CH-12-BIOTECHNOLOGY AND ITS APPLCATIONS notes

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CHAPTER-12-BIOTECHNOLOGY AND ITS APPLCATIONS

Introduction:
 Biotechnology essentially deals with large scale production of valuable products
(primary and secondary metabolites, proteins, enzymes, coenzymes, hormones,
second messengers, vitamins, antibiotics, drugs and pharmaceuticals) useful for the
society.
 Bacteria, fungi, plants and animals whose genes are manipulated (altered) for producing
valuable products and services are called “Genetically Modified Organisms” (GMO).
 The applications of Biotechnology include therapeutics, diagnostics, Genetically
Modified Crops (GMC) food processing, bioremediation, waste treatment and energy
production.

Three critical research areas of biotechnology are:


I. Providing the best catalyst in the form of GMO or pure enzyme
II. Creating optimal conditions through engineering for a catalyst to act.
III. Down streaming processes to purify the proteins/organic compound.

12.1 Biotechnological Applications in Agriculture:


 Food production can be increased by using the following three options:
1. Agrochemical based agriculture.
2. Organic agriculture.
3. Genetically engineered crop based agriculture.
1. Agrochemical based agriculture:
 The Green Revolution is a period of agricultural transformation that took between 1940
and 1960. During this period, several other countries including India experienced triple
increase in the crop production.
 The term green revolution was given by William Gaud (1968), Director of United
States Agency for International Development (USAID) at that time.
 Norman E. Borlaug initiated and was successful in producing high yielding, disease
resistant and semi-dwarf wheat and maize varieties in Mexico. For his contributions to
green revolution, he was awarded with a Nobel peace prize in 1970 and regarded as
“Father of Green Revolution”.
 Dr. M.S. Swaminathan is known as “Father of Green Revolution in India” for his
contributions to green revolution in India.
 PITIC-62, PENJAMO-62, LERMA ROJO-64, SIETE CERROS, SONORA-64 and
SUPER- X were the semi-dwarf varieties of wheat, produced by Norman Ernest
Borlaug during the Period of Green Revolution in Mexico.
 Increased crop yield during green revolution period is partly due to the use of improved
crop rarities, but mainly due to the use of better management practices and use of
agrochemicals (Fertilizers and Pesticides).
 Now a days, green revolution is not in practice in India and other
countries because the agrochemicals have become too expensive to buy by the
farmers and they showed negative impacts on non-target organisms and environment.
 Further increase in crop yield with these exciting varieties is not possible using
conventional breeding methods.
2. Organic agriculture (Farming) - Conventional crop food Vs Organic food:
 Organic farming depends on techniques such as crop rotation, green manure,
compost and biological pest control.
 Organically grown food is superior in mineral and vitamin content to the conventional
crop food.
 Organic food is free from the toxic chemicals such as fertilizers and pesticides.
 Organically grown food tastes better than that of conventionally grown food.
 Organically grown foods will have a longer shelf time than those of conventional crops.
3. Genetic engineering crop based agriculture - Traditional or conventional breeding
methods Vs Genetic engineering crop based agriculture:
 It is not possible to transfer foreign genes between two unrelated organisms and
also between prokaryotes and eukaryotes by traditional or conventional breeding
methods.
 Bacteria, fungi, plants and animals whose genes have been altered by manipulation are
called Genetically Modified Organisms (GMO).
 The gene which is transferred to another organism artificially by genetic
engineering is called Transgene. Plants with transgenes are called Transgenic Plants
(TP) or Genetically Modified Crops (GMC).
Benefits of genetic modification:
i. Made crops more tolerant to abiotic stresses (cold, drought, salt, heat etc.).
ii. Reduced reliance on chemical pesticides (pest-resistant crops).
iii. Helped to reduce post harvest losses.
iv. Increased efficiency of mineral usage by plants which prevent early
exhaustion of fertility of soil.
v. **Enhanced nutritional value of food. Eg: Golden rice. (Vitamin A enriched
rice)
vi. **Improved shelf-life. Eg: Flavr-Savr Tomatoes.
vii. Improved resistant to pesticide, herbicide and other chemical compounds.
viii. Improved crop yield.
ix. Produced the crops which can be used for vaccine production. Pharmaceutical
compounds can be produced.
x. GM has been used to create tailor-made plants to supply alternative resources to
industries, in the form of starches, fuels and pharmaceuticals.
xi. Production of pest resistant plants, which could decrease the amount of
pesticide used.
xii. **Bt toxin is produced by a bacterium called Bacillus thuringiensis (Bt for short).
xiii. **Bt toxin gene has been cloned from the bacteria and been expressed in plants to
provide resistance to insects without the need for insecticides; in effect
created a bio-pesticide. Examples: Bt cotton, Bt corn, rice, tomato, potato and
soya bean etc.
Genetically Modified Crops (TRANSGENIC PLANTS):
 Plants with desirable characters created through gene transfer methods are called
transgenic plants.
 **Ti plasmid of Agrobacterium tumefaciens has been widely used as an effective
vector for obtaining transgenic plants.
 When a transgene is introduced into the genome of an organism, it can achieve one of
the following properties:
 Produces a normal protein or over expresses a protein of interest.
 Produces the phenotype of interest.
 Modifies an existing metabolic pathway so as to produce a modified or a new
product.
 Suppresses the expression of an existing gene endogenously.
A. INSECT RESISTANT TRANSGENIC PLANTS:
 **Examples: Bt-cotton, Bt-corn, Bt-rice, Bt-tomato, Bt-potato and Bt-soya bean etc.

