What is the NEED for Genetically Modified Organisms?

Man is dependant on animals for milk, meat, eggs, wool, leather etc.Climate change will make it increasingly difficult to feed the world.With the global population expected to reach more than nine billion by2050, however, the world might soon be hungry for such varieties.Drought, damaging storms, and very hot days are already taking a toll oncrop yields.Climate change is likely to make the problem far worse, bringing highertemperatures and, in many regions, wetter conditions that spreadinfestations of disease and insects into new areas. Drought, damagingstorms, and very hot days are already taking a toll on crop yields, and thefrequency of these events is expected to increase sharply as the climatewarms. For farmers, the effects of climate change can be simply put: theweather has become far more unpredictable, and extreme weather hasbecome far more common.

http://www.technologyreview.com/featuredstory/522596/why-we-will-need-genetically-modified-foods/

TRANSGENESISTransgenesis refers to the phenomenon of introduction of exogenous DNA into the genome to create and maintain a stable heritable character.

The foreign DNA is called the TRANSGENE.

The animal whose genome is altered is called TRANSGENIC.

IMPORTANCE OF TRANSGENIC ANIMALS OR GENETICALLY MODIFIED ANIMALS:

Improvement of Genetic characters.Models for understanding human diseases.Medical and pharmaceutical applications.Important fro improving the quality and quantity of milk, meat, eggs, and wool production.In understanding Molecular biology, genetics, immunology and cancer,

TRANSGENIC ANIMALS

First transgenic animal was a mouse.

SUPER MOUSERat gene for Growth

Hormone

Into Mouse genome

Offspring larger than Parents

CHARACTERS MOUSE RAT

Size Sparrow sized Nearly 1 pound

Best known species Common House Mouse (MusMusculus)

Black Rat (RattusRattus); Brown Rat (Rattus Norvegicus)

Genetic Differences Mice have 20 chromosome pairs & 2.6 million base pairs

Rats have 21 chromosome pairs & 2.75 million base pairs

Ears Ears are large relative to the head.

Ears are small relative to the head

tail A mouse is small and has a skinny tail.

A rat is bigger and has a thicker tail.

Produces more eggs

Researcher Friendly animal

Small in size. Easy to handle

Why Mouse???

METHODS OF INTRODUCING A FOREIGN GENE

1.Retroviral Vector Method2.Microinjection Method3.Embryonic Stem Cell Method

1. Retroviral Vector Methodretrovirus is any virus belonging to the viral family Retroviridae. All

The genetic material in retroviruses is in the form of RNA molecules,while the genetic material of their hosts is in the form of DNA. Whena retrovirus infects a host cell, it will introduce its RNA together withsomeenzymes into the cell. This RNA molecule from the retrovirusmust produce a DNA copy from its RNA molecule before it can beconsidered part of the genetic material of the host cell. Retrovirusgenomes commonly contain these three open reading frames thatencode for proteins that can be found in the mature virus. Group-specific antigen (gag) codes for core and structural proteins of thevirus, polymerase (pol) codes for reverse transcriptase, protease andintegrase, and envelope (env) codes for the retroviral coat proteins .Retroviral vectors are created by removal op the retroviral gag, pol,and env genes. These are replaced by the therapeutic gene. In order toproduce vector particles a packaging cell is essential.

Packaging cell lines provide all the viral proteins required for capsidproduction and the virion maturation of the vector. These packaging cell lineshave been made so that they contain the gag, pol and env genes. Earlypackaging cell lines contained replication competent retroviral genomes and asingle recombination event between this genome and the retroviral DNAvector could result in the production of a wild type virus. Following insertionof the desired gene into in the retroviral DNA vector, and maintainance of theproper packaging cell line, it is now a simple matter to prepare retroviralvectors . One of the problems of gene therapy using retroviruses is that theintegrase enzyme can insert the genetic material of the virus in any arbitraryposition in the genome of the host. If genetic material happens to be insertedin the middle of one of the original genes of the host cell, this gene will bedisrupted (insertional mutagenesis). If the gene happens to be one regulatingcell division, uncontrolled cell division (i.e., cancer) can occur. This problemhas recently begun to be addressed by utilizing zinc finger nucleases or byincluding certain sequences such as the beta-globin locus control region todirect the site of integration to specific chromosomal sites.

