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Development of cDNA Microarrays for Gene Expression Research in Floriculture Crops. Dave Clark University of Florida Environmental Horticulture Dept. UF Floriculture Biotech Lab (est. 1995). Our main goal: To bridge the gap between basic molecular biology and applied floriculture
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Development of cDNA Microarrays for Gene Expression Research in Floriculture Crops Dave Clark University of Florida Environmental Horticulture Dept.
UF Floriculture Biotech Lab(est. 1995) • Our main goal: • To bridge the gap between basic molecular biology and applied floriculture • Florida produces 20% of US floriculture crops
UF Floriculture Biotech Development: • Phase 1 – 1995-2000 (then) • Biotechnology • Phase 2 – 2000-2005 (now) • Functional Genomics • Phase 3 – 2005-2010 (next) • Gene Function
Phase 1 – 1995-2000 • Establish a biotech system • Petunia hybrida • Proof of concept research • Expansion of available technology • Build critical personnel mass • Recruit good people • Make strong collaborations
Phase 1 – 1995-2000 • Transgenic Plants - hormones • Gibberellins • Dwarf plants • Cytokinins • “Stay Green” leaves • Ethylene • Long lasting flowers
Internal Growth Regulators AtGA2ox7 Makes dwarf tobacco Schomburg et al. 2003
Internal Growth Regulators AtGA2ox7 - Makes dwarf petunia
Stay Green Leaves sag-IPT Makes leaves stay green longer in tobacco Gan & Amasino 1995
sag-IPT Makes leaves stay greener longer in petunia Clark et al. in press
Long Lasting Flowers Etr1-1 ETR1-1 - Makes plants ethylene insensitive
Long Lasting Flowers Control Etr1-1
Problems with 35S::etr1-1 • etr1-1 petunias – commercially limited rooting pathogens – etr1-1 ‘MD’ etr1-1
Conclusions – Phase 1 • We have a good experimental system • We have good people in the lab • We have good collaborators • We needdifferent genes • We needbetter promoters
Phase 2 – 2000-2005 • Functional Genomics • New tools for genetics research • Exploration for new technologies • New and novel genes • Controlling gene expression • Petal specific promoters
Functional Genomics • Tools that help discover the biological function of genes • Uncovers how groups of genes work together in a biological process • Encompasses many traditional biological and genetic approaches
Petunia EST Project (EST = Expressed Sequence Tags) mRNA extraction Tissue collection Bioinformatics and Microarrays library synthesis EST sequencing
Petunia Flower Genes • Petunia gene sequencing: Floral development library 2774 C2H4-treated flower library 3264 Pollinated flower library 1824 Other libraries 1046 • Total genes sequenced 8908
Microarray Construction 4200 unique petunia genes
Microarray analysis – Ethylene Regulated Genes Green A>B Yellow A=B Red A<B Control (A) Ethylene (B) A+B
Ethylene Regulated Genes • We have isolated approximately 50 genes that are regulated by ethylene • Some of these genes are thought to be involved in making floral scent volatiles • Benzoic acid carboxyl methyltransferase
Phase 2 – Still going • We still bridge the gap between basic molecular biology and applied floriculture • We have a petunia EST database • Microarrays are working well • We have many new and novel genes • We have good promoters
Phase 3 – 2005-2010 • Gene Function • We are ahead of schedule: • Experimental system (phase 1) • Adopted by several groups • New genetic tools (phase 2) • Available to floriculture researchers • Understanding gene function
Flower Scent Composition - Petunia Benzoic acid carboxyl methyltransferase (BAMT) - makes methyl benzoate in petunia
Stop and Smell the Roses! US consumers bought almost 130 million roses on Valentine’s Day 2003 US consumers spent over $7.5B on cut flowers in 2002
Opportunity Knocks! • Commercial cut rose varieties don’t have much fragrance
The Future • We plan to maintain and grow the genetics database • We plan to make transgenic plants expressing new genes • We plan to knock out 1 gene a week – functional analysis • We plan to continue making new collaborations
Who supports us? • Intellectually • Harry Klee – UF • Bill Farmerie – UF • Don McCarty – UF • Michelle Jones – OSU • Jim Giovannoni – USDA/Cornell • EU PetNet Group • Financially • USDA Floriculture Initiative • American Floral Endowment • Fred Gloeckner Foundation • Florida FIRST • The Scotts Company