Bt-cotton:
 **Some strains of Bacillus thuringiensis produce proteins that kill certain insects
such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and
dipterans (flies, mosquitoes).

 B. thuringiensis forms protein crystals during a particular phase of their growth


contain a toxic insecticidal protein.
 **Actually, the Bt toxin protein exist as inactive protoxins but once an insect ingest
the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of
the gut which solubilise the crystals.
 The activated toxin binds to the surface of midgut epithelial cells and creates pores that
cause cell swelling and lysis and eventually cause death of the insect.

 Specific Bt toxin genes were isolated from Bacillus thuringiensis and incorporated into
the several crop plants such as cotton.
 **The choice of Bt toxin genes depends upon the crop and the targeted pest, as most
Bt toxins are insect-group specific.
 The toxin is coded by a gene cryIAc named cry.
 These proteins forms crystalline inclusions on bacterial spores and hence are called
Crystal proteins (cry proteins).

 These cry-proteins are encoded by Crystal genes (Cry-genes) which arc classified mainly
into Cry-I, Cry-II, Cry-III and Cry-IV groups.
 **CryIAc and CryIIAb genes via their proteins control the cotton bollworms
(Helicoverpa sp) and CryIAb genes via its proteins control corn borers (Ostrinia
nubilalis).
Toxin coded by cry gene Useful to control
i. cry IAc and cry IIAb  Cotton bollworms
ii. cry IAb  Corn borer.
 The gram-negative, soil bacterium Agrobacterium tumifaciens is a phytopathogen
that causes crown gall disease (tumours) at the collar region in dicot plants.
 Crown galls formation is due to the T-DNA (Transfer DNA) present in tumour
inducing (Ti) plasmids of Agrobacterium tumifaciens
 T-DNA enters into plant genome, integrates with the plant genome and produces
abnormal amounts of auxins and cytokinins which result crown galls in plants.
 The size of T-DNA is approximately 10-30 kbp.
 **Ti plasmids are the largest plasmids (150-250 kbp) of the bacterial kingdom and
hence are called megaplasmids.
 The size of Ti plasmids is reduced and oncogenes (tumour forming genes) are removed
during the construction of Ti plasmid vectors.
 **The removal of oncogenes from Ti-plasmids is known as disarming.

Fig: Formation of crown galls in dicot plants.

Fig: Process of inactive cry proteins into active cry proteins.


B. PEST (NEMATODE) RESISTANT TRANSGENIC PLANTS - RNAi technology:-
 Several nematodes parasitise a wide variety of plants and animals including human
beings.
 **A nematode Meloidegyne incognitia infects the roots of tobacco plants and causes a
great reduction in yield.
 A novel strategy was adopted to prevent infestation which is based on the process of
RNA interference (RNAi).
 **RNAi is called with various names in various plants such as Post Transcriptional
Gene Silencing (PTGS) Co- suppression, Quelling and Gene Knockdown.
 RNAi technology was first developed by Andrew Fire and Craig Mello (1998) in the
nematode Canorhobditis elegans and later found in a wide variety of other organisms
including mammals.
 **RNAi takes place in all eukaryotic organisms as a method of “cellular defense”.
 **This method involves silencing of specific m- RNA (homologous gene expression) in
presence of complementary ds RNA.
 This ds RNA binds with m- RNA and inhibits the translation of m- RNA (PTGS).
 **The source of this complementary RNA (ds RNA) could be from an infection by
viruses having RNA genomes or mobile genetic elements (transposons) that replicate
via RNA intermediate.
 **Transposons or transposable elements (TE) or jumping genes are short movable
DNA sequences that can change its relative position (self-transpose) within the
genome of a single cell.
 Barbara Mc Glintock’s discovery of these jumping genes early in her career earned her a
Nobel Prize in 1983.

