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Genetically Modified Organisms in Agriculture. In traditional breeding large pieces of chromosome are moved (by chromosome crossing over) often bringing in undesirable traits (e.g., poor product quality) with the desirable (e.g., pest resistance).

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Genetically modified organisms in agriculture
Genetically Modified Organisms in Agriculture

  • In traditional breeding large pieces of chromosome are moved (by chromosome crossing over) often bringing in undesirable traits (e.g., poor product quality) with the desirable (e.g., pest resistance).

  • Genetic engineering of plants does what plant breeders have been doing for thousands of years (i.e., moving genes around) but does so much more precisely.


Selection and traditional breeding versus genetic engineering
Selection and Traditional Breeding versusGenetic engineering

  • Food we eat has been genetically modified, through centuries of crosses, both within and between species.

  • For most of the last century through mutations induced by bombarding seeds with chemicals or radiation. In each of these techniques, large numbers of genes of unknown function are transferred or modified to produce new food varieties.

  • Gene splicing is the most refined, precise and predictable method of genetic modification because the function of the transferred gene or genes is known.



Hybrid wheat gave birth to agriculture and some say civilization itself.

— Jacob Bronowski, author The Ascent of Man


Teosinte
Teosinte civilization itself.

Modern corn


Plant biotechnology civilization itself.

Commercial variety

New variety

Desired gene

Using plant biotechnology, a single gene may be added to the strand.

(only desired gene is transferred)

=

(transfers)

Desired gene

Traditional plant breeding

Commercial variety

New variety

Traditional donor

DNA is a strand of genes, much like a strand of pearls. Traditional plant breeding combines many genes at once.

(many genes are transferred)

=

X

(crosses)

Desired Gene

Desired gene


  • PLANT GENETIC ENGINEERING civilization itself.

  • Bacteria contain plasmids - small circles of DNA in addition to the chromosome.

Bacterial chromosome DNA

Plasmid

  • Bacteria will take up plasmids from their surrounding medium.


  • Plant Transformation civilization itself.

  • Agrobacterium tumefaciens is a bacterium causing crown gall, a tumorous disease of plants.

  • Tumors free of the bacterium can also be found on infected plants.

  • This is because the tumor inducing (Ti) plasmid of the bacterium has been incorporated into the plant's DNA.



  • It will then stick to the ends of the cut plasmid so that the DNA is inserted into the plasmid.




  • It "fires" particles coated with DNA into plant cells.

  • The DNA becomes incorporated into the plant DNA.

  • Useful on plants not infected by Agrobacterium.


  • Gene Selection reproduce.

  • Total plant DNA cut into fragments - a genomic library - contains all the DNA.

  • extract mRNA, use reverse transcriptase to make cDNA giving a cDNA library - contains only structural DNA.

  • Put into plasmids in bacteria and grow colonies.

  • Hybridize plasmid containing above DNA with 32P labelled cDNA made from mRNA extracted from the plant (Northern blot).



Gene microarray
Gene Microarray radiolabelled.

Determines when many genes are switched on or off


  • Antisense radiolabelled.

  • Technology

  • The opposite matching nucleotide sequence of DNA is synthesized as the transcribed strand.

  • When the antisense mRNA is synthesized it binds to the sense mRNA and prevents the biosynthesis of the particular enzyme, effectively preventing its action.


Rnai rna interference

Promoter radiolabelled.

Sense

Antisense

RNA loop

Strands unwind

mRNA

RNAi (RNA interference)

  • More recently RNA has become the technique of choice.

  • The DNA sequence matching part of the required gene, followed by a spacer, and then the antisense matching the first sequence is incorporated into the plant.

  • The RNA forms a loop back onto itself, making double‑stranded (ds) RNA (matching the required mRNA).

  • This is processed into 21–23‑nucleotide 'guide sequences‘ by an enzyme called dicer already in the plant cells.

  • The guide RNAs are incorporated into a nuclease complex: the RNA- induced silencing complex (RISC); here the strands are unwound.

  • This destroys mRNAs that are recognized by the guide RNAs through base‑pairing interactions.

