Modern Ag Biotech Applications

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Modern Ag Biotech Applications Powered By Docstoc
					Martina Newell-McGloughlin
Director, UC Systemwide Biotech Research
and Education Program
•   Agronomic Traits                    Crop Biotechnology
     – Biotic Stress
         • Insect Resistance – Bt, cystatin
         • Disease Resistance
              – Viral- coat protein protection (Papaya ringspot virus)
              – Bacterial, Fungal, Nematode (Rice blight, rice blast)
         • Weed- herbicide tolerance (Striga, orobanchia)
     – ABiotic Stress
         • Drought, Cold, Heat
         • Poor soil
              – Salinity – tomatoes with transport protein
              – Aluminum -Citric acid
•   Yield
         • Nitrogen Assimilation – nodulation by rhizobia, GDH metab eng
         • Starch Biosynthesis, O2 Assimilation, photosynthesis/Rubisco
•   Quality Traits
     – Processing
     – Post harvest loss reduction
     – Reproduction: sex barriers, male sterility, seedless fruit
     – Nutrients (Nutraceuticals)
         • Macro: Protein (Cassava-ASP), Carbohydrates, Fats, Fiber
         • Micro: vitamins, minerals, phytochemicals
         • Anti-nutrients: Phytase, Toxin removal
•   Novel Crop Products
     – Proteins: nutraceuticals, therapeutics, vaccines
•   Renewable resources: Biomass conversion, feedstocks, biofuels,
•   Biotech Crops 2008: 125 million hectares (310 million acres up 9.4%) X74 –
•   25 countries (15 LDC) 12% increase over 2007, 13.3 M farmers (12 M 2007)
•   90% resource-poor LDC farmers (12.3 M - 11.0 M 2007) most Bt cotton
•   New: Egypt, Burkina Faso, Paraguay , Uruguay.
•   India Bt cotton up to 7.4 M Ha.                                 Source: ISAAA
           Snap shot
– $44 billion 1996 to 2007, 44% yield gains, 56%
  reduction costs (including a 359,000 tonne a.i. in
  pesticides); gains of 141 million tons, would have
  required 43 million additional hectares
– Environmental pesticide footprint down by 15.4
  %. GM reduction in 286 million kg of CO2
  emissions equivalent to removing 6 M cars from
  the roads (Brookes 2008)
– HT- increase in no- till: reduction in erosion,
  soils much healthier, organic matter, less soil
  compaction, fuel use down by 20 gals/acre
  (Fawcett & Towery 2005 )
– CP papaya saved Hawaii papaya industry (and
  helped organic farmers!) may be the outcome for
  plum pox –C5 PTGS insurance against typhoid
  Mary in nurseries
    Farmers Choice
  Papaya ringspot virus
• No natural resistance anywhere
  so could never breed resistance
• Removes viral reservoir thus
  protects all growers
Field trial of transgenic 'UH
  Rainbow' and 'UH SunUp' was
  established in Puna in 1995.
  Gonsalves, Mansardt, Ferreira

Plums highly resistant to PPV -
  System is totally resistant as
  virus is not harbored
  unknowingly – Tolerant non
  biotech trees can harbor virus
– China BT rice GM used pesticides fewer than 1/season;
  conventional rice used pesticides 3.7 times ( Rozell, 2005)
  Pesticides cost applied to the conventional rice was 8 to 10
  times as high as GM. 80% reduction in pesticide use.
  Significant decrease in adverse health effects – Lives saved
– India Bt cotton decrease insecticide use 70% and increased
  productivity in 58% (737 kg/Ha)

– Organisms in “Bt crops” fields fared better in trials than
  those with insecticides Monarch butterflies increase

– Engineering Modified Bt Toxins to Counter Insect
  Resistance - pink bollworms cadherin mutation bypass
  with modifed Cry protein Science, Nov. 2007

– BT corn 90% reduction in mycotoxin fungal fumonisins -
  total US benefit estimated at $23m annually. (Wu, 2006)

– Blight-resistant potato (BASF -Rpi-blb1 and Rpi-blb2
  NBS-LRR) -UI study concluded for the major potato-
  producing regions of the world would be $4.3 billion.
The Argentinean Case: GM crops an engine of
economical development
  Number 2 in GM production.

  17% of the global area of GM plants.

  In 2007, 98% of soybean in Argentina was GM.

  Yield have reached over 6 tonns per hectare
  In 1994-95 production costs were 182 dolars/Ha; in
   2007 are 117 dolars/Ha

  In 1994-95 farmers spent 78 dolars/Ha in
   herbicides; today they spend 37 dolars/Ha and
   insecticide use has decrease 90%.

