Cardineau guy pep talk 11, jan 12,2012

Guy A. Cardineau, Ph.D.

Higher Accumulation of

F1-V Recombinant Fusion Protein in Plants After

Induction of Protein Body Formation

Director, Centro de AgrobiotecnologíaDepartamento Agrobiotecnología y AgronegociosTecnológico de Monterrey, Campus Monterrey

ASASU Centennial Professor, Emeritus

Research Professor, Emeritus & Faculty Fellow

Center for Infectious Disease and Vaccinology

The Biodesign Institute,

The School of Life Sciences and

The Sandra Day O’Connor College of Law

Arizona State University

Biotechnology Drug Approvals 1982-2008While the number of approved biotech-based products approved per year is variable, the trend is upward. Biotechnology drugs appear the fastest-growing sector for drug development, and it is predicted that biotech drugs will comprise over 50% of all drug approvals by 2015 and more than 75% by 2025. These predictions are supported by the expected benefits of increased understanding of drug targets and the molecular and genetic bases of disease, as well as the declining conventional small-molecule drug pipelines in most major pharma companies. BioWorld Today Sept 1,2009

The table to the left represents information from an article published in BioWorld Today in late August 2009, written by Michael Harris,

2

late August 2009, written by Michael Harris, Executive Editor, about the top 25 biotech drugs currently on the market. The data provided includes revenues for each of these biotech drugs in 2008 (>$70B US), the date each drug product was first approved by the FDA and when patents protecting each drug are due to expire. It should be kept in mind that one feature of all these drugs is that they have been approved for more than one ndication; Harris reports that Genentech's Avastin is being tested in more than 450 clinical trials for treating more than 30 different types of cancer. It should also be kept in mind that 7 of the 25 "biotech" drugs are small molecules, and another 6 are antibodies.

Historically, Plants Have Been Routinely Used to Produce Pharmaceuticals, Naturally

� Global over-the-counter sales of plant-derived drugs are estimated at $40 billion per yearWell established regulatory systems are in place for these products

� Estimated one-quarter of the prescription drugs sold in the US, Canada and Europe contain active ingredients derived from plants

� Tens of thousands of plants are used for medicinal purposes

� Well established regulatory systems are in place for these products

Drug/Chemical Action/Clinical Use Plant Source Cocaine Local anaesthetic Erythroxylum coca Codeine Analgesic Papaver somniferum Digitalin, Digitoxin Cardiotonic Digitalis purpurea Quinine Antimalarial Cinchona ledgeriana Taxol Antitumor agent Taxus brevifolia Vinblastine, Vincristine Antitumor, Antileukemic Catharanthus roseus

SUMMARY from Large Scale Biology, Inc.

• Hormones and immune modulators

• Monoclonal antibodies - IgG

• Subunit vaccines

• Enzymes

Classes of New Protein Drug ProductsClasses of New Protein Drug Products

Production Systems in UseProduction Systems in UseProduction Systems in UseProduction Systems in Use• Bacterial fermentation

• Mammalian cells in fermentation

• Yeast

• Insect cells (GSK’s cervical cancer vaccine; 2005/6)

• Green plants – Stable and Transient Transformation,

Whole Plants and Plant Cells

One approved product in the market in plant cells

• Bacterial fermentation

• Mammalian cells in fermentation

• Yeast

• Insect cells (GSK’s cervical cancer vaccine; 2005/6)

• Green plants – Stable and Transient Transformation,

Whole Plants and Plant Cells

One approved product in the market in plant cells

Early Patent Filings on

Plant Made

Pharmaceuticals

5

Original Concepts of Therapeutic Protein,Vaccine Antigen, and Antibody Expression in Plants

Dow AgroSciences/ASU collaboration

developed a Newcastles Disease Virus

subunit vaccine in tobacco NT1 cells.

United States Patent 7,132,291, Cardineau, et al., November 7, 2006 (Canadian counterpart CA 2524293)

Vectors and cells for preparing immunoprotective compositions derived from transgenic plants AbstractThe invention is drawn towards vectors and methods useful for preparing genetically transformed plant cells that express

immunogens from pathogenic organisms which are used to produce immunoprotective particles useful in vaccine preparations. The

invention includes plant optimized genes that encode the HN protein of Newcastle Disease Virus. The invention also relates to

methods of producing an antigen in a transgenic plant.

