Special Research Report #304: Breeding & Genetic Engineering Floriculture Genomics: Basic Tools for Improvement though Biotechnology David G. Clark, Professor, Environmental Department University of Florida, Gainesville, FL 32611-0670

BACKGROUND biological function. Results 3: Expression of these have been promising to date, senescence-related genes was leading to the isolation of genes characterized in order to involved in the synthesis of identify components of the several interesting senescence signaling pathways biochemicals: methylbenzoate, and determine the most

FUNDING INDUSTRY SOLUTIONS a chemical giving a appropriate genetic targets for THROUGH RESEARCH AND EDUCATION sweet fragrance (Negre et al., the manipulation of

Phone: 703-838-5211 2003; Underwood et al, 2005); senescence. Fax: 703-838-5212 isoeugenol - clove oil – a E-mail: [email protected] chemical giving flowers a spicy RESULTS Website: www.endowment.org fragrance; benzylbenzoate, In order to satisfy all objectives

used commercially as an set forth in this proposal, the Four years ago, we insecticide ; 2-phenylethanol following significant started an effort in functional (rose oil) and beta-ionone, the milestones of this project were genomics and DNA sequencing two major constituents of rose achieved: has led to the development of fragrance. As a result of these one of the largest collections of efforts, we have produced genes expressed in ornamental 1 – Construct Petunia cDNA transgenic with altered plants (petunia) in the world. libraries from various floral fragrances that are perceived tissues – This milestone was After conducting focused differently by humans, as fully achieved in years 1 and 2 microarray experiments, we measured by sensory panelist of the project with the have isolated several genes that analysis of engineered flowers. production of three we have identified to have cDNA libraries. These potential utility for use in libraries contained genes from genetically engineering plants MATERIALS AND flowers at different with altered hormone METHODS developmental stages, flowers responses, altered floral treated with ethylene, and fragrance, and a variety of 1: DNA sequence data was pollinated flowers as a means other potentially valuable collected from approximately to supply all genes expressed characteristics. We have also 11,500 genes expressed in during development and floral isolated several genes that petunia flowers. A database of senescence. However, we appear to be expressed in clones and sequences has been added to this objective by specific floral tissues (ie. produced that is now available constructing additional cDNA ), and many of these to other floriculture libraries from petunia contain DNA sequence that researchers. , early development shares little or no similarity to (1-5 days after ) and other genes of known function 2: DNA microarrays were ripening fruit (16-24 days after in any other plant. We are constructed to allow for pollination). currently engineering many of identification of groups of these genes to either over- genes transcriptionally 2 – Obtain DNA sequence of express and/or knock out their regulated during ethylene 10,000 random cDNAs – This expression in transgenic plants regulated flower senescence. milestone was fully achieved in in an effort to uncover their years 1-3 of the project with the continual sequencing of the project to produce our own expression and analyzing random cDNAs isolated from DNA sequence database. In phenotypes in transgenic our flower cDNA libraries. year 3, we worked with the plants - Last year, the Clark Additional sequence was Solanaceous Genome Network lab was able to successfully obtained from these libraries at Cornell/USDA to make these prove the function of two genes over the last two years, data available to a broader involved in floral fragrance, resulting in the production of scientific community. Last and we currently have three DNA sequence data from an year, we were successful in additional genes that we additional 1500 cDNAs. This making all of the DNA believe will prove to be now constitutes the world’s sequence data, along with involved in floral scent largest collection of DNA potential functional data for biosynthesis. Once these sequence information for the each gene available for public transgenic plants are made, we species Petunia. use through TIGR. This group hope to use them in was responsible for experiments to determine their 3 – Construct cDNA constructing a petunia gene biological function. Work is microarrays for use in index, and for annotating the still ongoing in the Jones lab to isolating flower specific genes sequence for submission to isolated genes involved in – This milestone was fully NCBI GenBank. floral senescence, but several achieved in years 2-3 of the senescence related genes project (see significant 5 – Isolate DNA promoter encoding cysteine proteases accomplishments below), but elements from flower specific have been isolated as a result of we continue to proceed with genes – This was not an microarray experiments that construction of microarrays for original goal of the project, but are logical candidates for use in gene expression analysis plans to obtain promoter having a role in protein for a broader range of research elements were added later. degradation during collaborators. In the past year, Last year, we achieved our first senescence. the data produced as a result of progress for this objective with this project in cooperation with the isolation of 3 flower petal IMPACT TO THE The Institute for Genomics specific promoter elements INDUSTRY Research (TIGR) has been used from genes that were to produce a set of 70-base pair determined to be expressed This project will produce an oligonucleotides representing exclusively in petunia flower unprecedented number of the entire set of petunia genes petals. We are currently fusing approaches for experiments with collaborators at the Ohio these promoter elements to focused on determining the State/USDA-Wooster. These genes involved in ethylene function and regulation of oligonucleotides will be used to perception to determine if we virtually any gene expressed in produce a standard petunia can increase flower longevity a petunia plant. By microarray that can be by reducing ethylene sensitivity determining the function of distributed to a wider range of exclusively in flower petal interesting genes expressed in scientists for use in the next tissues of transgenic plants. If petunia flowers throughout all year. that proves to be a successful stages of development and approach, we will continue to senescence, we can now get a 4 – Make DNA sequence fuse these promoters to other better understanding of their available for public use – This genes of interest. role in important physiological was not an original objective of processes in floriculture . the project. However, we were 6 – Prove the in-vivo function This knowledge will also help fortunate enough to work with of petunia genes with us to determine the potential the computer engineering potential commercial utility for manipulation of particular department at UF in year 2 of by knocking out their genes for commercial utility through the production of the of the Ethylene III. M. Vendrell, H. Klee, J.C. next generation of transgenic Clevenger, D.J., J.E. Barrett, Pech, F. Romojaro (eds.). IOS plants. As a result of this H.J. Klee and D.G. Clark Press, Amsterdam pp. 370-372. project, we will continually 2004. Factors affecting develop new technologies that production in transgenic Chang H., M.L. Jones, G. will be useful in developing ethylene-insensitive petunias. important research Banowetz and D. Clark 2003. J. Amer. Soc. Hort. Sci. Overproduction of Cytokinins collaborations that are vital to 129:401-406. supplying the floriculture in Petunia Flowers

