Development of c DNA Microarrays for Gene Expression

Development of c. DNA 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 At. GA 2 ox 7 Makes dwarf tobacco Schomburg et al. 2003

Internal Growth Regulators At. GA 2 ox 7 - 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 Etr 1 -1 ETR 1 -1 - Makes plants ethylene insensitive

Long Lasting Flowers Control Etr 1 -1

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

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 – 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) m. RNA extraction Tissue collection Bioinformatics and Microarrays library synthesis EST sequencing

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

Petunia EST Database

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

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. 5 B 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 Mc. Carty – UF Michelle Jones – OSU Jim Giovannoni – USDA/Cornell – EU Pet. Net Group

UF Floriculture Biotechnology 2002 -2003

Questions?