Transgenic approaches to disease resistance (1).pptx

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TRANSGENIC
APPROACHES TO
DISEASE RESISTANCE
Submitted to,
Dr. Aghil Soorya. A
Assistant Professor
Dept of Botany
St Teresa’s college
Ernakulam
Submitted by,
Silpa Selvaraj
Roll no:14
I Msc Botany
St Teresa’s college
Ernakulam
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◤ TRANSGENIC PLANTS
▪ Transgenic plants are the those plants whose genome is integrated with a desired
foreign DNA using genetic engineering techniques.
▪ The aim is to introduce a new trait to the plant which does not occur naturally .
▪ The inserted gene sequence is known as the transgene.
▪ The purpose of inserting a combination of genes in a plant, is to make it as useful
and productive as possible.
▪ This process provides advantages like improving shelf life, higher yield, improved
quality, pest resistance, tolerance to heat, cold and drought resistance, against a
variety of biotic and abiotic stresses.
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Presented by Silpa Selvaraj
Transgenic approaches to disease resistance

◤ HISTORY
▪ The first transgenic plants were reported in 1983.
▪ It was developed through the insertion of nptII bacterial
antibiotic resistance gene into tobacco.
▪ Since then, many recombinant proteins have been
expressed in several important agronomic species of plants
including corn, tomato, potato, banana, alfalfa and canola.
▪ Tobacco plants were generally used, however potatoes and
bananas are also considered, for the purpose of vaccines for
human beings.
Alfalfa seeds and sprouts
Canola
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DEVELOPMENT OF TRANSGENIC PLANTS
Most transgenic plants are generated by ;
☆ Biolistic method (Particle gun method)
☆ Agrobacterium tumefaciens mediated transformation method.
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Presented by Silpa Selvara

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1. BIOLISTIC METHOD
▪ In this method, DNA of interest is coated with Gold or Tungsten, which is
subsequently shot into the target cells, under high pressure using a gene gun.
▪ The DNA separates from the coated metal and it integrates into the plant genome
inside the nucleus.
▪ This method has been applied successfully for many crops, especially monocots,
like wheat,maize etc for which transformation using Agrobacterium tumefaciens has
been less successful.
▪ The major limitation of this technique is that the DNA is randomly introduced into
the target cell.
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Particle gun method
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2. Agrobacterium tumefaciens MEDIATED
TRANSFORMATION METHOD
▪ It involves the use of soil-dwelling, gram negative bacteria,
known as Agrobacterium tumefaciens .
▪ It cause crown gall disease.
▪ In addition to the bacterial genome, it contains an extra
chromosomal DNA called Ti plasmid which can replicate on
its own .
▪ The Ti plasmid contains major three regions; T DNA region,
Virulence region and Opine catabolism region.
Crown gall disease
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◤ Ti PLASMID
1. T-DNA region:
▪ This region has the genes for the biosynthesis of auxin (aux), cytokinin (cyt) and
opine (ocs) and is flanked by left and right borders.
▪ These three genes-aux, cyt and ocs are referred to as oncogenes, as they are
the determinants of the tumor phenotype.
2.Virulence region or vir region
▪ Vir region codes for proteins involved in T-DNA transfer.
▪ At least nine vir-gene operons have been identified. These include vir A, vir G, vir
B1, vir C1, vir D1, D2, D4, and vir E1 and E2.
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3. Opine catabolism region:
▪ The opine catabolism region is the region from where the bacteria sources
its nutrients for the whole process.
▪ Opines are derivatives of amino acid or sugar phosphates that can be
catabolized to use in the form of nutrients.
▪ The types of opines found in Ti-plasmid are nopaline and octopine types.
Besides the above three, there is ori region that is responsible for the origin of
DNA replication.
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▪ The importance of this plasmid is that, it contain T-DNA region where the genes that
cause crown gall formation are removed and replaced with the genes of interest,
which can be transferred to a plant cell.
▪ In addition, Agrobacterium is capable of transferring large fragments of DNA very
efficiently.
▪ It also helps in the transfer of DNA pieces with defined ends.
▪ One of the biggest limitations of Agrobacterium is that, it has narrow host range
▪ This method works well for the dicotyledonous plants like potatoes, tomatoes and
tobacco .
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T-DNA transfer and integration:
1. Signal induction to Agrobacterium:
▪ The wounded plant cells release certain chemicals- phenolic compounds
and sugars which are recognized as signals by Agrobacterium.
2. Attachment of Agrobacterium to plant cells:
▪ The Agrobacterium attaches to plant cells through polysaccharides,
particularly cellulose fibres produced by the bacterium.
