Field Testing Transgenic Grapevine Expanded Version - Mid

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Transcript Field Testing Transgenic Grapevine Expanded Version - Mid

Genetic Engineering of Grapevine and
Field Testing for Bacterial & Fungal
Disease Resistance
D J Gray, Z T Li, S A Dhekney, M Dutt, D L Hopkins
Mid-Florida Research & Education Center
University of Florida/IFAS
T W Zimmerman
Biotechnology & Agroforestry
University of the Virgin Islands
www.mrec.ifas.ufl.edu/grapes
Field Testing Transgenic Grapevine for
Bacterial and Fungal Disease Resistance
Objectives
To test GM grapevines in Florida and the US Virgin
Islands under USDA/APHIS approved conditions
Evaluate for Pierce’s disease & fungal disease resistance
Evaluate for commercially-useful qualities
Begin to assess environmental risks of GM grape
Determine extent of gene flow via pollen
Determine if weedy hybrids occur
www.mrec.ifas.ufl.edu/grapes
Grapevine & Genetic Improvement
The Opportunity:
• World’s most valuable fruit crop
• 10 – 12th most valuable agricultural crop in US
• But - Grape is particularly difficult to improve genetically
– Genetic self-incompatibility makes breeding new varieties difficult
– Long grape lifecycle – years to make recurrent crosses
– Fine discrimination of wine type makes breeding a new “Cabernet” impossible
• These obstacles have led to little improvement of desirable
varieties, such as adding disease resistance to established
wine varieties
– An exception has been for table grape breeding where improved fruit types
have been created by laborious breeding
Why Grape Research in the Subtropics?
The Virgin Islands
The Virgin Islands must import all grape products
Therefore, like Florida, a large untapped local market exists
Grape is a high-value crop suited for small farm production
A new source of agricultural income
BUT - Disease-resistant cultivars are needed
www.mrec.ifas.ufl.edu/grapes
Why Grape Research in the Subtropics?
Florida
Florida is the US’s # 2 consumer of wine & grape products
Existing muscadine-based industry satisfies
less than 1% of market
Therefore a large untapped local market exists
Conventional varieties are needed for wine & seedless fruit
But - All such varieties will die from Pierce’s disease &
various fungal diseases if grown in Florida
Conventional breeding cannot be used to create resistant
versions of desirable varieties
www.mrec.ifas.ufl.edu/grapes
Genetic Engineering of Grapevine
Insertion of genes for disease resistance into otherwise
desirable varieties might result in grapevines that can be
grown in the Virgin Islands and Florida and address the
existing market
Agrobacterium-mediated genetic transformation of certain
Vitis vinifera varieties and rootstocks is now routine & efficient
Li et al. 2006 In Vitro Cell. Dev. Biol. Plant 42:220-227
Dhekney et al. 2007 ACTA Hort 738:749-753
Therefore all needed technology is in place to evaluate use of
genetic transformation in grapevine improvement
www.mrec.ifas.ufl.edu/grapes
Diseases of Grapevine
Bacterial Disease
• Pierce’s Disease – Florida & California
• Prohibits production of Vitis vinifera in
the Southeastern US & is increasing in Ca
Fungal Diseases – Florida, Virgin Islands & Worldwide
• Powdery Mildew – Most important disease of grape
worldwide
• Anthracnose
• Downy Mildew
• Ripe Rot
• Many others
Viral Diseases – Worldwide
• Grape fan leaf virus – most severe & widespread virus
• Leaf roll, corky bark, many others
Pierce’s Disease
• Endemic in Florida (the southern coastal plain) &
California
• Caused by Xylella fastidiosa
• Xylem limited bacterium that infects a wide range of
vascular plants
• Transmitted by xylem-feeding insects
• Primarily leaf hoppers & sharpshooters – Glassy
Winged Sharpshooter new in CA
• Lethal to all Vitis vinifera & other non-native varieties
Pierce’s Disease
Total loss of
vineyard results
Pierce’s Disease Tolerant Vines
Anthracnose of Grapevine
Powdery Mildew of
Grapevine
Typical chlorotic lesions
Powdery
Mildew
infects leaves,
stems & fruit
Aerial hyphae
Genetic Engineering of Grape
• Direct insertion of genes (DNA)
• Allows integration of single traits without
disturbing desirable characteristics
– Important for grape where varieties are highly prized
and change is resisted (e.g. wine varieties)
• Adds traits that normally are not found in grape
– Such as novel types of disease resistance
Genetic Engineering of Grape
• Modern techniques of molecular biology
allow DNA to be analyzed and manipulated
• Functional DNA, including genes, literally
can be cut and pasted together into new
and useful combinations
• Potentially, unlimited possibilities result
Understanding Genetic Control Elements
Basic definitions:
Promoter
Enhancer
Element
A segment of DNA that causes genes to become active.
Promoters serve as a binding site for the enzyme RNA
polymerase, which performs the first step of transcribing DNA in
the adjacent gene.
A segment of DNA that influences the activity of promoters by
serving as a binding site for specific proteins. Enhancers may
be physically separated from the promoter, but still influence it.
