation and characterization of Dendrobium floral-specific promoters. The full sequences of two Dendrobium flavonoid biosynthetic genes, Dihydroflavonol 4-reductase (DFR) and Chalcone synthase
(CHS), have been deposited in Genbank with accession numbers AY741318 and AY741319, respectively. The expression profiles of the two genes were tested using northern and RT-PCR analyses. Results clearly indicated that DFR is expressed specifically in floral tissue with the highest expression seen in medium sized unopened buds. In contrast, CHS is expressed in floral tissue as well as vegetative tissue with the exception of pseudobulbs. We have used a Genome Walker kit to produce a mini-library of Dendrobium Jaquelyn Thomas Uniwai Prince (UH503) genomic DNA. This library was used to amplify the upstream DNA sequences of the DFR gene through PCR. We have isolated 3 fragments of 300 bp, 450 bp and 3kB using the Genome Walker kit mini library. These fragments are being ligated into promoterless expression vector pBI 426 using Hind III and BamHI sites. These vectors will be used to evaluate the efficacy of each promoter region in transient expression of orchid petals and also by stable
transformation into the model crop, Petunia.
Impacts
Overall goal of this research project is to equip the molecular breeding program of Dendrobium with genetic tools to understand and control flower phenotypes. This will be achieved through isolation of floral specific promoters and characterization of the regulatory elements that control the two most important floral traits: flower shape and color. The University of Hawaii is a leader in classical and molecular breeding of the tropical orchid Dendrobium. The pedigree data from the classical breeding along with new molecular and biochemical analyses of floral traits from this project will also provide invaluable tools to strengthen the University's orchid biotechnology program. Strong and tissue specific expression of the new genes targeted to the flower is important in producing new varieties through bioengineering. Isolation of a floral specific promoter will fulfill this requirement allowing us to strengthen the biotechnology program to produce an exciting new array
of flower colors and shapes. The University helps the U.S. Pacific Basin orchid growers to remain globally competitive by providing new varieties for cut flowers and potted plants. It is envisaged that success of this project will ultimately produce new varieties for the U.S. Pacific Basin orchid grower.
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 October 1, 2003.
Impacts
Understanding the genetic elements that regulate flower color in orchids will allow the generation of new varieties with desirable flower colors and shapes.
Publications
- No publications reported this period
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