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3. Production of virulence proteins:
▪ To start with, signal induction by phenolics stimulates vir A which in turn activates
vir C.
▪ This induces expression of virulence genes of Ti plasmid to produce the
corresponding virulence proteins (D1, D2, E2, B, etc.).
▪ Certain sugars (e.g. glucose, galactose, xylose) that induce virulence genes have
been identified.
4. Production of T-DNA strand:
▪ The right and left borders of T-DNA are recognized by vir D1/vir D2 proteins.
▪ These proteins are involved in the production of single-stranded T-DNA, its
protection and export to plant cells. The ss T-DNA gets attached to vir D2.
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5. Transfer of T-DNA out of Agrobacterium:
▪ The ss T-DNA-vir D2 complex in association with vir G is exported from the bacterial
cell.
▪ Vir B products form the transport apparatus.
6. Transfer of T-DNA into plant cells and integration:
▪ The T-DNA-vir D2 complex crosses the plant plasma membrane.
▪ In the plant cells, T-DNA gets covered with vir E2. This covering protects the T-DNA
from degradation by nucleases.
▪ T-DNA-vir D2-vir E2-plant protein complex enters the nucleus through nuclear pore
complex.
▪ Within the nucleus, the T-DNA gets integrated into the plant chromosome .
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Agrobacterium tumefaciens mediated transformation method
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HERBICIDE RESISTANT PLANTS
▪ Weeds are unwanted & useless plants that grow along with the crop plants .
▪ They compete with the crops for light & nutrients.
▪ It is estimated that the worlds 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 called herbicides.
▪ The plants which develop resistance against herbicides are called herbicide resistant
plants.
▪ Eg : Avena fatua (Wild oat), Conyza canadensis (Horseweed)
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▪ An ideal herbicide is to posses the following characters : -
1. Capable of killing weeds with out affecting crop plants .
2. Not toxic to animals & microorganisms .
3. Rapidly degraded in the soil .
▪ Commercially available herbicides cannot discriminate weeds from the
crop plants . For this reason , crops are also affected by herbicides
hence there is need to develop herbicide resistance plants
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Glyphosate
▪ It is a broad spectrum herbicide .
▪ Effective against 76 of world’s worst 78 weeds .
▪ Less toxic to animals .
▪ Easily degradable.
▪ The American company (Monsanto) market it as round up .
▪ Mechanism of Glyphosate action :-
Capable of killing the plants in low concentrations
Rapidly transported to growing tissues .
Glyphosate is a competitive inhibitor of EPSPS(5-enolpyruvylshikimate-
3-phosphate synthase)
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• EPSPS is blocked by the herbicide
glyphosate.
• This prevents the plant from producing
the amino acids necessary for its growth
thereby blocking the growth of the plant.
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Enol

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STRATEGIES FOR ENGINEERING
HERBICIDE RESISTANCE
▪ Over expression of EPSPS gene
▪ Use of mutant variety of EPSPS gene .
▪ Detoxification of herbicide by a foreign gene.
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1.Over expression of EPSPS gene :-
▪ An over expression of EPSPS gene was detected in Petunia .
▪ Gene from Petunia was isolated & introduced into other plants .
▪ The transgenic plants can tolerate glyphosate 2 -4 times higher than that
required to kill wild type weed plants.
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Petunia

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2.Use of mutant EPSPS gene
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3.Detoxification of glyphosate :-
▪ The soil microorganisms possess an enzyme glyphosate oxidase
reductase that converts glyphosate to glyoxylate and
aminomethyl phosphonic acid .
▪ The gene was isolated from ochrobactrum anthropy & was introduced
in to crop plants. e.g: oil seed rape
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PEST RESISTANT PLANTS
▪ There is clearly a benefit to farmers, if transgenic plants are developing resistance to a
specific pest.
▪ There may also be a benefit to the environment, if the use of pesticides is reduced.
▪ Transgenic crops, containing insect resistance genes from Bacillus thuringiensis, have
made it possible to reduce significantly the amount of insecticide, applied on cotton in
the USA.
▪ Eg ; Bt cotton, Bt brinjal etc.
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INSECT RESISTANT PLANTS
▪ Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect pests.
▪ Its lethal effect is mediated by a protein toxin it produces.
▪ Through recombinant DNA methods, the toxin gene can be introduced directly into the
genome of the plant, where it is expressed and provides protection against insect
pests of the plant.
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◤ VIRUS RESISTANT PLANTS
▪ TMV resistant tobacco and tomato plants are produced by introducing viral coat
proteins.