Strategy of nematode resistant transgenic tobacco:


 In this method, nematode specific genes (viral DNA or Transposon DNA) are
introduced into the host with the help of disarmed Ti plasmid of Agrobacterium.
 The introduction foreign DNA can produce both sense and antisense RNAs in host
plant cells.
 These two RNAs, (sense and antisense) being complementary to each other, form a ds-
RNA in host cells that initiates RNAi.
 **This unusual ds-RNA in the cells is recognized by a ribonuclease-III (endonuclease)
called as DICER.
 The Dicer specifically binds with ds-RNA and cleaves it into small ds-RNAs of 20-25
nucleotides in size. These RNAs are called SiRNA (Short interfering RNA).
 In SiRNA, the sense strand is called Passenger strand and antisense strand is called
Guide strand.
 Then, the Dicer is associated another complex known as RISC (RNA Induced
Silencing Complex). This RISC complex contains an endonuclease called
ARGONAUT (Ago).
 Then, Ago specifically degrades the passenger strand of SiRNA.
 When nematode infects the tobacco plant the guide strand of SiRNA guides the RISC
and Ago to bind with the mRNA of the parasite.
 After biding with mRNA, Ago degrades the nematode mRNA and inhibits its translation
inside the tobacco cells.
 The consequence was that the parasite could not survive in a transgenic host expressing
specific interfering RNA.
 The transgenic plant therefore got itself protected from the parasite.

Fig: Mechanism of RNA-interference (RNAi).


 Several transgenic plants have been produced to meet specific needs of agriculture and
human life.
 Some of these are given below.
C. TRANSGENIC PI.ANTS SUITABLE FOR FOOD PROCESSING TECHNOLOGY:
(Flavr - Savr – Tomato - Bruise Resistant Tomato)
i. **The ‘Flavr Savr’ tomato is the first genetically engineered whole food to be sold in
commerce.
ii. Transgenic tomato ‘Flavr Savr’ is bruise resistant i.e., suitable for storage and transport due
to delayed ripening and offers longer shelf life.
iii. Tomato fruit ripening is due to the degradation of pectin cell walls by polygalacturonase
(PG) enzyme.
iv. The expression of PG is suppressed (endogenous suppression) with the help of a transgene
by using anti-sense RNA technique.
v. These transgenic tomatoes are rich in flesh, soluble solids and flavour, show longer shelf-
life and transport life and bruise (damage)-resistant because of delayed ripening.

Fig: Anti-sense RNA technology in Tomato.


D. TRANSGENIC PLANTS WITH IMPROVED NUTRITIONAL VALUE.
Golden Rice - ‘Taipei’ variety:
i. Transgenic golden rice obtained from ‘Taipei’ is rich in vitamin A and
prevents blindness.
ii. Golden rice is a transgenic rice (Oryza sativa) having a beta carotene
(precursor of vitamin-A) gene.
iii. Beta carotene gene is made specifically to express in the endosperm of the
rice.
iv. Golden rice was created by Ingo Potrykns and Peter Beyer (2000).
v. Golden rice was created by transforming 3 beta-carotene biosynthesis genes:
Type of genes… Code for the enzyme… Isolated from …
i. psy gene  Phytoene synthase  Daffodil plant - Narcissus
ii. Lyc gene  Lycopene cyclase pseudonarcissus (eukaryotic
cell).
iii. crt 1 gene  Phytoene desaturase &  Soil bacterium - Erwinia
Carotene desaturase uredovora (prokaryotic cell)

Fig: β- carotene biosynthesis.


HIRUDIN - An Anticoagulant is expressed in Brassica napus plant:
 **Hirudin is a protein that prevents blood clotting.
 Its gene was chemically synthesized (in vitro) and was transferred into Brassica napus
where hirudin accumulates in seeds.
 Hirudin is extracted and purified and used as medicine.

