Transgenic crops and application

Submitted to:
Dr. K. P. Pachchigar
Submitted by:
Gami Pankajkumar B.
M.Sc.(Agri.)GPB 1st sem.
MBB501: Principles of Biotechnology

What Are Transgenic Plants?
• A transgenic crop plant contains a gene or genes which
have been artificially inserted instead of the plant
acquiring them through pollination.
• The inserted gene sequence (known as the transgene)
may come from another unrelated plant, or from a
completely different species: for example transgenic Bt
corn, which produces its own insecticide, contains a gene
from a bacterium.
• Plants containing transgenes are often called genetically
modified or GM crops, although in reality all crops have
been genetically modified from their original wild state
by domestication, selection and controlled breeding over
long periods of time.

What is the need of transgenic plants?
• A plant breeder tries to assemble a combination of genes
in a crop plant which will make it as useful and
productive as possible.
• The desirable genes may provide features such as higher
yield or improved quality, pest or disease resistance, or
tolerance to heat, cold and drought.
• This powerful tool enables plant breeders to do what
they have always done - generate more useful and
productive crop varieties containing new combinations
of genes - but this approach expands the possibilities
beyond the limitations imposed by traditional cross-
pollination and selection techniques.

Procedure in production of Transgenic crops
The novel transgene is identified ,isolated and
clonned
Designing of gene
The target plants are transformed
(by vector mediated or direct method)
Selection of transgenic plant tissue/ cells
Regeneration of the transgenic plant by the
process of plant tissue culture

TRANSFORMATION
Gene transfer or uptake of DNA refers to the process
that moves a specific piece of DNA into cell.
The directed desirable gene transfer from one organism
to another and the subsequent stable integration &
expression of foreign gene into the genome is referred
as genetic transformation.

TRANSFORMATION METHODS:
Chemical
• Lypofection
• Protoplast based
• Polyamine based
• Silicon carbide
• Pollen based
Physical
• Gene gun
• Electrophoration
• Microinjection
• Macroinjection
Biological
• Agrobacterium
• Agroinfection

Applications:
 Crop Improvement
The following traits are potentially useful to plant genetic
engineering for abiotic and biotic stress resistance.
 Genetically Engineered Traits: The Big Six.
1. Herbicide Resistance
2.Insect Resistance
3. Virus Resistance
4. Altered Oil Content
5. Delayed Fruit Ripening
6. Drought tolerence

1. Herbicide Resistance
 Over 63% of transgenic crops grown globally have
herbicide resistance traits. Herbicide resistance is achieved
through the introduction of a gene from a bacterium
conveying resistance to some herbicides.
• In modern agriculture, the herbicides have been taken the
major role in weed control. Though the uses of herbicide
offers several advantages, i.e., permitting economic weed
control, increasing the efficiency of crop production
resulting in higher crop yield and biodegradability etc., they
are endowed with several limitations as well, i.e., lack of
selectivity is one of the most important limitation.

Genetic engineering offers the scope of modifying
plants through integration of genes providing resistance
to broad spectrum herbicides.
Three approaches have been followed in the
production of herbicide resistant plants:
i) over production of herbicide sensitive biochemical
compounds;
ii) structural alteration of a biochemical target
compound resulting in reduced herbicide affinity, and
iii) detoxification or degradation of the herbicide
before it reaches the biochemical target inside the
plant cell.

A) Glyphosate Resistance
 One of the most famous kinds of GM crops are
"Roundup Ready", or glyphosate-resistant. Glyphosate,
(the active ingredient in Roundup) kills plants by
interfering with the shikimate pathway in plants, which is
essential for the synthesis of the aromatic amino acids
like a phenylalanine, tyrosine and tryptophan.
More specifically, glyphosate inhibits the enzyme 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS).
The gene encoding EPSPS has been transferred from
glyphosate-resistant E. coli into plants, which allow
plants to be resistant.