WHY ORALLY DELIVERED PLANT-MADE VACCINES?

� Plant-derived vaccines are cost-effective andstable at room temperature.

� Plants provide both an encapsulated antigenand an oral delivery system that stimulatesthe mucosal immune system resulting in bothsecretory and circulating antibodies.

� The mucosal immune system is the first lineof defense against most pathogens.

� Oral vaccines are potentially safer, require noneedles and may not require trained medicalpersonnel to administer.

� Several Phase I Human Clinical Trials with plant-made vaccines have been run resulting in positive immune responses.

WHY INCREASE F1-V FUSION PROTEIN ACCUMULATION IN PLANTS?

�Our primary objective is to produce plant-derived heat stable vaccines that can be delivered orally.

�We have been using F1-V, a fusion between two antigens from the plague bacterium Yersinia pestis, as our model antigen in production improvement studies.pestis, as our model antigen in production improvement studies.

�We are assessing parameters that affect expression of F1-V fusion protein in plants and plant cells to be used as both a production and delivery system of vaccines and potentially other biopharma proteins.

�High antigen accumulation is required to compensate for partial proteolysis in the gut upon oral delivery.

Protein accumulation in plant tissues reflects a

balance between protein synthesis and degradation

• To date, most efforts have focused on increasing protein

synthesis.

– enhanced transgene expression can be obtained by optimizing

regulatory elements including stronger promoters, transcriptional enhancers, translational enhancers, alternative polyadenylation signals,

using synthetic genes with codons that have been optimized for gene

expression in target plants, overcoming RNAi and silencing

• Unfortunately, high transgene expression does not always

guarantee high levels of recombinant protein accumulation

since proteins may be expressed successfully but

subsequently degraded.

• It has been demonstrated that post-synthesis and/or post-

secretion instability and degradation are critical factors

contributing to low foreign protein yield.

25000

30000

35000

preboos t

pos tboos t

ANIMAL TRIALS: PRIME-BOOST STRATEGY

PRIME: s.c. 15 µg bacterially derived F1-V

BOOST: 2 g non-transgenic tomato (n = 5) on days

BOOST: 2 g F1-V transgenic tomato (n = 6) on days 21, 28, 35 (300 ug) and 42 (1200 ug)

[Ug/m

l]

250

300

350

preboost

postboost

[Ug/m

l]

0

5000

10000

15000

20000

25000

30000

35000

F1-spec if ic IgG1 V -spec if ic IgG1

preboost

postboost[Ug/m

l]

F1-specific IgG1 V-specific IgG1

0

50

100

150

200

250

300

350


preboost

postboost[Ug/m

l]


0

5000

10000

15000

20000

F1-spec if ic IgG1 V -spec if ic IgG1

Combined F1-V and V-specific IgG1 titerscorrelate with protection in mouse model(Williamson et. al., Clin. Exp.Immunol., 1999, 116; 107-114.)

tomato (n = 5) on days 21, 28, 35 and 42)


50

100

150

200

F1-specif ic IgG2 V-specif ic IgG2F1-specific IgG2a V-specific IgG2a

CHALLENGE (s.c. 20 LD50 Y. pestis)

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Days post-infection

% s

urv

ival

TG

WT

CONTROLSChallenge of the vaccinated mice

with s.c. Y. pestis

Alvarez & Cardineau Biotechnology Advances

2010, 28 (1): 184-196

% o

f su

rviv

al

Days post-infection

CHALLENGE (s.c. 20 LD50 Y. pestis)

12



• There are several possible sites and mechanisms of foreign

protein degradation in plants. Cytoplasmic proteases contribute

significantly to product losses within plant cells.

• Proteolytic degradation of foreign proteins can be minimized by

targeting synthesis to the endoplasmic reticulum (ER) rather than

the cytosol, but this doesn’t always work. the cytosol, but this doesn’t always work.