  Economical benefits of GM soybean USD$ 20 billion
   + 1 million jobs

  Problems, yes. Due to the economic success of GM
   soybean and maize, President Cristina Fernádez de
   Kirchne created a new tax on Gm soybean exports
   that producers oppose
China is awakening

                      Between       2001-05    the      chinese
                       government spent 15 billion US dólars
                       on AgBiotech projects; 2006-10 a 400%
                       increase has been projected
                      National Biotechnology program work
                       on the development of over 130
                       varieties of GM rice and 55 varieties of
                       GM cotton
                      10 GM products have been aproved for
                       human      consumption   (rice,    maize,
                       soybean and potato). Bt and disease
                       resistent rice is commercially planted in
                      GM cotton was used by 7.1 million small
                       farmers in 3.8 million hectares in 2007
                       with an economical benefit of USD$ 817
                       million in 2006.
 Spain: farmer Jose Victor Nogues”          Bt corn farmers earn about $85
  After 5 years of GM crops in the area,     more per acre, ISAAA (2006), while at
  most people can appreciate the huge        the same time producing a healthier
  benefits and lack of negative effects -    feed that is better for the environment.
  Introducing GM maize was definitely
  the way forward

                                             Romania: Buzdugan farmers reported
                                             price premiums of up to 10 percent for
                                             biotech soybeans due to fewer weed
 France: Thierry de l'Escaille,             impurities. Average price gain 2%.
  European Landowners' Organization,
  - “wide-scale adoption of these three      Production gain 29-33.5K M tonnes
  biotech crops in Europe could              (16-19% ) Earnings increase 35
  significantly increase annual              million and 62.4 million euros (2003).
  production, improve farmer income          I can tell you that soybean farmers in
  by more than 1 billion Euros
  (US$1.18 billion) and reduce spraying      Romania are very interested in biotech
  practices. With results like these, it's   seeds," “Although the seeds are 10 to
  easy to understand why farmers want        15 percent more expensive, the income
  access to this new technology," said       gains make the extra cost more than
     Blight-resistant potato
– Transgenic breeding lines preferred because resistance can be
  introduced into commercial lines with greater speed. The
  BASF GM potatoes involves the use of two broad spectrum
  resistance genes, Rpi-blb1 and Rpi-blb2. These two genes have
  a structure associated with regulatory genes called nucleotide
  binding site-leucine rich repeat (NBS-LRR) class of regulatory
  proteins. Many disease resistance genes code for proteins of
  that class. Numerous plant NBS-LRR genes are present in the
  typical plant genome, each protein is specific for a particular
  pathogen signaling a defense response, frequently a localized
  plant cell death called hypersensitive response. The C
  terminus of the protein containing LRR recognizes a ligand
  feature of a pathogen activating the NBS signaling module to
  initiate the defense response. The blight fungus suppresses the
  potato defense genes in sensitive plants but is thwarted by
  successful defense genes.
– Blight-resistant potato (BASF -Rpi-blb1 and Rpi-blb2 NBS-
  LRR) -UI study concluded for the major potato-producing
  regions of the world would be $4.3 billion.
    “Resistance” Genes natural and otherwise
• Rootknot nematodes R in tomato (Mi) and (aphids). Alternate to
  fumigation (Williamson UC Davis)
• Xylella fastidiosa Diffusible signal factor (Dsf) for disrupting Xf
  colonization, Inhibition of Xf polygalacturonase (PG). Targeting
  other Xf proteins required for virulence. protein/peptide-based
  inhibitors of pilins and adhesins – alternate to malathion vector
• Xa21 rice R gene confers resistance to several Xoo. Defense
  response triggered by Xo molecule, AvrXa21. Transgenic more
  resistant due to copy number (Ronald)
• Apple Fireblight (E. amylovora) -antibiotic sprays, cecropin lytic
  peptide analog worked well in field trials (Norelli)
• Apple scab (V. inaequalis) Multiple applications of fungicides used
  exclusively to control this disease. 'McIntosh' trees expressing
  either the endo- or exochitinase gene or both increased resistance
  to apple scab
• Use of apoptosis inhibition to protect plants from mycotoxin
  damage (Gilchrist, UC Davis)
• Sclerotinia-resist sunflower oxalate oxidase Pioneer wheat
• Zinc Fingers Dow/Sangamo
Natural Disease Resistance Genes Have Been Cloned

Xa21 gene has
been cloned from
an African rice
variety and
introduced into
modern cultivars to
confer resistance
to rice leaf blight.
Pam Ronald, Plant
Pathology, UC Davis
Use of apoptosis inhibition to protect plants from mycotoxin damage
David Gilchrist, Plant pathology, UC Davis
David Gilchrist, Plant pathology, UC Davis
       Rice staple food for 2.4 billion people
.   • Fungal diseases destroy 50 million metric
      tons of rice per year; varieties being
      developed resistant to fungi - proteins with
      anti-fungal properties.
    • Insects cause a 26 million tons loss of rice
      per year; insecticidal proteins
      environmentally friendly control.
    • Viral diseases devastate 10 million tons of
      rice per year; Tungro virus genome
      transgenes defense systems. Cassava
      Mosaic Virus similar protection system as
      papaya working in Kenya
    • Bacterial diseases cause comparable losses
      - transgenes such as cecropin lytic peptide
      basis for inbuilt resistance.
• Striga (Scrophulariaceae) is a
• genus of obligate root-parasitic
• flowering plants.

