1. Applications of plant Genetic Engineering
KOMAL JAMIM SHEHZADI
Asim Farooq
Basma Sarfraz
ASAB; NUST

2. Goals of Genetic Enginnering
Improvement of agronomically important crops.
Production of oils and lubricants.
Modification of lipid composition of seed crops.
Mnufacturing of industrially important chemicals.
And pharmeceutically active compounds.
Development of stress tolerant plants.

3. Genetically Engineered traits (Big Six)
Six genetically traits which are
Herbicide resistance
Insect resistance
Viruses resistance
Altered oil contents
Delayed fruit ripening
pollen control

4. Herbicides resistance
Weeds reduces the yields.
Compete for nutrients,sunlight,and water etc.
Herbicides reduces the impact of weeds.
Mode of action of herbicides
Herbicides disrupts disrupt one or more metabolic pathway or other vital processes to kill the weeds.

6. Disadvantage of using Herbicides
Weeds still reduce crop productivity by approximately 12%.
Some traditional crops are more sensitive to herbicides.

7. Solution Modification of crops with foreign genes.
Reduction of number of application of herbicides.
Using only single chemical.
Using biodegradable or less toxic herbicides.

9. Critics If chemical approach to weed control continues there will be
Incresed amount of chemicals in our environment.
Incresed dependency on toxic chemicals
Weeds may become resistance to herbicides.

11. Types of GE herbicides resistance
There are two types
1)modification of target (protein) so that it no longer binds to herbicides.
2) production of new protein that inactivates the herbicides.

13. Herbicides and resistance modified crops
Crops plants have been engineered to be resistance to four herbicides.
Herbicide: Resistance modified crops
Glyphosate: soyabeans,corn,canola,cotton
and sugarbeets
Glufosinate: soyabeans,canola,cotton,corn
sugarbeets rice.
Bromoxynil: cotton
sulfonlurea: cotton and flax

14. Glyphosate resistance
Most commonly used herbicide.Known as Roundup
Mode of Action
Inhibits EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) in the shikimate pathways in both plants and bacteria.
Gene encoding EPSPS in a glyphosate resistance E.coli strain was isolated.
Under control of plant promoter transferred into plant cells.
e.g soyabeans,corn,canola and cotton.

15. Glufosinate resistance
Market name Basta,and Liberty.
Mode of Action
Active ingredient (phosphinothricin) in glufosinate mimics the structure of glutamine.
This blocks the activity of glutamine synthase(required for nitrogen metabolism).
Gene is isolated from the bacterium streptomyces,whose protein products inactivates phosphinothricine in the herbicide.
e.g soyabean, corn.

16. Bromoxynil resistance
Nitrile herbicide marketed as Buctril,and bronate.
Mode of Action
Inactivates photosynthesis in plants.
Bromoxnil resistance plants are producedby transferring the gene encoding the enzyme bromoxnil nitrilase (BXN).
Gene is obtained from soil bacterium klebsiella pneumoniae.
Nitrilase inactivates bromoxynil(e.g cotton).

17. Mode of Action

18. Sulfonlurea
Environmentally friendly herbicide.Discovered by Dupont crop protection in 1975.
Mode of Action
Blocks an enzyme acetolactase required for the synthesis of amino acid(valine,lucine,and isolucine).
Herbicide resistance is developed by modifying the enzyme by gene mutation and then transferred into plants.
e.g cotton.

19. Non Resistant
Resistant

20. Insect Resistance Biopesticides
Bt-based insect resistance; successfully engineered Bt gene into different plant varieties
Protease inhibitors
PI’s are present in Storage Tissues contributing to the total of 10% of protein content (Huma and Khalid, DOB, UOK, Aug, 2007)
First observed in larvae growing on soya bean products
Transgenic plants produced; cowpea trypsin inhibitor gene, potato protease inhibitor, α-amylase inhibitor
One gene killing more insects in different plants

21. Continued…. Resistance of insects against Bt toxin and PIs
Two methods of PI resistance
High level expression of soybean-trypsin-inhibitor gene in transgenic tobacco plants failed to confer resistance against Helicoverpa armigera
Delaying insect resistance to transgenic crops
High dosage refuge strategy can be used to somehow delay the process of insect resistance (Bruce E.T. Department of Entomology, University Of Arizona)

