Genetic Engineering of Potato

PlSc 490 – Potato ScienceLecture 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

• 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 – ABPT5.5x – ABPT x 4x – S. tuberosum6x – ABPT

Hermsen 1985

Somatic Fusion

• Combining of somatic cells of uncrossable species

• Single hybrid cells regenerated in culture

Doubled ploidySubject to somaclonal variationExpensive process

GMO = Genetically Modified Organism (also GM)

Also genetically engineered (GE), transgenic, cisgenic, or intragenic

Genetic Engineering

• Transgenic/Cisgenic CropsA 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

• TransgeneThe 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

Organisms• Plants• Animal• Microbes

Genes

Traditional Breeding

Genetic Engineering

• Plants• Animal• Microbes

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

0 20 40 60 80 100

Extra gene/same vegetable

Extra gene/different vegetable

Multi genes/different vegetable

Animal gene

Fungal gene

Bacterial gene

Viral gene

Percentage of respondents that would eat:

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 TransformationAgrobacterium-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 TransformationPhenotypic 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: 2016-2017Cold-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

Song et al. 2003

• Katahdin transformed with RB• late blight resistance R gene from S.

bulbocastanum

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. 2013About 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