The document discusses the genetic manipulation of herbicide resistance in transgenic plants. It describes how foreign DNA can be introduced to plants to acquire new traits like herbicide resistance, which is then passed down to offspring. Specifically, it outlines strategies for developing resistance to glyphosate herbicide by overexpressing or mutating the target EPSP synthase enzyme or introducing alternative detoxification genes. The environmental impacts of widespread adoption of herbicide-resistant crops are also summarized.

1. THE GENETIC MANIPULATION OF
HERBICIDE RESISTANCE

2. TRANSGENIC PLANTS
 Plants containing introduced DNA, are known as
transgenic plants or genetically engineered plants.
 They have acquired a new trait from the introduced
DNA and inherit the trait for many generation.
 In some transgenic plants, the introduced DNA
blocks the normal functioning of certain original
genes of the plants.

3.  Goal of transgenics (involves introduction,
integration, and expression of foreign genes) is to
improve the crops, with the desired traits.
 Some of them are
 Resistance to biotic stress i.e. resistance to disease
caused by insect, viruses, fungi and bacteria
 Resistance to abiotic stresses- herbicides,
temperature (heat, chilling, freezing), drought, salinity,
ozone, intense light
 Improvement of crop yield, and quality e.g. storage,
longer shelf life of fruits and flowers.
 Transgenic plants with improved nutrition.
 Transgenic plants as bioreactors for the manufacture
of commercial products e.g. proteins, vaccines, and
biodegradable plastics

4. ENVIRONMENTAL STRESSES TO PLANTS
 Various types of external stresses influence the
plant growth
 Based on their characters external stresses are
grouped into
1. Biotic stress
2. Abiotic stress

5.  All most all the stresses, either directly or indirectly,
lead to the production of reactive oxygen species
(ROS) that create oxidative stress to plants.
 The production of ROS in high amount damages the
cellular constituents of plants which is associated with a
reduction in plant yield.
 Hence the major objective of plant biotechnology is to
develop plants that are resistant to biotic and abiotic
stresses.

6. Scientists have developed transgenic plants having-
 Herbicide resistance
 Insect resistance
 Virus resistance
 Improved storage proteins
 Improved oils and fats
 Male sterility
 Altered flower colors
 Environmental stress resistance
 Enhanced photosynthetic efficiency
 Novel proteins

7. NEED AND USE OF HERBICIDE IN MODERN
AGRICULTURE
 Weeds/wild herbs are unwanted and useless plants
that grow along with the crop plants
 Weeds compete with crops for light and nutrients,
besides harboring various pathogens
 It is estimated that the world’s crop yield is reduced
by 10-15% due to the presence of weeds
 To tackle the problem of weeds, modern agriculture
has developed a wide range of weed killers which are
collectively referred to as herbicide

8.  The use of herbicides is a well-established necessity in
modern agricultural practice.
 In recent years, the demand for environmental safety
has made it necessary to develop less toxic
compounds, and competition in this field has resulted in
the development of several new, better and safer
herbicides, including a number of selective compounds.
 In general, majority of the herbicides are broad spectrum
as they kill a wide range of weeds

9. Characteristics of a good/ ideal herbicide
 Capable of killing weeds without affecting crop plants
 Non-toxic to animals and microorganisms
 Rapidly translocate within the target plant
 Rapidly degrade in the soil
But none of the commercially available herbicides fulfill all the
above criteria.
The major limitation is that they cannot discriminate weeds from
crop plants
Examples: Glyphosate, Sulfonylurea, Phosphinothricin

10. HERBICIDE RESISTANT PLANT
 Major for this reason, the crops are also affected by
herbicides, hence the need to development of
herbicide- resistant plant
 So this plants provide an opportunity to effectively kill
the weeds (by herbicides) without damaging the crop
plants
 Many transgenic plants with herbicide resistance have
been developed by using genetic engineering.
 Such transgenic plants tolerate the herbicide an be
safe in the field, when the herbicides are applied in the
field.

11. GENETIC MANIPULATION OF HERBICIDE
RESISTANCE
 The use of herbicides to control weeds plays a
pivotal role in modern agriculture.
 More progress has been achieved in herbicide
resistance as single genes govern the resistance.
 Three approaches have been followed:
I. Over-production of a herbicide sensitive biochemical
target
II. Structural alteration of a biochemical target resulting
in reduced herbicide affinity,
III. Detoxification –degradation of the herbicide before it
reaches the biochemical target inside the plant cell.