22. Virus Resistance
Every year, tons of plants and plant products are lost due to viral infections
Different chemicals supplied to kill viral vectors
Viral DNA multiplies within the cell, thousands of copies are produced, virus infects next cell(s)/Plant(s)
Two genetic techniques are involved:
Coat protein-mediated resistance
RNA mediated virus resistance

23. Coat Protein-Mediated Resistance
Plant is genetically engineered with a particular viral coat protein gene
Roger N. Beachy from Donald Danforth Plant Science centre, St. Louis, USA carried out studies on TMV an reported that transgenically expressed CP interferes with disassembly of TMV particles in the inoculated transgenic cell. Using the known coordinates of the three–dimensional structure of TMV, mutant forms of CP were developed that had stronger inter–subunit interactions, and confer increased levels of CP–MR compared with wild–type CP

24. Continued…
Numerous transgenic plant varieties have been produced for resistance against Cucumber mosaic virus, alfalfa mosaic virus, tobacco rattle virus etc.
Resistance genes for a many other disease causing agents like fungi and bacteria have also been produced
Genes from tomato have been isolated against P. syringae and C. fulvum. A flax gene develops resistance against fungal rust disease
Transgenic varieties of Yellow Squash and Papaya

25. RNA-Mediated Virus Resistance
Directs the used of genetically altered RNA to stop viral multiplication within the cell
49 plants were generated that express the 5' untranslated region of the tobacco etch potyvirus (TEV) genome ligated to a mutated version of the TEV coat protein gene sequence that rendered it untranslatable. Eight different transgenic plant lines were analyzed for transgene expression and for resistance to TEV (Dougherty et al, DOM, Oregon State University)

26. Altered Oil Content
Various types of oils are produced in plants which differ due to difference in the fatty acid content etc
Plant oils are largely used commercially as well as domestically
Genes with altered properties of improved oil traits are cloned into soybean. These genes are selected through the particular gene promoters.
Reduced palmitic acid content, 80% oleic acid and 30% stearic acid content
Considerable change in fatty acid content

27. Delayed Fruit Ripening
Basically a hormone regulated process
Genetically engineered tomatoes were marketed, the only delayed fruit of the times
Technique not much developed
The ACC synthase gene obtained from fruits was engineered in antisense form in the papaya tissue through the particle gun. The gene translated to protein which resulted in delayed fruit ripening (Evelyn Mae, IPB, University of Philippines)

28. Flavr Savr Tomato First genetically modified food
Polygalacturonase gene was blocked
Resulted in delayed ripening of tomato
Antisense gene technology was used
FDA approved in May 1994
Produced only for 1 year

29. Pollen Control
Due to improved qualities, hybrid crops are used in agriculture
Hybridization must be in a controlled manner
Male flower part is removed before pollen release
Production of male sterile plants by inserting a gene from Bacillus amyloliquefaciens , producing barnase
Barstar gene technique is also used
Barstar pollen fertilizes male sterile barnase plant and a fertile hybrid is produced
Corn and chicory have been genetically engineered through this technique

30. GE foods- a big splash…
The initial objective was to improve crop protection.
through the introduction of resistance against plant diseases.
through increased tolerance towards herbicides.
Plant gene can be transfer via agrobacterium or Biolistics (gene gun).
By altering the genetic composition of the plant genome new proteins are introduced into the human and animal food chains. This means that human beings are now consuming products that have never before been considered foodstuffs. There is concern that these new proteins could potentially cause toxic or allergic reactions, or other health effects. Unfortunately, there is no easy way to predict the allergenic potential of GA foods since allergic reactions typically occur only after the individual consuming the food is sensitized by initial exposure to the allergen.

