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
8
10
12
14
16
18
20
22
24
-1 0 +3 +5 +7
Time (Days)
Ch
loro
ph
yll a
+b
(m
g/c
m 2 )
V26
I97-1-7
I97-3-18
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
pA
0
100
200
300
400
FID1 A, (DEC13\003F0301.D)
Be
nza
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ali
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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
min10 15 20 25 30 35 40 45
pA
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50
100
150
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FID1 A, (OCT26\012F1201.D)ci
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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?