development of genetically engineered crops why would we ...€¦ · genetically modified organisms...

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A. How are genetically engineered crops produced?

B. How do they compare to conventionally-bred crops?

C. What are the implications for safety assessment?

Development of genetically engineered crops

and relationship to conventional breeding

Rebecca Grumet, Michigan State University

Why would we want genetically engineered crops?

Farmers face many challenges to produce food and other agricultural products:

destruction by insectslosses to diseasecompetition from weedsenvironmental stresses (drought, frosts). . .

What are the options to deal with these challenges?Pesticides (insecticides, fungicides)

Manual or mechanical methods (weeding, removing diseased plants)

Integrated crop management (rotations, sanitizing, biological controls)

Improved varieties that can resist the challenges

** Most cost-effective and environmentally favorable approachSaves cost and impact of chemicals, labor, time, fuel…

conventional breedinggenetic engineering

Whether using conventional breeding or genetic engineering, the basis for crop improvement results from genes

Genes carry the information that is passed from generation to generation. They are the blueprint for (instructions to make) living organisms.

Anything that is alive has genes.(therefore, anything you eat has genesor was produced from an something that has genes)

The information of genes is carried in the form of DNA molecules.

The ‘language’ of DNA, is the same regardless of the organism – A key point for genetic engineering

DNA

molecule

Improving crop varieties

An insulin gene from a human will still make insulin if it is put into a different animal, or a plant, or a bacterium

Greater than 90% of the insulin currently produced for diabetes treatment is a product of genetic engineering (is produced in bacteria)

The human insulin gene has been transferred to bacteria so it the insulin can be made in processing vats in large quantities

Why?-- Cleaner, safer-- Less expensive than

raising animals-- It is human form of insulin

Recombinant DNA technologies - Put DNA together in new combinations

(gene construct)

Gene transfer technologies- Introduce DNA into a new organism

The primary technologies required for genetic engineering are:

The resultant products are genetically engineered

or genetically modified

Improving varieties by genetic engineering

Genetically engineered crops are crops into which one or more genes have been introduced to confer a useful trait

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Genetic modification is not limited to genetic engineering technologies

Genetic engineering is a new tool to add to a long line of genetic modifications

• crop domestication• development of land races• hybridization and modern

plant breeding• genetic engineering

Humans have genetically modified plants and animals for as long as they have cultivated crops or kept livestock.

GENETICALLY MODIFIED ORGANISMS - GMOs

Dog breeds

Plant and Animal Breeding – Genetic Modification

Maize Teosinte

Our crop species have been domesticated from wild relatives and bred for high productivity and other desirable features (disease resistance; uniformity)

Effective plant breeding predates the earliest dates set by archaeological records

Ancient

Peruvian

city-

dweller’s

beans were

10-100-fold

larger than

wild beans

in the area.

Photo from: www.teaching-abroad.co.uk

-- Selection

The primary tool available to early breeders

- Intentional selection(size, flavor, ease of cooking)

- Inadvertent selection(seed shattering, environment – land races)

Breeder’s Tools Selection: Choosing the best (largest, sweetest, most interesting, best

growing) to use for the next generation

Cabbage, Cauliflower,Broccoli, Brusslesprouts

are all the same speciesthat have been selectedfor different traits

Broccoli and cauliflower are flower mutants,Cabbage and brusslesproutsare stem mutants

Brasica oleracea

Selection can be very powerful!

Cauliflower

Brussels sprouts

Broccoli

Cabbage

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Late blight of potato, caused by Phytophtora infestans, was responsible for the Irish potato famine of the 1840’s

For selection to work, the trait must exist in the population

What if the trait does not exist in the population?