31. Controversies generated…
Few incidents:
In 1989, GE supplement L-tryptophan… outbreak of Eosinophilia Myalgia Syndrome (EMS)… 5000 people suffered.
In 1999, GA potatoes… poisonous to mammals… when fed to rats… caused highly weight reduction of many organs, impairment of immunological responsiveness and signs suggestive of viral infection.
Cornell University scientists reported recently that 44% of monarch butterfly larvae died within
four days when fed milkweed that had
been dusted with pollen from GA corn.
There has already been at least one known health disaster regarding genetically altered products. In 1989 the Japanese company Showa Denko marketed a GA version of the supplement L-tryptophan. After the release an estimated 5000 people suffered from an outbreak of Eosinophilia Myalgia Syndrome (EMS
Earlier in 1999, research findings that GA potatoes, spliced with DNA from the snowdrop plant and the Cauliflower Mosaic Virus (CaMv), are poisonous to mammals. When fed to rats, these GA potatoes, caused highly significant reduction in the weight of many organs, impairment of immunological responsiveness and signs suggestive of viral infection.
Cornell University scientists reported recently that 44% of monarch butterfly larvae died within four days when fed milkweed (their exclusive food) that had been dusted with pollen from GA corn, while all the caterpillars fed normal corn pollen survived. Agn a point to ponder

32. Potential risks to human health
The safety assessment of GM foods generally investigates:
direct health effects (toxicity).
tendencies to provoke allergic reaction (allergenicity).
specific components thought to have nutritional or toxic properties.
the stability of the inserted gene.
nutritional effects associated with genetic modification.
any unintended effects which could result
from the gene insertion.
Outcrossing. The movement of genes from GM plants into conventional crops may have an indirect effect on food safety and food security.
Many children in the US and Europe have developed life-threatening allergies, possibly due to the genetically engineered plant. to peanuts and other foods
That may create a new allergen or cause an allergic reaction in susceptible individuals.
possibility Involuntary produced Monsanto-engineered strains appears when GM crops were cross-pollinated.
One way to combat possible patent infringement is to introduce a "suicide gene" into GM plants.
after this the plant would be viable for only one growing season and would produce sterile seeds that do not germinate. Farmers would need to buy a fresh supply of seeds each year. However, this would be financially disastrous for farmers in third world countries who cannot afford to buy seed each year

33. Soybean…
The first genetically modified soybeans were planted in the US in 1996, that was Roundup Ready (RR) soybean, was developed by the biotech giant Monsanto.
In 2007, 216 million tones of soybeans were produced worldwide. The world’s leading soybean producers are the US(33%), Brazil (27%), Argentina (21%), and China (7%).
Each year, the EU imports approximately 40 million tones of raw soy products, primarily destined for use as cattle, swine, and chicken feed.
In addition, soybeans are used to produce numerous food ingredients , oils and additives. Lecithin, for example, is used as an emulsifier in
chocolate, ice cream, margarine, and
baked goods.
Over half of the world's 2007 soybean crop (58.6%) was genetically modified, a higher percentage than for any other crop.

34. Yield enhanced…
Even a small change in the amount of oil a soybean produces could affect industry profits marginally, the main aim was creating higher oil content without disrupting protein content.
Enhancement of soybean oil content was achieved by the introduction of a seed-specific transgenic for a DGAT2-type enzyme from the oil-accumulating fungus Umbelopsis ramanniana.
In these studies, the oil content was increased from approximately 20% of the seed weight to approximately 21.5%.
Though 1.5% does not sound like much, but given the size of the soybean industry and increase millions of dollars more in sales.

35. DuPont and Monsanto
The high-oleic soybean, developed by DuPont is one of the first in a wave of bioengineered cash crops that are being altered for nutritional purposes by removing primary antigen( protein P34) from soybean.
Currently, Monsanto has also introduced two varieties of biotech soybeans.
1. By promising to eliminate trans fats.
2.And produce oil with omega-3 fatty acid for use in yogurt, granola bars and spreads.
Modify the nutritional value of soybean oil , Fish oil
represent a move by DuPont and Monsanto to broaden the crop traits engineered in their seeds beyond simple properties like pest resistance to complex areas -- like nutritional value, drought tolerance and nitrogen fixation -- that are often influenced by multiple genes. These long-promised traits previously found little purchase in the seed giant's business plans

36. Disadvantage
For the first time since 1996, acres of Roundup Ready genetically modified soybeans could drop as more farmers decide to plant non-GMO.
“Farmers upset with Monsanto”.
Hamsters Experiment showed devastating results- made upset.
rising prices for genetically modified soybean seed are leading American farmers to plant more acres of non-GMO soybeans this year.
After feeding hamsters for two years over three generations, those on the GM diet, and especially the group on the maximum GM soy diet,
Monsanto’s Roundup Ready GM soybean seeds Technology fee. 
. By the third generation, most GM soy-fed hamsters lost the ability to have babies. They also suffered slower growth, and a high mortality rate among the pups. In 2005, Irina Ermakova, also with the Russian National Academy of Sciences.