– ER retention of soluble transport-competent proteins is inducible by the

carboxy-terminal retention/retrieval signal KDEL or HDEL, which is

recognized by a receptor located in the Golgi complex.

– Upon binding, the receptor retrieves C-terminal tagged proteins back into the

ER. Localization within the ER via the addition of KDEL or HDEL increases

the accumulation of foreign proteins in transgenic plants.

– However, the ER retention via KDEL is mediated by a KDEL receptor.

When the receptor is saturated with KDEL ligands, the KDEL-tagged recombinant protein either secretes or is transported to the lytic vacuole



• Some KDEL-tagged recombinant protein can be also misfolded

and delivered for degradation through an ER-dependent

mechanism named ‘‘unfolded protein response’’ or UPR, which

functions for both endogenous or heterologous proteins

• The K/HDEL system is common to all eukaryotes, but plants can

use a different ER localization system in seeds consisting of

specialized organelles called protein bodies (PB), which stably specialized organelles called protein bodies (PB), which stably

accumulate seed storage proteins within the ER.

• The maize 27 kD γ-zein seed protein is not secreted even though

it bears an N-terminal signal sequence and lacks a canonical

KDEL/HDEL ER-retention signal; it is able to form ER-localized

PB not only in maize endosperm but also when expressed in

storage or vegetative tissues of transgenic Nicotiana tabacum,

Hordeum vulgare and Arabidopsis thaliana plants, respectively.

• PB formation can lead to higher protein accumulation in the ER

possibly because of the exclusion from the normal ER turnover

The Rules of Science

WHAT IS ZERA?

� Cereal grains have evolved to store large amounts of proteins:γ-Zein is the major storage protein in maize.

� Zera (γ-Zein ER-accumulating domain) is the N-terminalproline-rich domain of γ-zein that is sufficient to induce theassembly of protein bodies.

� Zera adopts an extended helix conformation where polar� Zera adopts an extended helix conformation where polarresidues (histidines) are located on one side of the helix andhydrophobic residues (leucines and valines) on the oppositeside of the helix.

� This conformation provides high solubility in aqueous mediaand the ability to self-assemble both in hydrophobic andhydrophilic environments.

� The Zera domain retains its ability to developprotein bodies after being fused with an exogenousprotein of interest.

� Zera contains two targeting signals:

ZERA PROTEIN BODIES

Organelles surrounded by a membrane derived from the ER. Organelles surrounded by a membrane derived from the ER.

� Zera contains two targeting signals:

1- A signal peptide that internalizes Zera fusionprotein inside the ER

2- The Zera domain itself that oligomerizes coatingthe ER membrane and inducing the protein bodyformation.

The basis of Zera® technology

Nature knows how to assemble and store proteins in seeds in Protein Bodies

Zera® is a natural peptide from a corn storage protein, γ -Zein, that has assembling properties

Zera® can be used as a tag, in fusion with the protein of interest

1. Protein bodies are obtained

directly from the biomass

Zera ®Recombinant

product

© ERA Biotech SA | January 12 18

� Effects on expression level

� Formulation / Protection / Stability

� Activity, even in fusion, even assembled

directly from the biomass

3. When needed, a cleavage

can be done by proteases or

inteins*

4. Pure protein is obtained by

classical chromatography

technique

2. Solubilisation under mild

conditions

The benefits of Zera induced protein bodies (PBs)

� Zera fusion proteins inside PBs escape the ER degradation

pathway allowing higher accumulation rates.

� The accumulation of the Zera fusion proteins in PBs also

protect the plant cell from toxic proteins.protect the plant cell from toxic proteins.

� Post-translational modifications of Zera fusion proteins inside

PBs: ER classical processing (N-glycosylation). Absence of

Golgi complex glyco-modifications.

� The easy isolation of the protein body-like organelles makes

them an extraordinary enrichment tool.

