Presentation by Prof Chris Leaver, Oxford University Delivered at the B4FA Media Dialogue Workshop, Kampala, Uganda - November 2012 www.b4fa.org

1. Genetic Engineering, Crop
Improvement and Biotechnology
Chris Leaver
chris.leaver@plants.ox.ac.uk

2. Map of the world showing the major centres of origin
of crops, which are distributed mainly in tropical
regions

3. Plant Improvement using Breeding and Selection
- HISTORICAL PERSPECTIVE 8000 BC (5 million people)
Domestication of cereals and Pulses
2000 BC (50 million people)
Domestication of rice, Potato, Oats,
Soybean, Grape, Cotton, Banana.
1583 (500 million people)
Sexuality in plants Described
1742
First Company (Vilmorin) Devoted to Plant
Breeding and New varieties
1799
First Cereal Hybrid Described
1927
X-Rays Used for Mutation Breeding
1983 (5 billion people)
First Use of Gene Technology for Plants
2012 (7 billion people)
160 plus million hectares of GM Crops
grown in 29 Countries by 16 million farmers

4. The evolution of maize (corn)
The wild
ancestor
Domestication
Teosinte
First corns
America
Mexico
The adaptation
to Europe
Populations
South of Europe
Introduction
Extension of corn
crop
areas
Hybrids
First creation of
hybrids in France
SOURCE: GNIS (Groupement National Interprofessionnel des Semences)

5. Maize Domestication
Mutations
affecting
architecture
&
resource
allocation
What are they?
Mutations in 5 Genes were
Teosinte
Maize
responsible for these changes
Zea mays ssp. parviglumis
Zea mays ssp. mays
Zea mays ssp. mexicana

6. Genetic modification arose as a consequence
of cultivation and selection of the best plants
Planting seeds from
“good” plants increased
their representation in
subsequent generations
Natural
variation
within
population
Image courtesy of University of California Museum of Paleontology, Understanding Evolution - www.evolution.berkeley.edu

7. F1 Hybrid Seed Production in
self-pollinating crop species –
a basis for crop improvement and
the development of heterosis or
hybrid vigour
F1 hybrid seed production in a range of major crops
including Maize, Rice, Sorghum, Sunflower, Sugar Beet,
Carrot, Onions, Brassica’s etc

8. F1 Hybrid Seed Production in Maize
Pollen - male parent
Female parent
Tassel removed
X
F1 Hybrid
Ear
Inbred Parental Corn
Lines
Hybrid Vigour

9. F1 Hybrid Maize Production Field
Detasseled Maize

10. Hybrids

11. What traits/characteristics were
selected?
•
•
•
•
•
•
•
•
•
•
•
Nutrition
Yield
Rate of growth
Self-pollinating
Reduced pod shatter
Able to harvest & store the fruit
Palatability
Taste
Reduced toxins
Reduced / negligible dormancy requirement
Disease resistance
THESE TRAITS ARE ENCODED BY GENE(S)

12. Selection and Plant Breeding was Applied to a
Range of Important Crops we Grow Today
Teosinte
Rice
Corn
Tomato
The Creation of Corn
The corn that Columbus received was
created by the Native Americans some
8,000 years ago by domestication of a
wild plant called teosinte. They used
‘genetic engineering’ in a quite remarkable
way to produce a more productive variety.

13. PRODUCTS OF MODERN BREEDING
Tomatoes
Peppers
Potato

14. Mankind depends on a few crop species for our food

15. Sustainable food security is facing a major bottleneck
• Since the beginning of agriculture, humans have cultivated 7,000 plant species
• Today only 150 plant species (2%) are agriculturally relevant for food and clothing
• Only 10 plant species are cultivated today to provide 95% of food and feed
Cultivated today
95% of food and feed
Total cultivated since
the beginning of agriculture
Total kultiviert
Heute kultiviert
95% der Ernährung

16. The top four – Global yield
(UN-FAO Statistics)
Soybean
Wheat
2nd
4th
Maize
Rice
1st
3rd

17. Improved green-revolution plants led to
dramatically increased crop yields
The introduction of
disease-resistant, semidwarf varieties turning
countries from grain
importers to grain
exporters
Dwarf wheat was
developed at CIMMYT –
the International Maize
and Wheat
Improvement Center
Source: FAO via Brian0918

18. The myth of natural food
The food we eat comes from
plants already extensively
modified from their original
form. Even heritage varieties are
extensively genetically modified.
Credit: Nicolle Rager Fuller, National Science Foundation

19. Building Increased Productivity
and Sustainability into the Seed by
Plant Breeding and Biotechnology
The scientific basis of all crop improvement is
identification of the genes that encode and
regulate specific phenotypic characteristics or
traits of use to the farmer:
Genetic modification by marker assisted
breeding (MAB) and GM technology where
appropriate:

20. SOME PLANT BREEDING TARGETS - Traits
• YIELD
• QUALITY - NUTRITION
• AGRONOMICS
• RESISTANCE TO PESTS and DISEASES
• TOLERANCE TO STRESS (Heat,Drought,Flooding)
• TIME TO MATURITY
•ABILITY TO HARVEST AND TO STORE

21. A Quick reminder:
Traditional plant breeding
Commercial variety
Traditional donor
New variety
DNA is a strand of genes,
much like a strand of pearls.
Traditional plant breeding
combines many genes at once.
(many genes are transferred)
X
Desired Gene
=
(crosses)
Desired gene
21

22. PLANT BREEDING HAS BEEN
VERY SUCCESFUL BUT
HISTORICALLY IT HAS
BEEN AN IMPRECISE ART
THE NEW MOLECULAR
TECHNOLOGIES
ARE CHANGING THIS
The scientific basis of all crop improvement is
identification of the genes that encode and regulate
specific phenotypic characteristics or traits.
These genes can now be transferred more easily via marker
assisted breeding (MAB) - non GM or directly through genetic
engineering - GM.
The current challenge is to identify these genes.
Gene
Trait

23. Conventional Plant Breeding has been very successful but yield gains
are now slowing. The new molecular technologies allow more precise
and rapid crop improvement by marker assisted selection breeding
and GM approaches. This requires the identification of the gene(s)
that underlie the traits and then combination with native traits using
molecular markers and/or GM to improve the crop– these include:
•Avoidance of losses from pests-insects,bacteria,fungi,viruses
•More effective water use-drought tolerance
•Increased tolerance towards temperature stress
•Increased yield
•Time to maturity – shortened growing season
•Growth on marginal soils-salinity, pH, metal toxicity
•More effective fertiliser use-nutrient(NPK) use efficiency
•Increased flooding tolerance
•Competing with weeds
•Improved nutritional quality-biofortification (eg.Vitamins,Iron)
•Sustainable production with a low carbon footprint

24. NEW TOOLS FOR CROP
IMPROVEMENT
Elite
Germplasm
Marker Assisted
BREEDING
Gene
Sequencing
Seeds
Better
Varieties,
Faster
Seed
Production
GENOMICS
Functional
Genomics
Trait
Development
PLANT
BIOTECH
Plant
Transformation
Traits
New Traits

25. The Three Genomes of Plants
Nucleus
Mitochondria
Chloroplasts
DNA APPEARS BLUE
Genome sequencing in Arabidopsis thaliana
Size
Genes
•Vacuole
•Nucleus
115,400 kb ca.25,400
•Mitochondrion
•Plastid-chloroplast)
•Peroxisome
•ER/Golgi
•Plasma membrane
•Cytosol
367 kb
154 kb
ca. 58
79

26. Maize
Genome sequence data are available for many
important plants

27. Modern plant breeders use molecular
methods including DNA sequencing and
proteomics as well as field studies
Photo credits Scott Bauer USDA; CIMMYT; IRRI; RCMI; Duke Institute for Genome Sciences and Policy

28. DN
A
TRAIT

29. The Challenge: Finding the genes that provide the foundation of new
traits and crop improvements for farmers
A Central Role for Omics, BioInformatics and Systems Biology
Genome Sequencing
Technology
Platforms
Bioinformatics
Modelling physiology
Process
Grain filling
leaf 3
0
Transcriptomics
Molecular profiling
Metabolomics
Proteomics
Time post anthesis
Phenomics-
TRAIT ANALYSIS

30. What is Genetic Modification?
Genetic modification is the addition, alteration or removal of
genetic material, usually single genes, in order to alter an
organism’s characteristics.
Living organisms contain 5,000-30,000 genes arranged in linear
order in chromosomes which are long strands of DNA.
Genes are heritable segments of DNA that contain the code
for an individual protein molecule.
Nucleus
ca.25,000
Genes
Chloroplasts
ca.80 Genes
Mitochondria
ca.60 Genes
Genetic Information in a Plant Cell

31. Transgenic Plant Technology
Commercial variety
Desired gene
New variety
Using plant biotechnology, a
single gene may be added to
the strand.
(only desired gene is transferred)
=
(transfers)
Desired gene
31

32. S = gene for susceptibility to
pest
25,000 genes
Single gene
25,000 genes
R = gene for resistance to pest
Repeated
Backcrossing
and selection for
desired traits

33. Genetic modification is the addition, alteration or removal of genetic
material usually single genes, in order to alter an organism’s
characteristics. The genes can be from any donor organisms
Microorganisms
Plants
DNA
Animals
Approximately
30% of animal,
plant and fungus
genes are similar
Man
A large percent of our
genes are the same as
those of simple organisms
such as bacteria and
viruses