Duplicating enhancers or modifying their DNA sequences can
dramatically alter activity.
Gene
A heritable sequence of DNA that determines a particular
characteristic in an organism. A gene may code for a wide
range of functions. Most commonly, proteins are produced .
Gene
Terminator
A segment of DNA that signals the end of a gene.
Terminators serve to stop transcription by detaching RNA
polymerase.
RNA to Protein
Also known as “transcription” and “translation” . RNA
polymerase binds at the promoter and moves along the DNA
strand, transcribing the sequence into Messenger RNA. When
the terminator is reached, mRNA detaches and is translated
into a protein, the structure of which is dictated by the original
DNA sequence.
A Conventional Genetic Control
Element
RNA
*
Located on
Chromosome
Enhancer
Element
Chromosome
(Double-Stranded
DNA)
Protein
Core
Promoter
Gene
Gene
Terminator
Bidirectional Dual Promoter Complex
US Patent # 7,129,343 2006
Protein
Gene
Terminator
RNA
Transgene - 1
Core
Promoter
Duplicated Elements in
Divergent Orientation
RNA
Enhancer
Element
Duplicated
Enhancer
Enhancer
Element
Core
Promoter
Protein
Transgene - 2
Conventional Arrangement
of Gene Controlling
Elements
The patented BDPC is a new way to arrange Genetic Control
Elements. The BDPC provides more efficient control of genes
and results in better protein production.
Gene
Terminator
Assembly of DNA for Insertion into Grape
Antibiotic Resistance
(To select GE cells)
GFP
Functional Gene
(To see GE cells)
(Improved Trait)
Source Genes
Molecular Biology
Antibiotic Resistance
GFP
Functional Gene
Unique DNA Sequence for Genetic Engineering
Bi-Functional Fusion Gene
+
Burk et al. 2001
Yang et al. 1996
EGFP
NPTII
Reporter
Selectable
A bi-functional marker gene
Functional Genes Tested in Grape at
MREC
• Vitis vinifera thaumatin-like protein gene
– Tested for fungal resistance
• Grape albumen protein gene
– Tested for fungal resistance and seedlessness
• Lytic peptide genes
– Tested for PD and fungal resistance
• Hybrid resistance genes
– Tested for PD and fungal resistance
Example of Transformation Vector:
A Lytic Peptide-Containing Transformation
Vector Based on a Bidirectional Promoter
Complex (BDPC)
CsVMV-BDPC
Transgene - 1
Term
Lyt Pep
Transgene - 2
EGFP/NPTII
Core
Promoter
Core
Promoter
Modified Enhancer
Term.
Grape Transformation System
• Embryogenic cultures
– Totipotent cells of somatic
embryos are the target
• Agrobacterium
mediated
transformation
• Kanamycin selection of
transgenic cells
– Uses NPTII Gene
Selection of Transgenic Grape
• Transient GFP
expression
– First visualized at 2 days
and faded by 20 days
• Stable GFP
expression
– Apparent within 20 days
• GFP-positive
embryogenic cultures
– Isolated within 6 weeks
• Plants produced from
embryos are
Green Fluorescent Protein Expression
in Vitis vinifera & Vitis rotundifolia
A. Embryos
B. Leaf/stem/tendril
A
C. Flowers
E. Anther/stigma
B
A-C = V. vinifera ‘Thompson
Seedless’
D-E = V. rotundifolia ‘Alachua’
C
D
www.mrec.ifas.ufl.edu/grapes
E
Preparing Transgenic Grape
Lines for Greenhouse Testing
Plants from tissue
culture are propagated
to produce multiple
clones
Plants are arranged into
populations to study disease
resistance
Screening for Powdery MildewResistant Transgenic Grapevines
Transgenic vines
producing “Thaumatinlike” protein are grown
with non-transgenic
controls in an area of
high powdery mildew
incidence and without
chemical control.
Vines are rated 3x’s per
week for disease
development throughout
the growing season.
Experimental population of
transgenic and control
vines
Powdery Mildew
Test Site in UF/IFAS Greenhouse
Powdery Mildew-Resistant
Transgenic Grapevines (January
2005)
Certain transgenic vines
grown in an area of high
powdery mildew
pressure remained
vigorous and show
resistance to PM
throughout the growing
season.
Initial Powdery Mildew
Test Site in
Greenhouse
Resistant
Susceptible
Control Vines Transgenic Vines
Powdery Mildew Resistant Transgenic
Grapevines Selected in Greenhouse at MREC
Selected transgenic vines
that express Vitis vinifera
thaumatin-like protein
(VVTL-1) exhibit an 8 day
delay in visible lesion
development compared to
control vines
VVTL-1 is an endogenous
gene from grapevine
5 resistant lines have been
selected for field trials
Susceptible ‘Thompson
Seedless’
www.mrec.ifas.ufl.edu/grapes
Transgenic ‘Thompson
Seedless’
Testing Transgenic Plants for
Resistance to Pierce’s Disease
The Xylella bacterium, which causes Pierce’s disease, is
injected directly into the xylem tissue of transgenic grape
plants and control plants. The plants are then evaluated for
resistance.