 Also some micro-organisms have a version of EPSPS
that is resistant to glyphosate inhibition. One of these
was isolated from an Agrobacterium strain CP4 (CP4
EPSPS) that was resistant to glyphosate.
 It produce an enzyme that inactivates glyphosate.
Glyphosate is rapidly metabolized by Glyphosate
oxidoreductase (GOX)

Glyphosate Sensitive Plants
Without amino
acids, plant dies
X
Phenylalanine

Glyphosate Resistant Plants
With amino
acids, plant
lives
Glyphosate
oxidoreductase
Phenylalanine

b) Glufosinate Resistance
 Glufosinate (the active ingredient in phosphinothricin)
mimics the structure of the amino acid glutamine, which
blocks the enzyme glutamate synthase.
Plants receive a gene from the bacterium Streptomyces
that produce a protein that inactivates the herbicide.
c) Bromoxynil Resistance
 A gene encoding the enzyme bromoxynil nitrilase
(BXN) is transferred from Klebsiella pneumoniae
bacteria to plants.
 Nitrilase inactivates the Bromoxynil before it kills the
plant.
d) Sulfonylurea.
 Kills plants by blocking an enzyme needed for synthesis
of the amino acids valine, leucine, and isoleucine.
 Resistance generated by mutating a gene in tobacco
plants, and transferring the mutated gene into crop
plants.

2. Insect Resistance
Progress in engineering insect resistance in transgenic
plants has been achieved through the use of insect
control protein genes of Bacillus thuringiensis.
Insect resistance was first reported in tobacco (Vaeck et
al., 1987) and tomato (Fischhoff et al., 1987). Today
insect resistant transgenes, whether of plant, bacterial or
other origin, can be introduced in to plants to increase
the level of insect resistance.
 More than 400 genes encoding toxins from wide range
of B. thuringiensis have been identified so far and
approximately 40 different genes conferring insect
resistance have been incorporated into crops.

Genes conferring insect resistance to plants have
been obtained from microorganisms: Bt gene from
Bacillus thuringiensis; ipt gene (isopentyl transferase)
from Agrobacterium tumefaciens, cholesterol oxidase
gene from a streptomyces fungus and Pht gene from
Photorhabdus luminescens.
Bt toxin gene:
Bacillus thuringiensis (Bt) is an entomocidal
bacterium that produces an insect control protein.
Bt genes code for the Bt toxin.
Most Bt toxins are active against Lepidopteran larvae
but some are specific for Dipteran and Coleopteran
insects. The insect toxicity of Bt resides in a large
protein.

The toxins accumulate as crystal proteins (CS-
endotoxins) inside the bacteria during sporulation. They
are converted to active form upon infection by
susceptible insect, thereby killing the insect by
disruption of ion transport across the brush borders/
membranes of susceptible insect.
Based on their host range Hofte and Whiteley classified
Bt toxins into 14 distinct groups and 4 classes (Hofte
and Whiteley 1989) viz.
- CryI (active against Lepidoptera)
- CryII (Lepidoptera and Diptera)
-CryIII (Coleoptera) and
- CryIV (Diptera).

Cry proteins are toxic to insects (mainly against
lepidopteran, coleopteran, dipteran, and nematodes), but
non-toxic to human and animals (BANR, 2000).

Toxic Action of Cry Proteins
When Cry protein ingested by lepidopteran insect larvae ,
a protoxin is solubilized by the high pH of the gut lumen
and solubilization of the protoxin is activated through
cleavage by digestive enzymes into a smaller (~60kDa)
fragment (Hofte and Whiteley, 1989; OECD, 2007).
Then, activated toxic fragment can binds to receptors on
the membrane of the insect’s midgut epithelial cells (Bravo
et al., 1992) and follows the activation of an apoptotic signal
cascade pathway (Zhang et al., 2006), causing formation of
pores . This leads to osmotic shock, cell lysis and insect
death (Lorence et al., 1995).

• Mode of action of Cry toxin
Mode of action of Bacillus thuringiensis in lepidopteran caterpillar: 1. ingestion
by bacteria; 2. solubilization of the crystals; 3. activation protein; 4. binding of
proteins to the receptors 5. membrane pore formation and cell disruption
(Modified from: Schünemann et al., 2014).

Credit:Transgenic plants: resistance to abiotic and biotic stresses :Akila Wijerathna-
Yapa ;Journal of Agriculture and Environment for International Development -
JAEID 2017, 111 (1): 245-275DOI: 10.12895/jaeid.20171.643

3. Virus Resistance
 Chemicals are used to control the insect vectors of
viruses, but controlling the disease itself is difficult
because the disease spreads quickly.
 Plants may be engineered with genes for resistance to
viruses, bacteria, and fungi.
 Virus diseases of cultivated plants cause substantial loss
in food, forage and fiber crops throughout the world. No
large scale methods exist for curing plants once they
have become virus infected.
 The development of molecular strategies for the control
of virus diseases has been especially successful owing
to small genomic size of plant viruses which make them
particularly amenable to molecular techniques for
cloning.