1. Microinjection Method

• Female mice administered with FSH

• Follwed by Chorionic Gonadotrophin

• Produces 30-35 eggsSuperovulation

• Mating of female and male

• Eggs removed from the Fallopian tubes after fertilizationIsolation of Eggs

• DNA is injected into the male pronucleus of the fertilized egg woth the help of micrp pippette.

• Microscope is used to identify male pronucleus (Larger one)Micromanipulation

• Eggs with transgenes are kept overnight in incubator to develop to a 2-cell stage.

• Next, implanted into Foster mother

• After 3 weeks, mother delivers pups.

Implantation

A pronucleus is the nucleus ofa sperm or an egg cell duringthe process offertilization, afterthe sperm enters the ovum, butbefore they fuse. Sperm and eggcells are haploid, meaning theycarry half the numberof chromosomes. The male andfemale pronuclei don't fuse,although their genetic materialdo. Instead, their membranesdissolve, leaving no barriersbetween the male and femalechromosomes. Theirchromosomes can thencombine and become part of asingle diploid nucleus in theresulting embryo, containing afull set of chromosomes.

EMBRYONIC STEM CELL METHOD

Inner Cell mass of Blastocyst

These are pluripotent or ES cells

Introduction of foreign DNA into ES cell

Done by Microinjection or Electroporation

Identified by Marker gene or PCR analysis

Transfected cells are Cultured

Microinjection into Blastocyst

Implant into Foster Mother

Transgenic founder Mice are produced

Mammary gland cells from a donor ewe were isolated

Subjected to total nutrient deprivation for 5 days

An ovum was taken from another ewe and its nucleus was removed

Fusion of Mammary gland cell and Ova by pulse electricity

Fused cell is totipotent and develops into an embryo

Embryo implanted in Surrogate Mother

5 months later, Dolly was born!

NEED FOR TRANSGENIC PLANTS

To improve crops with the desired traits.Resistance to diseases caused by insects, viruses and fungi and bacteria.Resistance to herbicides, temperature, drought, salinity, ozone, intense light.Imrovement of crop yield, quality, storage, longer shelf life of fruits and vegetables.Transgenic plants as bioreactors for manufacture of commercial products like proteins, vaccines and biodegradable plastic.

The major objective of plantbiotechnology is to develop plantsthat are resistant to biotic andabiotic stresses.

Biotic Stress can be described in 3 major categories:

1.INSECT RESITANCE2.VIRUS RESISTANCE3.FUNGAL AND BACTERIAL RESISTANCE

RESISTANCE TO BIOTIC STRESS:

Common Name of Pest

Scientific Name Crops damaged

Cotton bollworm Helicoverpa zea Cotton

Cotton leafworm Spodopteralittiralis

Rice, cotton, maize

Tobacco Budworm Heliothis virescens Tobacco, cotton

Tomato Fruit worm

Heliothis armigera Tomato, cotton

Brown plant hopper

Nilapavarta lugens Rice

INSECT RESISTANCE

•The damage to crops are mainly caused by insect larvae and pests.•E.g.: Bollworms, Beetles, Grasshoppers, Aphids

•95% of pesticide sprayed is washed away.•Chemical pesticides cause pollution as they are not degradable.•Toxic to plants, human and animals.

RESISTANCE GENES FROMMICROORGANISMS

Bacillus thuringiensis (Bt) toxin:

Discovered by Ishiwaki 1901

Gram negative, soil bacterium

Produces parasporal crystalline protein with insecticidal toxin i.e, ICP- Insecticidal Crystalline Protein.

These proteins are also called Cry proteins.

MODE OF ACTION OF CRY PROTEINS:

Toxin of Cry1

(Mol mass=130KDa)

ParasporalCrystal

Ingested by Target insect

Acyivates in gut. Alkaline Ph and

proteolytic enzymes

Prototoxin gets activated

Active protein gets itself inserted into membrane of gut

epithelial cells

Ion channells are formed through

which excess loss of ATP occcur

Cellular Metabolism

ceases

Insect stops feeding, gets dehydrated

Finally dies

ADVANTAGES OF TRANSGENIC PLANTS WITH BtGenes:

Bt genes could be expressed in all parts of theplant including roots, stem and fruits.Bt toxin rapidly degrade in environment.Toxin produced kills only target insect. Safe forhuman consumption.Reason: Conversion of prototoxin to toxinrequires alkaline Ph and specific proteases andthese are absent in humans and animals.