▪ Other viral resistant transgenic plants are
▪ (a) Potato virus resistant potato plants
▪ (b) RSV resistant rice
▪ (c) YMV resistant black gram
▪ (d) YMV resistant green gram.
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FUNGAL & BACTERIAL RESISTANT PLANTS
▪ The human lysozyme gene, was introduced into tobacco (Nicotiana tabacum) by the
Agrobacterium-mediated method.
▪ The introduced human lysozyme gene was highly expressed and the gene product accumulated in
the tobacco plants. They showed enhanced resistance against the fungus Erysiphe
cichoracearum.
▪ Growth of the bacterium Pseudomonas syringae pv. Tabaci was also strongly retarded in the
transgenic tobacco, and the disease symptoms were reduced to 17% of that observed in the wild-
type tobacco.
▪ Thus, the introduction of a human lysozyme gene is an effective approach to protect crops against
both fungal and bacterial diseases.
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◤ GOLDEN RICE
▪ Rice is an important crop for nearly 4 billion people all around the world. At the same
time it is deficient in vitamin A.
▪ Deficiency of vitamin A leads to eye disorders.
▪ The most effective solution for this problem is to genetically engineer rice in such way
that beta carotene ,the precursor of vitamin A will be produced in rice endosperm.
▪ Such beta carotene rich rice grains will have a characteristic yellow or orange colour. It
is called as golden rice.
▪ It was developed by two research teams, one headed by Dr.Ingo Potrykus and the
other by Dr. Peter Beyer.
▪ They genetically engineered a Japanica rice variety by incorporating three genes,
two from daffodil (Narcissus pseudonarcissus) and other one from bacterium Erwinia
uredovora.
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Advantages of golden rice;
▪ It primarily benefits the economically poor and the disadvantaged section of the
society.
▪ It can be sown every year.
▪ Satisfies urgent nutritional needs.
▪ Sustainable and cost free solution for vitamin A deficiency.
▪ No side effects.
▪ Neither adversely affects natural biodiversity nor does it reduce agricultural
biodiversity.
▪ No negative environmental impact or any conceivable risk to consumer’s health.
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Objections against golden rice
▪ Although golden rice is claimed to be potentially effective in solving the problems related
to vitamin A deficiency, there are strong apprehensions about its acceptability.
▪ Environmental groups like Greenpeace, describe it as a “Trogen horse” which would
open the door for other GM crops, gradually displacing the traditional crops.
▪ It is argued that golden rice is quite insufficient to solve the problems of vitamin A
deficiency all by itself.
▪ Only a mixed diet consisting of several beta carotene containing items would solve the
problem of vitamin A deficiency.
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FLAVR SAVR TOMATO
▪ Among the first GM crops to reach the market in 1994 were the Flavr Savr
tomatoes.
▪ Flavr Savr tomatoes were launched by the biotechnological company, Calgene
(California).
▪ It is a transgenic variety of tomato, characterized by reduced and delayed fruit
softening.
▪ They are very tasty, remain attached to the plant for much longer time and retain their
flavour and freshness for a long time even after harvesting.
▪ They are developed by applying the antisense RNA technology which minimizes the
production of fruit ripening enzyme polygalacturonase.
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▪ Decrease in the synthesis of this enzyme reduces and delays fruit softening. This enables
the marketing of fresh tomatoes.
▪ Flavr Savr tomatoes were first launched and marketed under the brand name
“Macgreger tomatoes”.
▪ Even though no health risks were found, it was not widely accepted by the consumer
sector .
▪ The major reasons for this are high price, unattractive texture of skin, undesirable
compositional change, objection from environmentalists etc
▪ Gradually it was withdrawn from the market.
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Flavr Savr tomatoes
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◤
Bt CROPS
▪ Bt crops are pest resistant transgenic crops which contain the bacterial Bt toxin
gene integrated to their genome.
▪ Bt toxin is a toxin produced by Bacillus thuringiensis bacterium.
▪ This bacterium was discovered by Japanese bacteriologist Shigetane Ishiwata in
1901 in diseased silkworms.
▪ It has adverse effect on insect larvae.
▪ The bacterium produces an insecticidal crystal protein (ICP) called as delta
endotoxin.
▪ The gene which encodes this endotoxin is called cry gene.
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▪ When the ICP reach the midgut of the larvae, it gets proteolytically cleaved,
releasing active fragments.
▪ This fragments interact with the receptors in the midgut membrane.
▪ This results in the flow of cations into the cells which leads to the lysis and
destruction of the midgut membrane.
▪ The endotoxin even at very low concentrations are lethal to the insect larvae.
▪ But they are non toxic to other animals like mammals.
