development of transgenic plants with desired traits and description of herbicide resistant and pest resistant plants with the strategies

1. DEVELOPMENT OF TRANSGENIC PLANTS WITH
DESIRED TRAITS
D.INDRAJA

3. Why genetically engineered plants???
• To improve the agricultural, horticultural (or) ornamental value of a crop plant
• Resistance to certain pests, diseases and environmental conditions
• Reduction of spoilage
• Resistance to chemical treatments (Eg- Resistance to herbicide)
• Improving the nutrient profile of the crop

4. HERBICIDAL RESISTANCE PLANTS

5. Introduction
• Weeds are unwanted and useless plants that along with the crop plants.
• Weeds compete with the crops for light and nutrients, besides harbouring various pathogen.
• So it is estimated that the worlds crops yield is reduced by 10-15% due to the presence of
weeds.
• Herbicides are broad spectrum as they can kill wide range of weeds
What are Herbicides?
• Herbicides are chemicals that are sprays on the garden used to kill weeds
• They have several advantages and disadvantages.

6. An ideal herbicide is to posses the following characters:
• Capable of killing weeds with out affecting crop plants.
• Not toxic to animals and microorganism.
• Rapidly trans located with in the target plant.
• Rapidly degraded in the soil
• But none of commercially available herbicide do not fulfil the above characters
Effects
• when herbicides are sprayed on fields, they cannot distinguish from crops and weeds.
• Scientists have developed GM crops that are resistant to Herbicide resistant crops.
• Several classes of herbicides are effective for broad spectrum weed control. Act by inactivating vital
enzymes (involved in photosynthesis).
• To face this problem, herbicide resistant plants are generated

7. Herbicide resistant plants:
• Herbicide resistant plants are the plants having
the ability to reduce the herbicide-sensitive
target in the plant which binds to the herbicide.
• Genes for resistance against certain herbicides
have been introduced into crop plants so they
can thrive even when exposed to herbicides

8. Strategies for engineering herbicide resistance
1. Over-expression of the target protein
• If the target protein, on which the herbicide acts, can be produced in large quantities by the plant, then
the effect of the herbicide becomes insignificant
• It can be achieved by integrating multiple copies of genes by using a strong promoter

9. 2. Improved plant detoxification
• The plants do posses natural defense system against toxic substances
• Metabolism within the plant is one mechanism a plant uses to detoxify a foreign compound such as an
herbicide.
• A weed with the ability to quickly degrade an herbicide can potentially inactivate it before it can reach
its site of action within the plant.
3.Detoxification of herbicide using a foreign gene
• By introducing foreign gene in to crop plant that can be effectively detoxified

10. 4.Mutation of the target gene
• The target protein that herbicide is acted is modified
• This changed protein should be capable of changing the function of native protein but is resistant to
inhibition by herbicide this mutant protein is introduced in to plants and herbicide resistant plants are
developed
Plant dies
Plant grow

11. 1. Glyphosate Resistance:
• Glyphosate, initially produced and marketed by Monsanto under
the trade name Roundup®, is widely used as non-selective
herbicide. It effectively kills 76 of the world’s 78 worst weed
species.
• Majority of the weeds in the world can be destroyed by using
broad-spectrum herbicide- glyphosate. And it is a glycine
derivative
• Glyphosate (a herbicide) act by inhibiting one of the enzymes that
is necessary for the synthesis of amino acids in the chloroplast.
• This potential herbicide able to kill the weeds by acting as
competitive inhibitor of the enzyme 5-enol pyruvyl shikimate-3
phosphate synthase (EPSPS). The glycine derivative glyphosate
binds tightly to the EPSPS shikimate-3 phosphate complex.

12. Mechanism of action
• The enzyme EPSPS play a key role in the
biosynthetic pathways of the aromatic amino
acids phenylalanine, tyrosine and tryptophan.
• Glyphosate inflicts damage on the plant by
inhibiting the biosynthetic process of
aromatic amino acids and other products of
shikimate pathway
• Shikimate is one of the important
intermediate compounds formed in shikimic
acid pathway. Once shikimate is formed it is
then phosphorylated to produce shikimate-3-
phosphate.
• This acts as substrate for EPSPS. The EPSPS
binds the phosphoenol pyruvate side chain to
shikimate-3-phosphate to form EPSP.
• In the subsequent step, chorismate is formed
by the elimination of phosphate from EPSP.
Chorismate acts as a precursor of the phenolic
and indole rings of the aromatic amino acids