• All of the cultivated food-crop
• cereals in Africa are parasitized
• by one or more Striga spp.

•   Striga spp. in the savanna
•   regions alone account for
•   $7 billion and are
•   detrimental to the lives of over
    100 million African people.

      Host plants release factors required by parasitic plants Striga causes
      massive losses to crops in Africa:control strategy to inactivate host
      recognition factors - John Yoder, UC Davis,
  Host plants release factors required by parasitic plants:
   control strategy to inactivate host recognition factors

         Host plants                     Parasitic plants

   maize                                                 Striga
  cowpea                                              Orobanche
Arabidopsis                                           Triphysaria

                                                  Seed germination


                                    0h     12 h       24 h
               DMBQ              Haustorium development
Abiotic stress limiting factor for crops                    Abiotic Stress:
reaching genetic potential                             Drought, ColdHeat, Salinity
    Improved water conservation -Fewer crop                 Drought Stressed Rice
     losses -Higher yields on all acres through
     improved water utilization -Expand in
     drylands - Nuclear Factor Y B subunit
    Crops limited by salinity on 40 % world's     Control     + Gene D   Control + Gene E
     irrigated land (25 % US)
    Cold: Engineering with COR15a Tf, role
     in freezing tolerance.
    Plants engineered with Choline oxidase
     (codA) soil tolerated saline and cold
    Homeodomain-leucine zipper (HD-Zip)
     transcription factors respond to H2O &
     osmotic stress, exongenous abscisic acid
    Transport protein. Grow and fruit even in
     irrigation water that is > 50X saltier than
     normal. > 1/3 salty as seawater.
     Blumwald and Zhang)
                Increased Yields
         - Improve Nitrogen Assimilation
         - Increase Sucrose hydrolysis,
                  Starch biosynthesis
         - Increase O2 availability
         - Modify photosynthesis

                                  This short-day sorghum plant was
                                  used to map the Ma-1 gene (genes
                                  which modify photoperiodic
                                  behavior and thus maturity). This
                                  gene which works in other cereals
                                  would offer particular benefits to
Yield Gene       Control          biomass and forage crops in which
                                  flowering is undesirable

• LDC 30 % global energy. Growth driven by population and economic . Of
    the world's 47 poorest countries, 38 are net oil importers, and 25 import all
    of their oil, consuming much of their national income to pay for it.
•   The challenge: 5-10 times more efficient - 2001 $5/gal -2005 18c/gal
•   Biomass Conversion: Organic polymeric material, lignin, starches celluloses
    bioconverted ethanol; hemicellulose hydrolyzed to sugars, xylose , glucose .
•   Modify Plants and algae to improve enzymatic conversion.
•   Modify enzymes to improve conversion and fermentation
•   Maize other cereals, Switch grass, elephant grass poplars
•   Biodiesel is biodegradable and non-toxic - alkyl esters made from the
    transesterification of vegetable oils or animal fats., (60% less CO2)
•   Rapeseed, Botryococcus braunii (Bb) colloidal microalgae
•   Concerns: Food trade off – Efficiency of production – ecological impact
            Seeing the wood for the Trees
• Agronomic: HT, Disease resistance : increased productivity
  reduction of plantation establishment , reduced tree losses
• Poplar, Aspen, Pine, Walnut, Cottonwood trialed 57% timber
• Cell Cycle: LFY gene, PTFL gene from Populus able to induce
  early flowering in poplar.
• Phytohormones increase size, biomass, wood quality. GA 20-
  oxidase (AT) aspen fast growth (D/H) increas biomass (Eriksson)
• Paper: low lignin, faster growing ( Sederoff/Chiang)
• Biofuels: low lignin (ArborGen), faster growing
• Concerns: robustness/health, gene flow, longevity, similar trees.
  China lost track of Bt poplars.
• Male Sterility/Flowering inhibition: restrict gene flow, grow faster
  and produce more wood, since energy not wasted reproducing
• Tapetum barnase gene prevent pollen development promoters
  could lead to damage of non-reproductive tissue, (long generation
  times of conifers are considered).
• RNAi down regulation (full suppression often difficult)
• Tissue specific cytotoxic stilbene synthase (STS) competes
  chalcone synthase (CHS). Somatic cells STS no competition
  resveratrol anti-fungal (Höfig et al. 2006)
•   Simultaneously restores soil health and revegetates economical
•   University of Georgia poplar trees with merA, from mercury-resistant
    bacteria. Soil-borne and selected for heavy metals. Absorb from soil,
    convert to a relatively inert form, and release as vapor. 10X Mercury
•   Richard Meagher gene from the E. coli soybeans to Arabidopsis pumps
    arsenic from the soil and stores it in its leaves,
•   Bioremediation: Wilfred Chen Riverside developing high-affinity microbial
    bioadsorbents for heavy metal removal using engineered E. coli with
    surface-expressed peptide analogues (ECs) of phytochelatin - microbial
Modify Fruit Ripening