Susceptible parent Resistant breeding line

Development of Fusarium race 2 resistant celery lines

Hybridization

Mixing together different types of traitsBringing in new traits from other sources

Celery(Apium graveolens var. apii

Celeriac (Apium graveolens var. dulce)

Breeder’s Tools

-- Selection

-- Hybridization

- Mixing together different types of traits

- Well established by the 1700s

- Formalized in the 1900’s

Selection + Hybridization were the primary basis of plant breeding in the 20th century

Resulted in great gains in crop productivity and quality

what if the trait is not available in a species that can be readily crossed with the crop of interest?

e.g. cultivated potatoes are tetraploid (4n) many wild potatoes are diploid (2n)

(different numbers of chromosomes – will not produce fertile offspring)

There is still much improvement to be done for many crops and many traits

Breeder’s Tools

-- Selection

-- Hybridization

-- Recombinant DNA / Gene transfer

Through biotechnology techniques it is possible to introduce a new gene or small number of genes that are not be available through conventional breeding methods -

With conventional breeding we are limited to those species that are closely related and capable of crossing to produce fertile seed

With genetic engineering, the genes can come from anywhere

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1

5

4

3

2

How are genetically engineered crops produced?

1. Get the gene of interest and engineer into a form for expression in the new plant

2. Transfer the engineered gene into cells/chromosomes of the desired plant

3. Regenerate cells with the new gene back into whole plants

4. Test the transgenic plants for the desired gene and traits in the laboratory, greenhouse and field

5. Incorporate the new gene into useful high performing cultivars by conventional breeding


The hardest part can be figuring out what gene(s) will give the desired trait!

A typical plant has 20,000 – 40,000 genes.

The genes specify all of the information that is needed to allow a plant to:

- form leaves, roots, flowers, fruits, seeds…- germinate, and grow; carry out

photosynthesis and respiration; adapt to environmental conditions such as heat, cold, drought

- make storage compounds (starches, oils), pigments, flavors and fragrances

- make compounds to defend itself against insects and diseases

The information contained in a rice plant’s DNA, if written out letter by letter as nucleotide bases (i.e., ATGC)would fill 40 volumes, each with 1000 pages.

A single typical gene would fill less than one page.


Physiology, molecular biology, genetics, genomics, biochemistry, recombinant DNA technology

The hardest part is figuring out what gene(s) will give the desired trait

Wang et al., 2004

Weng et al., 2015

Colle et al., 2017

Mansfeld et al., 2017

Recombinant DNA

technology

Initial methods allowing

for manipulation of DNA

(copying, cutting,

pasting, moving) were

developed in the 1970’s

and 1980’s.

1. Get the gene of interest and engineer into a form for expressionin the new plant

Gene

transfer

technology

Common

methods

include:

Agrobacterium-

mediated

transfer

and particle

bombardment.

Source: North Dakota State Univ. Extension Service, www.ext.nodak.edu

2. Put the engineered gene into cells/chromosomes of

the desired plant

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Gene transfer technology

Source: North Dakota State Univ. Extension Service, www.ext.nodak.edu

Agrobacterium-mediated gene

transfer

Agrobacteria can be viewed as

natural genetic engineers

Part of the natural infection

process by Agrobacteria, is

to insert some of their genes

directly into the host

chromosome(discovered in the 1980’s –

breakthrough for plant genetic

engineering)

genes the bacteria would

transfer are replaced with

genes we want to transfer


Source: North Dakota State Univ.

Extension Service,

www.ext.nodak.edu

Particle bombardment

Direct gene transfer

- Use force of acceleration

to penetrate cell wall

DNA is taken up and either:

transiently expressed

hangs around long enough

to get some mRNA and

protein expression

(primarily for research)

or,

incorporated into the

genome

DNA enters nucleus and is

integrated into a

chromosome

CRISPR/Cas9 –

Breakthrough of

the year – 2015


Primary uses of

CRISPR/Cas9 for plants:

- Gene knock out

- Targeted gene insertion

CRISPR/Cas9 is based on bacterial systems that are used to detect and chop up foreign DNA.

There are two distinct components to this system: (1) a guide RNA (gRNA)

Where to cut?(2) an endonuclease, CRISPR-associated (Cas) nuclease, Cas9.

