Martina Newell-McGloughlin Director, UC Systemwide ...super4/35011-36001/35751-35761.pdfMartina...

Martina Newell-McGloughlinDirector, UC Systemwide Biotech Research

CO

Economic sustainability could be created by GMOs

CO2

Renewable Resources:

Value

Resources: Biomass conversion, feedstocks, biofuels, phytoremediationValue p y

Plants as Factories Pharmaceuticals/ Industrial products (V i Ri L f i L(Ventria – Rice Lactoferin Lysozyme Peru 30% Less Diarrhea, Quicker recovery 3/6 days, 1/3 less recurrence

Quality Traits - ($210B by 2015) Improved shelf life, processing, tasteImproved Nutrition –Improved Functionality

Agronomic Traits – $30B Biotic- pest/disease/weeds/

Abi ti St /Yi ld

p p yMacro: protein, oils, carbs, fibre

Micro: Vitamins, minerals, Phytochemicals – Antioxidants

1st Wave 2nd Wave 3rd Wave 4th Wave

Abiotic Stress /Yield Remove Antinutrients/allergens/ Toxins

US has the most productive and cost efficient food s ppl chainefficient food supply chain

Food Supply Chain t ib t $1 2Tcontributes over $1.2T

in GDP from value added products and

Expenditures % Productivity %25

20 5%

260

260%Farm Productivity(Output less Input) pservices

Each U.S. farmer feeds 20

20.5%200

( p p )

over 129 people

U.S. consumers spend 15150

7-10% of income on food versus other countries:10

10%Food Expenditure(% of Disposable Income)

100100% countries:

– 13% High income nations

(% of Disposable Income)100%

– 29% Middle income– 47% Low IncomeSource: ERS\USDA

Following are from “trusted” Sources?Following are from “trusted” Sources?•• FAO? Ecology Groups?FAO? Ecology Groups?

• FAO: global demand for food 2.5 –3X in poorest countries by2050 (FAO, Rome).

• 17% of land under cultivation degraded by human activity from 1945 to 1990. Ag land shrinks by 20 000 hectares yearlyland shrinks by 20,000 hectares yearly. (World Bank, 1997)

• Without yield increase land use will d bl b 20501997 double by 2050

• Without greater productivity China/India will need 4X land area

1997 acreage

• Latin America: greatest yield increase had lower land use (less deforestation)

• High yield “land sparing” better than “wildlife”-friendly inefficient land use farming

Green, Royal Soc. Bird Protection African Society Ornithology 2005)

The Crop Technology Timeline Crops

200010,000yrs 0

graphWild plants

X X XX

X

X

1M

Human Cultures

DomesticationAgriculture

Primitiveman

Hunters and Gatherers10M

8,000 BC19thCEa 20th C

CultivationSelective Cross breedingCell culture

Md 20th C1930s1940s

Somaclonal variationEmbryo rescuePolyembryogenesis1940s

1950s1970s1980

y y gMutagensis and selectionAnther cultureRecombinant DNA

Assyrian relief carving from870 B C showing artificial1980

1980s1990s2000

Recombinant DNAMarker assisted selection-omics, bioinformaticsEpigenetics/RNAi/Paramutation

870 B.C. showing artificial pollination of date palms.

2000200021st C

Epigenetics/RNAi/ParamutationAdaptive technology/transgenomicsSystems Biology

Biotech is UnnaturalBiotech is UnnaturalIs Agriculture Natural?Is Agriculture Natural?

Chile

gg

North America

Paris BotanicalGarden, 1766

Some images courtesy of Wayne Parrott

And Risk is relative !

Mutation BreedingMutation Breeding“Gold Nijusseiki”

i t t t bl kresistant to black spot disease.

The gamma field is a circular field f 100 di i h 88 8TB In 1957 at The Atomic Energy

Maythorpe �Golden Promiseof 100m radius with 88.8TBqCo-60 source at the center. 2,252 new plant varieties in 158 species,

In 1957 at The Atomic EnergyResearch Establishment, Harwell

including Italian durum wheat, 70% of the area under cultivation. wheat, barley, oats, rice, soybeans, , y, , , y ,string beans, navy beans, potatoes, onions, cherries, apples, grapes

CA

Pearradiation

bredCA

http://www.irb

This diagram shows the various stages of anther and isolated pollen culture. The stages of anther culture from anther to haploid plantlet can be described as follows: a) an unopened flower bud, 1b) anthers, 1c) the anthers in culture, 1d) and 1e) proliferating anther, 1f) haploid callus, 1g) differentiating callus, h) haploid plantlet. Isolated pollen culture is as follows: a) an unopened flower b d 3b) i l t d ll f lt d th 3 ) ll lt 3d)bud, 3b) isolated pollen from a cultured anther, 3c) pollen culture, 3d) multinucleate pollen, 3e) and 3f) pollen embryo. Homozygous plants can be obtained by treating the haploid plantlets with colchicine

Radish Cabbage

A Russian scientist named Karpechenko promised Stalin that he could double productivity by crossing a cabbage p y y g gwith a radish

R di h C bbRadish Cabbage

R h b iRaphanobrassica

Instead Karpechenko introduced a brand new species to science and went to Siberia for his efforts!!(Traditional crossbreeding selection, mutation breeding, somaclonal selection and wide crosses allow no control at the genome level and it may take years of backcrossing to remove unwanted effects)