Fruit ripening modified    Caffeine-free coffee plants
to Improve Quality and     and controlled ripening
Reduce Postharvest         coffee plants.
Losses by altering the     Coffee and tea plants with
activity of cell wall      improved disease resistance
enzymes, such as           and tolerance of
polygalacturonase,         environmental stresses such
involved in softening      as cold and drought.
and deterioration.         Controlling the ripening
                           process will allow the farmer
The biosynthesis of        to both control and lengthen
ethylene, the fruit-       the harvest season.
ripening hormone, has
also been blocked in
several ways to delay
fruit ripening. Ethylene
can then be applied to
induce ripening when
desired, as is currently
done with tomatoes and
    Modern Biology / Genomics -
A new Research-Paradigm in modern Biology

      Phenotype            Genotype / Genes / DNA
 Appearance and Traits      Inherited Information
    of an Organism          Defining an Organism

   Improved                     Genomics
  Crop Plants              = the Totality of the Information
                           of all Genes and their Functions
        From Genomics to Improved Crops
                   The 2 Phases of Biology

              Reverse Genetics                         Phase 2
                Forward Genetics
                                                     Molecular Breeding
Map    Transcriptome                                    New Plant Traits

                            Metabolome                 Transgenics

           Phase 1
   Metabolic Pathways are the target for complex modifications





Unraveling protein collaborations could change the way
pathways are manipulated for improvement of valuable traits
in plants.
Attempts to modify storage proteins or secondary metabolic
pathways more successful than have alterations of primary
and intermediary metabolism
                            Plant Metabolic Engineering
Beware the flux! High                                           • Mod storage proteins or
  Lysine maize                 Enzymes and Intermediaries         2nd metabolic paths >
• Photo: Fruc-1, 6-            known- Little known on             success than alt 1ry and
  Bisphosphatase               controls and integration           intermed metabolism
  Phosphoribulokinase                                             (ILSI, 2004/ 2008).
  ↓3X ↓10X, minor
  photosynth rate
                                    Metabolic                  • Best: Mod single genes or
• Plastid aldolase,                                              series ind enzy. steps.
  catalyzes reverse                 Pathway                    • Conversion of exist comp
  reaction not subject                                           to another rather than
  to allosteric regulate,          Engineering                   change flux through path
  signif ↓↓ in
  photosynth rate & C
                                                                • ID rate-limiting steps in
  partitioning (Haake
  et al., 1998)                 Transcription Factors! Maize    • Target to channel metab
                                 C1 and R, reg flavonoids         flow into new paths,
  Other Considerations           aleurone , accumul rate        • Gene-silencing reduce or
• Site of synthesis              anthocyanins activating the      eliminate undesirable
• Site of activity               entire path (Bruce al 2000)      comps, traits, or switch off
• Charge and size               Wheat Rescue ancient TFs          genes to inc desirable
                                                                • Direct DNA cassette Paul
• Redundancy                     NAC (Uauy 2006)
                                                                  Christou 2009
            Improved Nutritional Content
Many common food crops not perfect for nutritional requirements of
 humans or animals.
Proteins: Maize, wheat, Sweet potato and cassava
WHO: 800 million people suffer from malnutrition, Protein-energy
  malnutrition (PEM), the most lethal form, affects 1 in 4 children:
  150 M (26.7%) underweight; 182 M stunted.
70% live in Asia, 26% Africa, 4% Latin America, Caribbean
• Grains low in Lysine – LDCs food - Feed Rations/pollution
• High Lysine maize: Use non feedback- enzyme (5X ppm)
• N assimilation modified pathway GDH 12% increase protein
• SRP Nonallergenic Amaranthus Albumin for potato
• High Protein: Cytokinin rescue flower pair kernels fused single
  kernel two embryos - high protein/oil low CHO

• NAC Tfs (NAM) senescence and nutrient remobilization leaves to
  grains, RNAi delay senes 30% protein, Zn, Fe
• Artificial Proteins:
    • ASP-1-sweet potato 67% increase protein (EAA 80%)
                Improved Nutritional Content
• Starch High Amylose (resistant starch) inhibit 2 SBE
• Sorbitol role in fruit carbon metabolism and affects quality
  attributes sugar-acid balance and starch accum
• Wheat puroindoline genes in rice better starch/flour
Fibre – Humans increase
• Polymers, Inulins, Fructo-oligosaccharides (FOS)
• SC Fructans sucrose taste: GI Tract health- fermented colonic
  – bifidobacteria (compete pathogenic bacteria)
  SC Fatty acid – anticancer/ inhibit HMG-CoAR less LDL
• SC fructans 1-SST Jerusalem artichoke. 90% sucrose
  converted "fructan beets“ (Koops, 2000)
• Potato synthesize the full spectrum of inulins from globe
  artichoke roots
• Lignans: enterodiol/lactone estrogen-dependent cancer
Fibre – Animals Decrease
• Brown midrib (COMT)–Decreased lignin increase
  digestibility better feed conversion, livestock prefer
    Improved Nutritional Content
Oils and Fatty acids
• Altering chain length and saturation level