  • This is far more effective than antisense constructs.


Advantages of genetically engineered crop plants
Advantages of Genetically Engineered Crop Plants radiolabelled.

  • Desirable traits can be introduced without accompanying undesirable traits from e.g., wild plants.

  • The incorporation of disease or insect resistance decreases the use of toxic pesticides.


Advantages of Genetically Engineered Crop Plants radiolabelled.

  • Reduction in toxic pesticide use

    • “Roundup-ready” crops: the resistance to glyphosate means that a very specific, environmentally-benign herbicide can be used.


Genetically Engineered Crop Plants radiolabelled.

Phosphoenolpyruvate

+ erythyrose 4-phosphate

Shikimate

Phosphoenolpyruvate +

shikimate-3-phosphate

5-enolpyruvylshikimate

-3-Phosphate

Chorismate

Aromatic amino acids,

phenolics, lignin

EPSP synthase

  • Herbicide resistance

  • The herbicide glyphosate (“Roundup”) inhibits the enzyme EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase).


Herbicide resistance
Herbicide resistance radiolabelled.

  • Glyphosate resistant (Roundup Ready®) soybeans have a single added protein: a glyphosate-tolerant enzyme (CP4-EPSPS) from a bacterium, under the control of a constitutive promoter.

  • These plants are unaffected by glyphosate.

  • Glyphosate resistance has also been transferred to cotton, canola and maize, and wheat is about to be released.


  • Tomato Ripening radiolabelled.

  • Tomato fruit have to be fully mature when picked in order for a full color and flavor to develop in the fruit.

  • Most are typically harvested green and immature to prevent overripening in transit; they do not develop a full flavor or color on being ripened by ethylene.

  • An attempt was made to delay the softening of the ripe fruit so that they can be picked at a later stage.

  • A gene for the wall softening enzyme polygalacturonase was isolated, the antisense gene synthesized, and plants transformed with this antisense gene.

  • However it did not enjoy commercial success because the tomato variety that was transformed was not a high quality variety from the point of view of its other characteristics.


  • Tomato plants transformed with the antisense genes for either of two enzymes of ethylene biosynthesis (ACC synthase and ACC oxidase) produce no ethylene and do not ripen, but they do progress to the mature green stage.


If picked at this stage they can be shipped and then can be made to undergo full flavor ripening by exposure to ethylene


  • Insect resistance through Bt toxin made to undergo full flavor ripening by exposure to ethylene

  • Bacillus thuringiensis(Bt), produces a toxin, called Bt toxin, that is lethal to many insects; it inhibits an enzyme in insect gut.

  • The gene for Bt toxin has been isolated and used to transform cotton and other crops, with a constitutive promoter so that all tissues contain Bt toxin.

  • This offers season-long protection against insects, reducing or eliminating the need to spray for insect control.

  • The downside appears to be the build up or populations of insects resistant to Bt toxin.

  • Border planting of non-Bt corn is recommended.


Advantages of Genetically Engineered Crop Plants made to undergo full flavor ripening by exposure to ethylene

Nutrition

  • Desirable human nutritional traits can be introduced that are not found in normal plants.


Golden Rice made to undergo full flavor ripening by exposure to ethylene

  • Many people in tropical areas have insufficient vitamin A in their diet leading to blindness in children.

  • Golden rice is rice is genetically engineered to contain the needed series of genes for the biosynthesis of carotene, a precursor of vitamin A.

  • Dr. Potrykus is trying to release the seed free to those who need it, but it has been held up by arguments over patents on the techniques used in its creation.

  • Will golden rice be accepted by those who consider white rice more desirable?


  • Vaccines from plants made to undergo full flavor ripening by exposure to ethylene

  • One new promising venture in the field of human medicine is to incorporate vaccines into plants.

  • The vaccine would be an antigen from the coat protein of a virus or bacterium.

  • Potatoes and bananas have therefore been transformed with genes for antigens for several diseases.


