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The New Zealand Institute for Plant & Food Research Limited
Manipulating Plant Genomes
Richard Espley
The New Zealand Institute for Plant & Food Research Limited
Plant & Food Research
• Based in New Zealand
• Government-owned Crown Research Institute
• Over 900 employees (650 research staff)
The New Zealand Institute for Plant & Food Research Limited
PFR - our science
Our science combines market insight with a fundamental
understanding of the biology of our food resources.
The New Zealand Institute for Plant & Food Research Limited
Breeding and genomics
» New cultivars with identified consumer and producer traits
» Identified molecular mechanisms controlling key traits
» Breeding programmes using integrated genomics tools
ZESPRI® GOLD Kiwifruit
» Sweet, yellow-fleshed kiwifruit
» More than 26 million trays sold in 2009
JAZZ™ apples
» Tangy, crunchy, juicy apples with flavour
» Rated by consumers in Europe and US as outstanding
» Excellent shelf life
Moonlight potato
» Smooth white skinned, white fleshed general purpose potato
» High yield, good eating
Conquest wheat
» Improved milling wheat variety
» Competitive as an import replacement variety
The New Zealand Institute for Plant & Food Research Limited
Breeding has been going on for a long time…..
The New Zealand Institute for Plant & Food Research Limited
We have modified plant genomes for
thousands of years......
The New Zealand Institute for Plant & Food Research Limited
History of crop development
• Major crops were domesticated ~ 10,000 – 5000 years ago
• The development of human civilizations correlates with development of agriculture
• Plant domestication followed the end of the most recent glacial period
Glacial retreat
Glacial advance
Wild gathering Pre-domestication
cultivation
Domestication
Neolithic
Revolution
thousand
years ago
The New Zealand Institute for Plant & Food Research Limited
Plants were domesticated in parallel in several regions
Reprinted by permission from Macmillan Publishers Ltd.: [Nature] Diamond, J. (2002). Evolution, consequences
and future of plant and animal domestication. Nature 418: 700-707, copyright 2002.
Wheat, barley, pea, lentil
~ 13,000 years ago
Rice, soybean
~ 9000 years ago
Rice, bean
~ 8500 years ago
Corn, squash, bean,
potato
~ 10,000 years ago
The New Zealand Institute for Plant & Food Research Limited
Selection leads to genetic modification
Natural variation
within population
Planting seeds from
“good” plants increased
their representation in
subsequent generations
The New Zealand Institute for Plant & Food Research Limited
Improvements in corn from Mexico
7000
years ago
500
years ago
Cobs from archeological sites in the Valley of Tehuacan, Mexico
The New Zealand Institute for Plant & Food Research Limited
From teosinte to modern corn…
The New Zealand Institute for Plant & Food Research Limited
The history of crop improvement
Pamela Ronald, University of California, Davis: “Tomorrow‟s
Table: Organic Farming, Genetics and the Future of Food.”
The New Zealand Institute for Plant & Food Research Limited
Norman Borlaug – father of the green revolution
"More than any other single person of this age, he has helped provide bread
for a hungry world. We have made this choice in the hope that providing
bread will also give the world peace." Novel Committee.
Bred semi-dwarf, high-yielding varieties of wheat
Combined with disease resistance
The New Zealand Institute for Plant & Food Research Limited
Plant breeding relies on genetic diversity
The New Zealand Institute for Plant & Food Research Limited
Genetic tools for use in breeding
• Marker assisted selection
• Genome-wide association studies
• Recombinant DNA technology and transgenic plants
The New Zealand Institute for Plant & Food Research Limited
Phenotyping vs. genotyping
Genotype: sequence of all the
genes in a genome
The New Zealand Institute for Plant & Food Research Limited
Plant breeding – Marker Assisted Selection
Introgression of a desired gene using markers associated
with the genomic region linked to the trait
Elite tomato Wild tomato with high colour
but poor taste etc
We want to add a
high colour trait to
an “elite” tomato
plant.
The New Zealand Institute for Plant & Food Research Limited
We cross the two plants.