• Novel genes to produce unusual fatty acids in oilseed
• MUFA: High Oleic Acid: more stable than PUFA heat/
  oxidation resistant, little or no postrefining
  (hydrogenation): AS oleate desaturase soybean gave >80%
  oleic acid (23%), Less SF milk/meat of animals
• MCT: medical foods, ergogenic aids. Acyl-ACPT canola,
  increase in capric (C10) and caprylic (C8)
• High-CLA: Antiox- free radicals heart disease/cancer
• Omega -3 DHA-EPA “Fish Oil” CV/thrombosis/ Cancer/
  Arthritis/ Cognitive/Mental/ premies -    D6
  Desaturase: Canola/soybean precursor SDA 3.6X ALA in
  generating EPA
• GLA safflower oil (C18:3n-6) anti-inflammatory effect,
  improved skin health and maintaining weight loss
• Sitostanol: phytosterol phospholipid Block cholesterol
Slide courtesy of Bruce Chassy
Improved Nutritional Content
          enzyme     Micro nutrients Vitamins/ minerals:
         (maize)     • Vit A Golden rice II b-carotene-Rice 25X (CBP)
        (bacteria)       • Biofortified cassva flour- Field trial Nigeria (Sayre)
                     • Vit B Folate increase in rice (pregnancy deficinicies)
                     • Vit E a-tocopherol g-TMT; Vit C increase corn DHAR
                     Minerals: Ferritin (bean S protein), Metallothionein (Rice,
                       wheat). Ca/proton antiporter (sCAX) Ca transport into
                       vacuoles. Ca-fortified carrots enhanced absorption.
                     Multi vitamin Corn
                     • Combinatorial direct DNA transformation rapid production of
                       multi-complex metabolic pathways
                     • transferred 5 constructs controlled by different endosperm-
                       specific promoters into white maize. Different enzyme
                       combinations show distinct metabolic phenotypes – resulting
                     • 169X beta carotene (60 mg/g v. 14 by breeding)
                     • 6X vitamin C, and
                     • 2X folate (Christou, 2009)
                    Improved Nutritional Content
Functional components - greater than nutrient value alone

• Carotenoids: Golden Rice, Sweet Potato, Cassava - (sight,
• Lycopene: polyamine Tomato – (reduce LDL, cancer)
• Isoflavones: genistein and daidzein; Isothiocyanates
• Phenolics: resveratrol antioxidant Sirtuins (anti-aging)
  polyphenol oxidase : help sequester protein during ensiling,
• Gallic acid hydrolyzable tannins, sequester protein in the
  rumen, more efficiently absorption
• Flavanols: Catechins, Flavones: quercetin (less adjuncts)

Anti-nutrients: Trypsin Inhibitors; oxalic acid; furans;
  Phytate, Bioavailability Phosphate, divalent ions: Phytase
  (Rice, alfalfa)

Allergens: soy P34 removal; peanut; gluten digestion
Toxins: Glycoalkaloid (potato) AS solanine
• Cyanogenic glucoside (cassava) hydroxynitrile lyase
   Increased b-Carotene in Rice Grains
• Over 120 million children                             Introduced
worldwide are deficient in                                enzyme
vitamin A. Rice engineered                                (source)
to for b-carotene,                                                              Normal rice
converted to vit A in the
body. This trait in rice                                 (daffodil)
cultivars distributed                                    (maize)
worldwide could prevent 1
to 2 M deaths each year.
• Golden Rice 23X rice 1.       Phytoene                                        “Golden” rice
                               Carotenoids               (daffodil)
                               are Elevated
Ferritin, an iron-rich bean    40 Times Oil

