Manipulating Plant Genomes - Maurice Wilkins Centre...Pamela Ronald, University of California, Davis: “Tomorrow‟s Table: Organic Farming, Genetics and the Future of Food.” The

The New Zealand Institute for Plant & Food Research Limited

Manipulating Plant Genomes

Richard Espley


Plant & Food Research

• Based in New Zealand

• Government-owned Crown Research Institute

• Over 900 employees (650 research staff)


PFR - our science

Our science combines market insight with a fundamental

understanding of the biology of our food resources.


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


Breeding has been going on for a long time…..


We have modified plant genomes for

thousands of years......


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


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


Selection leads to genetic modification

Natural variation

within population

Planting seeds from

“good” plants increased

their representation in

subsequent generations


Improvements in corn from Mexico

7000

years ago

500

years ago

Cobs from archeological sites in the Valley of Tehuacan, Mexico


From teosinte to modern corn…


The history of crop improvement

Pamela Ronald, University of California, Davis: “Tomorrow‟s

Table: Organic Farming, Genetics and the Future of Food.”


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


Plant breeding relies on genetic diversity


Genetic tools for use in breeding

• Marker assisted selection

• Genome-wide association studies

• Recombinant DNA technology and transgenic plants


Phenotyping vs. genotyping

Genotype: sequence of all the

genes in a genome


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.


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

http://www.ars.usda.gov/is/graphics/photos/jul03/k10498-2.htm


We can use markers to

look at their DNA and

identify those with the

colour gene.

Faster and easier than

waiting for fruit phenotype



Is this an elite, high colour tomato?

No, half of its genes are from the

poor tomato so poor taste etc



We have to repeatedly

cross back to the elite

tomato, using markers

to identify plants with

the colour gene



Plant breeding – introgression of a (colour) gene

After several

generations, elite,

high colour tomato

Markers greatly

accelerate breeding

programs


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)


MAS also used in drought tolerant plants

70% of water usage is for agriculture


MAS also used in drought tolerant plants

Water optimized corn


Plant genomes

Maize


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


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


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.

+ =


Routine transformation by Agrobacterium


GM applications – Bt gene

Wild-type peanut plant Peanut plant expressing the Bt gene


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 effect of Bt toxin is highly specific

Bacillus


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 effect of Bt toxin is highly specific

Bacillus


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


Other GM targets

Herbicide tolerance

Insect resistance

Drought tolerance

Biofortification:

High lycopene tomatoes

Vitamin A rice

Resveratrol in letuce…..


Global

Biotechnology


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






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


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)

http://upload.wikimedia.org/wikipedia/en/7/72/Gros_Michel_Bananas.jpg


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)


GM tamarillo trial - Kerikeri trial site (1998-2000)


GE Free NZ 2003

New Zealand: National day of action against the lifting of the GE moratorium

//upload.wikimedia.org/wikipedia/commons/1/10/GE-free_march_Auckland_2003.JPG


Consumer attitudes in NZ

John Knight

Nature Biotechnology 2007

Choice…

Price…


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:


Case study - apples


Arctic apples

• PPO knockouts

• No browning

• Regulatory submission complete (APHIS/USDA)

• Bulking up planting material

http://www.okspecialtyfruits.com

http://www.okspecialtyfruits.com/


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


Drivers for apple quality

Taste

Texture

Novelty

Health

Colour

• Anthocyanin

• Plant health

• Marketing

• Human health

Photo courtesy of ENZA


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 =


Origins of apple


Naturally occurring red-fleshed apple germplasm


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


Pigment accumulation in tobacco –

transient transformation


Agrobacterium transformation of apple cells

with a transcription factor


Transformed apple


Transgenic apple – grafting for fast fruiting


Containment facilities in Auckland



Rapid generation of 35S:MYB10 fruit


Transgenic trees are highly pigmented

A

BA

B


Fruit contains 20 x anthocyanin


Sensory – what do they taste like?

California - 50 screened panellists

(gender, age, diet, ethnicity, income etc)

Double blind x 4 sets


Does high colour affect health properties?

Reduced risk of cancer

Reduced risk of CVD

Pulmonary function

Antioxidant effects


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


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


Manipulating plant genomes….

….using genetic tools

Marker Assisted Selection – single gene traits

Whole Genome Selection – multiple and/or complex traits

Genetic modification – targeted changes


Resources…

Twitter: @plantandfood

http://www.linkedin.com/company/the-new-zealand-institute-for-plant-&-food-research-ltd


www.plantandfood.com

Documents

Manipulating Plant Genomes - Maurice Wilkins Centre...Pamela Ronald, University of California, Davis: “Tomorrow‟s Table: Organic Farming, Genetics and the Future of Food.” The