Genetic Engineering of Potato
PlSc 490 – Potato Science
Lecture
Joe Kuhl
March 27, 2014
Overview
• Traditional potato modification
• Define Genetically Modified
• Transformation methods
– Agrobacterium, ballistic, advanced methods
• Transformation variables
• Potato genetic engineering, examples
• GM Testing
Potato Genetic Manipulation
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Wide crosses
Ploidy manipulation
Mutatgenesis
Somaclonal variants
Somatic fusion
Embryo rescue
Solanum L.
• ~1400 species (largest genus in the
Solanaceae)
• “Wild” potatoes: ~160 species
• Section Petota (Potato)
– Subsection Estolonifera (2 Series)
– Subsection Potatoe (19 Series)
• Includes tomato
– Solanum lycopersicum
• Diploid to hexaploid (x = 12)
– 2x, 3x, 4x, 5x, 6x
Wide Crosses - Origin of ABPT
4X – S. acaule (A) x 2x – S. bulbocastanum (B)
3x – AB (4x – AB not useful)
Doubling
6x – AB x 2x – S. phureja (P)
Bottle next! ±4x – ABP x 4x S. tuberosum (T)
4x – ABPT
5.5x – ABPT
x 4x – S. tuberosum
6x – ABPT
Hermsen 1985
Somatic Fusion
• Combining of somatic cells of uncrossable
species
• Single hybrid cells regenerated in culture
Doubled ploidy
Subject to somaclonal variation
Expensive process
GMO = Genetically Modified
Organism (also GM)
Also genetically engineered (GE),
transgenic, cisgenic, or intragenic
Genetic Engineering
• Transgenic/Cisgenic Crops
A transgenic crop plant contains a gene or genes which
have been artificially inserted instead of the plant
acquiring them through pollination.
Define by how a new variety is generated, not by what
the variety is.
Genetic Engineering
• Transgene
The inserted gene sequence may come from related or
unrelated plant, or from a completely different
species.
Example: transgenic papaya produces the PRSV coat
protein
Example: transgenic Bt cotton contains a gene from a
bacterium
Genes
Dr. Joe Kuhl - University of Idaho
Gene X
Traditional vs. Genetic Engineering
1. Only genes from closely related species are
involved with traditional methods
2. Traditional methods mixes large sets of genes
of mostly unknown function, as opposed to
one or a few well-characterized genes with
genetic engineering
Ronald and Adamchak 2008
Traditional Breeding
Organisms
• Plants
• Animal
• Microbes
Genes
• Plants
• Animal
• Microbes
Genetic Engineering
Organisms
• Plants
• Animal
• Microbes
Genes
• Plants
• Animal
• Microbes
Cisgenics
Simplot Company: “All potato in potato”
• Agrobacterium-based methods that utilize a
plant-derived transfer DNA and a novel
transient selection system to insert only native
DNA into plants
• Marker free approach
• Selection against backbone incorporation
Percentage of respondents that would eat:
Extra gene/same vegetable
Extra gene/different vegetable
Multi genes/different vegetable
Animal gene
Fungal gene
Bacterial gene
Viral gene
0
20
40
60
80
100
Lusk and Sullivan (2002) Food Technology
Hunter 2014
Modification Methods
• Biological (DNA transfer)
– Agrobacterium (stable vs. transient)
• Physical (DNA transfer) – gene gun
• Targeted genetic modification
Modification Methods
• Gene(s) Transfer
– Biological
• Agrobacterium tumefaciens
mediated T-DNA transfer
Crown Gall
Agrobacterium-mediated Transfer
Modification Methods
• Gene(s) Transfer
– Physical
• Particle Bombardment
• Microprojectile-mediated
Gene Silencing
• Exploits plant regulatory mechanism
– RNA Interference (RNAi)
• Targets specific plant gene(s)
• Decrease or eliminate expression
• May use siRNA or miRNA
– Short-interfering RNA (siRNA)
– Micro-RNA (miRNA)
Gene Silencing
• Exploits plant resistance mechanism
– RNA-mediated anti-viral defense
• Modify virus vector to carry specific plant gene
targets
• RNAi (interfering RNA from dsRNA)
• siRNA-mediated (~22bp oligonucleotide dimers)
• Post-transcriptional gene silencing
– Decrease or eliminate expression
Silencing of Polyphenol Oxidase
(PPO)
Rommens et al. 2004
Targeted Genetic Modification
• Engineered nucleases or meganucleases
– Create DNA double-stranded breaks at specific
genomic locations
– This activates DNA repair mechanisms
– With or without homologous template
• Modify native plant genes in directed and
targeted ways
– modify endogenous genes
Targeted Genetic Modification
Novel Restriction Enzymes:
• Homing endonucleases
• Zinc finger nucleases (ZFNs)
• TALE nucleases (TALENs)
– Transcription Activator-Like Effectors (TALEs)
• CRISPR
– Clustered regularly interspaced short palindromic
repeats
Plant Transformation
• Gene(s) Transfer
– Integration of transgene(s) into the plant genome
Trait
Plant Transformation
• Variables (for each “event”)
– Copy number
– Location in the plant cell
– Location in the plant genome
– Content of transferred genetic information
– Resulting phenotype
Plant Transformation
• Gene(s) Transfer – Plant Breeding and Testing
• Desired trait(s)
– Activity of the introduced gene
– Stable inheritance of the gene
– Avoid unintended effects on plant growth, yield,
and quality
Potato Transformation
• Lengthy breeding programs, tetrasomic
inheritance, asexually propagated
• High in vitro regeneration capacity
• Excellent host for Agrobacterium tumefaciens
• One of the first crops to be successfully
transformed (Ooms et al. 1986), A. rhizogenes
• Stiekema et al. 1988, A. tumefaciens
Potato Transformation
• Ultimate objective – transfer of a gene into an
existing cultivar to produce an enhanced
version
• Silencing – interferes with the operation of the
naturally occurring gene, to switch off, reduce
activity, or delay natural operation
Potato Transformation
Agrobacterium-mediated Transfer
• Copy number, one or more copies
• Diploid regenerates doubled their
chromosome number
• Some tetraploid regenerates were male-sterile
• “Random” insertion
• Create large populations of independent
transformants
Potato Transformation
Phenotypic Changes
• Off-types: genotype (cultivar) dependent
– 15-80%
– Field grown
• Generate sufficient material and trial under
field conditions
– “Generally, majority of transgenic material was
phenotypically indistinguishable from control
plants, and stable over several generations” (S.