37. Conclusion
GM foods have the potential to solve many of the world's hunger and malnutrition problems.
It helps protect and preserve the environment by increasing yield and reducing reliance upon chemical pesticides and herbicides.
Yet there are many challenges ahead for governments, especially in the areas of safety testing, regulation, international policy and food labeling.

38. Corn
Genetically engineered corn was first sold in 1996, and since then, scientists have introduced a variety of new genes.
In 2002, 32% of field in US was genetically engineered planted within 12.4 million hectares.
Most common is Bt insect resistant corn, e.g. from European corn borer and corn root worm.
Other include resistance to different herbicides. Disease-resistant corn crops may have lower levels of mycotoxins, potentially
carcinogenic compounds.
Sweet corn 3%, pop corn % , 24%, 4%
It was the second-most common GM crop.

39. Disadvantages
The study found that three strains of molded crops -- MON 810 and MON 863, which are resistant to pests, and NK 603, which is fortified to withstand weed killer -- significantly disrupted the blood chemistry of rats who ate them.

40. Canola
A genetically engineered rapeseed that is tolerant to herbicide was first introduced to Canada in 1995.
In 2008 genetically modified (GM) herbicide tolerant canola is being grown commercially in Australia for the first time.
Today 80% of the acres sown are genetically modified canola.
In 2003, Australia's gene technology regulator approved the release of canola altered to make it resistant to glufosinate ammonium, a herbicide.
Many other crops includes cotton, papaya, tomato, rice, sugar beet are also genetically modified for different purpose.
GM canola is as safe to human health, safety and the environment as non-GM canola

41. Nutritionally Enhanced Plant “Golden rice”
Rice that has been…
1. Genetically modified.
2. Contain beta carotene.
3. Produce vitamin a once consumed.
Produced by combining genetic material from
Daffodils
Peas
Japonica rice.
Reason for choosing rice Main staple food in third world countries.
Includes 118 countries in Africa and South-East Asia.

42. Developed by… Developed by two scientists in 1999.
Dr. Ingo Potrykus of the Institute for Plant Sciences at the Swiss Federal Institute of technology in Zurich, Switzerland.
Dr. Peter Beyer of the center for Applied Bioscience at the University of Freiburg in Germany.

43. How Does It Work?
The addition of 2 genes in the rice genome will complete the biosynthetic pathway:
1. Phytoene synthase (psy) – derived from daffodils.
2. Lycopene cyclase (crt1) – from soil bacteria Erwinia uredovora.
Produces enzymes and catalysts for the biosynthesis of carotenoids (β-carotene) in the endosperm.
Presence of pro-vitamin A gives rice grains a yellowish-orange color, thus,
the name ‘Golden Rice’.

44. Vitamin A :
Sources from: Fish oil, Liver Milk, Eggs Butter, Orange/Yellow fruits and vegetables.
Required for: Growth Healthy skin & cells, Good night vision.
Deficiency (VAD): Blindness ,weakened immune system, increased susceptibility to infection and cancer, anemia, deterioration of the eye tissue, cardiovascular disease and can increase the risk of maternal mortality.
Facts: Between 100 and 140 million children are vitamin A deficient each year. About 250,000 to 500,000 become blind, (1/2 dying within 12 months of losing sight), 600,000 women die from childbirth-related causes each year.

45. Limitations
Vitamin A in “Golden” rice is not sufficient for the daily recommended amount.
Even if it was enough, people require zinc, proteins, fats and other elements (which they also often lack) in their diets to convert the beta-carotene to vitamin A.
White rice is associated with culture and tradition ; golden rice may not be accepted .