34. Lessons from Molecular Evolution
 The living world is one
large gene-pool of
functional and
pseudogenes
 This gene-pool is
permanently evolving, this
is the basis of evolution
 Nature is one big genetic
laboratory
 It is very misleading to talk
about human gene, pig
gene, rat gene etc.
Animals share many genes in common with plants, fungi
and bacteria

35. Traditional plant breeding
Commercial variety
Traditional donor
New variety
DNA is a strand of genes,
much like a strand of pearls.
Traditional plant breeding
combines many genes at once.
(many genes are transferred)
=
X
Desired Gene
(crosses)
Desired gene
Transgenic Plant Technology
Desired gene
Commercial variety
New variety
Using plant biotechnology, a
single gene may be added to the
strand.
(only desired gene is transferred)
=
(transfers)
Desired gene
35

36. REASONS FOR UNDERTAKING ANY
GENETIC MODIFICATION
1 To improve the efficiency of a specific metabolic pathway so as
to improve the “efficiency” of the plant as a whole in terms
of its yield, nutritional quality or agronomic
characteristics(eg height, seed size)
2 To bypass some limiting such as intolerance to heat or
cold,drought,flooding, or to improve resistance to pests and
diseases
3 To change the nature of the harvested product – as a human
foodstuff; to provide a product of therapeutic value; to provide
industrial feed-stocks (e.g. the production of biodegradable
polymers) and biofuels.

37. Specificity of Genetic Modification
Identification and isolation of specific genes with
defined function
Insertion of specific genes into a crop species to
promote desirable characters
GM progeny can be selected for the product or
activity of specific genes with a defined function
There are no “surprises” from unknown genes
transferred along with the planned cross

38. Source of gene
(diseaseresistant plant)
Gene of interest
Isolate gene of interest
using molecular biology
methods
Once a gene is
introduced into
the plant genome
(DNA) it functions
like any other gene
Recombine into
recipient plant
DNA (Genome)

39. Two routes for the delivery of new traits and products
Genes can now be transferred more easily via marker assisted breeding (MAB) - non GM or directly through genetic
engineering - GM.
.
Products
Marker Assisted Breeding
MAB
Germplasm
Development
Traditional &
Molecular Breeding
Genetics
Genetic diversity
Analytical Screens
Biochemistry
Variety
Development
Yield Trials
Product Testing
Molecular Genetics
Marker Identification
by Trait, Crop,
species
GM
Transgenic Plant
Development
Cell Culture
Molecular Biology
Genetics
Gene Discovery
Plant Biology
Genomics
• 2 A I 3 7A to a dse u n e s
4 B 7 u m te q e c r
• 2 ,0 0L n p r w e c p c
0 0 a e e e k a a ity

40. Can Genetic Improvement of Crops
Help Feed the world?
• No single solution will solve this problem but
the new genetic technologies of plant breeding
developed during the last few years can helpthey are but one tool in the toolbox.
• They can can increase agricultural efficiencies
and save people from hunger in a sustainable
manner, particularly in African nations where
the need is greatest. Genomics, markerassisted screening, phenotype analysis,
computer modeling, and genetic modification
(GM) when required, have greatly accelerated
the breeding process.

41. The scientific basis of all crop improvement is identification of the
genes that encode and regulate specific phenotypic characteristics or
traits of use to the farmer.
REDUCED STRESSES
Biotic and Abiotic
• Drought or
• Pests and
Flooding
Diseases
• High or low
• Weeds
Temperature
• Saline or
. Phyto-remediation
acid soils
. Increased
greenhouse
gases- Tolerance
to climate change
IMPROVED NUTRITION
AND HEALTH
IMPROVED PLANT
PERFORMANCE
MORE
SUSTAINABLE
PRODUCTION
Environment
• Nutrient use efficiency
• Water use efficiency
• Control of flowering
• Plant architecture
• Heterosis
• Yield
Plant Gene
Technology
NEW
INDUSTRIES
Quality Traits
• Vitamins & Minerals
• Biofortification
• Post harvest quality
• Taste
• Proteins
• Oils and Fats
• Carbohydrates
• Fibre & Digestible
energy
• Bloat Safety
CHEMICAL
FEEDSTOCKS
• Biodegradable
Plastics
• Biofuels
PHARMACEUTICALS
• Vaccines
• Antibodies
• Diagnostics

42. Genetic transformation
of plants

44. Ideal Transformation Method
•
•
•
•
•
Can be applied to any genotype
Produces fertile plants
Has high efficiency
Introduces genes in single copy
Gene is stable and expressed over time
/generations in a Mendelian manner
• No background genetic changes

45. The steps involved in genetic modification
Identify the gene
an interesting gene
from a donor organism
Isolate
the interesting gene
Insert
the gene in a
genetic construction
Multiply
the genetic
construction
(bacteria,
plant ...)
Transfer the gene
Evaluate
Plant
regeneration
gene
expression
Add to other
varieties
by crosses
Selection of transformed cells
SOURCE: GNIS (Groupement National Interprofessionnel des Semences)

46. Gene Isolation by
standard techniques
of molecular biology
The first step is to isolate DNA
like you did yesterday.Then cut
the DNA into gene size pieces with
special enzymes and identify the
genes and what they do. The trait
or characteristic which they
contain the information for.