storage protein,
Phytase, an enzyme that breaks
down phytate making Fe                Ye et al. (2000) Science 287: 303-305.
                                                                               “Golden” rice 2
available, reabsorption of iron, a    A lack of dietary iron, zinc and
gene for a cystein-rich
                                      calcium results in unhealthy
metallothionein-like protein has
                                      increases in cadmium uptake into
been engineered into rice
                                      the kidney and liver
    Nutrigenomics Nutritional Genomics
"Leave your drugs in the chemist's pot if you can heal the patient
 with food." (Hippocrates)
• Nutrigenomics refers to the prospective analysis of differences
  among nutrients in the regulation of gene expression
• Nutrigenomics/Nutritional Genomics/ Nutrigenetics?:
  Genetically based, nutrition intervention that maximizes the
  health and effectiveness of each individual.
• Monogenic Diseases: 97% of the “disease-associated” genes
• Phenylketonuria, phenylalanine hydroxylase PHE -> TYR. Leads
  to neurological damage and mental retardation. PHE restricted
  TYR-supplemented diets -no Aspartame!
• Lactose Intolerance – Juvenile enzyme active into adulthood
• Polygenic diseases obesity, cancer, diabetes, and
  cardiovascular diseases,
• SNP in haemochromatosis linked gene (HFE) risk,
• MTHFR PM higher intakes of folic acid serum homocysteine
• Most dietary effect specific interactions on molecular level,
  regulation of gene expression directly and indirectly activity of
  transcription factors
Nancy Fogg-Johnson: Nutrigenomics will revolutionize health and
nutrition – It will inform how we prevent and treat disease and how
food is grown, processed, and made. Eventually nutrigenomics will
be able to discover diets that prevent or retard the onset of the most
serious and widespread of today's killer diseases, like cancer, as well
as degenerative diseases like Alzheimer's. When/if?
• Sitosterolemia (hyperabsorption of sterols hypercholesterolemia risk for
  atherosclerosis). Regulation of sterol uptake
• Mice was treated with a lipid metabolism-altering drug
• DNA microarray used for expression profiling of various tissues.
• Differential display with a control led to the discovery of an unknown gene.
• Computer simulation found that two proteins gene regulated reverse
  transport of dietary sterols out of the apical surface of intestinal cells.
• Exploring human gene databases, found a human homologue
• This explained why dietary sterols, which are structurally similar to
  cholesterol, are not absorbed in normal individuals.
• By scanning sitosterolemic individuals for this gene, it was found that all of
  them had a mutation in this gene responsible for their uncontrolled
  hyperabsorption of dietary sterols.
   Nutrigenomics Nutritional Genomics
• Number of genes regulate lipid metabolism/insulin
  sensitivity, affect susceptibility to T2 diabetes mellitus.
• SREBP-1c (sterol response element binding protein)
  mutations led to fatty livers, hypertriglyceridemia, severe
  insulin resistance, type 2 diabetes mellitus. One
  polymorph expression highly induced in mice on high
  fructose diets. Two missense mutations in exons
  domain of SREBP-1c were found in individuals
  displaying severe insulin resistance. Another
  association was found between an intronic single
  nucleotide polymorphism (C/T) between exons 18c and
  19c and the onset of diabetes in men, but not in women.
• T2DM Asian/Hispanic populations insulin resistance
  rather than b-cell dysfunction. In African-Americans the
• Hyperlipidemia: E4 allele in the apolipoprotein E higher
  LDL compared with the other (E1, E2, E3) for same fat
  intake levels
   Nutrigenomics Nutritional Genomics
• One single nucleotide polymorphism (-75 G/A) in the
  apolipoprotein A1 gene in women is associated with an
  increase in HDL . ApoA1 women showed increase in HDL
  with increase in PUFA compared to G variant taking
  similar amounts of PUFA.
• Haplotype (HapK) in leukotriene A4 hydrolase (LTA4H)
  risk of myocardial infarction (MI) European and African
  Americans. MI significantly greater in African-Americans
  HapK. (n6/n3)
• Barbecue Heterocyclic aromatic amines acetylated to
  reactive metabolites bind DNA - colon cancers. N-
  Acetyltransferase NAT is a phase II metabolism enzyme
  that exists in two forms: NAT1 and NAT2. HAA can be
  activated through acetylation to reactive metabolites
  which bind DNA and cause cancers. Only NAT2 fast
  acetylators can perform this acetylation. Studies have
  shown that the NAT2 fast acetylator genotype had a
  higher risk of developing colon cancer in people who
  consumed relatively large quantities of red meat.
       Caveat Emptor!
Will there be implications for
your insurance if you have a
susceptibility to heart disease?
Will there be implications if
you fail to follow a diet to
retard the onset of symptoms?
• "Nutrigenetic Testing: Tests Purchased from Web Sites Mislead Consumers.“
• Government Accountability Office (GAO) commercial "nutrigenetic" testing
  dubious clinical validity of commercial genetic tests, and unethical practices
• Investigators posed as 14 clients used the DNA from just 2 - man (48)-girl
  (9mnth). Despite this, the test 'results' were contradictory and warned of risks
  for various conditions. osteoporosis, heart disease, diabetes and more. Affiliated
  companies then offer nutritional supplements to stave off these predicted
  sicknesses — but the pills turn out to be little more than multivitamins, offered
  with a hefty dose of misleading medical advice. Cost to you a mere $89 to $395!
  (Nature, 2006)
                    How to Produce a Bio-therapeutic
Glycosylation Folding         Volume       System             Comments
              Complexity      required
                                                           • System well
                        HIGH Volume> 100   Transgenic unproven
                                 KGs         Animals • Appropriate for
                                                             Mabs, fusion
                 Protein                                     proteins
                Complexity                     Cell        • Well characterized
                            Volume< 100      Culture         System
          YES           LOW     KGs         (mammalian,      (Mammalian)
                                            Insect, plant) • Limited capacity
                                                             Opt Manufacturing
Glycosylation                                                strategy
                             Volume> 100   Transgenic • System early
           NO           HIGH     KGs         Plants • Appropriate for
                                                           Mabs, new
                 Protein                                   direction
                Complexity    Volume<100              • Well characterized
                                 KGs         Microbes System
                        LOW                           • Excess capacity
 The Cow Pock or the Wonderful Effects of the   Pasteurization
New Inoculation! James Gillray (1757-1815)
Vide--The Publications of ye Anti-Vaccine
Society, June 12, 1802, Library of Medicine
Vaccines create antibodies to neutralize the causative virus,
bacteria or toxin.Antigens may be introduced by:Killed

Killed vaccines designed to create antibodies without the
negative effects of infection so are generally considered to be
safe. However, during the inactivation process, some of the
surface antigens needed to create the desired antibodies may
be destroyed thus reducing their effectiveness.

