Unit 2: APPLICATION OF BIOTECHNOLOGY

What is Genetic Engineering?

• Genetic engineering also called genetic modification, is the direct manipulation of an
organism's genome by removing or introducing DNA using biotechnology.
• The organism created through genetic engineering is called as genetically modified
organism.
• Creation of genetically modified organisms requires recombinant DNA. Recombinant
DNA is a combination of DNA from different organisms or different locations of the
same organism.
• If genetic material from another species is added to the host, the resulting organism is
called transgenic.
• If genetic material from the same species or a species that can naturally breed with the
host is used the resulting organism is called cisgenic.

Biotechnology in Agriculture:
• Agricultural biotechnology also known as agritech is an area of agricultural science that
involves the use of scientific tools and techniques such as genetic
engineering, molecular cloning, vaccines, and tissue culture in order to modify plants
to procure highest possible yield.
• Many plant species are usually infected with virus, bacteria, fungi, insects etc which
results in an overall reduction the crop yield.
• Several methods can be used in order to create transgenic plants: Electroporation,
Particle Bombardment, Microinjection, Agrobacterium mediated gene transfer, virus
mediated transformation.

Application of Biotechnology in Crop and Livestock Improvement

Animal Breeding:
Though traditional breeding programmes have been around for many years now, their
application remains limited. They are not very specific, as conventional breeding would result

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in a cross between two animals where many genes can get transferred simultaneously. One of
the major applications of this technique is the development of new breeds of productive cows
that can produce more nutritious milk. The milk of an ordinary cow lacks lactoferrin, an iron-
containing protein, which is significant for infant growth. A transgenic bull, Herman, has been
microinjected with the human gene forlactoferrin. The breeding of Herman and its progeny
will prove to be a new source of nutritious milk.

Pesticide-Resistant Crops
Not to be confused with pest-resistance, these plants are tolerant of allowing farmers to
selectively kill surrounding weeds without harming their crop. The most famous example of
this is the Roundup-Ready technology, developed by Monsanto. First introduced in 1998 as
GM soybeans, Roundup-Ready plants are unaffected by the herbicide glyphosate, which can
be applied in copious quantities to eliminate any other plants in the field.

Stress Resistant Plants

Less than 20% of the earth is arable land but some crops have been genetically altered to make
them more tolerant of conditions like salinity, cold, and drought. The discovery of genes in
plants responsible for sodium uptake has lead to the development of knock-out plants able to
grow in high salt environments. Up- or down-regulation of transcription is generally the
method used to alter drought tolerance in plants.

Improving Animal Nutrition

Animal nutrition is another major concern that can be addressed through biotechnological tools.
Many bacterial cells have been efficiently used to over-express proteins for medicinal
applications. Similarly, animal proteins like somatotropins can be over-expressed in bacteria
and generated in larger quantities for commercial purposes. Giving small amounts of these
proteins to animals like sheep and cows has already shown an increase in the animal’s feed
conversion efficiency. Another growth hormone – Bovine Somatotropin (BST) is given to dairy
cows to improve their milk production by as much as twenty per cent.

Synthetic Seed
Synthetic seeds are defined as artificially encapsulated somatic embryos, shoot buds, cell
aggregates, or any other tissue that can be used for sowing as a seed and that possess the ability
to convert into a plant under in vitro or ex vitro conditions, and that retain this potential also
after storage, This also helps in maintaining the uniformity.

Nutrient Supplementation

Molecular Pharming

High vitamin content in crops

Insect resistant crops

GM Food

• Genetically modified foods are foods produced from organisms that are genetically
altered by genetic engineering.

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• Genetic engineering techniques allow for the introduction of new traits as well as
greater control over traits than previous methods such as selective
breeding and mutation breeding.
• GM food includes crops that are resistance to certain pests, diseases, stressful
environmental conditions, resistance to chemical treatments, improving shelf life and
the nutrient profile of the crop.