Perform the cut

CRISPR/Cas9 technology

Following cut, broken DNA is recognized and DNA repair mechanisms are activatedEnzymes will put DNA back together, but now will be missing some sequence,gene is disrupted – “knock out” gene

http://sites.tufts.edu/crispr/genome-editing/homology-directed-repair/

Alternatively, a new gene can be inserted into the location of the cut

The advantage relative to other methods is we can direct exactly where the new gene goes

In either case, a mechanism such as Agrobacterium or particle bombardment is still needed to deliver the CRISPR/Cas9 machinery into the cell

With gene knockout, however, after the cutting has been done,there is no new DNA that is added

In any of these cases (Agrobacterim, particle bombardment, gene introduction, gene knockout), the genetic change is made in a single cell

The next step is to regenerate the single cell into a whole plant

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3. Regenerate cells with the new gene back into whole plants

Plant tissue culture

Conditions have to be identified for each

species, and often each variety, by

experimentation

Often the variety you can regenerate is

not the best to grow and produce a crop

This step may include the use of a selectable marker, such as antibiotic resistance or herbicide resistance, to distinguish between the cells that have the new genes and those that do not

Has the new DNA been successfully transferred – is it present in the plant?How many copies are present?

1. PCR (polymerase chain reaction)2. Southern analysis3. Mendelian inheritance – transfer to next generation

Mar

ker

AP3-

3AP3-

8C

RC

5C

RC

11C

RC

15W

Tp-

AP3-

etr1

-1

p-C

RC

-etr1-

1

Mar

ker

AP3-

3AP3-

8C

RC

5C

RC

11C

RC

15W

Tp-

AP3-

etr1

-1

p-C

RC

-etr1-

1

CRC-etr1AP3-etr1

Little, Papadopolou, Grumet, 2007

PCR verification of introduced At-etr1-1 gene and respective

stamen/petal or carpel-specific promoter

4. Verify that the regenerated plants are transgenic and

test for the traits in the laboratory, greenhouse and field

Molecular verification

Is the new gene being expressed? (DNA RNA protein)

Western verification of expression of full-length or truncated ZYMV coat protein gene (Fang et al., 1993)

Northern verification of ACS expression in transgenic melon (Papadopoulou et al., 2005)

WT AZ ACS1 ACS3 ACS4

1. RNAnorthern analysis, RT-

PCR, qRT-PCR

2. Protein western analysis, ELISA (typically antibody

based methods; sometimes can look for new or enzyme activity)

4. Verify that the regenerated plants are transgenic and test for the traits in the laboratory, greenhouse and field

ZYMV-CP WTAtCBF

ACS

“Proof of concept”

Does the gene do

what it was

supposed to do?



Disease resistance? Salt stress resistance?

Earlier fruit production?



At the commercial scale, as many as hundreds of individual transgenic plants may be produced and tested to find the one that meets all desired criteria such as:

- level of gene expression- single gene integration - location of integration - desired trait conferred - no unintended undesired traits occur

Cultivar

Development

5. Incorporate the new gene into useful high

performing cultivars by conventional breeding

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Cultivar DevelopmentField testing for performance

in a range of environments

By 2011, there were more than 880 Bt cotton hybrids available in India (Katage and Qaim, Proc Natl Acad Sci, 2012)

Length of growing season,

daylength, temperatures, water

availability, growing season,

pests, diseases….

How do genetically engineered (GMO) crops compare to crops developed by conventional plant breeding?

• Source of genes• Amount of change

• Extent of characterization

How does genetic engineering compare to conventional plant breeding?

•Source of genes (where do the genes come from?)

Conventional Breeding: Genes within species orclosely related species

X

celery

celeriac


•Source of genes

The language of DNA is the same, regardless of the organism it comes from

Conventional Breeding Genes within species orclosely related species

Genetic Engineering Genes from anywhere

Regulation of recombinant DNA research and products (U.S.)