46. BENEFITS May solve VAD in third world countries.
Can improve amount of Vitamin A.
May influence the acceptance of GMOs if successful.
Golden Rice may not be the complete solution to VAD, but it is a start and a contribution to the problem.
GMOs have actually reduced the use
of herbicides and pesticides.
Now, is the time to experiment and not when a natural disaster (Ex. overpopulation) plagues us and it is too late. The Truth is…

47. Free Distribution
Funded by the Rockefeller Foundation, the Swiss Federal Institute of Technology, and Syngenta, a crop protection company.
Golden Rice Humanitarian Board-responsible for the global development, introduction and free distribution of Golden Rice to target countries.

48. Molecular farming (biopharming)
It is the use of genetically engineered crops to produce compounds with therapeutic value , including antibodies, blood products, cytokines, growth factors, hormones, recombinant enzymes and human and veterinary vaccines.

49. Uses
Several PDP products for the treatment of human diseases are approaching commercialization, including
Recombinant gastric lipase for the treatment of cystic fibrosis.
Antibodies for the prevention of dental caries etc.
Potential products include the development of antigens for vaccines that might be mass-produced in plants such as corn and used to fight such diseases as cancer and diabetes.
. Considerable attention is focused, therefore, on the restraint and caution necessary to protect both consumer health and environmental biodiversity.

50. Antibodies to HIV
Today in London, UK regulators announced the approval of Europe’s first clinical trial of an anti-HIV product produced in genetically modified tobacco plants.
Plant-produced antibody designed to stop transmission of HIV when applied directly to the vaginal cavity.
The active ingredient is an antibody called P2G12 – it recognizes proteins on the surface of HIV to block infection. More specifically, it’s a monoclonal antibody made from immune cells for one specific role.

51. Advantages
Commercial scale-up involves simply planting seed rather than using costlier fermenters.
Plants do not carry pathogens that might be dangerous to human health.
On the level of pharmacologically active proteins, there are no proteins in plants that are similar to human proteins.
Like animals, plants are complex, multicellular organisms and therefore their process of protein synthesis is more similar to that of animals than those of bacteria or yeast.
Purification of the desired product from plants is often easier than from bacteria, which can be labour and cost intensive.
Transgenic plants can be grown on an agricultural scale requiring only water, minerals and sunlight.
Plants offer advantages over live animals and animal cell cultures in terms of safety, cost, time involved, and storage and distribution issues.
By microorganism current production capacity will soon be overwhelmed, it would be very expensive for the pharmaceutical companies, proteins of interest are too complex to be made by microbial systems.

52. Expressions System Expressions System Yeast Bacteria Plant viruses
Transgenic Plants
Animal Cell Cultures
Transgenic Animals
Cost of maintaining
inexpensive
expensive
Protein yield
High
Medium
Very high
Medium to high
Therapeutic risk
Unknown
yes
Type of storage
-2.0°C
RT*
N2**
N/A
Gene size (protein)
Restriction
Limited
Not limited
Production cost
Low

53. Raising industry
Today Molecular Farming is considered "big business". According to the Canadian Food Inspection Agency, in a recent report, says that U.S. demand alone for biotech pharmaceuticals is expanding at 13 percent annually and to reach a market value of $28.6 billion in Molecular Farming is expected to be worth $100 billion globally by 2020.

54. Edible vaccines Defination:
A vaccine that is based on the genetically engineered expression of an antigenic protein by an edible plant. Following consumption, the protein is recognized by the immune system.
History:
The first journal publication describing edible vaccine was published in 1992.
The studies was conducted by Mason, Lam and Arntzen at Texas A&M University.
A tobacco plant was successfully transformed and Hepatitis B surface antigens was expressed.

55. Unique Properties
It hold great promise as a cost-effective, easy-to-administer, easy-to-store, fail-safe and socioculturally readily acceptable vaccine delivery system, especially for the poor developing countries.
It involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins.
Initially thought to be useful only for preventing infectious diseases, it has also found application in prevention of autoimmune diseases, birth control, cancer therapy etc.
Edible plant-derived vaccine may lead to a future of safer and more effective immunization.
Resistance to genetically modified foods may affect the future of edible vaccines.