20

TRANSIENT EXPRESSION OF F1-V FUSION PROTEIN IN N. benthamiana

pCaSFV

5’ CsVMV3’ Ag7 5’ NOSLB RB

NPT2 F1-V fusion

5’ vspA SP

3’ vspB

pCaSFV

5’ CsVMV3’ Ag7 5’ NOSLB RB

NPT2 F1-V fusion

5’ vspA SP

3’ vspB5’ CsVMV3’ Ag7 5’ NOSLB RB

NPT2 F1-V fusion

5’ vspA SP

3’ vspB5’ CsVMV3’ Ag7 5’ NOSLB RB

NPT2 F1-V fusion

5’ vspA SP

3’ vspB

pCFV

RB5’ CsVMV 3’ vspB5’ NOS

LBF1-V fusion3’ Ag7 NPT2

pCFV






LBF1-V fusion3’ Ag7


LBF1-V fusion3’ Ag7 NPT210 2

35S

:Zera

-F

1-V

35S

:F1-V

CsV

MV

-F1-V

CsV

MV

-SP

-F

1-V

ng bacterialrF1-V

W.TRB5’ CsVMV 3’ vspB5’ NOS







LBF1-V fusion3’ Ag7



p35SF1V

RB5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS

LBBar F1-V fusion

TEV-5’ UTR

p35SF1V


LBBar F1-V fusion

TEV-5’ UTR

RBRB5’ CaMV35S 3’ vspB3’ Ag7 5’ NOS

LBBar F1-V fusion

TEV-5’ UTR

5’ CaMV35S 3’ vspB3’ Ag7 5’ NOSLB

Bar F1-V fusion

TEV-5’ UTRp35S:Zera-F1V

TEV-5’ UTR


LBBar F1-V fusionZera

p35S:Zera-F1V

TEV-5’ UTR



TEV-5’ UTRTEV-5’ UTR





10 2 2

V

W.T.

Zera-F1-V

(67 kDa)

F1-V(56 kDa)

Zera-F1-V

dimers

NT1 TRANSFORMATION: Zera-F1-V vs. F1-V

3-week old

Selection of the healthiest NT1 calli

3-week old calli

Liquid culture of NT1 cells

Freeze-dried NT1 cell culture.

Selection of the elite lines by Western-blot

8 weeks after transformation

200

250N

um

ber

of

calli re

co

vere

d

F1VLBA4404 / F1-V

NT1 TRANSFORMATION: ZERA-F1-V vs. F1-V

200

250N

um

ber

of

calli re

co

vere

d F1V

LBA4404/ ZeraF1V

LBA4404 / F1-V

200

250N

um

ber

of

calli re

co

vere

d F1V

LBA4404/ ZeraF1V

GV3101 /ZeraF1V

LBA4404 / F1-V

150

200

250N

um

ber

of

call

i re

co

vere

d F1V

LBA4404/ ZeraF1V

GV3101 /ZeraF1V

F1-V: 49 calli

Zera-F1-V:

2 calli

transformation

0

50

100

150

6 7 8 9 10 11 12 13 14 15

Time [weeks]

Nu

mb

er

of

calli re

co

vere

d

0

50

100

150

6 7 8 9 10 11 12 13 14 15

Time [weeks]

Nu

mb

er

of

calli re

co

vere

d

0

50

100

150

6 7 8 9 10 11 12 13 14 15

Time [weeks]

Nu

mb

er

of

calli re

co

vere

d

0

50

100

150

6 7 8 9 10 11 12 13 14 15

Time [weeks]

Nu

mb

er

of

call

i re

co

vere

d

GV3101 /ZeraF1V

EHAO105 / ZeraF1V

IMMMUNO-ELECTRON-MICROSCOPY OF ZERA-F1-V TRANSGENIC NT1 CALLI

Immunocytochemistry using anti-Zera or anti-F1-V antibody

F1-V FUSION PROTEIN

ACCUMULATION IN NT1 CALLI

0

5000

10000

15000

20000

25000

Zera-F1-V NT1 F1-V NT1

Ba

nd

in

ten

sit

y [

A.U

.]

F1-V fusion protein accumulation: >3X

higher in Zera -

F1-V than in F1-V NT1 calli

ALFALFA TRANSFORMATION: ZERA-F1-V vs. F1-V

ZeraF1-V

F1-V

1 month after transformation

ZERA-F1-V F1-V

day 0

1 month

19 elongated leaves (5% of explants)

144 elongated leaves (58% of explants)

ZeraF1-V

2 months

1 month

4-5 months

F1-V FUSION PROTEIN ACCUMULATION IN ALFALFA

F1-V fusion protein accumulation: >3X

higher in Zera-F1-V than in F1-V alfalfa.