- Laboratory and greenhouse (contained experiments)(Institutional Biosafety Committee)

- Field research (confined field trials)(University, State and Federal (USDA) approvals)

- Commercialization/Crop production(USDA, FDA, and EPA approvals)

agriculture food environment

The access to genes from virtually any organism led to development of regulatory systems to assure safe use of the technology


• Amount of change

Conventional breedingLarge numbers (100s) of genes,

May be both desirable and undesirable

Progeny of celery X celeriac cross:thin, stringy, brittle, bitterlong series of backcrosses to regain celery quality + new resistance

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Could unsafe traits, be introduced by conventional plant breeding?

(e.g., psoralens in celery)


• Amount of change

Conventional breedingLarge numbers of genes,

May be both desirable and undesirable

Genetic engineeringOne or very few genes

How do the extent of changes caused by GE compare to conventional breeding?

Directly compared wheat with increased gluten (seed storage protein important for bread making)

produced by

- Genetic engineering - Conventional breeding

Near isogenic lines (almost identical) developed by backcrossing After 8 generations of backcrossing – 99.6% identical(generation 1- 50%, 2- 75%.... generation 7, 99.2%, generation 8 – 99.6%)

Baudo et al., 2006; Plant Biotechnology Journal

(University Bristol, University Nottingham, UK)

Experimental evidence

note: if 30,000 genes, 99.6% same; = 0.004 or 0.4% different

= 120 genes

Plant & Soil Sciences eLibraryPRO

Amount of change – experimental evidence comparing GE and conventional breeding

Number of significant differences in gene expression for transgenic and conventionally bred wheat for increased wheat gluten___________________________________________

Transgenic vs. non-transgenic standard 5

Conventionally bred high gluten vs. conventionally bred standardnear isogenic lines 92

___________________________________________

changes in gene expression (RNA)

Baudo et al., 2006; Plant Biotechnology Journal

(University Bristol, University Nottingham, UK)

Desiree + transgenes

Maris Piper

PHU.4637

Cultivar comparisons

Amount of change – experimental evidenceProtein composition

Principal component analysis of GM potato expressing SAM decarboxylase or glucan branching enzyme and comparison among potato cultivars.

Lehesranta et al. 2005, Plant Physiol. (Univ. Kuopio Finland and Scottish Crop Research Institute)

Transgene comparisons in Desiree background

Wild type parent (Desiree)

Biological vs. statistical significance


• Extent of characterization (what do we know about the genes?)

Conventional Breeding: Known by speciesGene product(s) not

generally known

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* Extent of characterizationGenetic Engineering: Completely characterized

for nucleotide and amino acid sequence

Encoded protein is expressed in the laboratory

Function verifiedTested for toxicity and allergenicity

All of these tests are done before release of any GE product


* Extent of characterizationGenetic Engineering: Completely characterized

for nucleotide and amino acid sequence

integration into the genome can only influence expression of the currentgenetic capacity of the species

Site of integration unplanned(This is changing with new technologies,

i.e., CRISPR/Cas9)

For conventional breeding or genetic engineering new varieties must be tested for yield, quality,performance in a wide range of conditions,and safety

The key factor is not how it is produced, but what is produced and how will it be used?

The key questions are: which gene is introduced? into which crop?what trait does it confer? where will it be grown?

Safety concerns are not new, nor are they unique to genetic engineering

U.S. National Academy of Sciences – May 2016Society of Toxicology – Sept. 2002 – Consensus position statementNational Research Council – Institute of Medicine – Sept. 2004National Research Council – National Academy of SciencesAmerican Medical AssociationInstitute of Food TechnologistsAmerican Dietetic Association

European and International StatementsFrance – French Academy of Medicine – 2003Italy – Eighteen scientific associations – October 2004

(including National Academy of Science, Societies for Toxicology, Microbiology, Nutrition, Biochemistry) signed consensus statement on safety of GMO crops

FAO – Food and Agriculture OrganizationWHO – World Health OrganizationInternational Council for Science – 2005, 2010

(111 National Academies of Science and 29 scientific unions)

GE technologies are not inherently risky, decisions should be made case by case

How do the risks of genetic engineering compare to conventional plant breeding?