E. TRANSGENIC PLANTS USEFUL FOR HYBRID SEED PRODUCTION.


i. Male sterile plants of Brassica napus are produced. This will eliminate the problem
of manual emasculation (directly used as female parent) and reduce the cost of
hybrid seed production.
ii. Male sterility was produced in crucifers (Brassicaceae) by using rol-B and rol-C genes
from the bacterium Agrobacterium rhizogenes or barnase gene from another
bacterium Bacillus amyloliquefaciens.
iii. These genes were transformed into host plant by using disarmed Ti plasmid-
expression vectors.
iv. These genes code for an RNAs which is cytotoxic to tapetal cells and inhibits pollen
development in cruciferae plants.
E. TRANSGENIC PLANTS TOLERANT TO ABIOTIC STRESSES CAUSED BY
CHEMICALS, COLD, DROUGHT, SALT, HEAT etc.,
i. **Basmati variety of rice was made resistant against biotic and abiotic
stresses.
ii. **Roundup Ready soya bean is herbicide tolerant.
 Glyphosate (Gulfosinate) (Round Up) is a herbicide which inhibits Enoyl Pyruvate
Shikimate Phosphate Synthase (EPSPS), a regulatory enzyme in the synthesis of
aromatic amino acids like phenylalanine, tryptophan and tyrosine in plants.
 When these herbicides are sprayed to eliminate weed plants, normal plants are also
affected along with weeds in most of the cases.

 Some E. coli cells possess Glyphosate resistant EPSPS genes.


 This herbicide resistant gene is introduced into the plants with the help of disarmed Ti
plasmid- expression vectors to produce glyphosate resistant transgenic plants.
 Examples: Roundup Ready Soyabean (Glycine max) is a genetically engineered
variety of glyphosate-resistant soyabean produced by Monsanto Company, USA in
1994.
G. TRANSGENIC CROP PLANTS RESISTANT TO PATHOGENS AND PESTS:
i. Transgenic papaya is resistant to papaya ring spot virus.
ii. Transgenic tomato plants are resistant to the bacterial pathogen Pseudomonas.
iii. Transgenic potato plants are resistant to the fungus Phytophthora (late blight of
potato).
 Besides these, genetically modified crops have evolved as alternative resources to
industries, in the form of starches, fuels and pharmaceuticals.
 Transgenic plants have been shown to express the genes of insulin, interferon, human
growth hormones, antibiotics; antibodies etc., these biochemicals produced by plants
are as good as or sometimes better than those produced in bacteria.
 **Utilization of plants as biofactories or bioreactors for obtaining commercially useful
products, specialized medicines, chemicals and antibodies on a large scale is described
as “Molecular farming”.

ETHICAL ISSUES:
 Ethics includes ‘moral principles’ that control or influence a person’s behaviour.
 This is connected with beliefs and principles about what is right or wrong, morally
correct or acceptable.
 This includes a set of standards which evaluate the morality of all human activities that
might help or harm living organisms.
 Thus bioethics makes a set of standards which is used to regulate our activities in
relation to the whole bio-kingdom.
 Now a day, biotechnology, particularly recombinant DNA technology, is used for
exploitation of the biological world by various ways.
 Genetically modification of organisms can have unpredictable results when such
organisms are introduced into the ecosystem.
The major bioethical concerns connected to biotechnology are:
i. There is a fear of transferring allergins from genetically modified food to
sensitive humans and animals.
ii. Due to molecular farming there is a risk of changing the fundamental nature
of vegetables.
iii. There is a risk whether they pose harmful effect on biodiversity and overall
impact on environment.
iv. There is a risk of gene pollution (genetic contamination), which may result in
the development of super weeds.
v. Use of animals in biotechnology is cruelty towards animals which causes great
suffering to them.
vi. Transfer of human genes into animals or vice versa is great ethic threat for
humanness.
vii. Biotechnology has no respect for living beings. Its only goal is to exploit them
for commercial use in benefitting human society.
 Therefore, the Indian Government has set up organizations such as GEAC (Genetic
Engineering Approval Committee), which will make decisions regarding the validity
of GM research and the safety of introducing GM-organisms for public services
BIOPATENT:
 A patent is the right granted by a government to an inventor to prevent others from
commercially using his invention.
 It is the legally granted right on intellectual property (property of mind) called
Intellectual Property Rights (IPR).
 At present, patents are being granted for biological entities and for products derived
from them. These patents are called biopatents.
Biopatents are awarded for the following:
i. New strains of microorganisms.
ii. Cell lines (cultures of animal cells that can be propagated repeatedly and
sometimes indefinitely.)
iii. Genetically modified of plants and animals.
iv. Novel biotechnological procedures.
v. Novel nucleic acid sequences.
vi. Novel products and biomolecules.
vii. Novel product applications.
 Biopatents are being granted by the Government for biological entities and products
derived from them.
 **Countries like USA, Japan, members of European Union are granting biopatents.
 Biopatents help in economic growth for individual involved and also country
concerned.
 Biopatents are being done due to their ethical and political values also.
 Biopatents sometimes become ‘absurd’ and problem causing if they are done in broad
sense.
 Example: Biopatent of all transgenic plants of Brassica family will provide monopoly (A
market structure characterized by a single seller, selling a unique product in the
market.) to few powerful corporate only.
SUPER BUG:
 Ananda Mohan Chakrabarthy, an Indian born American scientist created a
recombinant Multi Plasmid Hydrocarbon Degrading Psuedomonas putida .
 This recombinant strain was cloned with Octane, Xylene, Camphor, Naphthalene, 2,
4-D, 2,4,5-T (weedicides) and chlorobenzene degrading plasmid genes. These strains
are applied to clean oil spills in an efficient manner.
 In USA, before 1980 Me forms were not permitted to claim patent. But, in 1988, Dr. A.
M. Chakrabarthy was the first scientist to get a US biopatent for creating Oil Eating
Bug.
BIOPIRACY (Biocolonialism/biological theft)
 **Biopiracy is the term used to refer to the use of bio-resources by multinational
companies and other organisations without proper authorisation from the countries
and people concerned without compensatory payment.