Relative Size of Genomic i b l i lIntrogression by Classical

Breeding and Molecular geneticsBreeding and Molecular genetics

1,000,000 2,000,000 3,000,000 4,000,000 5,000,0000

Genome-wide analyses of introgression -oaks to fruit flies - substantial fraction of genomes malleable Hybridization gives rapid genomic changesof genomes malleable. Hybridization gives rapid genomic changes, chromosomal rearrangements, genome expansion, differential expression, and gene silencing (transposable elements). Baack 2008

Transcriptomic and Metabolomic studies show greater variation between conventional bred cultivars and even growth locations than between GM and

t l i t ( t f f th i t d d difi ti !) diffparental variety (except of course for the intended modification!) - differences between sites were generally greater than differences between linesWheat ( Baker 2006), Potato (Catchpole 2005)

Natural Variability Natural Variability –– Reference HybridsReference Hybrids

30

yy yy7 Varieties, 6 Locations, 1 Year7 Varieties, 6 Locations, 1 Year

25

30

20

10

15

5

10

0

Asp Thr Ser Glu Pro Gly Ala Cys Val Met Iso Leu Tyr Phe His Lys Arg TrpAsp Thr Ser Glu Pro Gly Ala Cys Val Met Iso Leu Tyr Phe His Lys Arg Trp

Amino acidsAmino acidsSlide courtesy of Bruce Chassy

Wide Crosses

Tomatoes are members of theDeadly nightshade family

Lycopersiconchmielewskii

•High solids = More L. esculentum

gsauce: Two approaches one end: “Natural” cross withNatural cross with high solanine “toxic” wild tomato

Back-cross

•Using antisense, switch off existing

series

Tomato Cultivar

gene – no introgression of genes from the toxic plant

Method of Delivery of Method of Delivery of DNA Into a Plant CellDNA Into a Plant Cell

1. Transformation1. Agrobacterium tumefaciens -mediated

transformation cultured cells, explants (bits leaves/stems)transformation cultured cells, explants (bits leaves/stems)2. Direct methods

Gene Gun

2 E i2. Expression1. Promotor Constitutive (CaMV 35S),

tissue/time – specific (RuBisCo, glutenin, α-amylase, patatin) --Turning on disease resistance genes only when the--Turning on disease resistance genes only when the pathogen appears, to counter pathogen adaptation

-- Delaying expression of transgenes when the new protein interferes with early plant growth.

2. Codon usage

3. SelectionAntibiotic/herbicide (negative) – Positive selection (nutrientAntibiotic/herbicide (negative) – Positive selection (nutrient

deficiency)

4. ReporterGUS ( l id ) GFP ( fl i )GUS (β-glucuronidase), GFP (green fluorescent protein),

LUX (luciferase)

Method of Delivery of Method of Delivery of DNA Into a Plant CellDNA Into a Plant CellDNA Into a Plant CellDNA Into a Plant Cell

1 Agrobacterium -mediated transformation1. Agrobacterium -mediated transformationAgrobacterium tumefaciensAgrobacterium rhizogenes (Hairy Root)g z g ( y )

- cultured cells, wounded explants, vacuum infiltration

2. Direct methods

-protoplast polyethyleneglycol (PEG) method-protoplast electroporation-silicon carbide fibers-silicon carbide fibers-protoplast microinjection-particle bombardment

C G ll i l t di f ll d ib d i l t i t th tThe beginning

Crown Gall is a plant disease formally described in late nineteenth century

In 1902 the USDA reported crown galls as one of the most serious enemiesof fruit growersg

1907 Agrobacterium tumefaciens is determined as the causal agent ofcrown gall (Smith and Towsend, USDA report). Although there is previousreport in Italian by Cavara in 1897

Crown gall resulting when b t i l DNA i t llbacterial DNA is naturally transformed into this Davis CA treeCA tree

Discovery of the Ti plasmid

In 1974-75 the group of Jeff Schell and Marc Van Montagu, at the State University of Ghent, made two crucial discoveries:

1) Virulent Agrobacterium strains contained a 200kb plasmidthat was absent in non-oncogenic strains.

2) Using a plasmid-free, non-oncogenic recipient strain andthe “Kerr cross” conjugation system, they demonstrated thatacquisition of tumor-inducing ability was due to the transferq g yof the 200 kb plasmid.They named this plasmid Tumor-inducing plasmid (Ti).

Similar results were independently produced by the group of Milton Gordon, Mary Dell Chilton and Eugene Nester at Washington University in Seattle

Could the Ti plasmid be used for plant genetic engineering?

In the late 1970s Agrobacterium started emerge as apotential system for introducing foreign DNA into plant cells,however several questions remained unanswered.

Did plant cells containing T-DNA or other foreign sequencesmaintain totipotency?

Was there a mechanisms whereby plants could identify andeliminate foreign DNA during meiosis?

Could the presence or expression of foreign DNA disruptgametogenesis or fertilization?

The first disarmed Ti plasmid derived vector is produced, 1983

P. Zambryski, et al. EMBO J 1983

Foreign genes can be expressed in plant cells

Attempts to detect expression of bacterial transposon borngenes in plants failed.genes in plants failed.

In 1983, three papers reported the successful expressionf b t i l i l t llof bacterial genes in plant cells

Herrera-Estrella et al. Nature 303, 209-213; May 1983Bevan at al Nature 304, 184-187; July 1983Fraley et al PNAS 80: 4803-48-07; August 1983(ups, Monsanto enter the scene)( p , )

NATURE Vol 303 19 May 1983 pp 209 213NATURE, Vol. 303 19 May 1983. pp 209‐213Luis Herrera‐Estrella, et al

The first genetically engineered plantsg y g p

In 1984 the first fully fertile transgenic plants thatIn 1984 the first fully fertile transgenic plants thatfunctionally expressed bacterial genes were reported.The kanamycin resistance gene used for these studiesy gwas shown to be transferred as a simple mendelianfactor to the progeny most of the regenerated transgenicplants.