Agrobacterium Gene Transfer

• Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in plants.
• It has the ability to introduce new genetic material into the plant cell.
• The genetic material that is introduced is called T-DNA (transferred DNA) which is
located on a Ti plasmid. This natural ability to alter the plant’s genetic makeup was the
foundation of plant transformation using Agrobacterium.

• The Agrobacterium-mediated transformation process involves a number of steps:

a. Isolation of the genes of interest from the source organism
b. The development of a functional transgenic construct including the gene of interest,
promoters, markers facilitate screening.
c. Insertion of the transgene into the Ti-plasmid
d. Introduction of the T-DNA-containing-plasmid into Agrobacterium
e. Allowing the interacting of the transformed Agrobacterium with plant cells to enable
the transfer of T-DNA into plant chromosome
f. Regeneration of the transformed cells into genetically modified (GM) plants
g. Testing for trait performance or transgene expression (Screening)

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Biolistic Method for Gene Transfer (Particle Bombardment)

• The basic principle of this method is to use a high velocity microprojectile to
incorporate the introduced gene. Microprojectiles are DNA coated gold or tungsten
particles.
• Microparticles penetrate the walls and release the exogenous DNA inside the cell where
it will be integrated in the genome.

GM Tomato: Flavr Savr Tomato

• A genetically modified tomato or a transgenic tomato is a tomato that has had
its genes modified by using genetic engineering.
• Flavr Savr Tomato was the first commercially available genetically modified food
produced by Calgene in California.
• In fresh market, tomato is usually picked up before it is ripened to facilitate long
distance shipping. Later the tomatoes are ripened by treating them with ethylene gas.
This altered the taste and texture of the fruit.
• Pectin is a part of plants cell wall that provides firmness to the plant. Fruit softening
occurs in tomatoes during ripening due to breakdown of pectin by enzyme
Polygalacturonase (PG) making it very difficult to store tomatoes.
• To combat these problems Flavr Savr tomato was created that displayed delayed
softening.
• This was achieved by artificially transferring an ‘antisense PG gene’. The gene was
excised from E. coli and transferred into the plant using Agrobacterium tumefaciens.
• This resulted in a reduction in the expression of PG enzyme thus improving the shelf
life and delayed ripening without altering the flavour and colour of tomatoes.
Advantage:
• Longer shelf life
• Easier to ship
Disadvantage:
• Safety and health risk
• Ethical problems
• Only developed countries can afford it

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GM Papaya: Rainbow
• Plant viruses cause severe disease on crop plants and result in yield loss.
• A major problem for papaya production worldwide is papaya ringspot virus (PRSV).
The virus is a non-enveloped, flexuous rod-shaped particle that is between 760–800 nm
long and 12 nm in diameter.
• There are two major types of this virus: Type P (PRSV-P) that is virulent and Type
W (PRSV-W) that is harmless.
• When infected by the virus, papaya exhibits yellowing, leaf distortion, water-soaked
spots and streaks (ringspot) on the trunk and petioles, etc.
• In order to genetically engineer resistance in plants two approaches are used: a.
Pathogen Derived Resistance B. Pathogen Independent Resistance
• To combat this problem, Dennis Gonsalves and his team decided to develop a
genetically modified papaya that was resistant to the virus. The transformed papaya was
called Rainbow Papaya.
• In order to do that, coat protein gene of PRSV was isolated cloned and introduced into
the embryos of papaya by using the gene gun (biolistic).
• This resulted in the insertion of a part of the viral gene into the plants own DNA thus
resulting in the development of pathogen-derived resistance (PDR).
• After screening it was eluded that 17 plants were able to produce the viral protein. These
plants were then propagated in the field along with PRSV.
• Out of the 17, a single line designated line 55-1 displayed continuous resistance to the
virus.
Advantage:
• Crops that are resistant to the virus were produced
• GM papayas resulted into sustainability
Disadvantage:
• Safety and health risk
• Ethical problems
• Only developed countries can afford it

