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 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 Modern corn
Plant biotechnology 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 • 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 • 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.
Plasmids can be cut at a specific position by restriction enzymes which often cut "on the diagonal" leaving "sticky ends" which can re-anneal.
Plant DNA can also be cut with the same restriction enzymes. • It will then stick to the ends of the cut plasmid so that the DNA is inserted into the plasmid.
Plasmids are now engineered to remove the tumorous property. • However they will still transfer into the plant DNA any genes added to the plasmid in the plasmid location that is inserted into the plant DNA.
Another transformation method is the gene gun. • 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 • 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).
Only DNA from colonies with those genes becomes radiolabelled. • To see if specific or not, to ascertain tissues, etc., compare with cDNA from another tissue. • If not there, then you have a tissue-specific DNA in the plasmid.
Gene Microarray Determines when many genes are switched on or off
Antisense • 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.
Promoter 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 • 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 • 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 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 • 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 • 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.
To ripen tomato fruit have to be exposed and respond to naturally-produced ethylene. • 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 • 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 Nutrition • Desirable human nutritional traits can be introduced that are not found in normal plants.
Golden Rice • 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 • 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 • 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 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 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 *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 Source: International Service for the Acquisition of Agri-biotech Applications
Source: International Service for the Acquisition of Agri-biotech Applications
Food ProductionEffect 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 corn – 3.5 billion pound yield increase and $125 million in additional income • 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
Oranges resistant to citrus canker • Disease-resistant sweet potatoes • Disease-resistant bananas Products in the pipeline Agronomic benefits • Pest- and disease-resistant cassava
Tomatoes enriched with flavonols • 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 pipeline 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
Bananas to deliver a hepatitis vaccine • Apples to protect against Respiratory Syncytial virus • Potatoes to protect against cholera, E. coli and Norwalk virus Products in the pipeline Functional foods
Source: Conservation 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 • 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.
RISKS AND ETHICAL QUESTIONS 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 • 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 • 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 • 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!!