Plant genetic engineering was born

De Block, Herrera-Estrella, Zambrisky et al. EMBO 1984Horsch, Rogers et al. Science 1984

Selectable markerBinary vectors facilitated gene transfer

for plants

Cloning sites

Virulence25bp

g

25bp

T-DNA-lessTi plasmid

VirulenceRegion T-DNA

Ori

O f l f l A d E lOrigin of replication functional in Agro and E.coli

Certain compounds such as Acetosyringon which are released bywhich are released by wounded ]act as a recognition for the AgrobacteriumAgrobacterium tumefaciens in order to link to wounded

llcells.

Insertion of DNA into cells via biolistics (“gene gun”)

Why Transform?Why Transform?

A. Testing function of genes or parts of genes

- DNA complementation of mutants- protein dissection- determining place and timing of gene expression

reporter genes:GUS (β-glucuronidase),GFP (green fluorescent protein), LUX (luciferase),CAT (chloramphenicol acetyltransferase),Barnase (RNAase- kills cells expressing it)( p g )

Uses of transformationUses of transformation

A. Testing function of genes or parts of genes

Hairy roots (Agrobacterium rhizogenes),- The production of bioactive compoundsp p- Express root-specific pathways and have shown

stable production of alkaloids, polyacetylenes, sesquiterpenes, naphtoquinones, and other natural products

- For the over-production of secondarymetabolites and biotransformation of chemicals

- Hairy roots usually store secondary metabolites in vacuoles inside the cells.

Why Transform?Why Transform?

B. Modifying Expression of Endogenous GenesIncreasing E pression- Increasing Expression

Transgenomic switch on of silenced genes- Turning genes off

-antisense, RNAi, ribozymes-insertional mutagenesis using T-DNA

-e.g. tomato polygalacturonaseg p yg

Why transform?Why transform?

C. Moving genes from other organismsg g g

Single gene novel Expressed TraitBt toxin for insect resistance-Bt toxin for insect resistance

Single gene modification of existing trait- Phytate- male sterility

Modified Metabolic PathwaysModified Metabolic PathwaysSingle gene

- herbicide resistance -glyphosate M lti lMultiple genes

- Beta Carotene rice- Lipid metabolic pathwaysp p y- Nitrogen assimilation


B. Cloned DNA to be introducedB. Cloned DNA to be introduced

1. promoter (constituitive or inducible),p ( ),coding region, polyadenylation site

2 l bl k2. selectable marker gene- kanamycin or G148 resistance: neomycin,

phosphotransferase (NPTII),- hygromycin B:hygromycin phosphotransferase

(HygB) gentamicin: gentamicin acetyltransferase- streptomycin: streptomycin phosphotransferasestreptomycin: streptomycin phosphotransferase- Bialophos: BAR

Method of Delivery of Method of Delivery of DNA Into a Plant CellDNA Into a Plant Cell

1. PromoterPromoter (constitutive or inducible), coding region, a terminating signal, ( ), g g , g g ,

polyadenylation siteTransgenes may be introduced into plants tissues on separate plasmids or

cointegrative vectors. In the latter case multiple genes including thecointegrative vectors. In the latter case multiple genes including the selectable or screenable markers are present on the same plasmid.

Commonly used promotersConstitutive promoterConstitutive promoter

- CaMV 35S : suitable for expression of foreign genes in dicots:-The maize ubiquitin promoter, also a constitutive promoter whichdrives strong expression of transgenes in monocots.

Organ/ tissue specific promotersVicilin and phytohemaglutinin glutenin promoters seed specific- Vicilin and phytohemaglutinin, glutenin promoters seed specific

expression,- α-amylase promoter for expression in the aleurone of cereal grains;grains;- Patatin promoter for tuber specific expression in potatoes and the -- RuBisCo promoter for green tissue specificity


PromotersI d ibl d T i t E iInducible and Transient Expression

Some transgenes are known to impact plant growth or yield,i i d h l h d dso it is prudent to turn them on only when needed.

- Inducing male sterility for hybrid seed productionwithout the need for a restorer line,,

- Turning on disease resistance genes only when the pathogen appears, to counter pathogen adaptation,

Delaying expression of transgenes when the new protein- Delaying expression of transgenes when the new protein interferes with early plant growth.

iInducible: alcohol dehydrogenase, EthanolTransient: Tobacco Mosaic Virus (TMV) (LSB Geneware now owned by

Kentucky )

Uses of transformationUses of transformationUses of transformationUses of transformation

D. Concerns in use of transgenic plants

- Chloroplast transformation- Selectable marker- Site Specific Integration- Transgenomics


2. Selectable marker gene1. Removable selectable marker gene

- genes using the Cre-lox system or transposable elements

Site-specific recombination systems have been demonstrated to be valuable tools for marker gene removal to delete genomic DNA regulate genetools for marker gene removal , to delete genomic DNA, regulate gene expression, recombine chromosomes, and target new DNA into designated transgenic loci.