47. PROTEIN/ENZYME

48. Schematic
representation of the
two main ways to
create transgenic plants
Agrobacterium Method
Particle Gun Method

49. Agrobacterium Method
Agrobacterium tumefaciens-a common soil bacterium

50. Nature’s original genetic engineer
Gall
formation
Agrobacterium
Crown Gall
The soil bacterium Agrobacterium is able to infect plants
and make them produce the food it needs to live on. The
bacterium does this by inserting a small piece of its own
DNA into the genome (DNA) of the plant. Scientist have
modified this naturally occur process to make genetically
modified plants.

52. Agrobacterium-mediated plant transformation
Agrobacteria
containing
recombinant Ti plasmid are
multiplied in liquid culture
Cocultivation:
Agrobacterium
culture is added to callus culture
(e.g. rice) in Petri dish. Agrobacteria
infect the callus cells. T-DNA
excises from the Ti plasmid and
integrates into chromosomal DNA in
the nucleus of the callus cell.
In planta transformation: Flowering Arabidopsis is
inverted so that flowers dip into the Agrobacterium
culture in a bell-jar. Application of vacuum helps
bacterial infiltration. Plants are removed and grown.
Flowers are allowed to self and seeds are germinated in
selection agent so that only transformed seedlings
(about 10% of the total) develop.
Selection: transformed cells
(white) are resistant to
selection agent (herbicide or
antibiotic. Non-transformed
cells (color) eventually die.

53. Transformed Callus
Growing on an
un-supplemented medium

54. Totipotency:
Regeneration of a New Plant from a
Single Cell

55. More recently techniques have been developed in whereby Agrobacterium is vacuum
infiltrated into developing floral buds of a number of different plant species

56. Vacuum Infiltration of Floral Buds
In planta transformation: Flowers of the plant iare
inverted so that flowers dip into the Agrobacterium
culture in a bell-jar. Application of vacuum helps
bacterial infiltration. Plants are removed and grown.
Flowers are allowed to self and seeds are
germinated in selection agent so that only
transformed seedlings (about 10% of the total)
develop.

60. Advantages of Particle Bombardment
• Simple procedure
• Broad application range (relies on physical rather than
genetic parameters; thus often genotype-independent)
• Transformation restricted only by competence of plant
tissue to take up DNA and regenerate
• Can be used to transform organized tissues e.g. plant
embryos
• Multiple genes can be introduced simultaneously
• No plasmid backbone sequences are required (clean
transgene integration)

64. Broad Leaved
Crops
Cereal
Transformation

65. Crop Transformation
• High efficiency transformation protocol
• Output > 25,000 transformed plants per year

67. From laboratory to
commercialisation
specific gene transfer in the lab. followed by subsequent
testing in the field
this is the only plant breeding technology which requires
regulatory approval (and, in some countries, labelling of all the
food products derived from modified plants):
• testing for food toxicity, nutritional value, composition and allergenicity – includes animal feeding
trials
• characterisation of the transferred gene as well
as its effects on the host genome
•an environmental audit as well

68. A quick reminder
Conventional breeding
Elite variety
Breeding line
During conventional breeding, genes
are always mixed and newly assorted.
This often results in non-desired traits
of elite crop varieties.The desired
improvement is obtained by many years
of selection in the field.
New variety
=
X
(Cross)
Favorite gene
Favorite gene
Non-desired gene
Gene technology
Using gene technology, it is possible to
transfer only a favorite gene into an elite
crop variety. All other traits of the the
elite crop variety will be preserved.
Favorite gene
Elite variety
New variety
=
(Gene transfer)
Favorite Gene

69. Why are GM methods used sometimes
and molecular breeding others?
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
2. Genetic resources not
required
3. Plant can be propagated
vegetatively
Photo credits: Gramene.org ETH Life International

70. How have we fared thus far?
Rice genome
Sequenced
Plant
Transformation
1983
1865
Mendel’s Discovery
of Genes
1905
Genetics
1953
Structure of DNA
1001
Arabidopsis
genomes
sequenced
2002 2011
1995 2000
Crop Circles
‘Synteny’
2010
First Plant NGS
Genome
Sequence