Transformed with Psag12-IPT industry with new innovations. Clark D.G., C. Dervinis, J. Delays Corolla Senescence and It is our ultimate goal to take Barrett, H. Klee, and M.L. Decreases Sensitivity to promising new technologies Jones 2004. Drought-induced Ethylene. discovered during this research leaf senescence and 132: 2174-2183. into other floriculture crops in horticultural performance of the not too distant future, and Psag12-IPT Petunias. J. Amer. For additional information make flowers smell better, last Soc. Hort. Sci. 129:93-99. longer on retail shelves, and contact Dave Clark at

[email protected] have fewer insect and disease Negre F., C.M. Kish, J. pests. Boatright, B. Underwood, K. Shibuya, C. Wagner, D. Clark REFERENCES and N. Dudareva 2003. Jones, M.J., G.B. Chaffin, J.R. Regulation of floral scent Eason, and D.G. Clark 2005. emission after pollination in Ethylene-sensitivity regulates snapdragon and petunia proteolytic activity and flowers. The cysteine protease gene 15:2992-3006. expression in petunia corollas.

Journal of Experimental Clark D.G., B. Underwood, K. 56:2733-2744. Shibuya, H. Loucas, D.

Tieman, and H. Klee 2003. Microarray Analysis of Floral Underwood, B.A., D.M. Senescence in Petunia. Tieman, K. Shibuya, H.M. Proceedings of the NATO Loucas, R.J. Dexter, A.J. Advanced Research Workshop Simkin, C.A. Sims, E.A. on Biology and Biotechnology Schmelz, H.J. Klee and D.G. Clark 2005. Ethylene- of the Plant Hormone Ethylene III. M. Vendrell, H. Klee, J.C. regulated floral volatile Pech, F. Romojaro (eds.). IOS synthesis in petunia corollas. Press, Amsterdam pp. 137-141. Plant Physiology 138:255-266.

Ciardi J., K. Barry, K. Shibuya, Chen, J-C, C-Z Jiang, T.E. S. Nourizadeh, J. Ecker, H. Gookin, D.A. Hunter, D.G. Klee and D. Clark 2003. Clark, M.S. Reid 2004. Increased flower longevity in Chalcone synthase as a reporter petunia through manipulation 2006 © Copyright in virus-induced gene silencing of ethylene signaling genes. American Floral Endowment. studies of flower senescence. Proceedings of the NATO All Rights Reserved. Plant Molecular Biology Advanced Research Workshop 55:521-530. on Biology and Biotechnology