2. Selectable marker gene

1. Removable selectable marker gene- genes using the Cre-lox system or transposable- genes using the Cre-lox system or transposable

elements

2 P iti l ti2. Positive selection.- BOGUS - exclusive energy source - cellobiuronic acid, a

disaccharide when transported into the cell, is metabolized to glucose by beta-glucuronidase, the technique produces healthier, more genetically stable plants and eliminates concern about

d i l t ith tibi ti i tproducing plants with antibiotic resistance.- PMI (phosphomannose isomerase) Plant cells

without this enzyme are unable to survive in a tissue i i i 6culture medium containing mannose-6-phosphate as

a sole carbon source.

DNA TRANSFER The explants are placedAfter 6 weeks the

DNA TRANSFER

The explants are co-cultivated with the

The explants are placed on a medium containing antibiotics for selection and control of the

cotyledon explants are discarded.Shoots are beginning to cultivated with the

agrobacterium for 48 hours to allow time for the agrobacterium to transfer

and control of the Agrobacterium. The medium also contains the growth regulator zeatin

g gform and are transferred to fresh MSZ media.The shoots haveagrobacterium to transfer

the engineered DNA to the wounded plant cells.

growth regulator, zeatin riboside.Here the formation of an

The shoots have regenerated from the calli and are ready to betransferred to rootinginitial callus can be seen

on the explant. Time point: 3 weeks

transferred to rooting media. Time point: 9 weeks

These are fully differentiated transgenic y gtomato plants rooting in MS media with kanamycin. Time point: 11 weeksRooted plantlets are then transferred toRooted plantlets are then transferred to boxes containing soil. The plants must be acclimated to the air, or "hardened off". The process takes 4 5 days Time point: 13 weeksprocess takes 4-5 days. Time point: 13 weeks

TO THE GREENHOUSE

These transgenic plants are ready for transfer to the greenhouse. Shown are two cultivars of tomato; Moneymaker (left), and Motelle (right). Both contain engineered genes.contain engineered genes.

Time point: 15 weeks

A GREENHOUSE OF TRANSGENIC PLANTS.

Plants and growth conditions are carefully monitored and controlled.The plants are used for analysis seed production or are transplantedThe plants are used for analysis, seed production, or are transplantedto the field.

α Amylase promoter which is

Reporter gene under tissue specific promoterα-Amylase promoter which is endosperm specific controllinga bacterial reporter gene, gusA, i i i Th blin transgenic rice. The blue stain, is expressed to peak levels at day 6 in the germinating transgenic seed.

Luciferase (firefly) in arabadopsis

Green Fluorescence ProteinProtein (jellyfish)Tobacco

Production of transgenic red clover plants (Trifolium pratense) for alfalfa mosaic virus (AMV), white clover mosaic virus (WCMV) virus resistance Agrobacterium-mediated transformation using cotyledonary explants and chimeric nptII gene as g y y p p gselectable marker. T-DNA of binary vector containing the chimeric nptII gene and the WCMV coat protein gene (WCMV4). (Spangenberg et al 2001)


Transient ExpressionThe basic principle of GENEWARE® is use of a safe vector modified p p

from the TMV to place any gene (or a large number of genes) within a tobacco plant. The target protein is then efficiently collected and purified in KBP’s pilot and commercial bioprocessing facilities. Kilogram amounts of a wide array of complex proteins can be produced rapidly with low capital investment relative to conventional biotechnology manufacturing operations.

http://www.kbpllc.com/GENEWARE/tabid/86/Default.aspx

Cloning using insertional Cloning using insertional mutagenesismutagenesis

- The concept of gene identification and cloning using insertional mutagenesis is well established. Many genes have been isolated using T-DNA transformation or transposable elements. Maize transposable elements p phave been introduced into heterologous plant species for tagging experiments. \Site-specific recombination systems from lower organisms have also been shown to function efficiently in plant cells. Combining transposon and site-specific recombination systems in plants would create the possibility to induce chromosomal deletions. This 'transposition-deletion' system could allow the screening of large segments of the genome for interesting genes and may also permit the cloning of the DNA corresponding to the deleted material by the same site-specific recombination reaction in vitro. This methodology may

id i t t t lib i f l DNA lprovide a unique means to construct libraries of large DNA clones derived from defined parts of the genome, the phenotypic contribution of which is displayed by the mutant carrying the deletion.

• A major challenge in genetically engineering small grain cereals, is maintaining expression of the genes of interest over time Grains aremaintaining expression of the genes of interest over time. Grains are surprisingly adept at stifling the implanted gene's activity, a process known as gene silencing. Lemaux and her colleagues devised a way to surmount this h dl b i i t hit hhik bil i f DNA khurdle, by engineering genes to hitchhike on mobile pieces of DNA, known as transposons aided by the enzyme, transposase. By removing transposase during genetic segregation, the transposed gene can be stabilized in a location that is devoid of selectable marker genes and plasmid backbone DNA. Expression of the transposed gene is more stable with only ~10% of events becoming silenced after four generations, rather than the 80-90% that can be seen in lines developed by other methods.Counteracting the effects of apoptosis following Agrobacterium infection by heat treatment,increased sorghum transformation frequency to nearly 8% (Gurel et al., 2008).

• Lemaux introduced into barley the maize transposable element, Ds, and the transposase gene. When individual plants were crossed, Ds was activated and transposed to new locations in the genome with preference to insert into genic regions – an advantageous trait for large genome species. Using the Ds sequence as a tag, the sequence can be used as a priming site to identify the gene into which Ds transposed and to map its location (Cooper et al., 2004; Si h t l 2006) B id tif i D l t th t l t h tSingh et al., 2006). By identifying Ds elements that map close to phenotypes or genes of interest the element can be reactivated and Ds generally will transpose to nearby locations. One gene tagged by this approach was a wall-associated kinase gene or WAK shown to be a 125 member family in riceassociated kinase gene or WAK, shown to be a 125-member family in rice (Zhang et al., 2005) that, based on studies in Arabidopsis, is involved in biotic and abiotic stress tolerance (with Z-H He, San Francisco State).