12.  Resistance to glyphosate and sulfonylurea herbicides
has been obtained by using genes coding for the
mutant target enzymes 5-enolpyruvylshikimate-3-
phosphate synthase(EPSPS) and acetolactate
synthase (ALS) respectively. These two enzymes are
involved in amino acid biosynthesis pathway.
 Resistance to glyphosate has been achieved by using
gox gene (glyphosate oxidase), which detoxifies the
herbicide. This gene has been isolated from
Achromobacter bacterial strain.

13.  Plants resistant to glufosinate ammonium have
been obtained by using genes derived from
bacteria that encode phosphinothricin
acetyltransferase (PAT), which converts
phosphinothricin into its acetylated form.

14. GLYPHOSATE RESISTANCE
 It is a Glycine derivative
 It acts as a broad-spectrum herbicide and is
effective against 76 of the world’s worst 78 weeds
 It is less toxic to animals and is rapidly degraded by
micro-organisms
 It has a short half-life
 The American chemical company Monsanto
markets glyphosate as Round Up

15.  Example: Glyphosate resistant petunia, tobacco,
tomato, corn, etc.
 Glyphosate is the active ingredient of many
commercial herbicides such as Glyphos, Tumble
weed, Roundup, etc.
 It is a competitive inhibitor of EPSP synthetase,
blocking the bio-synthesis of tryptophan and phenyl
alanine.

16.  EPSP sythease gene was isolated from the bacterium
Salmonella typhimurium and glyphosate resistant cell
line of petunia and introduced into plant cells using Ti
plasmids.
 Transgenic plant cells are resistant to the herbicide
glyphosate by over producing the enzyme EPSP
synthetase or by producing glyphosate tolerant EPSP
synthetase.

18. MECHANISM OF ACTION OF GLYPHOSATE
 Glyphosate is rapidly transported to the growing
points of plants
 It is capable of killing the plants even at a low
concentration
 Glyphosate acts a competitive inhibitor of the
enzyme 5-enoyl-pyruvylshikimate 3-phosphate
synthase (EPSPS)
 EPSPS is a key enzyme in shikimic acid
pathway that results in the formation of aromatic
amino acids ( trypatophan, phenylalanine and
tyrosine), phenols and certain secondary
metabolites.

19.  The enzyme EPSPS catalyses the synthesis of 5-
enoylpyruvylshikimate 3-phosphate from shikimate
3-phosphate and phosphoenol pyruvate
 Glyphosate has some structural similarity with the
substrate phosphoenol pyruvate.
 Consequently, glyphosate binds more tightly with
EPSPS and blocks the normal shikimic acid
pathway
 The lethal action of glyphosate is primarily because
of the starving of the cells of aromatic amino acids,
resulting in the disruption of protein synthesis.
 In this way, the herbicide glyphosate inhibits the
biosynthesis of aromatic aminoacids and other
important products.

22. GENETIC MANIPULATION OF GLYPHOSATE
HERBICIDE RESISTANT CROP:
 Three possible methods of producing glyphosate-
resistant plants by genetic engineering
1. Transfer of a glyphosate-sensitive EPSP synthase
under the control of a powerful promoter causing
overexpression of the protein
2. Transfer of a gene which codes a mutated,
glyphosate-resistant EPSP synthase
3. Transfer of gene which provides an alternative for
EPSPS

23. TRANSFER OF A GLYPHOSATE-SENSITIVE EPSP
SYNTHASE CAUSING OVEREXPRESSION OF THE
PROTEIN
 Using a petunia cell line which overproduces
EPSP synthase by gene amplification, a cDNA of
the EPSP synthase gene was isolated and a
chimeric gene under the control of the 35S
cauliflower mosaic virus (CaMV) promotor was
constructed.
 CaMV 35S promoter has been transformed into
other crop plants and over expressed
 Petunia cells transformed by this construction
exhibited a 40-fold increase in EPSP-synthase
activity.
 The transgenic plants were significantly tolerant to
glyphosate.

25. GENETIC MANIPULATION USING CP4-EPSPS
GENE
 Since 1985 many researchers have identified many
genes from different sources with same or different
mode of action which help plants combat glyphosate
 CP4-EPSPS gene which is isolated from
Agrobacterium strain.
 CP4, is best suited for transformation as it is insensitive
to glyphosate
 CP4-EPSPS and sensitive EPSPS have identical
binding site for substrate glyphosate.
 CP4- EPSPS have high affinity for PEP then glyphosate,
which allow the shikimate pathway to function normally,
as it ‘bypass’ the endogenous EPSPS.