Millam)
Potato Transformation
Trait
Potato - Commercial Lines
• Commercial lines: 1995-2001
• ‘New Leaf’ – Baccillus thuringiensis (Bt) CryIIIA
gene, Colorado potato beetle resistance
– R. Burbank, Atlantic, Superior
• ‘New Leaf Plus’ – Bt resistance plus PLRV
resistance
• ‘New Leaf Y’ – Bt resistance plus PVY
resistance
Simplot
Generation 1: 2014+
Low acrylamide, low bruise
Generation 2: 2016-2017
Cold-sweetening resistance
Generation 3: 2018+
Late blight resistance, PVY resistance
Target cultivars (initially): Ranger Russet, Russet
Burbank, Atlantic, Snowden
Current GM Crops
Gruskin 2012
Marshall 2012
Potato - Potential Traits
• Disease and pest resistance
– Colorado potato beetle (cryIIIA)
– Potato tuber moth (cryV, cryI Ac9)
– Potato cyst nematodes (chicken egg white cystatin)
– Viruses, e.g. PLRV and PVY (sense and antisense)
– Bacteria and fungi
• Erwinia and Phytophthora infestans
Colorado Potato Beetle Resistance
• Bt Insect-Resistance
– “Bt” short for Bacillus thuringiensis, a soil
bacterium whose spores contain a crystalline (Cry)
protein
– Cry breaks down in insect gut to release a toxin
(delta-endotoxin) – toxic to some insects
cryIIIA
Late Blight Resistance
• Katahdin transformed with RB
• late blight resistance R gene from S.
bulbocastanum
Song et al. 2003
Useful Traits
• Tuber Quality
– Anti-bruise (down-regulate PPO)
– Reduced glycoalkaloid content (down-regulate Stg1)
– Starch
• High amylopectin and high amylose
– Reducing sugars (over-express ADPglucose
pyrophosphorylase)
Potato - GM Traits
• Gene silencing of vacuolar acid invertase
using RNAi
Bhaskar et al. 2010
Useful Traits
• Nutritional value
– Inulin (express artichoke genes)
– Carotenoids (down-regulate zeaxanthin epoxidase,
express Erwinia phytoene synthase)
• Pharmaceutical
– Vaccines, others…
GM Testing
GM crops are the most extensively tested crops
ever added to the food supply.
• GM plants must be shown to shown to be the
same as the parent crop from which it was
derived
• If a new protein trait has been added, the
protein must be neither toxic nor allergenic
GM Information
• Source of the gene
• Characterization of the insert
• Compositional analysis
– Plant toxins, anti-nutrients, and allergens
– Unintended up- or down-regulation of critical molecules
Of 129 transgenic crops submitted to FDA (1995-2012) - All
failed to detect any significant differences, or any believed
to have biological relevance (engineered vs.
nonengineered or reference species)
DeFrancesco 2013
NAS 2004
• American Association for the Advancement of
Science – October, 2012
“Indeed, the science is quite clear: crop
improvement by the modern molecular techniques
of biotechnology is safe.”
• European Commission – 2010 report
“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.”
• Nature Biotechnology Editorial, Sept. 2013
About GM Food:
“…the World Health Organization, the US National
Academy of Sciences, the European Commission [and] the
American Medical Association, have come out with
ringing endorsements of their safety. The fact is, negative
attitudes remain entrenched and widespread. And
changing them will require a concerted and long-term
effort to develop GM foods that clearly provide convincing
benefits to consumers - something that seed companies
have conspicuously failed to do over the past decade.”
Regulatory systems – U.S.
• Institutional Biosafety Committee (IBC)
• U.S. Department of Agriculture - Animal and
Plant Health Inspection Service (APHIS)
• U.S. Environmental Protection Agency (EPA)
• Department of Health and Human Services Food and Drug Administration (FDA)
• International agreements
http://www.colostate.edu/programs/lifesciences/TransgenicCrops/index.html
Regulatory systems – U.S.
• Institutional Biosafety Committee (IBC)
• U.S. Department of Agriculture
• under the Federal Plant Pest (Protection) Act
as ‘plant pests’ – if perceived threat of them
becoming pests
• Genes taken from plant pests are used
• Glyphosate resistance from Agrobacterium
• 35S promoter – from CaMV
• A. tumefaciens used to deliver transgene
Regulatory systems – U.S.
• U.S. Environmental Protection Agency (EPA)
• under the Federal Insecticide, Fungicide and
Rodenticide Act (FIFRA), if pest-resistant, they can
be interpreted as ‘plant pesticides’
Regulatory systems – U.S.
• Department of Health and Human Services Food and Drug Administration (FDA)
• treats GM food crops as equivalent to conventional
food products and no special regulations were
added, but a pre-market consultation process for
GM and other novel foods is voluntary
• International agreements