Cloning using transposon tagging• The molecular isolation of transposable elements now permits the• The molecular isolation of transposable elements now permits the

cloning of genes in which the element resides. The major advantage of this system is that genes whose function is not known can be cloned The first step in this procedure is to identify a plant stock thatcloned. The first step in this procedure is to identify a plant stock that is mutant for a specific trait because a transposable element has been inserted into and inactivated the gene. Next, a genomic library (often in bacteriophage lambda) of the plant stock is created. This library isin bacteriophage lambda) of the plant stock is created. This library is then screened with a clone for the transposable element. Any clone that is selected from the screening will contain the element. In the clone, sequences for the mutated gene will lie adjacent to the , q g jelement. A sublcone containing sequences from the gene is then developed from the non-transposable element DNA of the original clone. This clone is then used to screen a genomic library containing g y gDNA from a normal plant. In this manner, any clone that is selected should contain a full, normal copy of the gene.

• It has been demonstrated that these elements can be induced to move from one location to another in the new species. If this movement is coupled with the appearance of mutant phenotype, then the gene responsible for the phenotype can cloned in that particular species. These techniques have now allowed the use of transposon tagging in plant species in which active transposable elements have not been identified.

TILLINGTILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that identifies single-gbase-pair allelic variation in a target genetarget gene. Facilitates high-throughput and independent ofindependent of genome size, reproductive system or generation time.

Bacillus thuringiensis (Bt) produce insecticidal Cry and Cyt proteins. The 3d-CryBacillus thuringiensis (Bt) produce insecticidal Cry and Cyt proteins. The 3d Cry toxins are pore-forming toxins that induce cell death by forming ionic pores into the membrane of the midgut epithelial cells. Ingestion of the protoxin, activation by midgut proteases to produce the toxin Cry1A fragment and the interaction with the g p p y gprimary Cadherin receptor promotes an extra proteolytic cleavage, where helix α-1 of domain I is eliminated and the toxin oligomerization is induced, forming a structure of 250 kDa. The oligomeric structure binds to a secondary receptor, aminopeptidase N or alkaline phosphatase. The secondary receptor drives the toxin into detergent resistant membrane microdomains forming pores that cause osmotic shock, burst of the midgut cells and insect death.

Phosphoenolpyruvate (PEP) carboxylase (photosynthetic gene)

Roundup functions by inhibiting activity of the enzyme 5-enolpyruvylshiki-mate-3-phosphate synthase (EPSPS). The EPSPS enzyme is critical aromatic amino acids phenylalanine, tyrosine, and tryptophan. All plants synthesize the amino acids needed for plant growth and function. A blocked pathway results death.

An alternate form of the EPSPS enzyme, called CP4-EPSPS, that is not inhibited by glyphosate A single point mutation in the gene switched the nucleotideby glyphosate,. A single point mutation in the gene switched the nucleotide guanine for cytosine, which causes the amino acid alanine to be substituted for glycine and prevents glyphosate from binding the enzyme, allowing the Shikimate pathway to function normally. The gene (plus promoter) was inserted into soybean to create the Roundup Ready soybean. Since the soybean contains the CP4-EPSPS enzyme, which is not inhibited by glyphosate, the modified "Roundup Ready" soybean will not be killed by Roundup.

(bacteria)

(maize)

(bacteria)

(daffodil)(daffodil)

T-DNA in pSYN12424 - Golden Rice 2. selectable marker cassette maize polyubiquitin (Ubi1)maize polyubiquitin (Ubi1) promoter with intron, phosphomannose isomerase (pmi)

Combinatorial direct DNA transformation

Multi vitamin Corn

• Combinatorial direct DNA transformationCombinatorial direct DNA transformation rapid production of multi-complex metabolic pathwaysp y

• Christou ransferred 5 constructs controlled by different endosperm-specific promoters into white maize. Different enzyme combinations show distinct metabolic phenotypes – resulting in

• 169X beta carotene (60 µg/g v. 14 by breeding)

• 6X vitamin C, and2X f l t (Ch i t 2009)• 2X folate (Christou, 2009)

Combinatorial direct DNA transformation

• Since no vector backbones are required, preventing the integration of potentially recombinogenic sequences, they remain stable across generations. q y gThese groups’ constructions facilitate effective manipulation of multi-gene pathways in plants in a single transformation step effectivelya single transformation step effectively recapitulating the bacterial operon model in plants.

• This system has an added advantage from a commercial perspective in that these methods i t bl ith t diti l hcircumvent problems with traditional approaches

which not only limit the amount of sequences transferred, but may disrupt native genes or lead to poor expression of the transgene, thus reducing both the numbers of transgenic plants which must be screened and the subsequent breeding and Introgression steps required to select a suitable commercial candidate.

