BIOTECHNOLOGY: Engineering plants for the future
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BIOTECHNOLOGY: Engineering plants for the future. beer, wine, cheese through micro-organisms. for food quality, yield, resistance to pathogens, produce metabolites, secondary products new varieties as plant breeders Involves manipulation of biochemistry, physiology, and development

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BIOTECHNOLOGY: Engineering plants for the future

  • beer, wine, cheese through micro-organisms.

  • for food quality, yield, resistance to pathogens, produce metabolites, secondary

  • products

  • new varieties as plant breeders

  • Involves manipulation of biochemistry, physiology, and development

  • Advent of DNA technology revolutionized

  • -Components of recombinant DNA technolgy

  • -Tissue and protoplast culture

  • -Use of agrobacterium as vectors for genetic engineering

  • -selected examples


ENGINEERING PLANTS WITH THEIR OWN GENES

  • Ethylene induced fruits lack the texture and flavor of vine ripened appearance

  • Pectin degradation by Polygalacturonase (PG) cause softening

  • Antisense technology: sense RNA binds with antisense RNA

  • Takes twice as long as normal tomato

  • Was not commercially successful


Steps in producing Transgenic

  • A source of foreign DNA containing the desired gene

  • A vector that carries the gene

  • Means to introduce the vector into host plant


Methods for isolating/cloning genes

  • -Restriction enzymes from bacteria

  • cut double stranded DNA at specific

  • sequences

  • -EcoRI cut at CTTAAG

  • Different enzymes cut at different

  • location, so can use combination of

  • enzyme to isolate fragment of DNA

  • Or make cDNA from mRNA using

  • reverse transcriptase enzyme

  • Each piece of cDNA represent a gene



  • Electroporation

  • Biolistic methods for direct entry

  • Protoplast fusion

  • Micropropagation-virus free


PLANT PROTECTION opines for bacteria

  • Competition from weeds: nutrient, space

  • Attack by viruses, fungi, and predatory insects

  • Extensive use of herbicide, fungicides, insecticides

  • Carryover of agricultural chemicals along with food product, effect on ecosystems

  • Transgenic provides opportunities with decreased reliance on dangerous chemicals


  • Weeds cause $12 b crop loss annually opines for bacteria

  • Mixture of herbicides to kill pre- and post-emergence weeds

  • Multiple spraying, heavy chemical load on crops and soil

  • Herbicide-resistant crops are solution!

  • Roundup resistant soybean.

  • Glyphosate inhibits Enzyme EPSP Synthase (aromatic amino acids)

  • EPSP pathway is only found in plants and in microorganisms

  • Humans need aromatic amino acids in their diet


  • Herbicide resistant can be achieved by over-expression of tolerant enzymes

    • Find a gene that encodes an enzyme variant that is resistant to the herbicide

    • Find a strong promoters

  • Cells expressing large amounts of enzyme will tolerate higher doses of enzyme inhibitors

  • Strategy: Petunia cells in culture screened for glyphosate resistance

  • Most cells died, few survived

  • clone variant gene (EPSP synthase inhibitor tolerant)

  • express in crops with strong promoter (35S) by agrobacterium vector

  • More than half of the soybeans planted in North America are glyphosate resistant

  • Other examples: Glufosinate (Liberty) resistant.

  • Herbicide resistant weeds: undesirable side effects from selection pressure


Bacillus thuringiensis (Bt) resistance tolerant enzymes

  • Economic loss from pathogens $100b

  • Insecticides are not specific, may kill the pollinators; now hire commercial pollinators

  • Bt toxin ingested by insects, degraded, binds to receptors in gut and interference

  • with normal digestion

  • - Humans do not have the receptor

  • - Resistant insects?? Plant along with non-transgenic plants.


Disease Resistance tolerant enzymes

  • Express chitinase to degrade chitin: a fungal cell wall constituents

  • Vaccination of plants with cDNA from TMV coat protein: TMV resistance

  • C4 genes in C3 plants, net carbon gain, reduce rubisco oxygenase activity

  • Improvement of nitrogen fixation ability: Less reliance on N2 fertilizers


GOLDEN RICE tolerant enzymes

  • White rice no beta carotene:

  • precursor of vitamin A

  • -Vitamin A deficiency: Blindness in

  • young children

  • Also iron rich

psy (phytoene synthase) from daffodil (Narcissus pseudonarcissus)

crtl from the soil bacterium Erwinia uredovora


  • - Erucic acid to zero in canola oil. tolerant enzymes

  • - Increase unsaturated fatty acids in oil

  • Tobacco plants to produce drugs: use male sterile plants so no seeds

  • no transfer of genes.