Is there scientific consensus? The key factor is not howit is produced, but whatis produced and how it will be used

2010 report fromThe European Commission Directorate-General for Research:EC Sponsored Research on Safety of Genetically Modified Organisms. A Decade of EU Funded Research (2001-2010). I. Economidis, D. Cichocka, J. Hogel (eds)http://ec.europa.eu/research/biosociety/pdf/a_decade_of_eu-funded_gmo_research.pdf

How do the risks of genetic engineering compare to conventional plant breeding? is there scientific consensus?

The main conclusion to be drawn from the efforts of more than 130 research projects, covering a period of more than 25 years of research, and involving more than 500 independent research groups, is that biotechnology, and in particular GMOs, are not per se more risky than e.g. conventional plant breeding technologies.

CONCLUSIONSA crop is a combination of thousands of genes and traits that have been modified and refined by humans over the eons

Genetic engineering is a additional tool to add new genes or traits to a crop

Both for conventional breeding and genetic engineering new varieties must be tested for yield, quality, safety, and performance in a wide range of conditions

Thank you!

http://ec.europa.eu/research/biosociety/pdf/a_decade_of_eu-

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Whether using conventional breeding or genetic engineering, the basis for crop improvement results from genes

Genes carry the information that is passed from generation to generation. They are the blueprint for (instructions to make) living organisms.

Anything that is alive has genes.(therefore, anything you eat has genesor was produced from an something that has genes)

The information of genes is carried in the form of DNA molecules.

The ‘language’ of DNA, is the same regardless of the organism – A key point for genetic engineering

DNA

molecule

Improving crop varieties


Greater than 90% of the insulin currently produced for diabetes treatment is a product of genetic engineering (is produced in bacteria)

Genetically engineered crops are crops into which one or more genes have been introduced to confer a useful trait


Greater than 90% of the insulin currently produced for diabetes treatment is a product of genetic engineering

The human insulin gene has been transferred to bacteria so it the insulin can be made in processing vats in large quantities

Why?Cleaner, saferLess expensive than raising animalsIt is human form of insulin

Recombinant DNA technologies - Put DNA together in new combinations

(gene construct)

Gene transfer technologies- Introduce DNA into a new organism

The primary technologies required for genetic engineering are:

The resultant products are genetically engineered

or genetically modified

Improving varieties by genetic engineering

CRISPR is based on bacterial systems that are used to detect and chop up foreign DNA. There are two distinct components to this system:

(1) a guide RNA (gRNA)Where to cut?

(2) an endonuclease, CRISPR-associated (Cas) nuclease, Cas9. Perform the cut


Following cut, broken DNA is recognized and DNA repair mechanisms are activatedEnzymes will put DNA back together, but now will be missing some sequence, gene is disrupted – “knock out” gene

Following cut, broken DNA is recognized and DNA repair

mechanisms are activated

Enzymes will put DNA back together, but now will be missing

some sequence, gene is disrupted – “knock out” gene


https://www.systembio.com/crispr-cas9-plasmids

Still need to be able to regenerate cells with knocked out genes,

but don’t need to incorporate new genes

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For Cas9 to successfully bind to DNA, the target sequence in the genomic DNA must be complementary to the gRNA sequence and must be immediately followed by the correct protospacer adjacent motif or PAM sequence. (canonical PAM: 5'-NGG-3')

Genome sequencing and bioinformatics tools are used to identify the endogenous PAM sequences present in the target genome

https://www.addgene.org/CRISPR/guide/#PAM

System was first

successfully

demonstrated in 2012

in a bacterial system

Since then, have been

demonstrations in

animal and plant

systems

tracrRNA – trans-activating CRISPR RNA, forms stem loop structure that enables the Cas9-crRNA complex to locate the targeted DNA

development of genetically engineered crops why would we ...€¦ · genetically modified organisms...

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