Some of their progeny
inherit the novel colour,
some don‟t – how can we
tell the difference?
Photo by Stephen Ausmus USDA
Plant breeding – introgression of a gene
The New Zealand Institute for Plant & Food Research Limited
We can use markers to
look at their DNA and
identify those with the
colour gene.
Faster and easier than
waiting for fruit phenotype
Plant breeding – introgression of a gene
The New Zealand Institute for Plant & Food Research Limited
Is this an elite, high colour tomato?
No, half of its genes are from the
poor tomato so poor taste etc
Plant breeding – introgression of a gene
The New Zealand Institute for Plant & Food Research Limited
We have to repeatedly
cross back to the elite
tomato, using markers
to identify plants with
the colour gene
Plant breeding – introgression of a gene
The New Zealand Institute for Plant & Food Research Limited
Plant breeding – introgression of a (colour) gene
After several
generations, elite,
high colour tomato
Markers greatly
accelerate breeding
programs
The New Zealand Institute for Plant & Food Research Limited
MAS used to develop Swarma-Sub1 rice
After several
generations,
Swarna-Sub1
Swarna Sub1
•IRRI used MAS to develop submergence-tolerant rice
•Swarna is a high-yielding rice variety but sensitive to flooding
•Sub1 donor with poor yield but good submergence-tolerance
•Now accounts for 25% rice grown in India (monsoon areas prone to flooding)
The New Zealand Institute for Plant & Food Research Limited
MAS also used in drought tolerant plants
70% of water usage is for agriculture
The New Zealand Institute for Plant & Food Research Limited
MAS also used in drought tolerant plants
Water optimized corn
The New Zealand Institute for Plant & Food Research Limited
Plant genomes
Maize
The New Zealand Institute for Plant & Food Research Limited
Beyond MAS….
Whole genome assisted selection
1000 Genomes Project
Complex and multiple traits
1000s of markers
Whole plant fitness – predicts plant performance
Accurate phenotyping & genotyping of „training population‟
Predictive values (Genomic Estimated Breeding Values)
Arabidospis 1001 genomes
The New Zealand Institute for Plant & Food Research Limited
When to use MAS or GM breeding methods
Molecular breeding
1. Desired trait must be
present in population
2. Genetic resources
must be available
3. Plant should be
propagated sexually
GM
1. Gene can come from
any source
3. Plant can be
propagated vegetatively 2. Genetic resources not
required
The New Zealand Institute for Plant & Food Research Limited
Plant breeding by genetic modification
Transgenics: Genes from non-crossable plants,
viruses or bacteria with selection markers e.g.
antibiotic or herbicide resistance
Intragenics: Genes from own species or crossable
species re-arranged in vitro. Marker free. T-DNA
from compatible plant.
Cisgenics: Genes from own species or crossable
species but only using native gene (inc. promoter,
introns, terminator). Marker free. T-DNA from
non-crossable species.
+ =
The New Zealand Institute for Plant & Food Research Limited
Routine transformation by Agrobacterium
The New Zealand Institute for Plant & Food Research Limited
GM applications – Bt gene
Wild-type peanut plant Peanut plant expressing the Bt gene
The New Zealand Institute for Plant & Food Research Limited
Bacillus thuringiensis (Bt) bacteria produce
insecticidal proteins
Bacillus
thuringiensis expressing Bt toxin
Plant cell
expressing Bt toxin
Bacillus thuringiensis
expressing insecticidal Bt
toxin can be sprayed onto
plants
Or the plants can be
engineered to express the
Bt gene coding for Bt toxin
The New Zealand Institute for Plant & Food Research Limited
The effect of Bt toxin is highly specific
Bacillus
thuringiensis expressing Bt toxin
Plant cell
expressing Bt toxin
Intestine
The Bt toxin affects only some insects
because to be effective it has to be
processed and bind to a specific
receptor protein
Processing
Receptor
bindingIntestinal
lumen
The New Zealand Institute for Plant & Food Research Limited
The effect of Bt toxin is highly specific
Bacillus
thuringiensis expressing Bt toxin
Plant cell
expressing Bt toxin
After binding, the insecticidal proteins
assemble to form a pore in the lining of
the insect intestine which kills the insect
Pore
assembly
Intestine
The New Zealand Institute for Plant & Food Research Limited
Other GM targets
Herbicide tolerance
Insect resistance
Drought tolerance
Biofortification:
High lycopene tomatoes
Vitamin A rice
Resveratrol in letuce…..