BT crops
• Bt Crops are named after Bacillus thuringiensis (Bt).
• Bacillus thuringiensis is a bacterium that naturally produces a crystal protein that is
toxic to many pest insects.
• Bt crops are crops that are genetically engineered to produce the same toxin as Bt in
every cell of the plant with the goal of protecting the crop from pests.
• The cry genes of Bacillus thuringiensis (Bt gene) was found to express proteinaceous
toxin inside the bacterial cells.
• When specific insect species like Lepidoptera, Diptera etc ingest the toxin, they are
killed due to pores produced in the epithelium of the gut.
• When insects attack and eat these crop plant the Cry toxins enter the digestive system.
Since the Ph level in in the gut is high, the proteins dissolve. Cry ion channels are
formed that facilitate loss of ions which ultimately results in the death of cells by
forming pores within the gut.
• Based on their insecticidal activity against insects these proteins are classified into four
major classes: cryI, cryII, cryIII and cry IV.
• The Bt gene is isolated and introduced into a disarmed Ti plasmid of Agrobacterium
tumifaciens.

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• The genetically modified Agrobacterium tumefaciens is then allowed to infect the

desired plant.
• Many BT crops have been developed and commercialized like Bt brinjal, Bt cotton, Bt
canola, Bt cabbage etc.
Advantage:
• The toxins are highly specific and do not harm beneficial insects.
• Increase in the yield
• Less use of pesticide
Disadvantage:
• It does not have an effect of mites, spiders, maggots, rootworms etc.
• Ethical Issues
• Pollination of no GMOs with GMOs.

BT Cotton
• Bt cotton is a GMO cotton variety which produces an insecticide against bollworm.
• Bt crops are crops that are genetically engineered to produce the same toxin as Bt in
every cell of the plant, with the goal of protecting the crop from pests.
• The cry genes of Bacillus thuringiensis (Bt gene) was found to express proteinaceous
toxin inside the bacterial cells.
• When specific insect species like Lepidoptera, Diptera etc ingest the toxin, they are
killed due to pores produced in the epithelium of the gut.
• When insects attack and eat these cotton crops, the Cry toxins enter their digestive
system. Since the Ph level in in the gut is high, the proteins dissolve. Cry ion channels
are formed that facilitate loss of potassium ion which ultimately results in the death of
cells thus forming pores within the gut.
• Based on their insecticidal activity against insects these proteins are classified into four
major classes: cryI, cryII, cryIII and cry IV. Bt cotton contains cry1Ac gene that
provides resistance against bollworm.
• The Bt gene is isolated and introduced into a disarmed Ti plasmid of Agrobacterium
tumifaciens.
• The genetically modified Agrobacterium tumefaciens is then allowed to infect the
cotton plant to provide restraint against the pest.
• In 2002, a joint venture between Monsanto and Mahyco introduced Bt cotton to India.
95% of the cotton grown in India is Bt.
Advantage:
• The toxins are highly specific and do not harm beneficial insects.
• Increase in the yield
• Less use of pesticide
• Reduction in environmental pollution by the use of insecticides.
Disadvantage:
• High cost of Bt cotton seeds as compared to non Bt cotton seeds.
• Ineffective against sucking pests like aphids, whitefly etc.
• Ethical concerns

Bt Brinjal
• Brinjal is susceptible to many diseases and pests, including the eggplant fruit and shoot
borer (EFSB). EFSB is a medium-sized moth (Lepidoptera) whose feeding larvae cause
damage to the brinjal crop by boring into stems and fruits. EFSB has been described as