More than 50 biotech food products have been approved for commercial use in the united states
More than 50 biotech food products have been approved for commercial use in the United States

  • Canola

  • Corn

  • Cotton

  • Papaya

  • Potato

  • Soybeans

  • Squash

  • Sugarbeets

  • Sweet corn

  • Tomato

Products on the market


Four crops accounted for nearly all of the global biotech crop area
Four crops accounted for nearly all of the global biotech crop area

US agriculture

About 60% of acreage devoted to corn, soybeans and cotton is now planted with crops genetically modified to be resistant to insects and/or herbicides.

Source: International Service for the Acquisition of Agri-biotech Applications


Global Area of GM Crops crop area

Million Hectares

Source: International Service for the Acquisition of Agri-biotech Applications


Six countries accounted for 95 percent of the global biotech crop area up from 4 and 99 in 2002
Six countries accounted for 95 percent of the global biotech crop area – up from 4 and 99% in 2002

*Australia, Bulgaria, Colombia, Germany, Honduras, India, Indonesia, Mexico, Romania, South Africa, Spain and Uruguay accounted for the remaining 1 percent of biotech crop acres.

Source: International Service for the Acquisition of Agri-biotech Applications

Source: International Service for the Acquisition of Agri-biotech Applications


Global Area of GM Crops crop area – up from 4 and 99% in 2002

Source: International Service for the Acquisition of Agri-biotech Applications



Food production effect of biotech crops
Food Production Agri-biotech ApplicationsEffect of Biotech Crops

Net economic impact

Pesticide reduction

Yield increase

Current cultivars 4 billion pounds $1.5 billion 46 million pounds

Potential cultivars 10 billion pounds $1 billion 117 million pounds

Total 14 billion pounds $2.5 billion 163 million pounds

Benefits of biotechnology – More food


  • Bt cotton – 185 million pound yield increase and $102 million in additional income

  • Biotech soybeans – $1 billion in additional income through production cost savings

Source: National Center for Food and Agricultural Policy


Products in the pipeline

  • Disease-resistant sweet potatoes

  • Disease-resistant bananas

Products in the pipeline

Agronomic benefits

  • Pest- and disease-resistant cassava


Products in the pipeline1

  • Soybean and canola oils with higher levels of vitamin E

  • Vitamin-enriched rice

  • Decaffeinated coffee

Products in the pipeline

Enhanced nutritional qualities


Products in the pipeline2
Products in the pipeline million in additional income

Enhanced nutritional qualities

“I think in the long term we will have foods that are less hazardous because biotechnology will have eliminated or diminished their allergenicity.”

— Steve Taylor, Ph.D. Department of Food Science and Technology, University of Nebraska

Benefits of biotechnology – Better food


Products in the pipeline3

  • Apples to protect against Respiratory Syncytial virus

  • Potatoes to protect against cholera, E. coli and Norwalk virus

Products in the pipeline

Functional foods


Conservation tillage improves wildlife habitat water quality

Source: million in additional incomeConservation Technology Information Center

Conservation tillage improves wildlife habitat, water quality

Nearly three-fourths of no-till soybean acres and 86 percent of no-till cotton acres were planted with biotech varieties.

Benefits of biotechnology – Environment


Potential risks
Potential risks million in additional income

  • The potential risks associated with genetically modified foods result not so much from the method used to produce them but from the traits being introduced.

  • With gene splicing, only one or two traits at a time are introduced, making it possible to assess beforehand how much testing is needed to assure safety.


Could gm organisms harm human health or the environment

RISKS AND ETHICAL QUESTIONS million in additional income

Could GM organisms harm human health or the environment?

  • Genetic engineering involves some risks, but these have so far been shown not to occur.

    • Pollen from a transgenic variety of corn that contains a pesticide may stunt or kill monarch caterpillars

    • Possible ecological damage from pollen transfer between GM and wild crops

    • Could genetically modified crops could cause allergic reactions?

Figure 12.20A, B


  • Damage to non-pest insects million in additional income

  • Bt protein can be toxic to some caterpillars.

  • Cornell researchers found that monarch butterfly larvae that were fed milkweed leaves coated with high levels of pollen were harmed.


Damage to non-pest insects million in additional income

  • Field evaluations show that exposure of non-target organisms, such as monarch larvae, to Bt pollen would in fact be minimal:

  • Most pollen has much less toxic protein.