56. Currently searched plants vaccine
Tobacco- Human Papillomavirus.
Peanuts- Rinderpest Virus.
Papaya- Cysticercosis.
Potato- Rotavirus.
Rice- Helicobacter pylori.
Bananas- E .coli, HBV.
Tomato- Malaria virus.
That causes abdomanal tissue growth.
Cattle plague
Parasitic infection of the central nervous system.
Gastroenteritis; vomiting and diarhea
Ulcer in g.i. tract
Researchers in Ithaca, NY, are working to develop bananas to produce antigens so that they can be used as edible vaccines against diarrhea caused by the E. coli bacteria.
Recently, these researchers transformed potatoes to produce an E. coli protein that then produced immune responses in human volunteers who ate the raw potatoes.
These researchers are now trying to introduce the same antigens in raw bananas, a medium more palatable than raw potatoes.

57. Advantages of edible vaccines
Humanitarian- save millions of lives.
Cut down costs
- vaccines are given orally, no purification of protein is needed.
- elimination of medical professionals.
Renewable resource.
Palatability.

58. Unsafe but useful…
The concept is intriguing, in practice it could prove quite unsafe and risky.
Even the creator of the edible vaccine, Charles Arntzen, now concedes that the idea should be abandoned.
Research in this area now focuses on non-food crops where the vaccine is subsequently purified and packaged as a pill or capsule. It permits wider distribution and easier storage of the product.
As a result, developing countries would be capable of distributing much-needed vaccines that are impractical in a liquid form, which requires refrigeration or must be administered via injection.
Concern over allergies has been raised, as it has for GM crops, but in a reverse sense.
Many critics of GM crops argue that allergies could be triggered by inserting a gene from an allergen such as peanuts into another plant.
Despite scientists’ assertion that the inserted gene does not code for the allergen, the concern is not with the transgene, but with the host plant itself.
The plant could include contaminants that might induce an allergic response. For example, many vaccines in use now are produced in eggs, and individuals with an allergy to eggs are advised not to get such vaccines.

59. Biopolymer
BIOPOLYMERS look exactly like a conventional plastic and have similar characteristics. The difference is just that they are made from vegetable raw materials.
Biopolymers are produced from annual crops and plants like wheat, corn, potato and sugar cane and other new alternatives now being developed for the production of food packaging materials like film sheet and foam containers. i.e. PLA (Polylactic Acid).
It is difficult to imagine what life today would be like without the convenience of plastic products. But plastics need no longer be made from mineral oil derivatives.
As crude oil and gas becomes scarce and more expensive, chemists and plastics engineers all over the world look for new, sustainable raw materials and new technologies to produce polymers.

60. Types of Biopolymer Some examples:
The input materials for the production of these polymers may be either renewable (based on agricultural plant or animal products) or synthetic.
There are four main types of green polymer based respectively on:
1. Starch
2. Sugar
3.Cellulose
4.Synthetic materials
Not suitable for packaging liquids, Good oxygen barrier properties
Found as granules in plant tissue, from which it can easily be recovered
in large quantities
-Obtained from potatoes, maize, wheat
Medical applications. Examples include surgical implants which do not
require operative removal. not feasible to use polylactides for packaging
because of their high price turn made from lactose (or milk sugar) obtained from sugar beet,
potatoes, wheat, maize etc. Polylactides are water resistant
packaging for
confectionary and cigarettes wholly biodegradable
The relatively high price of biodegradable polymers of synthetic
substances,
Although these polymers are produced from
synthetic starting materials, they are fully biodegradable and
compostable.

61. Research demonstrated
Plants are able to produce PHBV using 3 genes from bacterium Alcaligenes eutrophus into canola for synthesis of PHB in seeds.
Poly b-hydroxybutyrate is produced in experimental plant Arabidopsis.
By bacterium requiring 3 bacterial genes.

62. Applications of green polymers
Every biopolymer has its own material-specific properties, e.g. barrier properties such as oxygen permeability.
The barrier properties are relevant to the choice of biopolymers for the packaging of particular products.
Bioplastics have very promising prospects for use in pesticide soil pins, for packaging in-flight catering products and for packaging dairy products.

63. The Biota Water bottle looks and feels like plastic, but it's actually made from corn.
The new, high-tech material is just as effective as plastic and it's biodegradable.
If you throw it in a landfill or compost pile, the bottle dissolves in as little as 80 days, according to Biota.