The New Zealand Institute for Plant & Food Research Limited
Global
Biotechnology
The New Zealand Institute for Plant & Food Research Limited
GM crops around the world
2011: 16.7 million farmers in 29 countries planted 160 m hectares. 8% increase on 2010
Source: Clive James ISAAA, 2012
The New Zealand Institute for Plant & Food Research Limited
The New Zealand Institute for Plant & Food Research Limited
The New Zealand Institute for Plant & Food Research Limited
The New Zealand Institute for Plant & Food Research Limited
The New Zealand Institute for Plant & Food Research Limited
Papaya
Hawaii papaya industry decimated by ringspot virus
Resistance RNAi - 1995
85% total crop GM
Rainbow = GM SunUp x non-gm Kapoho,
Kapoho field trial 1995
Dennis Gonsalves
The New Zealand Institute for Plant & Food Research Limited
Banana – the next fruit crisis?
Banana – triploid (sterile), clonal
Massive export plantations & essential smallholder production
Gros Michel wiped out by Panama disease
From 1960 - Cavendish (resistant to Panama)
Cavendish susceptible to new races of Panama
Panama now damaging plantations in Philippines, Indonesia (not
S America yet)
The New Zealand Institute for Plant & Food Research Limited
Banana – the next fruit crisis?
Arthur Kamenya, a banana farmer who lost a third of his plants to BXW (Xanthamonas wilt) disease
The Guardian March 2011 & Nature Biotech June 2011
Ugandan scientists grow GM banana as disease
threatens country's staple food
•Ban on GM crops waived after bacterial disease causes
annual banana crop losses of $500m
•Gene license holder waives royalties
•Field trials 2011 (HR response proteins)
The New Zealand Institute for Plant & Food Research Limited
GM tamarillo trial - Kerikeri trial site (1998-2000)
The New Zealand Institute for Plant & Food Research Limited
GE Free NZ 2003
New Zealand: National day of action against the lifting of the GE moratorium
The New Zealand Institute for Plant & Food Research Limited
Consumer attitudes in NZ
John Knight
Nature Biotechnology 2007
Choice…
Price…
The New Zealand Institute for Plant & Food Research Limited
Risk assessments for GM crops
Before release into the environment, GM crops are subject to
risk-assessment and risk-management measures to evaluate:
• Risks to human health (including toxicity and allergenicity)
• Risks of evolution of resistance in target pathogens or pests
• Risks to non-target organisms
• Risks from movement of transgenes
For local perspective see:
The New Zealand Institute for Plant & Food Research Limited
Case study - apples
The New Zealand Institute for Plant & Food Research Limited
Arctic apples
• PPO knockouts
• No browning
• Regulatory submission complete (APHIS/USDA)
• Bulking up planting material
http://www.okspecialtyfruits.com
The New Zealand Institute for Plant & Food Research Limited
New Zealand apple market
USA Europe
South America
• NZ‟s 3rd largest horticultural export
• NZ$400 million
• Fresh fruit exports & plant varieties
Breeding
Mapping
Genomics
Germplasm
The New Zealand Institute for Plant & Food Research Limited
Drivers for apple quality
Taste
Texture
Novelty
Health
Colour
• Anthocyanin
• Plant health
• Marketing
• Human health
Photo courtesy of ENZA
The New Zealand Institute for Plant & Food Research Limited
Breeding can be a lengthy process…
Traditional breeding: An improved variety
1984 Royal Gala x Braeburn cross
1988 9,600 seedlings
1990 Fruit evaluation
1996 Trials/PVR
2003 Commercial Jazz™ planting - NZ, France, USA