0

5000

10000

15000

20000

25000

30000

Zera-F1-V F1-V

Ban

d in

ten

sit

y [

A.U

.]

ANALYSIS OF NT1 CALLI AND ALFALFA

BY F1-V SOUTHERN-BLOT ANALYSIS

ALFALFA

F1-V PROTEIN ACCUMULATION vs. GENE COPY NUMBER

Plant tissue

Line

Recombinant

protein

µµµµg F1-V/g TSP (*)

Gene copy #

(**)

Alfalfa leaves

A-Z51 Zera-F1-V 230 ± 20 3

A-Z35 Zera-F1-V 150 ± 10 n.d.

A-Z21 Zera-F1-V 160 ± 20 1

A-Z54 Zera-F1-V 200 ± 30 2

A-FV30 F1-V 55 ± 4 1

A-FV57 F1-V 58 ± 6 2

A-FV24 F1-V 50 ± 3 1

A-FV23 F1-V 55 ± 4 n.d.

NT1 calli N-Z1 Zera-F1-V 3800 ± 300 n.d.

N-Z5 Zera-F1-V 8500 ± 200 1

N-Z8 Zera-F1-V 6100 ± 500 3

N-Z4 Zera-F1-V 4900 ± 300 1

N-FV1 F1-V 1300 ± 100 1

N-FV4 F1-V 1700 ± 100 2

N-FV6 F1-V 200 ± 20 n.d.

N-FV28 F1-V 2000 ± 200 3

CONCLUSIONS

� The F1-V fusion protein accumulation in NT1 cells and alfalfa was at least 3X higher using Zera technology.

� The accumulation of F1-V in ER-derived PB-like structures induced by Zera was confirmed by EM.

� The regeneration of alfalfa or NT1 calli expressing Zera-F1-V was delayed compared to F1-V likely due to the PB-like formation.

� These results confirm the potential of Zera technology as a strategy to increase value-added proteins in plants.

Expression of ZERA®-GFP in N. benthamiana by agroinfiltration

P1 TnosTL enh

D35S

HcProHcPro

ZERA®-Gfp TnosTL enh

D35S

Zera®-GFP


Zera®-GFP+

HcPro

Zera®-GFP

ZERA® technology can address unmet needs for therapeutic protein

developmentA highly efficient process is used to produce proteins based on ZERA® technology:

high expression levels and simple downstream process.Insect cells+X buffer

Homogenization by sonication

Centrifugation 10000g 20’

x3

H2O wash by sonication

Centrifugation 10000g 20’

x2

Sto

rPro

reco

ve

ry


Potential for improved shelf-live under non-refrigerated conditions

Tobacco: protein extraction from fresh and dried leaves

20

Zera®EGF Zera®Ct Zera®T20

Tobacco: protein extraction from fresh and dried leaves

20

Zera®EGF Zera®Ct Zera®T20

1009994 92

87

68

26

100104

108 110

117

95

66

0

20

40

60

80

100

120

0 5 10 15 20 25

Re

ma

inin

g a

ct

(%)

Time (min)

Stability at 45ºC comm GOX

zGOX PBs

Glucoxidase (Gox) fused to Zera® and accumulated in StorPro® is

more stable at high temperature than wt Gox.

Immediatly extracted from fresh leaves

1wk 37ºC & 5 months RT storage

ZERA® technology can address unmet needs for vaccine development

ZERA® technology induce significant cellular and humoral immune responses.

The cellular immune responses elicited by vaccines based on ZERA® Technology confer protection and are cytotoxic.