Golden rice is a variety of Oryza sativa rice produced from

genetic engineering

Main purpose is to provide pro-vitamin A to third world,

developing, countries where malnutrition and vitamin A

deficiency are common.

Who Began the Golden Rice Project?Started in 1982 by Ingo Potrykus-Professor emeritus of the

Institute for Plant Sciences

Peter Beyer-Professor of Centre for Applied Biosciences, Uni.

Of Freiburg, Germany

Funded by the Rockefeller Foundation, theSwiss Federal Institute of Technology, andSyngenta, a crop protection company.

Effects of Malnutrition Symptoms of vitamin A deficiency (VAD) include; night

blindness, increased susceptibility to infection and

cancer, anemia (lack of red blood cells or hemoglobin),

deterioration of the eye tissue, and cardiovascular

disease

Nearly 9 million children die from malnutrition each

year. A large proportion of those children die from

common illnesses that could have been avoided through

adequate nutrition

The reduced immune competence increases the

morbidity and mortality rates of children

Goals Mutate rice plants to produce carotenoids, or organic

pigments, specifically β-carotene (pro-vitamin A) in the

endosperm, the edible part of the grain

Make Golden Rice accessible locally, free of charge to farmers,

who are able to grow, save, consume, replant and locally sell

Golden Rice

Vitamin A(Retinol)

How Does It Work? The addition of 2 genes in the rice genome will complete

the biosynthetic pathway

1. Phytoene synthase (psy) – derived from daffodils

(Narcissus pseudonarcissus)

(Phytoene synthase is a transferase enzyme involved

in the biosynthesis of carotenoids. It catalyzes the

conversion of geranylgerany pyrophosphate to

phytoene.)

2. Lycopene cyclase (crt1) – from soil bacteria Erwinia

uredovora

Produces enzymes and catalysts for the biosynthesis of

carotenoids (β-carotene) in the endosperm

The psy and crt1 genes were transformed into the rice

nuclear genome and placed under the control of an

endosperm-specific promoter, so they are only expressed

in the endosperm.

The exogenous lyc gene has a transit peptide sequence

attached so it is targeted to the plastid, where

geranylgerany diphosphate formation occurs.

The bacterial crt1 gene was an important inclusion to

complete the pathway, since it can catalyze multiple

steps in the synthesis of carotenoid, 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.

Recent analysis has shown the plant's

endogenous enzymes process the lycopene to

beta-carotene in the endosperm, giving the

rice the distinctive yellow color for which it is

named. The original golden rice was called

SGR1, and under greenhouse conditions it

produced 1.6 µg/g of carotenoids.

Advantages

Golden rice give more quantity vitamin-A

Cheaper option to supply vitamin A requirement compared to

other supplementary measures

Sustainable option as once released for common cultivation can

be cultivated every growing season by farmer saved seeds,

therefore no need of yearly budgetary investment for

distribution

Health

May cause allergies or fail to perform desired effect

Supply does not provide a substantial quantity as the

recommended daily intake

Environment

Loss of Biodiversity. May become a gregarious weed and

endanger the existence of natural rice plants

Genetic contamination of natural, global staple foods

Culture

Some people prefer to cultivate and eat only white rice

based on traditional values and spiritual beliefs

DISADVANTAGE

Present scenario Farmland is decreasing..

Population is increasing in a very high rate.

Production is not increasing—stagnant.

Other problems in recent days.–

1) increased temperature

2) salinity

3) drought

4) biotic stresses

BRINJAL CULTIVATION IN INDIASolanum melongena (brinjal or eggplant) is a popular

vegetable.

Brinjal has been cultivated in India for the last 4000 years.

The area under cultivation is estimated to be around 5 lakh hectares.

The total production stands at around 82 lakh metric tonnes.