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Bt COTTON
▪ Bt cotton is a genetically engineered form of natural cotton.
▪ Bt toxin has to be ingested by the insect larvae to cause death.
▪ The toxin becomes active at the high pH of the insect gut.
▪ It then attacks the gut cells, punching holes in the lining.
▪ Bt spores spill out of the gut and germinate inside the insect causing the death
of the insect within a couple of days.
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Why Bt cotton is a good choice over
conventional cotton?
▪ Cotton is the most important commercial crop of India, It is called as white gold.
▪ In India it is grown in 5 percent of the total crop area but it uses upto 55 percentage of
all pesticides.
▪ Bt cotton has high resistance to pest attacks, gives high yield, environment safe and
cost effective.
▪ Since the plants produce toxins in their tissues, there is no need to spray synthetic
pesticides.
▪ Bt toxins do not destroy beneficial insects.
▪ Exposure of farm workers and non target organisms to pesticides also get very much
reduced.
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Bt BRINJAL
▪ Bt Brinjal is the first Genetically Modified food crop in India.
▪ It is developed by inserting a gene called cry1Ac from a soil bacterium
called Bacillus thuringiensis through Agrobacterium- mediated gene
transfer.
▪ Bt Brinjal was developed by Maharashtra Hybrid Seeds Company
(Mahyco).
▪ It gives resistance against insects such as the Brinjal Fruit and Shoot
Borer (Leucinodes orbonalis)
▪ Once a fruit and shoot borer larva feed on Bt brinjal plants, the cry1Ac
protein reacts with the alkaline insect gut, It disrupts the insect’s
digestive process, causing paralysis and eventually the death of the
larvae.
Fruit and shoot borer
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Advantages of Bt Brinjal
▪ Bt brinjal has been found to be safe for human consumption and safe for the
environment.
The Cry1Ac endo-toxin is a protein that breaks down when cooked. It is active
only in an alkaline medium, and since humans consume brinjal only when cooked,
it will not interfere with digestion.
▪ Additionally, as the stomach is acidic, the digestive process will not be affected by
the introduction of the Cry1Ac toxin. The toxin breaks down into common amino
acids in the digestive system, which are part of the normal diet and are neither
toxic nor allergic.
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▪ The Cry1Ac endo-toxin would only damage the fruit and shoot borer gut
which is alkaline.
▪ Bt brinjal increases marketable yields, thus resulting in higher incomes for
farmers.
▪ Farmers will be able to continue to save and re-use their seeds for the
hybrids varieties.
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CONCERNS ABOUT Bt Brinjal
▪ Environmental Impact: The use of pest-resistant Bt Brinjal may lead to the extinction
of species considered important for farm ecology.
Bt Brinjals can become costly: Companies that own Bt Brinjal seeds can charge a
high price for their procurement, as it is predicted that its high yield and increased
shelf life will fuel demand. If that is the case, it might lead to a scenario where small
and marginal farmers can ill afford Bt brinjal seeds.
▪ Scientists are of the opinion that Bt brinjal poses a serious health risk, as its
resistance to antibiotics can make medicines ineffective. There also might be adverse
effects of forming allergens and other anti-nutritions in foods.
M.S Swaminathan, the father of India’s Green Revolution, had asked for an
independent study on the long toxicity of Bt Brinjal before approving it for commercial
use.
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Some interesting transgenic plants
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Transgenic colorful corns
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Are blue java
bananas
transgenic???
Blue Java Banana
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Blue java bananas are
transgenic
Blue java bananas are
not transgenic
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Blue Java which owes its name to the characteristic blue
colour of the peel when it is unripe. However, no genetic
modification is involved – it’s all work of mother nature. Its
peculiarity is not so much in the colour, but precisely in its
sweet taste that is said to have the consistency of an ice
cream and the flavour that recalls vanilla.
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REFERENCE
1. https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.frontiersin.org
/articles.
2. https://www.google.com/url?sa=t&source=web&rct=j&url=http://www.igntu.ac.in/eCo
ntent/IGNTU-eContent-317364151984-M.Sc-EnvironmentalScience-2-
ManojkumarRai-Environmentalmicrobiologyandbiotechnology.
3. Kikkert, J. R., Vidal, J. R., & Reisch, B. I. (2005). Stable transformation of plant cells
by particle bombardment/biolistics. Methods in molecular biology (Clifton, N.J.), 286,
61–78.
4. Nakajima, H., Muranaka, T., Ishige, F., Akutsu, K., & Oeda, K. (1997). Fungal and
bacterial disease resistance in transgenic plants expressing human lysozyme. Plant
cell reports, 16(10), 674–679.
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THANK YOU
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