Modified live vaccines contain an attenuated or weakened
version of a disease agent. Modified live vaccines are
effective but can negatively impact the health of the animal.
Subunit vaccines use only the necessary parts of the virus to
stimulate immunity. Unlike modified live vaccines, subunit
vaccines stimulate the immune system to prevent disease
without stressing the animal. And unlike killed vaccines,
subunit vaccines do a better job of disease prevention as they
only contain concentrated amounts of the target antigen. These
qualities make subunit vaccines both safe and effective.
Plant-made vaccines are a new type of subunit vaccines.
           Rinder Pest
Devastating Disease 100% Fatal
Existing Vaccine: Problems
transport, lack of refrigeration, and
lack of a simple system for
administration. Recombinant
product, freeze-dried, abating
problems with transportation and
handling, administered to scarified
skin to regenerate the serum.
The vaccinia virus is attenuated,
The recombinant vaccine - two of
the viral surface antigens H and F
eliminates risk of contracting the
disease it is easy to determine if the
animal has been vaccinated and is
not just a survivor.
Vaccination of cattle results in a
high level of immunity, affording
protection against test inoculations
of 1000 times the lethal dose of
rinderpest virus.
Tilahun Yilma, UC Davis
                                                 Why Make
                                             Pharmaceuticals in
                                        • Supply the increasing
                                          demand for new biotech
                                          drugs (esp. antibodies)
                                           – 50 Mabs by 2010
                                        • Significantly decrease unit
                                        • Improve patients’ access to
                                          biotech medicines
• Plants are an efficient producer of proteins
   – Plants are scalable bioreactors
   – Plants provide cost advantages to mammalian cell culture systems
   – 3-5 times faster than mammalian systems
• Plant cells are similar to human cells
   – Similar protein synthesis machinery
   – Read the same genetic code
   – Assemble, fold and secrete complex proteins
                   Why Plants?
Produce protein ( primarily Mabs)
• Correct post-translational modification mostly (unlike Microbes)
• No propagation of human pathogens or other mammalian contaminants;
  no other mammalian contaminants de novo;
• Asepsis can begin at purification, not inoculation (less go wrong)
• Scale-up utilizes the same technology used in agriculture to-day.
• Faster, cheaper, more convent, more efficient than CHO cells

                 Why Seeds?
• Protein at the highest levels in the harvestable seed.
• Seeds are easier, more economical than whole plants to transport to a
  processing factory
• Proteins can be extracted and purified in prep. for packaging.
• Seeds can be stored for prolonged periods with protein intact.
• Hundreds of acres of protein-containing seeds could inexpensively double
  the production CHO bioreactor factory.
                     Plants as Chemical Factories
Malting grain model (Ventria, Inc., CA and defunct attempt in MO, now Kansas)

       Transform rice with
       desired protein
       by a-amylase promoter
       in the aleurone layer.
       proteins lactoferrin and
       lysozyme                                  Imbibe seeds to         Extract and
                  Grow rice crop                 induce expression of purify proteins
                  in the field,                  a-amylase promoter from
                  harvest grain at               & production of         germinating
                  maturity.                      desired protein.        seeds.
                                                         Rice Lactoferin Lysozyme
                                                         Peru 30% Less Diarrhea,
                                                         Quicker recovery 3/6 days,
Ray Rodriguez, Molecular and Cellular Biology, UC Davis  1/3 less recurrence
                             Plant Based Expression Systems for
                        Efficient Production of Human Therapeutics
  Human deficiency in alpha-1-antitrypsin (AAT)                AAT expression kinetics via agroinfiltration of
   results in non-smoking related emphysema                    Nicotiana benthamiana plants will be optimized

                                                   AAT is used as a
                                                    model human
                                                    protein; other
                                                     proteins will
                                                       also be
                                                    expressed and

 The optimized human AAT gene has been cloned into                                        scalable
 Agrobacterium and is expressed using a chemically                                       induction
inducible plant viral (CMV) replicon expression system                                    methods
  Inducer                                                                                  will be

        Inducible Promoter   CMV 1a                            750            Functional AAT (mg / kg FW tissue)
                                                  2a H
       Promoter     CMV 2a       CMV 2b            Replicase   500