The main growing areas are in the states of West Bengal, AP, Bihar, Karnataka, Maharashtra, Orissa, Tamil Nadu & UP .

The average yields of Brinjal in India is reported to be around 16 -17 Tons per hectare.

Major Problems

Shoot damage Fruit Damage

50 - 90% of damage is caused by fruit and shoot borer

Fruit borer Damage as identified by the consumer

What is Bacillus thuringiensis (Bt)

TMMBt

cottonEM view of a purified inclusion body

EM view of Bt with spores and crystals

Endospore

Crystal

• Soil bacterium; ubiquitous

• Different strains produce their own insecticide proteins.

•The protein is called as cry protein for its crystal form.

• Each cry protein selectively affects insects belonging to a

particular order (eg.,Lepidoptera,

Coleoptera, Diptera, etc) at their very early larval stage

How bt brinjal developed? Bt brinjal is a genetically improved brinjal

developed by inserting a cry1Ac gene into brinjal genome

The Cry1Ac protein gives the brinjal plant in-built resistance against lepidopteran insects like Brinjal Fruit and Shoot Borer

Use of Bt as insecticide not new – farmers using it for over 50 years as a spray

PLR1

Non-Bt Bt

Shoot Damage

Non BtBt

Fruit damage

Non bt brinjal Bt brinjal

Ingestion (occurs while feeding plant tissues)

Solubilization (Alkalinity)

Activation (pH >9.5)

Binding (Specific receptor)

Insertion (Damage gut wall)

Pore formation

Cell lyses

D e a t h

Selective action of Bt in insects

Bt has no toxic impact in higher animals

(HUMAN) because of…..

Acidic stomach

Very low pH (∽1.5 in humans)

Absence of required receptors

Bt is safe to non-target organisms-HUMAN

DOES IT AFFECT BIODIVERSITY?? Any new gene incorporation in a genome increases its

genetic diversity.

In nature, every evolution of a new species is due to some gene incorporation through cross pollination..

The idea that Bt brinjal will destroy biodiversity is nonsensical. In fact, since brinjal has no natural resistance (to pests), the introduction of Bt brinjal will help increase genetic diversity.

What are the safety concerns?

Toxicity

Allergenicity

Out-crossing / Gene flow

Effects on non-target organisms

Environmental impact

Pest resistance

All safety issues were addressed before regulatory approval was given

Safety to non-target organisms

Feed safety studies were conducted using high

dose of Bt-cotton seed-meal / protein on:

Fish

Birds

Mammals - goats, buffaloes, cows, rabbits

Honey bees

Earthworms

Biological control agents(Lacewing, Ladybird beetles etc)

Results revealed no ill effects & Bt is safe !

Benefits to ecology and environment

1) Reduction in pesticide residues in soil and water inbrinjal fields.

2) Lesser pollution of air and local environment due todecreased use of insecticides.

3) Protection of naturally occurring predators andparasitoids and other beneficial organisms due toreduced use of insecticides.

4) Reduction in soil and ground-water contamination.

5) Safeguarding soil microflora and invertebrates fromdamage caused by unintended and excessive use of

insecticides.

Benefits to farmersFarmers are expected to benefit at

multiple levels. Some of these include:

1) Considerable reduction in cost of production by saving on cost of insecticides and lower labor cost as a result of reduced spraying.

2) Manifold increase in yield per unit area by saving fruits from damage caused by Insects.

3) Significant improvement in marketable fruits thereby increasing income per unit area.

4) Reduction in direct exposure to insecticides leading to lesser health problems.

Cotton is a natural fiber harvested from the cotton plant.

Thousands of acres globally are devoted to the production of cotton

Cotton demand a large amount of both sunshine and water,which makes its production more effecttive in tropical and sub tropical areas

As a rule, the high demands of cotton in water, soil and sun mean that the planting of this cash crop frequently replaces the cultivation of sustenance crops

Cotton also carries environmental controversy, particularly in the developing world, where dangerous pesticides are heavily employed.