Technology to address Concerns in Technology to address Concerns in use of transgenic plantsuse of transgenic plantsuse of transgenic plantsuse of transgenic plants

Chloroplast transformation

- For the many crops in which chloroplasts are strictly maternally inherited, which is to say not transmitted through pollen transformation of the chloroplastthrough pollen, transformation of the chloroplastgenome should provide an effective way to containforeign genes.g g

- Also greatly increases transformation efficiency andlevel of gene expression

Technology to address Concerns in Technology to address Concerns in use of transgenic plantsuse of transgenic plantsuse of transgenic plantsuse of transgenic plants

Targeted Site Specific IntegrationIn mammalian cells- been applied to inactivate or modify

specific genes.Gene targeting can be done in plants as wellGene targeting can be done in plants as well

- low efficiency transfected DNA recombines with homologous sequences.

- Use of site-specific recombination systems may have great potential for overcoming this limitation.

Th C /l t i ti f bi t iThe Cre/lox system, consisting of a recombinase protein(Cre), short recombination sites (lox)- catalyses precise and stable insertional recombination y p

between the respective DNA target sites.- development of more efficient systems, applicable to many plant species that could target a transgene to amany plant species, that could target a transgene to a single pre-selected chromosomal site, thus eliminating variation in gene expression level.

Transplastomic plant

Exogenous cloned cpDNA biolistic gene gun method. DNA-coated gold particles are bombarded onto cells.

Plastid transformation is much more complex than cell nucleus transformation because there are up to 10,000 plastid genomes in each p

Upon its DNA is released from the particles and integrated into the chloroplast genome through

p , p gcell. Transplastomic lines are genetically stable only if all plastid copies are modified in the same way, i.e. uniformly. This is achieved

homologous recombination. The most versatile chloroplast selectable marker is aminoglycoside adenyl transferase

through repeated regeneration under certain selection conditions. Plastid transformation has already been successfully performed in

(aadA), which can be expressed in the chloroplast to confer resistance to spectinomycin or streptomycin.

tobacco, potatoes, oilseed rape, rice, Arabidopsis thaliana maize

Electron micrographs showed the presence of the insecticidalpresence of the insecticidal protein folded into cuboidal crystals in chloroplasts of transplastomic cabbage (A~E),

Appearances of nontransformed (A D) and T1 transplastomic bt cabbage (B E) after

p g ( ),but not in the nontransformed cabbage

Transplastomic bt cabbage

Appearances of nontransformed (A, D) and T1 transplastomic bt cabbage (B, E), after feeding with 20 Spodoptera litura for 7 days (A, B) or after feeding with 40 Plutella xylostellafor for 7 days (D, E). Appearances of Spodoptera litura (C) and Plutella xylostellafor (F) after feeding with nontansformed (C1 F4) or transplastomic bt cabbagexylostellafor (F) after feeding with nontansformed (C1, F4), or transplastomic bt cabbage leaves (C2, C3, F5).Overexpression of bt crystal proteins in the chloroplast of bt transformed cabbage was demonstrated by analytical electron microscope (AEM). Electron micrographs showed thedemonstrated by analytical electron microscope (AEM). Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals in chloroplasts of transplastomic cabbage (A~E), but not in the nontransformed cabbage

Where we have been and where we are going ?

Systems-Biology

Genomics

FunctionalGenomics

y gy

GeneKnowledge

DNAGenetics

FunctionalGenetics

1940 1970 1990 2000 2009

Time

A First Introduction to NCBINCBIDavid R. Nelsonrev. Jan. 10, 2008

Typed in 10- font, one human sequence would stretch more than 5,000 miles. Digitally formatted, can be stored on one CD-ROM. Biologically encoded, it fits within a single cell.

• Genomics: the analysis of genomes. A genome can be thought of as the complete set of DNA sequences that codes for the hereditary material that is p q ypassed on from generation to generation. These DNA sequences include all of the genes (the functional and physical unit of heredity)

• Comparative genomics Info from one organism can have application in another• Transcriptomics: analysis of all transcripts (expressed genes changes based on

external stimuli – stress- diet – aging = Functional genomics!)• Proteomics: Proteome is the set of all expressed proteins for a given organism.• Metabolomics: analysis of all metabolites produced by organism–can be used as

biological markers• Systems biology, involves the integration of all the “omics” information to

h l i f i B d di h l “create a whole system view of an entity. By understanding the complete “parts lists” in a genome, we gain a better understanding of complex biological systems and how they interact to make a functioning organism.

Bioinformatics involves the integration of computers softwareBioinformatics involves the integration of computers, software tools, and databases in an effort to address complex biological systems. Bioinformatics used for BIG BIG DATA: G i d t iGenomics and proteomics.

Modern Biology / Genomics -A new Research-Paradigm in modern BiologyA new Research-Paradigm in modern Biology

PhenotypeAppearance and

Genotype / Genes / DNA Inherited InformationAppearance and

Traitsof an Organism

Inherited InformationDefining an Organism

Improved Crop Plants

Genomics= the Totality of the InformationCrop Plants yof all Genes and their Functions

Gene expression profile for the entire genome of the baker's yeast from 2 cellbaker s yeast from 2 cell type.cDNA expressed in cell type #1 green signal, cDNA in g g ,cell type #2 is a red signal. Thus, genes induced in cell type #1 or #2 appear as Microarrays can be used to study how crops respond to green or red.Genes expressed at equal levels in both cells appear

Microarrays can be used to study how crops respond to infection by pathogens or adapts to stress [salt, drought, temperature]. Integrated understanding of coordinated multiple gene expression unlocks complex traits and allows as yellow spots.multiple gene expression unlocks complex traits and allows engineering resistance to pathogens and natural stress. Same approach can be used to optimize multi-trait nutritional value

Genomics Tools• Sequence desired crop cowpea, banana, sorghum, wheat, • Genotype selected largest possible number of genes for traits of economic

importance - disease and pest resistance, quality traits. Selected after consultation with resource persons, especially breeders .p , p y

