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 – (then) – Biotechnology Phase 2 – (now) – Functional Genomics Phase 3 – (next) – Gene Function

Phase 1 – 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 – 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

sag-IPT – Drought Tolerance

Long Lasting Flowers Etr1-1 ETR1-1 - Makes plants ethylene insensitive

ControlEtr1-1 Long Lasting Flowers

Problems with 35S::etr1-1 etr1-1 petunias – commercially limitedetr1-1 petunias – commercially limited ‘MD’ etr1-1 rooting pathogens – etr1-1

Conclusions – Phase 1 We have a good experimental system We have good people in the lab We have good collaborators We need different genes We need better promoters

Phase 2 – 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

Tissue collection mRNA extraction library synthesis EST sequencing Bioinformatics and Microarrays Petunia EST Project (EST = Expressed Sequence Tags)

Petunia Flower Genes Petunia gene sequencing: Floral development library 2774 C 2 H 4 -treated flower library 3264 Pollinated flower library 1824 Other libraries 1046 Total genes sequenced 8908

Petunia EST Database

Microarray Construction 4200 unique petunia genes

Microarray analysis – Ethylene Regulated Genes Control (A) Ethylene (B) Green A>B Yellow A=B Red A<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 – 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

Measuring Floral Scent

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

Flower Scent Composition - Rose

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? Financially –USDA Floriculture Initiative –American Floral Endowment –Fred Gloeckner Foundation –Florida FIRST –The Scotts Company Intellectually –Harry Klee – UF –Bill Farmerie – UF –Don McCarty – UF –Michelle Jones – OSU –Jim Giovannoni – USDA/Cornell –EU PetNet Group

UF Floriculture Biotechnology

Questions?