  • The majority of the heavy pollen moves only a short distance away from cornfields: within the first three meters 90 percent falls.

  • Corn fields typically contain a low concentration of weeds for insect food.


Damage to non pest insects
Damage to non-pest insects million in additional income

  • Exposure of monarchs would be limited only to larvae developing on milkweeds within the cornfield or very near to cornfields during pollen shed, and even here field studies show minimal effect.

  • Seldom does the pollen density reach damaging levels

  • The Bt pollen toxicity is degraded rapidly by sunlight and is washed off leaves by rain.

  • In addition less monarch-lethal insecticides are sprayed!!


Spread in nature
Spread in Nature? million in additional income

  • Genetically engineered does not make a plant any more likely to become an invasive or persistent weed.

  • Genetically modified potatoes, beets, corn and canola planted in natural habitats were as feeble at spreading and persisting in the wild as their traditional counterparts.


Allergic reactions
Allergic reactions? million in additional income

  • Some people develop allergic reactions to certain foods, such as shellfish and peanuts.

  • Proteins that are taken from commonly allergenic foods are presumed to be allergens.

  • The problem is avoided by not placing genes for allergic proteins into crop plants.

    • E.g., soybean that contained a protein from Brazil nuts was withdrawn.


  • Allergic reactions? Starlink corn: million in additional income

  • Starlink contains an insecticidal protein related to Bt.

  • The regular Bt and Cry9C proteins kill insects in the same way, by destroying the insect’s stomach cells.

  • The only major difference is that these two classes of proteins bind to different midgut binding sites.

  • Starlink appeared in human food before it was approved for such use, but it was later shown to have no allergic effects.


  • Antibiotic resistance markers million in additional income

  • Scientists use antibiotic resistance genes as part of the transforming genes as a means of selecting for transformed cells.

  • Can antibiotic resistance genes be inadvertently transferred from the plant cells to bacteria in the guts of animals and humans, making the bacteria resistant to antibiotics and thus rendering some antibiotics less useful for treating bacterial diseases?

  • Research by various world agencies has consistently concluded that these genes have never been shown to be transferred from crops derived through biotechnology to bacteria in nature.


  • Antibiotic resistance markers million in additional income

  • The DNA contained in food - including the antibiotic resistance genes - is broken down in the human gut during the digestive process.

  • Antibiotic resistance genes used in plant biotechnology were obtained from naturally occurring bacteria from human and animal guts or the environment.

  • Nonetheless researchers have now developed a way of cutting out the antibiotic resistance gene before the production of the final product.


  • To increase the amount of a specific DNA sequence (above), million in additional incomePCR (polymerase chain reaction) is used.

  • More DNA is formed by a high temperature DNA polymerase from primers consisting of base sequences from the ends of the DNA template sequence to be amplified.

  • A temperature alternation permits the two new strands to disassociate to form additional templates on which new DNA is made.

  • This can also be used as a measure of the amount of original mRNA in a tissue from which the cDNA template was made.


Pcr is used to amplify dna sequences

in excess million in additional income

in excess

Repeat Cycle

PCR is used to amplify DNA sequences

  • The polymerase chain reaction (PCR) can quickly clone a very small sample of DNA in a test tube to make enough to analyze


much C, G, T, A + million in additional incomeddC, ddG,ddT,ddA

ATGGGATTCCTAddG

ATGGGATTCCTAGddT

Electrophoresis direction

T

G

A

T

C

DNA - TGATC

  • Genome Sequencing

  • Gene sequencing is done by the synthesis of more DNA by PCR with DNA polymerase, using four different fluorescent-labelled dideoxy nucleotides together with regular nucleotides. The dd-nucleotides stop further elongation.

  • The resulting partial length DNAs are separated out by electrophoresis that can resolve the DNAs by one nucleotide.

  • The different bands are then laser scanned to tell which nucleotide has incorporated at each base of the sequence.


Protein degradation
Protein Degradation million in additional income

  • Ubiquitin attaches to protein and targets it for degradation in the proteasome


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