• Development of PCR-based markers (e.g., microsatellites) for marker-assisted selection: Resistance to Striga, root knot nematodes, aphids, maruca podborer,maruca podborer,

• Transgenes for resistance to viruses, parasitic weeds• Introgress into locally adapted speciesG G G G i A t itGenome Gene map Gene sequence Gene expression Ag traits

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From Genomics to Improved CropsFrom Genomics to Improved CropsThe 2 Phases of BiologyThe 2 Phases of BiologyThe 2 Phases of BiologyThe 2 Phases of Biology

Reverse Genetics Phase 2Gene RNA Proteins Metabolites Organism

Forward Genetics

Molecular BreedingDNA SequenceMap Transcriptome New Plant Traits

ProteomeMetabolome Transgenics

Profiling

Phase 1

GeneFunction Factory™

Structural genomics includes the genetic mapping, g g pp g,physical mapping and sequencing of entire genomes.

How to do Structural genomicsphysical mapping andphysical mapping and sequencing of entire genomes.

The diagram outlines a strategy for determining the sequence of

i l BAC YAC Sha single BAC or YAC. Shotgun libraries are constructed in M13 and plasmid vectors using hydrodynamically sheared BAC or YAC DNA. Subclones are sequenced randomly to q ygenerate an average coverage greater than 5-fold. The subclone sequences aresubclone sequences are assembled into contigs using phrap software. The gaps between contigs are closed bybetween contigs are closed by "primer walking" on plasmids or BACs.

This figure shows the BACs that were sequenced for Chromosome 1 ofChromosome 1 of Arabidopsis. The seventy seven seed BACs were selected by hybridization to characterized markers of h i ichromosome 1. Minimum overlapping clones were chosen by BAC‐endchosen by BAC‐end sequencing and fingerprinting. TIGR g p gcollaborated with the SPPC on sequencing chromosome 1 in 19991999.

Distances for markers displayed at the top of the map are always given relative to the nearest "framework" marker. In the example above, the marker O5629 is 3.0cM from g3715, PHB2 is 4.7cM from g3715 and PDC1 is 7.2cM from g3715 and is the top marker for this hchromosome

The figure summarizes the BAC contigs built by Marco Marra and Muno Sekhon of Washington University Following initial assembly of the BACs with the FPC software atWashington University. Following initial assembly of the BACs with the FPC software at high stringency, manual editing was performed to join contigs. The long bars represent each of the five A. thaliana chromosomes; numbered colored blocks each represent a different BAC contig

Arabadopsis Genome

Solexa:Ill i iIllumina sequencing technology

Solexa: Illumina sequencing technology

This platform is based on massively parallel sequencing of millions of fragments using Illumina reversible terminator-based sequencing chemistry. This novel sequencing y q gtechnology, together with the Illumina Genome Analyzer, offers a highly robust, accurate, and scalablehighly robust, accurate, and scalable system that sets a new standard for productivity, cost-effectiveness, and accuracy among next generationaccuracy among next-generation sequencing technologies.

Marker-Assisted Selection [MAS]- QTLs

Most traits are not controlled by a single gene, but rather by a set of genes acting in concert. Traits that are controlled by multiple genes, such as size, tend to have a more subtle, constant variation

Most traits are not controlled by a single gene, but rather by a set of genes acting in concert. Traits that are controlled by multiple genes, such as size, tend to have a more subtle, constant variation in the result phenotype, leading to a continuous distribution of phenotypic values. These are also known as "quantitative" traits. Traits controlled by one or a very few genes, such as certain di d h d i di h i

in the result phenotype, leading to a continuous distribution of phenotypic values. These are also known as "quantitative" traits. Traits controlled by one or a very few genes, such as certain di d h d i di h idiseases, tend to have more drastic, discrete changes in expression, and these are termed "qualitative" traits.

Single gene traits- many genes mapped to relatively precise locations

diseases, tend to have more drastic, discrete changes in expression, and these are termed "qualitative" traits.

Single gene traits- many genes mapped to relatively precise locationsSingle gene traits many genes mapped to relatively precise locations by observing the segregation of the trait in a mapping population.

Quantitative traits - too difficult to distinguish changes in phenotype, so special QTL mapping studies must be done

Single gene traits many genes mapped to relatively precise locations by observing the segregation of the trait in a mapping population.

Quantitative traits - too difficult to distinguish changes in phenotype, so special QTL mapping studies must be doneso special QTL-mapping studies must be done.

Set of intervals, not points, scattered over the genome, in which a gene or genes affecting the trait is thought to lie.

so special QTL-mapping studies must be done.Set of intervals, not points, scattered over the genome, in which a

gene or genes affecting the trait is thought to lie.Depending on how the study was done, these intervals may be

centered over a single marker, or lie in between two markers.h l f h d i li f k h i l ll l

Depending on how the study was done, these intervals may be centered over a single marker, or lie in between two markers.

h l f h d i li f k h i l ll lThe result of the study is a list of markers whose particular alleles tend to be associated with certain changes in the traits being evaluated.

The result of the study is a list of markers whose particular alleles tend to be associated with certain changes in the traits being evaluated.

MAS involves scoring indirectly for the presence or absence of aMAS involves scoring indirectly for the presence or absence of a

Marker-Assisted Selection [MAS]- QTLs

MAS involves scoring indirectly for the presence or absence of a desired phenotype or phenotypic component based on the sequences or banding patterns of molecular markers located in or near the genes controlling the phenotype

MAS involves scoring indirectly for the presence or absence of a desired phenotype or phenotypic component based on the sequences or banding patterns of molecular markers located in or near the genes controlling the phenotypenear the genes controlling the phenotype.