 Most of the industrialized nations are rich financially but poor in biodiversity and
traditional knowledge.
 In contrast the developing and the underdeveloped world are rich in biodiversity and
traditional knowledge related to bio-resources.
 Traditional knowledge related to bio-resources can be exploited to develop modern
applications and can also be used to save time, effort and expenditure during their
commercialization.
 There has been growing realization of the injustice, inadequate compensation and
benefit sharing between developed and developing countries.
 Therefore, some nations are developing laws to prevent such unauthorized
exploitation of their bio-resources and traditional knowledge.

i. Biopiracy of Neem (Azadirachta indica):


 Seed oil from Indian neem plant was patented by USA. But, Indians use neem oil and
its products in everyday life activities. This created a long controversy and India filed a
case on this biopiracy.
 After a long fight, European Patent Office (EPO) cancelled this patent in 2000.
ii. Biopiracy of Haladi (Turmeric - Curcuma domestica):
 In 1995, Us had got a patent on turmeric powder as a wound healing agent. This
patent was cancelled in 1997, at the complaint of CSIR (Council of Scientific and
 Industrial Research), New Delhi, India.
 India had been using turmeric powder and its products as natural antibiotics.
iii. Biopiracy of Brazzein - Pentadiplandra brazzeana:
 A West African plant produces a protein called brazzein, which is approximately 2000
times as sweet as sugar and is a low – calorie sweetener but the protein brazzein was
patented in U.S.A.
iv. Biopiracy of Basmati Rice:
 Rice is an Important food grain ,the presence of which goes back thousands of years in
Asia’s agricultural history
 **There are an estimated 200,000 varieties of rice in India alone. The diversity of rice
in India is one of the richest in the world.
 Basmati rice is distinct for unique aroma and flavour and 27 documented varieties of
basmati are grown in India.
 In 1997, an American company got patent rights on Basmati rice through the US
patent and Trademark office.
 Indian Basmati was crossed with semi - dwarf varieties and claimed as an invention
.this allowed the company to sell a ‘new ‘variety of basmati, in the US abroad.
 The Indian Parliament has recently cleared the second amendment of the Indian patents
bill, that take such issues into consideration, including patent terms, emergency
provisions and research and development initiative.
BIOWAR and BIOWEAPONS:
 Biowar or biological war is the issue of biological products (harmful microorganisms
and their products) as weapons against humans or their crops or animals.
 A biological weapon or bio weapon is a device that carries and delivers target
pathological agent or toxin derived from it.
For examples:
i. Anthrax powder – Bacillus anthacis (Familiarly used)
ii. Botulin (Botulinum toxin) - Clostridium botulinum.
iii. Plague - Yersinia pestis or Pestuerella pestis.
iv. Encephalitis Virus - Alpha virus.
Some Historical Bio-wars during 20th century:
i. In world war –I Germany used Bacillus anthracis (anthrax) , Bacillus mallei
(glanders) to infect livestock and animals feed exported to Allies.
ii. In 1932-1945 Japan conducted research on Bacillus anthracis, Shigella sp, Vibrio
cholera, etc.
iii. In 1942, Britain developed strategic amounts of anthrax.
iv. In 1940s Nazi prisoners infected with Rickettsia sp., hepatitis-A. plasmodia sp.,
and treated with investigational vaccines and some drugs.

Fig: The meaning of bio-war and its tools.

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