Testing Xylem Sap for
Lytic Peptide
The presence of
lytic peptide is
determined by ELISA
Pure xylem sap is exuded from
decapitated plants due to root
pressure
Test Samples
Purified Protein
CK
Transgenic Vines
Tracking Bacterial
Concentrations in Test
Plants
• The presence and
number of bacterial
colonies provides an
early estimate of plant
resistance
Susceptible Control
Transgenic
150
Dead
100
50
Seasonal dormancy
0
0
• Periodic sampling
provides information on
internal spread of
bacteria over time
Resistant Control
Dead
200
# Xylella Colonies
• After stem inoculation,
the xylem sap from leaf
petioles is placed on
Xylella-specific culture
media
100
110
135
Days after Inoculation
150
250
200
150
100
50
T.S. Control
Tampa Control
Line 14
July
Month
June
April
March
February
January
December
November
October
September
August
0
July
No. Xylella Colonies
Recovery of Xylella From Inoculated Grapevine
PD Resistant Transgenic Grapevines
Selected in Greenhouse at MREC
These vines were inoculated
in July 2004
Since even resistant controls
developed symptoms, the PD
test is considered to be
stringent
Lack of symptoms in
transgenic plants that contain
proprietary lytic peptide genes
suggest high level of
resistance
Approximately 100 highly
resistant lines have since
been selected, 15 of which
were propagated for field trials
Susceptible
Transgenic Vines Resistant Control
Control Vines
w Lytic Peptide
‘Tampa’
‘Thompson Seedless’
www.mrec.ifas.ufl.edu/grapes
Production of Transgenic Grapevines
at MREC
• Over 2,200 unique transgenic plants have been
produced
– 90%+ have been Thompson Seedless (the most widely
planted variety in the US)
• Nine different functional genes have been tested
– Most have been discarded due to poor response
• The hybrid LIMA gene appears to provide PD
resistance
• The native thaumatin gene appears to provide
fungal resistance
• Field tests must now be established
Selected Transgenic Plants Propagated For
Field Trial
www.mrec.ifas.ufl.edu/grapes
The Virgin Islands UVI Field Site
A protected site used for evaluation of GM plants
 5 lines containing VVTL-1, replicated 5-7 times
 29 transgenic vines plus 9 controls planted 1/2007
Approved through USDA APHIS notification
process in October 2006
www.mrec.ifas.ufl.edu/grapes
The Virgin Islands Field Site
University of the Virgin Islands, St. Croix
Established January 2007
Two ‘Thompson Seedless’ lines
containing VVTL-1 gene (June 2007)
www.mrec.ifas.ufl.edu/grapes
The Florida Field Site
Isolated from cross-fertile wild & cultivated vines
 15 lines containing either of 2 experimental lytic peptide,
replicated 4-5 times
 5 lines containing VVTL-1, replicated 8 times
Transgenic varieties used (180 plants = 60%)
 30% ‘Thompson Seedless’, 10% ‘Merlot’, 10% ‘Seyval Blanc’
 10% ‘Freedom’ rootstock
Non-transgenic controls (120 plants = 40%)
 Same scions and rootstocks as above (20%)
 PD-resistant hybrids ‘Tampa’ and BN5-4 (20%)
Approved through USDA APHIS notification
process in October 2006
www.mrec.ifas.ufl.edu/grapes
The Florida Field Site
UF/IFAS Mid-Florida Research & Education Center
www.mrec.ifas.ufl.edu/grapes
The Florida Field Site
Trellises constructed in March 2007
www.mrec.ifas.ufl.edu/grapes
The Florida Field Site
Planting April 2007
www.mrec.ifas.ufl.edu/grapes
The Florida Field Site
‘Thompson Seedless’, June 2007
www.mrec.ifas.ufl.edu/grapes
The Florida Field Site
July 6, 2007
www.mrec.ifas.ufl.edu/grapes
What’s Next?
Evaluation for disease resistance & clonal
fidelity are ongoing
Environmental risk assessment studies planned
Greenhouse screening of endogenous genes and
varieties will lead to new field planting in 2008-09
www.mrec.ifas.ufl.edu/grapes
Screening Endogenous Genes for use
in Powdery Mildew Resistance
‘Syrah’
before
veraision
Non-transgenic
Transgenic
www.mrec.ifas.ufl.edu/grapes
Screening Endogenous Genes for use
in Powdery Mildew Resistance
Non-transgenic
Transgenic
‘Syrah’
ripe
www.mrec.ifas.ufl.edu/grapes
Acknowledgments
Florida Department of Agriculture & Consumer
Services Viticulture Trust Fund
Long-term support of grape biotech research
USDA Tropical, Sub-Tropical Agricultural Research
Grants Program
Support for endogenous gene discovery and field tests
Florida Genetics LLC (www.flgenetics.net)
Support for patent costs and commercialization efforts
www.mrec.ifas.ufl.edu/grapes