The sequence polymorphism is indicative of the presence or absence of a specific gene or segment that is known to carry a desired allele.

DNA markers can increase screening efficiency – they provide:

near the genes controlling the phenotype.The sequence polymorphism is indicative of the presence or absence of

a specific gene or segment that is known to carry a desired allele. DNA markers can increase screening efficiency – they provide:DNA markers can increase screening efficiency they provide:• the ability to screen in the juvenile stage for traits that are expressed

late in the life of the organism (i.e. grain or fruit quality, male sterility, photoperiod sensitivity);

DNA markers can increase screening efficiency they provide:• the ability to screen in the juvenile stage for traits that are expressed

late in the life of the organism (i.e. grain or fruit quality, male sterility, photoperiod sensitivity);y, p p y);

• the ability to screen for traits that are extremely difficult, expensive or time consuming to score phenotypically (i.e. quantitatively inherited or environmentally sensitive traits such as root

y, p p y);• the ability to screen for traits that are extremely difficult, expensive

or time consuming to score phenotypically (i.e. quantitatively inherited or environmentally sensitive traits such as root ymorphology, resistance to quarantined pests or to specific races or biotypes of diseases or insects, tolerance to certain abiotic stresses such as drought, salt and mineral deficiencies or toxicities);

ymorphology, resistance to quarantined pests or to specific races or biotypes of diseases or insects, tolerance to certain abiotic stresses such as drought, salt and mineral deficiencies or toxicities);

• the ability to distinguish the homozygous from the heterozygous condition of many loci in a single generation without the need for progeny testing (as molecular markers are co-dominant);

h bili f i l MAS f l h

• the ability to distinguish the homozygous from the heterozygous condition of many loci in a single generation without the need for progeny testing (as molecular markers are co-dominant);

h bili f i l MAS f l h• the ability to perform simultaneous MAS for several characters at one time (or to combine MAS with phenotypic or biochemical evaluation).

• the ability to perform simultaneous MAS for several characters at one time (or to combine MAS with phenotypic or biochemical evaluation).

Circle of genes Syntenic relationships

The genomes of several grasses arranged so that regions carrying similar genes are aligned

Marker-Assisted Selection [MAS]- Techniques

Most genetic maps consist of anonymous molecular markers and not genes of known function.

Most genetic maps consist of anonymous molecular markers and not genes of known function.

Molecular markers can be generated by a variety of different techniques, each with their advantages and disadvantages, and different sorts of information will

Molecular markers can be generated by a variety of different techniques, each with their advantages and disadvantages, and different sorts of information will g ,accompany the different marker types.

These markers may pinpoint genes, (for example cDNA, RFLP or EST markers) or proteins (isozyme markers)

g ,accompany the different marker types.

These markers may pinpoint genes, (for example cDNA, RFLP or EST markers) or proteins (isozyme markers)RFLP or EST markers), or proteins, (isozyme markers), or they may mark genomic DNA (for example genomic RFLP, RAPD or microsatellite markers).

RFLP or EST markers), or proteins, (isozyme markers), or they may mark genomic DNA (for example genomic RFLP, RAPD or microsatellite markers).

They may be gel-based, or PCR-based; they may detect a single locus or multiple loci; they may or may not require sequence information; finally, they may vary in their d f li bili diffi l d d h

They may be gel-based, or PCR-based; they may detect a single locus or multiple loci; they may or may not require sequence information; finally, they may vary in their d f li bili diffi l d d hdegree of reliability, difficulty and expense, and the nature of the polymorphism they detect.

A user needs to be somewhat familiar with a particular

degree of reliability, difficulty and expense, and the nature of the polymorphism they detect.

A user needs to be somewhat familiar with a particular ptechnology to assess whether a database is providing adequate and useful information.

ptechnology to assess whether a database is providing adequate and useful information.

TGT AAT AGT TAT ATT TTCATT ATA AAT TGT GTT TGT AGA CAT CAT AAA TTT AAAACA TGG CTT TTT AAC CTGATA AAT CCT ACG AAT ATTTGT AAT AGT TAT GTT ATTGCA GTA AGT ACC GTT TGTGCA GTA AGT ACC GTT TGTATT ATA AAT TGT GTT CTG

TGT AAT AGT TAT ATT TTCATT ATA AAT TGT GTT TGT AGA CAT CAT AAA TTT AAAACA TGG CTT TTT AAC CTGATA AAT CCT ACG AAT ATTATA AAT CCT ACG AAT ATTTGT AAT AGT TAT GTT ATTGCA GTA AGT ACC GTT TGTATT ATA AAT TGT GTT CTG Which genes are turned off then on ? Courtesy of Dr. Young Moo Lee

Protein Function in the Post-genomic Era

a, Network of protein interactions and predicted functional links involving silencing informationsilencing information regulator (SIR) proteins. b, Network of predicted functional linkages involving the yeast prion protein Sup35.

DAVID EISENBERG, et al. Nature 405, 823 - 826

How proteins group together in a yeast cell. Unraveling proteincollaborations could change the way

h i l d f d (2000) http://genome-www.stanford.eduSaccharomyces).

pathways are manipulated for drug targets, improvement of valuable traits in plants and animals.

Gavin, A.C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature, 415, 141 - 147, (2002).

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