The approach is to identify those viral genes or gene
products which was present at an improper time or in wrong
amount ,will interfere with the normal functions of infection
process and prevent disease development.
Virus Coat Protein Mediated Cross Protection
The concept of cross protection is the ability of one virus to
prevent or inhibit the effect of a second challenge virus.
Transgenic tobacco expressing tobacco mosaic virus
(TMV) coat protein which resistance similar to that occurs in
viral mediated cross protection (Powell-Abel et al., 1986).
The resistance reduce numbers of infection sites on
inoculated leaves, suggesting that an initial step in the virus
life cycle has been disrupted.
 It has been demonstrated that TMV cross protection may
result from the coat protein of the protecting virus preventing
un-coating of the challenge virus RNA.

This approach has been used in several crops
like tobacco, tomato, potato, rice, maize, melons,
alfalfa, sugar beet etc.
Courtesy:Transgenic plants: resistance to abiotic and biotic stresses :Akila
Wijerathna-Yapa ;Journal of Agriculture and Environment for International
Development - JAEID 2017, 111 (1): 245-275DOI:
10.12895/jaeid.20171.643

4. Altered Oil Content
• Oil quality can be changed by genetic modification of crops
or by chemical modification of the fatty acids.
Conventional breeding has been remarkably successful in
modifying oil quality.
• Genetic engineering now offers unique opportunities to
produce novel fatty acids. The various strategies used for
modifying fatty acids are as follows:
• (i) introduction of a novel enzyme, e.g., an acyl transferase
or an acyl-ACP thioesterase, acyl-ACP desaturase, etc.,
• (ii) suppression of an enzyme activity, e.g., α acyl-ACP
desaturase),
• (iii) site-directed mutagenesis to alter the specificity, etc. of
an enzyme, e.g., acyl-ACP desaturase, etc., and
• (iv) creation of hybrid genes to generate novel enzyme
activities.

Perhaps the most successful example is the increased
lauric acid content of B. napus; a transgenic line name
'Laurical' has been released for commercial cultivation.
Lauric acid does not occur naturally in Brassica sp. oil. But
seeds of undomesticated California bay ( Ullibellularia
californica) accumulate laurate (12:0).
The gene encoding lauroyl-ACP thioesterase was isolated
from U. californica and transferred into B. napus. Some of
the transgenic lines showed upto 60% lauric acid in their
oils. Thereis some evidence that a part of the lauric acid in
transgenic B. napus is subjected to ß-oxidation.
Varieties of canola and soybean plants have been
genetically engineered to produce oils with better cooking
and nutritional properties.
Genetically engineered plants may also be able to produce
oils that are used in detergents, soaps, cosmetics, lubricants,
and paints.

5. Delayed Fruit Ripening
Allow for crops, such as tomatoes, to have a higher
shelf life.
Tomatoes generally ripen and become soft during
shipment to a store.
Tomatoes are usually picked and sprayed with the
plant hormone ethylene to induce ripening, although
this does not improve taste.
Tomatoes have been engineered to produce less
ethylene so they can develop more taste before
ripening, and shipment to markets.

• Transgenic tomato with delayed ripening:lower level of
ethylene production
• Reduced activity of the cell wall degrading enzymes, e.g.
polygalacturonases

–Produced by calgene by blocking the
polygalacturonase (PG) gene, which is involved
in spoilage. PG is an enzyme that breaks down
pectin, which is found in plant cell walls.
–Plants were transformed with the anti-sense PG
gene, which is mRNA that base pair with mRNA
which plant produces, essentially blocking the
gene from translation.
–First genetically modified organism to be
approved by the FDA, in 1994.