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the most serious and destructive pest to brinjal crops and may cause up to 90% losses
in yield.
• Bt brinjal is a GMO variety which produces an insecticide against EFSB.
• Bt crops are crops that are genetically engineered to produce the same toxin as Bt in
every cell of the plant, with the goal of protecting the crop from pests.
• The cry genes of Bacillus thuringiensis (Bt gene) was found to express proteinaceous
toxin inside the bacterial cells.
• When specific insect species like Lepidoptera, Diptera etc ingest the toxin, they are
killed due to pores produced in the epithelium of the gut.
• When insects attack and eat these crop plant the Cry toxins enter the digestive system.
Since the Ph level in in the gut is high, the proteins dissolve. Cry ion channels are
formed that facilitate loss of potassium ion which ultimately results in the death of cells
thus forming pores within the gut.
• Based on their insecticidal activity against insects these proteins are classified into four
major classes: cryI, cryII, cryIII and cry IV.
• The Bt gene is isolated and introduced into a disarmed Ti plasmid of Agrobacterium
tumifaciens.
• The genetically modified Agrobacterium tumefaciens is then allowed to infect the
desired plant.
• The cry genes of Bacillus thuringiensis (Bt gene) was found to express proteinaceous
toxin inside the bacterial cells.
• When specific insect species like Lepidoptera, Diptera etc ingest the toxin, they are
killed due to pores produced in the epithelium of the gut.
• When insects attack and eat these cotton crops, the Cry toxins enter their digestive
system. Since the Ph level in in the gut is high, the proteins dissolve. Cry ion channels
are formed that facilitate loss of potassium ion which ultimately results in the death of
cells thus forming pores within the gut.
• Based on their insecticidal activity against insects these proteins are classified into four
major classes: cryI, cryII, cryIII and cry IV. Bt cotton contains cry1Ac gene that
provides resistance against EFSB.
• The Bt gene is isolated and introduced into a disarmed Ti plasmid of Agrobacterium
tumifaciens.
• The genetically modified Agrobacterium tumefaciens is then allowed to infect the
brinjal plant to provide restraint against the pest.
Advantage:
• The toxins are highly specific and do not harm beneficial insects.
• Increase in the yield
• Less use of pesticide.
• Reduction in environmental pollution by the use of insecticides.
Disadvantage:
• High cost of Bt brinjal seeds as compared to non Bt brinjal seeds.
• Ethical concerns

Modification in Plant Quality

• With an ever-increasing global population, massive 1/3rd are suffering from hunger
with an estimation that a child dies for every two seconds worldwide from starvation;
this does not even take into account the number of people who are mal and
undernourished.

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• Thus, genetic engineering is a great promise to use technology to benefit not only the
farmers, but also societies worldwide.
• Improving the plant quality will facilitate proper nutrition and better health conditions
to the majority of the world population.

Golden rice
• It is a variety of rice produced through genetic engineering to biosynthesize beta-
carotene, a precursor of vitamin A, in the edible parts of rice.
• Two genes were inserted in order to make golden rice
1. psy (phytoene synthase) from daffodil (Narcissus pseudonarcissus)
2. crtI (carotene desaturase) from the soil bacterium Erwinia uredovora
• The psy and crtI genes were transferred into the rice nuclear genome and placed under
the control of an endosperm-specific promoter, so that they are only expressed in the
endosperm.
• Geranylgeranyl diphosphate, present in the endosperm is converted into Phytoene with
the help of the enzyme phytoene synthase.
• The bacterial crtI gene catalyses multiple steps in order to synthesis lycopene from
phytoene, while these steps require more than one enzyme in plants.
• The end product of the engineered pathway is lycopene, but if the plant accumulated
lycopene, the rice would be red.
• However, rice plant converts the lycopene into beta-carotene by using endogenous
enzymes giving the rice the distinctive yellow colour.
• In 2005, Golden Rice 2 was produced. In this rice, phytoene synthase gene was isolated
from maize instead of daffodils. This rice could produce 23 times more carotenoids
than golden rice 1.
• Golden Rice 1: 1.6 µg/g of carotenoid
• Golden Rice 2: 37 µg/g of carotenoid

Advantages:
• It benefits the poor and disadvantaged
• Comparatively cheap method to supply vitamin A than the conventional sources.
• It will help in controlling the deficiency of vitamin A
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• It overcomes ethical concerns from vegetarians and others

• Rice is a staple food in most countries therefore this can be the best approach to target a
large population.
Disadvantage:
• Nobody can predict the long-term effect of GMO
• Scientist fear that if the intake of golden rice will increase it will be loss to farmers who do
not produce it.
• It is feared that it will crossbreed with and contaminate wild rice.
• The potential development of herbicide-resistant weeds.