Vaccines made with ZERA® technology have a positive immunomodulatory effect.Case studies: Zera®-E2 (Classical Swine Fever), Zera®-E7SH (Human Papilomavirus) and Zera®NP (Lymphocytic

Choriomeningitis virus)

14,00

16,001000000

Citotoxic immune response Challenge against LCMV infection


0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

%C

D8

+/IF

Nγγ γγ

+

Z-NP particles induce specific CD8 T-cells

in the absence of any extra-adjuvant

1

10

100

1000

10000

100000

PBS Zera-NP LCMV

Zera-NP StorPro bodies are efficient

immunogens against LCMV infection

*

Log

10

pfu

/gr

*

ZERA® technology can address unmet needs for vaccine development

Effective DNA vaccines could be also made using ZERA® Technology. Case studies: Zera®-E7SH (HPV) and Zera®NP (LCMV)

Log

10

pfu

/gr

1000

10000

100000

1000000

Challenge against LCMV infection


Zera®-NP DNA vaccine protection is as efficient as LCMV in

challenge experiments

Log

10

pfu

/gr

1

10

100

1000

PBS Empty

vector

NP Zera-NP Zera LCMV

* *

S I2 I3 I4 P

PO

I

I2

10 %

20 %

I1

S

15 DAP

2 3 48 DAP 15 DAP

I1

I2

S10

20

Induced StorPro® in tobacco leafsNatural PBs in maize endosperm

Natural maize PBs and StorPro® bodies are dense organelles


Ze

ra®

-PO

I

BiP

I2

I3

P

I4

27 %

42 %

56 %

BiP

27γ27γ27γ27γZ

PB

I3

I2

I4

ER

30

46

52

w/w

P

StorPro® bodies are highly packed assemblies which can be recovered

effiently by density gradients

Su

cro

se s

tep

den

sit

y g

rad

ien

t

H S I2 I3 I4 P

Density gradient

purification

H S

IF2

StorPro® bodies are dense organelles


Su

cro

se s

tep

den

sit

y g

rad

ien

t

ZERA®-GFP

Centrifugation

80.000g 2h 4ºC

IF2

IF3

IF4

P

PBs

H H’ Pb S C RF

Zera-EGFZera-hGH

StorPro® bodies recovered by low-speed centrifugation

Some examples of Zera® fusion proteins recovered by low speed

centrifugation (1000-2500xg)

H H’ Sp W PB


hGH

Preclarified homogenate (H); Clarified Homogenate (H’); Soluble protein discarded (Sp); Wash step (W);

StorPro fraction (Pb); Solubilized fusion protein (S); Cleavage step (C); Reverse phase purification (Rf)

There is no need of density gradient to recover StorPro® bodies in highly pure

fraction

StorPro® bodies recovered by low-speed centrifugation

Additional examples of Zera® fusion proteins recovered by low speed

centrifugation (1000-2500xg)

1. Zera

2. Zera-Bivalirubin

3. Zera-EGF

4. Zera-Insulin

5. Zera-hGH

6. Zera-Gfp

7. Zera-Gfp

8. Zera-Xylanase

1 2 3 4 5 6 7 8


Value proposition: Zera® makes products better by accumulating more product

Industrial Enzymes

• Versatility to adapt to a broad spectrum of real industrial conditions.

• Readily immobilised purified enzymes while keeping the activity

• Capacity to produce multi-enzymatic StorPro bodies

0

50

100

150

Enz

Zera-Enz

Act

ivit

y

The Zera® technology improves the performance and properties of protein-based products and processes

– Versatility in terms of eukaryotic expression systems

– Versatility in terms of protein types (complex proteins, membrane proteins, etc)


Vaccines for human and animal health

• Strong cellular response without adjuvants

• Efficient antigen presentation and protection

• Stable at room temperature

Therapeutic Products

• High activity performance of Zera® fusion peptides

• Incorporation of post translational modifications

• Multiple formulations and delivery formats from a single construct

Proliferation ZERA-Peptide

1 10 100 1000 100000

25

50

75

100

125

Cell line 1)