The Flavr Savr Tomato
(First transgenic Plant Product)
Cited by:
https://www.google.com/search?q=flavour+saver+tomato&source=lnms&tbm=isch&sa=X&
ved=0ahUKEwjqlduvbThAhW57nMBHephCCMQ_AUIDigB&biw=1500&bih=711&dpr=0.9#im
grc=oSDrw5FBxewfTM:

Use of gene for 1-aminocyclopropane-1-
carboxylic acid(ACC) deaminase:
s-adenosyl methionine (SAM)
1-aminocyclopropane-1-carboxylic acid(ACC)
ethyleneOther
metabolites
SAM hydrolase
(bacteriophage T3)

6.Drought resistance:
 A number of such genes have been identified, isolated,
cloned and expressed in plants, which are potential
sources of resistance to abiotic stresses.
These genes include Rab (responsive to abscisic acid)
and SalT (induced in response to salt stress) genes of rice;
genes for enzymes involved in proline biosynthesis in
bacteria (proBA and proC in E. coli) and plants.
In plants, proline is preferentially produced from
ornithine under normal conditions. However, under stress
it is made directly from glutamate, the first two reactions
of the pathway being catalyzed by a single enzyme ∆1-
pyrroline-5-carboxylate synthetase (P5CS) .

The gene encoding P5CS (as well as that encoding P5CR,
A1-pyrroline-5-carboxylate reductase; a non-rate limiting
enzy me of the pathway) has been isolated from soybean
and mothbean, and cloned.
 The mothbean PSCS gene has been transferred and
overexpressed in tobacco. The transgenic plants produced
10- to 15-fold more proline than the control plants.
 The leaves of transgenic plants retained a higher osmotic
potential and showed a greater root biomass under water
stress than did the control plants. Thesefindings indicates
that overexpression of P5CS in plants enhances their
tolerance to osmotic stress.

Genetically Engineered Foods
1. More than 60% of processed foods in the United States
contain ingredients from genetically engineered
organisms.
2. 12 different genetically engineered plants have been
approved in the United States, with many variations of
each plant, some approved and some not.
3. Soybeans.
 Soybean has been modified to be resistant to broad-
spectrum herbicides.
 Scientists in 2003 removed an antigen from soybean
called P34 that can cause a severe allergic response.

4. Corn
 Bt insect resistance is the most common use of
engineered corn, but herbicide resistance is also a
desired trait.
 Products include corn oil, corn syrup, corn flour,
baking powder, and alcohol.
 By 2002 about 32% of field corn in the United
States was engineered.
5. Canola.
 More than 60% of the crop in 2002 was genetically
engineered; it is found in many processed foods,
and is also a common cooking oil.

6. Cotton.
 More than 71% of the cotton crop in 2002 was
engineered.
 Engineered cottonseed oil is found in pastries,
snack foods, fried foods, and peanut butter.
7.Nutritionally Enhanced Plants—Golden Rice:
 More than one third of the world’s population relies
on rice as a food staple, so rice is an attractive target
for enhancement.
 Golden Rice was genetically engineered to produce
high levels of beta-carotene, which is a precursor to
vitamin A. Vitamin A is needed for proper eyesight.
–

 Golden Rice was developed by Ingo Potrykus and
Peter Beyer, and several agencies are attempting to
distribute the rice worldwide
 Other enhanced crops include iron-enriched rice
and tomatoes with three times the normal amount
of beta-carotene
 Rice endosperm synthesize geranyl-geranyl
pyrophosphate which can be converted into ß-
carotene by 3 enzymes.
 Phytoene desaturase and Lycopene ß Cyclase
derived from daffodil(Narcissus pseudonarcissus)
and Carotene desaturase from Erwinia uredova.

 The transgenes providing these enzyme activities were
transferred into rice through Agrobacterium mediated
transformation.
 The resulting rice looks yellowish orange in colour
and contain ß-carotene kwnon as ‘Golden Rice’.

Advantages of Transgenic crops:
• Resistance to biotic
stress(insects,diseases,viruses)
• Resistance to abiotic stress
• Herbicide resistance
• Improved quality
• Male sterile crops
• High yield
• Cheap to maintain disease resistant
species
• High adaptability

Disadvantages
• Allergic reactions
• Reducing the numbers of pest insects in
farmland and impacting biodiversity.
• Significant negative impact on
wildlife(wider effects on food chains )
• Chance of developing resistance in weeds
(uncontrollable weeds)

• References books:
1.Introduction to plant biotechnology- H.S. Chawla
2.Biotechnology, expanding horizons - B.D.Singh

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