       Promoter     CMV 3a            AAT                      250
              RNA 3a
                                 RNA AAT
                                             AAT      AAT        0
                                                                      Negative Control    pCMV-SPAAT +      pCMV-SPAAT +
                                            AAT      AAT               (Healthy Plant)    Inducer; Cotton   Inducer; Syringe
         Genetic Engineering Technology
       Allows Production of Novel Products
                                    Passive Vaccines
                                    Ab enteric bacteria
                                    E.coli O157:H7 meat
                                    foodborne path

                                    Viruses PlantAb protected mice
                                    against genital herpes
Polyhydroxybuterate                 similar physical props to MCC
biodegradable plastic   Metabolic   remained stable in human
                        Pathways    exhibited no diff in affinity
Acetylenic &Vernolic                for binding, neutralizing HSV
Acid Containing
Chemical feedstocks

                                      Active Vaccines
                                      gastroenteritis virus
                                      Cholera/Hep B/banana
The Dow AgroSciences unique
Concert™ Plant-Cell-Produced System
uses plant cells, rather than whole
plants, in a culture medium comprised
primarily of water, sugar and salt. This
eliminates the possibility of
contamination and environment
concerns. The Concert™ Plant-Cell-
Produced System is totally bio-
contained as all production is done in a
sealed and sterile production process.
This means only the required inputs get
into the vaccine and only a safe, non-
replicating vaccine comes out.
          Alfalfa   Plasma Proteins, Foot-and-mouth disease vaccine
          Maize    Anti- HIV and Anti herpes Simplex Antibodies
                   Microbiocides for pulmonary infection                        Mabs
                   for cancer, autoimmune disease RA, Vaccines hepatitis B,
                   Norwalk virus (Travelers disease), Vaccines & Mabs for
                   animal, Aprotinin for blood loss, Gastric lipase cystic fibrosis,
          Lettuce Vaccines for Hepatitis B
          Moss     Factor IX for hemophilia B
          Rice     Lactoferrin Lysozyme for GI health, Alternatives to abs in
                   poultry diets, Topical infections, inflammations, B-cell
                   lymphoma vaccine
         Safflower Therapeutics and oil-based products for oral/dermal delivery
         Spinach Protective antigen for vaccine against Bacillus anthracis
         Soybean Tobacco extensin signal peptide - Anti-HSV-2 (IgG)
         Tobacco    Non-Hodgkins B-cell lymphoma, TGF-b glucocerebrosidase for
                    Gauchers Syndrome , Alpha galactosidase for enzyme
                    replacement therapy, IgGs for prevention of dental decay,
                    common cold, GAD 7 cytokines for type 1 Diabetes, Colon
                    cancer surface antigen – Fabrazyme fat-storage disorder
Tomato , Potato     Edible vaccines: Enterotoxigenic E. coli, Norwalk virus,
Banana (someday!)   Hepatitis B, Vibrio cholera, Rabies virus-intact Glycoprotein
                    Antimicrobe peptides,
Wheat               Carcinoembryonic antigen - Murine IgG signal peptide
Potato              Polyhydroxybuterate biodegradable plastic
• potential gene flow to food
  crops of the same species
• co-mingling of food and non-
  food crops
• worker exposure to plant
  material containing active
  pharmaceutical ingredients
• ProdiGene paid $3 million bond
  for violations in Nebraska and
  Iowa reimburse the USDA for
  the costs, involved in disposing
  of the contaminated crops.
• Now making brazzein ( a sweet
  protein) in maize!
                Principles of Confinement
Physical and biological
Confinement essentially means keeping the crop and its products on the
  land where it was grown until removed for processing, with no
  inadvertent exposure to the public and minimal exposure of products to
  workers and the environment.

Identity Preservation: A Closed Loop System
Preventing co-mingling or gene flow prime directive for industry as well as
   regulatory agencies. (No StarLink®, repetition!).
• Potential gene flow to food crops of the same species
• Vectors: Birds, insects, viruses
• Co-mingling of food and non-food crops
• Worker exposure active pharmaceutical ingredients (APIs).
Identity Preservation: A Closed Loop System
• Preventing co-mingling or gene flow prime directive for industry
  as well as regulatory agencies.
Physical and biological
• Confinement: physical, temporal and biological isolation, as well
  as appropriate spatial isolation, acreage limitations, dedicated
  equipment, facilities
• “Trap” Plant borders, Color coded kernels, Male sterile plants,
  work well - limited to a few species.
• Transformation plastid (not all pollen is plastid-free, illegit recomb
  none of the transgenes functional in nuclear genome)
• Genetic Use Restriction Technologies, or GURTs
Depending on the timing, expression of the lethal gene leads either to seeds that are
incapable of germinating or to death of the seedling.
During seed maturation stage lea promoters ( “late embryogenesis abundant”) activate the
genes whose products control and accompany the drying process. Shortly after the seeds
have swollen, germination-specific promoters activate the genes whose products break
down substances from the storage tissue and transport them to the young seedling.
T- GURT seed will germinate only when treated with this inductor chemical.

V-GURT seed treated with an inductor that activates the lethal gene. Produce seeds that
mature but that are no longer able to germinate and cannot therefore be used for re-sowing.

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