13. • Since it affects production of aromatic acids, protein synthesis stops growth of plants stops due to
inhibition of aromatic acids which is useful for synthesis of IAA (auxin) a plant growth hormone
• So finally it leads to death of plant occurs
• It shows less effects in animals due to absence of shikimic acid pathway they get their amino acids
through diet as they are essential type of amino acids
Strategies for glyphosate resistance
1. Glyphosate Resistance by over Expression of EPSPS Gene:
• Herbicide resistant EPSPS is found in petunia that can be resistant at even at high conc of glyphosate
• This gene is isolated and expressed in target plants and by amplification over expression is done
2. Use of mutant EPSPS gene
• An EPSPS mutant gene that is resistant to glyphosate was first detected in bacterium salmonella
typhimurium
• It was found that single base substitution C to T result in change of amino acid from proline to serine in
EPSPS . This modified enzyme can not bind to glyphosate and thus provide resistance
• This mutant gene is transferred in to tobacco plant with Ti plasmid vector. However this provides only
marginal resistance due to shikimate resistance should occur in chloroplast

14. • While glyphosate resistant EPSPS gene was produced in cytoplasm not transported in to chloroplast
• This problem is overcome by tagging with chloroplast specific transit peptide sequence
• By this it can enters in to the chloroplast and confer resistant against herbicide
Example : soyabean,tomato

15. 3. Detoxification of glyphosate
• The soil microorganisms possess glyphosate oxidase that converts glyphosate in to glyoxylate and
aminomethyl phosphonic acid
• This gene gas been isolated from soil organism ochrobacterium anthropic with suitable modification of
gene and it is introduced in to crop plants
Example : maize, soyabean
4. Combined strategy
• Providing both mutant EPSPS gene and glyphosate oxidase in to plants

16. 2. Phosphinothricin resistance/ glufosinate
• It is also broad spectrum herbicide. it is more effective against broad leaf weeds
• Phosphinothricin, produced and marketed by Hoechst AG under the trade
name Basta®
Phosphinothricin – natural herbicide
• It is a derivative of natural product bialaphos
• Certain species of streptomyces produce bialaphos (combination of
phosphinothricin with two alanine residues forming a tripeptide) by action of
peptidases it is converted in to active phosphinothricin

17. Mechanism of action
• Phosphinothricin is a herbicide that acts by inhibiting another enzyme necessary for amino acid
biosynthesis (glutamine synthetase) and nitrogen metabolism. This enzyme converts ammonia to
glutamate.
• Inhibiting the activity of this enzyme leads to rapid accumulation of ammonia within the plant cell.
Higher concentrations of ammonia are toxic to the cell. It also inhibits photosynthesis
Strategy
• Enzyme phosphinothricin acetyl transferase (also called bar gene) acetylates phosphinothricin and
deactivates it
• This enzyme was found in streptomyces hygroscopicus
Examples : maize, rice, wheat, cotton, potato, tomato, sugar beet
PAT= phosphinothricin acetyl transferase
(inactive)
(Active)

18. 3. Sulphonyl ureas and imidazolinones resistance
• The sulphonyl ureas and imidizolinones inhibit acetolactate synthase(ALS) a key enzyme in synthesis of
branched aminoacids namely isoleucine, leucine, valine
• Mutant form of this genes are identified and gained resistance to sulphonyl ureas and imadazolinones
Examples : maize, tomato, sugar beet
Environmental impact on herbicide resistant plants
• Disturbance in biodiversity due to eradication of weed plants
• May be evolution of super weeds can occur

19. PEST RESISTANCE PLANTS

20. INTRODUCTION
• About 15% of world cropyield is lost to insect or pest
• The damage to crops is mainly caused by insect larvae and to same extent adult insects. The majority of
insects that damage the crops are:
Lipidoptera-Boll worms
Coleoptera-Beetles
Homoptera-Aphids
Orthoptera-Grass hoper

21. • Chemical pesticides are used for pest control till sometimes ago but alternative to chemical pesticides are
looking due to some of the problems such as:
1. 95% of pesticide sprayed washed away from plant surface and accumulate in soil
2. Difficult to deliver pesticides to vulnerable parts of plants such as roots,stems and fruits
3. And it is not efficiently degraded in soil causing environmental pollution
4. And it is also toxic to non targeted organisms particularly humans and animals
• So prevent all these drawbacks transgenic plants with insect resistance transgenes have peen developed