26. Strategy for the development of glyphosate-resistant

27. STRATEGIES FOR ENGINEERING HERBICIDE
RESISTANCE
 Resistance to these broad-spectrum herbicides can
be generated by expressing transgenes in them
that serves any one of the following purposes:
1. Over expression of the target protein
2. Improved plant detoxification
3. Detoxification of herbicide by using a foreign
gene
4. Mutation of the target protein

28. 1. Over expression of the target protein
 The target protein, being acted by the herbicide can be
produced in large quantities so that the affect of the
herbicide becomes insignificant.
 Overexpression can be achieved by integrating
multiple copies of the genes and/or by using a
strong promoter.
 Example: Petunia cDNA clone and an E. coli gene which
encodes a highly glyphosate tolerant EPSP synthase
yielded transgenic tobacco plants which showed higher
tolerance to glyphosate than plants over - expressing the
wild type EPSPS gene.

30. 2. Improved plant detoxification
 The plants do posses natural defense system
against toxic compound
 Detoxification involves the conversion of toxic
herbicide to non-toxic/ less toxic compound
 By enhancing the plant detoxification system, the
impact of the herbicide can be reduced

31. 3. Detoxification of herbicide by using a foreign gene
 By introducing a foreign gene into the crop plant, the
herbicide can be effectively detoxified
Herbicide Gene product Mechanism
Glyphosate Glyphosate
oxidoreductase
Detoxification
Bromoxynil Nitrilase Detoxification
2,4-D Monooxygenase Detoxification
Glufosinate N-acetyl transferase Detoxification
Gene based examples of herbicide resistance in crop
plants which shows detoxification

32. 4. Mutation of the target protein
 The target protein which is being affected by the
herbicide can be suitably modified
 The changed protein should be capable of
discharging the functions of the native protein gene
but is resistant target protein gene is identified, it can
be introduced into the plant genome and thus
herbicide resistant plants can be developed
 For success in the development of herbicide resistant
plants, good knowledge of the target protein and
the action of herbicide is required

33. ADVANTAGES OF HERBICIDE RESISTANT
CROPS
 It is an excellent weed control, hence higher crop
yields
 Its flexibility because of its possibility to control the
weeds later in the plant’s growth
 It also reduces the numbers of sprays in a season
therefore reduction in the fuel use
 It’s also reduces soil compaction because of less
need to go on the land to spray
 Because of low toxicity compounds it do not remain
active in the soil

34. COUNTRIES THAT HAVE APPROVED MAJOR HT
CROPS FOR FOOD, FEED AND/OR CULTIVATION
Crop Countries
Cotton Argentina, Australia, Brazil, Canada, China, Colombia,
Costa Rica,EU, Japan, Mexico, New Zealand, Paraguay,
Philippines, Singapore, South Africa, South Korea, USA
Maize Argentina, Australia, Brazil, Canada, China, Colombia,
EU,Honduras, Indonesia, Japan, Malaysia, Mexico, New
Zealand, Panama, Paraguay, Philippines, Russian
Federation, Singapore, South Africa, South Korea,
Switzerland, Taiwan, Thailand, Turkey,USA, Uruguay
Potato Australia, Canada, Japan, Mexico, New Zealand,
Philippines, South Korea, USA
Wheat Australia, Colombia, New Zealand, USA
Rice Australia, Canada, Colombia, Honduras, Mexico, New
Zealand, Philippines, Russian Federation, South Africa,
USA

35. THE ENVIRONMENTAL IMPACT OF HERBICIDE-
RESISTANT CROPS
 The development of genetically modified herbicide-
resistant crops has undoubtedly contributed to
increase in the yield of crops.
 Farmers particularly in the developed countries (USA)
have started using these GM crops. Thus, the proportion
of herbicide from 17% in 1997 to 68% in 2001.
 The farmer is immensely benefited as there is a
reduction in the cost of herbicide usage.
 It is believed that the impact of herbicide –resistant
plants on the environment is much lower than the
direct use of the herbicide in huge quantities. There
are however, other environmental concern.

36.  Disturbance in biodiversity due to elimination of
weeds
 For example, the expansion of GM herbicide-tolerant
corn and soy, which are twinned with herbicides, has
destroyed much of the habitat of the monarch butterfly
in North America.
 Rapid development of herbicide-resistance weeds
that may finally lead to the production of super
weeds.
 In the past 20 years, 37 weed species have developed
resistance to the herbicide glyphosate.

37.  Increased Herbicide Use: Cultivation of GM
herbicide-tolerant crops has pushed up the use of
herbicides such as glyphosate.
 Herbicide sales in Canada rise by 199% between 1994
and 2016.
 A transgene that confers herbicide resistance represents a
new potential threat to the environment.
 In some crops such as canola (Brassica napus L.), the transgene
can introgress into weedy relatives.
 Its ecotoxicity as have side effects on soil
microorganisms and agricultural flora and fauna