Development of cDNA Microarrays for Gene

Expression Research in Floriculture Crops

Dave ClarkUniversity 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. 2003Schomburg et al. 2003

Internal Growth Regulators

AtGA2ox7 - Makes dwarf petunia

Stay Green Leaves

sag-IPT

Makes leaves stay green longer in tobacco

Gan & Amasino 1995Gan & Amasino 1995

sag-IPT

Makes leaves stay greener longer in petunia

Clark et al. in pressClark et al. in press

sag-IPT – Drought Tolerance

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Long Lasting Flowers

Etr1-1

ETR1-1 - Makes plants ethylene insensitive

ControlControl Etr1-1Etr1-1

Long Lasting Flowers

Problems with 35S::Problems with 35S::etr1-1etr1-1

• etr1-1 petunias – commercially limitedetr1-1 petunias – commercially limited

‘‘MD’MD’etr1-1etr1-1

rootingrooting pathogens – pathogens – etr1-1etr1-1

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 – 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

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

C2H4-treated flower library 3264

Pollinated flower library 1824

Other libraries 1046

• Total genes sequenced 8908

Petunia EST Database

Microarray ConstructionMicroarray Construction

4200 unique petunia genes

Microarray analysis – Ethylene Regulated Genes

Control(A)

Ethylene(B)

Green A>BYellow A=BRed 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 – 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

min10 15 20 25 30 35 40 45

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Benzoic acid carboxyl methyltransferase (BAMT) - makes methyl benzoate in petunia

Stop and Smell the Roses!

US consumers US consumers bought almost bought almost 130 million 130 million roses on roses on Valentine’s Valentine’s Day 2003Day 2003

US consumers US consumers spent over spent over $7.5B on cut $7.5B on cut flowers in flowers in 20022002

Opportunity Knocks!

• Commercial cut rose varieties don’t have much fragrance

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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 Biotechnology2002-2003

Questions?Questions?