Cell line 2

nM

%P

roli

fera

tio

n

Acknowledgements

Boyce Thompson Boyce Thompson Boyce Thompson Boyce Thompson

InstituteInstituteInstituteInstitute

Dan Dan Dan Dan KlessigKlessigKlessigKlessig

Joyce Van EckJoyce Van EckJoyce Van EckJoyce Van Eck

TishTishTishTish KeenKeenKeenKeen

XiurenXiurenXiurenXiuren ZhangZhangZhangZhang

Wendy Wendy Wendy Wendy VonhofVonhofVonhofVonhof

Jason Jason Jason Jason EibnerEibnerEibnerEibner

NoreneNoreneNoreneNorene BuehnerBuehnerBuehnerBuehner

Bryan MaloneyBryan MaloneyBryan MaloneyBryan Maloney

Arizona State University >> Arizona State University >> Arizona State University >> Arizona State University >> Lucrecia AlvarezAmanda Walmsley >Federico MartinDwayne Kirk >Emel TopalYuguang Jin Heidi PinyerdJacki Kilbourne Jason CrisantesAaron Hicks Manuela RiganoDavid Julovich Michael EwingJulia Pinkhasov Angela RojasEric Chandler Amber GustinLuca Santi Deborah PauleyHugh Mason Jilliane Miller

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Matt FantonTim Miller

Dow AgroSciencesDow AgroSciencesDow AgroSciencesDow AgroSciences

Steve WebbSteve WebbSteve WebbSteve Webb

Chuck MihaliakChuck MihaliakChuck MihaliakChuck Mihaliak

Jennifer RiceJennifer RiceJennifer RiceJennifer Rice

Butch MercerButch MercerButch MercerButch Mercer

University of ArizonaChieri KubotaRyo Matsudo

4040

Hugh Mason Jilliane MillerCharles Arntzen Andrew Koons

Essential Sponsors and CollaboratorsEssential Sponsors and CollaboratorsEssential Sponsors and CollaboratorsEssential Sponsors and Collaborators

Arizona State UniversityArizona State UniversityArizona State UniversityArizona State University BiodesignBiodesignBiodesignBiodesign Institute at ASUInstitute at ASUInstitute at ASUInstitute at ASU

Cornell U. Dept. of Food ScienceCornell U. Dept. of Food ScienceCornell U. Dept. of Food ScienceCornell U. Dept. of Food Science Dow Dow Dow Dow AgroSciencesAgroSciencesAgroSciencesAgroSciences

Benchmark Benchmark Benchmark Benchmark BiolabsBiolabsBiolabsBiolabs US Department of DefenseUS Department of DefenseUS Department of DefenseUS Department of Defense

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TecnologicoTecnologicoTecnologicoTecnologico de Monterreyde Monterreyde Monterreyde Monterrey FondosFondosFondosFondos ZHZHZHZH

FEMSAFEMSAFEMSAFEMSA

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Tec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de MonterreyTec de Monterrey

Israel RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael RamirezIsrael Ramirez Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Cecy Garcia Andrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea MartinezAndrea Martinez Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar Jose Manuel Aguilar

Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Valeria Lobos Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Veronica Rocha Federico LopezFederico LopezFederico LopezFederico LopezFederico LopezFederico LopezFederico LopezFederico Lopez Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia Sergio Garcia EchauriEchauriEchauriEchauriEchauriEchauriEchauriEchauri

Carlos Carlos Carlos Carlos Carlos Carlos Carlos Carlos OrigelOrigelOrigelOrigelOrigelOrigelOrigelOrigel Javier Garcia Javier Garcia Javier Garcia Javier Garcia Javier Garcia Javier Garcia Javier Garcia Javier Garcia Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Jesus Hernandez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez Ricardo Camilo Chavez

Paulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina CalderonPaulina Calderon Cristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina MoralesCristina Morales JoharisJoharisJoharisJoharisJoharisJoharisJoharisJoharis SalgadoSalgadoSalgadoSalgadoSalgadoSalgadoSalgadoSalgado Gonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo MendozaGonzalo Mendoza

Miguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel OrtizMiguel Angel Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Cesar Ortiz Axel Gomez Axel Gomez Axel Gomez Axel Gomez Axel Gomez Axel Gomez Axel Gomez Axel Gomez Miguel Miguel Miguel Miguel Miguel Miguel Miguel Miguel SuasteguiSuasteguiSuasteguiSuasteguiSuasteguiSuasteguiSuasteguiSuastegui

Cardineau Lab

Tec de Monterrey, Fall 2011

The Potential of Plants