Traditional breeding: Introgression of resistance gene
1946 M. floribunda x commercial varieties
1999 Apple scab resistant cultivars introduced
2006 Resistant strains
X =
The New Zealand Institute for Plant & Food Research Limited
Origins of apple
The New Zealand Institute for Plant & Food Research Limited
Naturally occurring red-fleshed apple germplasm
The New Zealand Institute for Plant & Food Research Limited
Understand the function of genes to develop markers
MYB10
WD40
bHLH
Anthocyanins
Environmental signals
Anthocyanin transcriptional complex
Light signal transduction
4-Courmaroyl + 3 Malonyl-CoA
Chalcones
Flavonones
Dihydroflavonols
Leucoanthocyanidins
Anthocyanidins
Anthocyanins
CHS
CHI
F3H
DFR
LDOX
UFGT
EBGs
LBGs
4-Courmaroyl + 3 Malonyl-CoA
Chalcones
Flavonones
Dihydroflavonols
Leucoanthocyanidins
Anthocyanidins
Anthocyanins
CHS
CHI
F3H
DFR
LDOX
UFGT
EBGs
LBGs
The New Zealand Institute for Plant & Food Research Limited
Pigment accumulation in tobacco –
transient transformation
The New Zealand Institute for Plant & Food Research Limited
Agrobacterium transformation of apple cells
with a transcription factor
The New Zealand Institute for Plant & Food Research Limited
Transformed apple
The New Zealand Institute for Plant & Food Research Limited
Transgenic apple – grafting for fast fruiting
The New Zealand Institute for Plant & Food Research Limited
Containment facilities in Auckland
The New Zealand Institute for Plant & Food Research Limited
The New Zealand Institute for Plant & Food Research Limited
Rapid generation of 35S:MYB10 fruit
The New Zealand Institute for Plant & Food Research Limited
Transgenic trees are highly pigmented
A
BA
B
The New Zealand Institute for Plant & Food Research Limited
Fruit contains 20 x anthocyanin
The New Zealand Institute for Plant & Food Research Limited
Sensory – what do they taste like?
California - 50 screened panellists
(gender, age, diet, ethnicity, income etc)
Double blind x 4 sets
The New Zealand Institute for Plant & Food Research Limited
Does high colour affect health properties?
Reduced risk of cancer
Reduced risk of CVD
Pulmonary function
Antioxidant effects
The New Zealand Institute for Plant & Food Research Limited
Red-fleshed apple in the diet
• 20 x more anthocyanin
• Increased antioxidant capacity
• Reduction in plasma cytokine levels*
• Increase in bacterial populations
• Pre-biotic effect?
Contol RG C RG P+C MYB10 C MYB10 P+C
0
1000
2000
3000
4000
5000
6000
7000
8000
Pro
sta
gla
nd
in (
PG
)E2 p
g/m
lDiet
7 Days
21 Days
PGE2 - Prostaglandin
* Increased fat in diet induces cytokines, can lead to chronic disease, type 2 diabetes
0
50
100
150
200
250
mm
ol T
rolo
x e
g/k
g D
W
Peel
Cortex
The New Zealand Institute for Plant & Food Research Limited
What have we learned from GM red-fleshed
apples?
• Understand genetic process of colour in apple
• Tree performance not affected
• Fruit crop load normal
• Storage is compromised
• Taste OK
• High level of bioactive compounds
• Reduces markers of inflammation
• Alters gut bacteria
• Provided markers for red-fleshed apple breeding project
The New Zealand Institute for Plant & Food Research Limited
Manipulating plant genomes….
….using genetic tools
Marker Assisted Selection – single gene traits
Whole Genome Selection – multiple and/or complex traits
Genetic modification – targeted changes
The New Zealand Institute for Plant & Food Research Limited
Resources…
Twitter: @plantandfood
The New Zealand Institute for Plant & Food Research Limited
www.plantandfood.com