  • Oral delivery of vaccines by edible plant tissue: norwalk virus coat protein

  • in potato. Fed mice produced antibody against Norwalk virus.

  • - Plants as alternate source of renewable fuels: ethanol by fermentation with yeast

  • Sunflower oil in place of diesel oil.

  • More reduced and more energy content , burn cleaner

  • Potential disadvantages: Displace crop plants, arable lands


Ethical arguments against genetically modified foods
Ethical arguments against genetically modified foods tolerant enzymes

  • GMOs are wrong because risks outweigh benefits

  • GMOs are wrong , no matter how great the benefits

Credit: Dr. Comstock, North Carolina State University


A. Unsafe for consumers tolerant enzymes

“Frankenfoods”


Unsafe for environments “superweeds” tolerant enzymes

  • Herbicide resistance - canola gene flows

    into weedy relatives

  • Bt toxin kills monarch butterfly larvae

  • Unfair to small farmers

    • “Rich get richer, poor get poorer”


Ethical arguments against gm foods
Ethical arguments against GM foods tolerant enzymes

GM foods are wrong no matter how great the benefits may be.


Gm foods are wrong because it s wrong to
GM foods are wrong because it’s wrong to: tolerant enzymes

1. Play God

2. Invent world changing technology

  • Cross species boundaries (Cold gene from fish

    But mules, hybrid wheat)

  • Reproduce by nonsexual means (Plant Cuttings)

  • Disrupt integrity, beauty, balance of nature

    (Monarch Butterfly)

    6. Harm sentient beings


  • Unsafe for consumers? tolerant enzymes

    Food allergens, toxins

  • Unsafe for environment?

    • Unintended effects on nontarget organisms

    • Gene flow, development of resistant weeds

  • Unfair to small farmers?

    Rich get richer, poor get poorer




Ethical arguments for gm foods
Ethical arguments FOR GM foods tolerant enzymes

Potential to improve:

  • Diets in developing countries

  • Efficiency of food production

  • Safety and purity of food

  • Agricultural sustainability

  • Diversity of agro-ecosystems


Enhanced nutrition
Enhanced nutrition tolerant enzymes

Vitamin A Rice

Iron Enhanced Rice

Amino Acid Balance


Insect resistance
Insect resistance tolerant enzymes

  • Bt corn

  • Insect resistance from Bacillus thuringiensis

  • Non-toxic to humans

  • Target insect: corn

    borer

  • 40% U.S. Corn crop

    Bt

  • Potential to reduce

    insecticide use


Disease resistance
Disease resistance tolerant enzymes

  • Potatoes

  • Squash

  • Tomatoes

  • Corn

  • Rice

  • Canola

  • Soybeans

  • Grapes

  • Cantaloupes

  • Cucumbers


Regulatory oversight in biotechnology http www aphis usda gov biotechnology index html
Regulatory Oversight in Biotechnology tolerant enzymeshttp://www.aphis.usda.gov/biotechnology/index.html

  • US Dept. of Agriculture

    • Plant pests

    • Plants

    • Veterinary biologics

  • US Food and Drug Administration

    • food, feed

    • food additives

    • veterinary drugs, human drugs and medical devices

  • US Environmental Protection Agency

    • microbial/plant pesticides

    • new uses of existing pesticides

    • novel microorganisms


  • Represent tolerant enzymes

    valid concerns

  • Demand scientific and political vigilance




  • During making transgenic plants, only a small population of plants get

  • Transformed. How do the plant molecular biologists select a transgenic plants?

  • Select for trait of interest

  • Select antibiotic resistant plants after transformation (1st generation)

  • Select antibiotic resistant plants after seed production (2nd generation)

  • Select antibiotic susceptible plants after transformation (1st generation)

  • Select antibiotic susceptible plants after seed production (2nd generation)


  • Glyphosate kills plants because it plants get

  • It prevents Electron Transport chain in photosynthesis

  • It prevents respiration

  • It prevents synthesis of secondary metabolites

  • It prevents synthesis of aromatic amino acids



Identify the wrong method of transforming plants with foreign DNA

1. Electroporation

2. Biolistic methods for direct entry (Gene Gun)

3. Sodium Chloride based channel opening

4. Agrobacterium infection




  • In our lab, DNA extraction from GMO/non-GMO samples requires negatively

  • Charged beads to

  • Exclude the proteins from the sample

  • Exclude magnesium (divalent cation) to protect DNA in the sample

  • Exclude RNA from the sample

  • Help PCR methods amplify magnesium regulated enzymes

  • Help reduce protein contamination (PCR inhibitor)



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