The underside of the leaves of African and American species of cotton produce a sticky-sweet nectar which attracts many pests, chief among them the bollworm (the boll weevil has been largely eradicated), the larvae of a small moth, Pectinophora gossypiella. Bollworms and budworms burrow into the boll or bud shortly before harvest and feed on the fibers or lint

Cotton is typically grown using heavy levels of pesticides

Endosulfan is the primary pesticide used to control the bollworm. It is toxic to human beings if touched or ingested and particularly lethal to children

A more common alternative today is to turn togenetically-modified crops produced by companiessuch as Monsanto. The most common species, “Bt”or Bacillus thuringiensis, is advertised as fullyresistant to the common bollworm. Early use showsthat this is true, as such large portions of cottonproduction in the US and China (20 and 30%respectively) now use Bt strains.

Bt Cotton is produced by inserting a synthetic version of a gene

from the naturally occurring soil bacterium Bacillus

thuringiensis, into cotton.

The primary reason this is done is to induce the plant to

produce its own Bt toxin to destroy the bollworm, a major

cotton pest.

The gene causes the production of Bt toxin in all parts of the

cotton plant throughout its entire life span. When the bollworm

ingests any part of the plant, the Bt cotton toxin pierces its

small intestine and kills the insect.

GENETIC ENGINEERING FOR EXTENDED SHELF LIFE OF FRUITS Extends shelf life, keeps

quality intact

Long distance transport easy without damage to fruit.

Slow ripening improves flavour

BIOCHEMICAL CHANGES DURING TOMATO RIPENING

0

0.5

1

1.5

2

2.5

3

3.5

4

days

RECOMBINANT FOOD Transfer of pig genes into sheep may offend the

sentiments of Jews and Muslims.

Introduction of animal genes into food plants may invite opposition by strict vegetarians.

Few people insist that the GM foods should be specifically labelled.

Few are totally against GM !

Most of the people have opinions somewhere between these two extremes.

RELEASE OF GENETICALLY ENGINEERED ORGANISMS It is feared that release of GMOs into the environment

could have far-reaching consequences.

This is because the living GMO’s proliferate, persist, disperse and sometimes may transfer their DNA into other organisms.

It is also feared that there exists a possibility of GMOs displacing the existing organisms, besides creating new species. This may lead to severe environmental damage.

RELEASE OF GENETICALLY ENGINEERED ORGANISMS There are two broad categories of release of GMOs into the

environment:

the experimental release of GMOs into the environment -that is, the introduction of GMOs into the environment for experimental purposes, also commonly known as field or clinical trials. These types of releases are mainly carried out for the purposes of study, research, demonstration and development of novel varieties. The behaviour of the GMO in an open environment and its interactions with other organisms and the environment are studied.

•release of GMOs into the environment by placing on themarket for commercial purposes - if the results of theexperimental release are positive, the company maydecide to place the GMO on the market, that is, make itavailable to third parties either free of charge or for a fee.The GMO may be placed on the market for purposes ofcultivation, importation or transformation of GMOs intoindustrial products.

RELEASE OF GENETICALLY ENGINEERED ORGANISMS

Legal requirements and obligations Any release of GMOs into the environment must comply

with the provisions of Legal Notice 170 of 2002.

The legal requirements and obligations of this legislation include:

observing the laid down principles for the environmental risk assessment

mandatory post-market monitoring requirements, including long-term effects associated with the interaction with other GMOs and the environment,

mandatory information to the public

Legal requirements and obligations a requirement for Member States to ensure labelling

and traceability at all stages of the placing on the market

first approvals for the release of GMOs to be limited to a maximum of ten years

phasing out the release of GMOs containing antibiotic resistance marker (ARM) genes, which may have adverse effects on human health and the environment

the possibility for the Council of Ministers to adopt or reject a Commission proposal for authorisation of a GMO by qualified majority.

National regulatory systems in IndiaMinistry of Environment and forests (MoEF)

Department of Biotechnology (DBT)

There are six competent authorities to handle various issues viz.,

Genetic Engineering Approval Committee (GEAC)

Review Committee on Genetic Manipulation (RCGM)

Recombinant DNA Advisory Committee (RDAC)

Institutional Biosafety Committee (IBSC)

State Biotechnology Coordination Committee (SBCC)

District Level Committee (DLC)