22. RESISTANCE GENES FROM MICROORGANISMS
1. Bacillus thuringiensis toxin
• It was first discovered by ishiwaki
• It is a gram negative soil bacterium. The bacterium produce parasporal crystalline proteinaceous toxin
having insecticidal activity
• The protein produced by bacillus thuringiensis is referred to as insecticidal Crystalline protein. These are
the endotoxins produced by sporulating bacteria Commonly called δ endotoxins

23. Bt toxin gene
• Several strains of B.thuringiensis produce wide range of crystal proteins
• They are named cry1-cry40 based on the size and sequence similarities
• Thus total no of genes producing Bt toxin (cry proteins) are more than 100
Mode of action
• They are active against lepidopteran larvae some are specific against dipterian and coleopteran insects
• The parasporal crystal on alkali treatment they are converted in to 250K Da subunit(protoxin) by
reduction using mercaptoethanol it converts to molecular weight of about 130K Da .
• This parasporal crystal when ingested by target insect get activated by alkaline pH (7.5-8.5) and
proteolytic enzymes
• This results in conversion of protoxin to active toxin 68K Da

24. • Cry protein has three domains
• Domain-I  7α helix at N terminal
 responsible for toxin membrane insertion and pore formation
• Domain-II  3 antiparallel β sheet
 toxin receptor interactions
• Domain-III  2 anti parallel β sheet
 receptor binding and pore formation
• The active form of toxin protein gets itself inserted in to membrane of gut epithelial cells of insect. This
result in formation of ion channels through which there occurs excessive loss of cellular ATP
• As a consequence cellular metabolism ceases insect stops feeding and become dehydrated and finally
dies
• Bt toxin open cation selective pores lead to inflow of cation in to cell cause osmotic lysis and destruction
of epithelial cells and death of insect
The Bt toxin is not toxic to humans and animals since conversion of protoxin to toxin requires alkali pH
and specific proteases (which are absent in humans and animals)

26. Bt toxin as biopesticide
• Preparation of Bt spores or isolated crystal have been used as organic biopesticide for 50 years
• But it is not much success due to
• Low persistence and stability (sunlight degrades) of toxin on surface of plants
• It can not effectively penetrate to various parts particularly roots
• Cost of production is high
2. Cholesterol oxidase
• Streptomyces species contain cholesterol oxidase. The filtrate from these species are found to be toxic to
boll worm larvae
• It has been introduced in to tobacco to develop transgenic plant
3. Isopentenyl transferase gene
• This gene is present in agrobacterium tumefaciens produce a enzyme cytokines.
• It is introduced in to tobacco and tomato plants as transgene tobacco horn worm

27. RESISTANCE GENE FROM HIGHER PLANTS
• These proteins called non Bt-insecticidal proteins
1. Protease inhibitors/ proteinase inhibitors
• Proteinase inhibitors are the proteins that inhibit the activity of proteinase enzymes. Certain plants
produce these inhibitors naturally to provide defence against herbivorous insects
• When these inhibitors are injected by insects they interfere with digestive enzymes of insect and result in
nutrient deprivation causing death of insects
• It is possible to control insects by introducing proteinase inhibitor gene in to crop plants that normally
produce these proteins
2. Cow pea trypsin inhibitor gene
• Wild species of cow pea plants in Africa were resistant to attack by wide range of insects. The
insecticidal protein was trypsin inhibitor that capable of destroying insects belonging to lepidoptera,
coleoptera, and it has no effect in mammalian trypsin because it is non toxic to mammals
• CPTi gene was introduced in to tobacco,potato and oil seed rape for developing transgenic plants

28. Advantages of proteinase inhibitors
• The insects not controlled by Bt can be effectively controlled
• Use of proteinase gene along with Bt gene will help to over come Bt resistance development in plants
Disadvantages
• High level of gene are required
• The expression should be very low in plant parts consumed by humans and expression should be high in
parts utilized by insects
3. α Amylase inhibitors
• The insect larvae secrete α amylase to digest starch by blocking this enzyme activity the larvae can be
starved and killed
• This gene has been isolated from bean and expressed in tobacco against coleopteran insects
4. Lectins
• These are plant glycoproteins and provide resistance to insect by acting as toxins
• This gene has been isolated from snowdrop (Galanthus nivalis) has been transferred and expressed in
potato and tomato
• It mainly acts on piercing and suckling insects and high doses are required

29. RESISTANCE GENE FROM ANIMALS
• Proteinase (from mammals), Bovine pancreatic trypsin inhibitor,α1-antitrypsin has been isolated from
animals and introduce to plants creating them transgenic and resistant to insects