Arbaminch College of health science

Department of Pharmacy
1

 Course Name: Immunological and Biological

Products
 Course Code: Phar3171
 Course ECTS: 3
 Course Hrs: 81 hrs
 Academic year: 2024

By; Fitsum.T( B.pharm, MSc in PSCM)

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 COURSE OBJECTIVES:
2

 This course is designed to introduce the students with the

role of biotechnology and allied technologies in the
development of a range of pharmaceutical products of
modern biotechnology, &
 Application of recombinant DNA derived drugs
(immunological and biological products) in
pharmaceutical care of a patient.

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 OUTLINE:
3

 Introduction to biotechnology

 Branches of biotechnology

 Stages of biotechnology development

 Career in biotechnology

 Applications of biotechnology

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 INTRODUCTION TO BIOTECHNOLOGY
4

 According to United Nations Convention on

Biological Diversity :
 “It’s any technological application that uses biological
systems, living organisms, or derivatives to make or
modify products or processes for specific use.”
 United States Congress’s Office of Technology
Assessment defined biotechnology as:
 “Any technique that used living organism to make or
modify a product, to improve plants or animals or to
develop microorganisms for specific uses”.
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 Introduction to Biotechnology…….
5
 In its most general meaning, it is the use of biologic
processes to create a product for human use and
benefit.
 An integrated application of scientific and technical
understanding of a biologic process or molecule to
develop a useful product.

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6
Introduction to Biotechnology…….
 Biologic processes of interest include cellular
activities such as;
 Protein synthesis,
 DNA replication & transcription,
 Receptor-ligand interactions &
 Fermentation of bacteria, yeast, and mammalian cells.

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 BIOTECHNOLOGY DEVELOPMENT
7

 Today, when people use the term biotechnology;

 the application of modern methods of manipulation of
DNA to make a product.
 In fact, biotechnology is ancient, providing the basis
for making a wide range of products, including bread,
cheese, beer & wine.
 In Ethiopia Tela, Tej, Injera & others produced by
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using the biotechnology.
 Biotechnology development……
8

 Egypt ~5000 B.C made wine from grapes.

 Ancient biotechnology relied on fermentation, the

breakdown of organic molecules by microorganisms,

particularly sugars, into simpler compounds, often

carbon dioxide.
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 Biotechnology development……

 In 1680; Leeuwenhoek observed yeast under

microscope (200x).
 In 1684; protozoa/fungi.
 Between 1866 and 1876; Pasteur established that
yeast and other microbes were responsible for
fermentation.
 In 1858; Rudolf Virchow, concluded that cell is the
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basic unit of life.
 GOAL OF BIOTECHNOLOGY
10

 To understand more about the processes of inheritance

and gene expression.
 To provide better understanding & treatment of various
diseases, particularly genetic disorders.
 To generate economic benefits, including;
 Improved plants & animals for agriculture.
 Efficient production of valuable biological molecules.
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 BRANCHES OF BIOTECHNOLOGY

 Pharmaceutical products:
Antibiotics, vaccines

 Aquatic & marine  Genetically modified

plants & animals

 Industrial biotechnology
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11
 Multidisciplinary nature of Biotechnology:

12

Biotechnology: Blend of Technologies

 Applied sciences
 Computer applications,
 Engineering,
 Agriculture,
 Pharmacy,
 Microbiology, Immunology, Molecular biology,
 Biochemistry, Virology, Physiology,
 Bioinformatics &
 Genomics, Proteomics, Metabolomics.

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 Multidisciplinary nature of Biotechnology……
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 APPLICATIONS OF BIOTECHNOLOGY
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 STAGES OF BIOTECHNOLOGY DEVELOPMENT

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A) Ancient biotechnology; early history as related to food and

shelter; includes domestication.

B) Classical biotechnology; built on ancient biotechnology;

fermentation promoted food production and medicine.

C) Modern biotechnology; manipulates genetic information in

organism; genetic engineering.

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 A) Ancient Biotechnology:
16

 History of domestication and agriculture.

 Nomadic lifestyle due to migratory animals and edible plant

distribution.

 People settled, sedentary lifestyles evolved.

 Cultivation of wheat, barley and rye (seed collections).

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 Ancient Biotechnology…….
17

 Sheep and goats: milk, cheese, butter and meat.

 Fermented foods and beverages.

 Louis Pasteur published his findings on the direct link

between yeast and sugars.

 Production of baker’s yeast: Saccharomyces cerevisiae.

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 B) Classical Biotechnology:

 Industry today exploits early discoveries of the fermentation

process for production of huge numbers of products.

 Different types of beer, Vinegar, Glycerol, Acetone, Butanol,

Lactic acid, Citric acid, Antibiotics (Penicillin, Cephalosporin's).

 Chemical transformations to produce therapeutic products.

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18
 C) Modern Biotechnology:

 Cell biology: Structure, organization and reproduction.

 Biochemistry: Synthesis of organic compounds, cell extracts for

fermentation (enzymes versus whole cells).

 Genetics: Reappearance of Gregor Mendel’s findings in 1866 &

1900s, theory of inheritance (ratios dependent on traits of parents),

theory of transmission factors.

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19
 What Are the Areas of Biotechnology?

 Organismic biotechnology; uses intact organisms; does not alter

genetic material.

 Molecular biotechnology; alters genetic makeup to achieve

specific goals.

 Transgenic organism; an organism with artificially altered genetic

material.
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20
 Areas of Biotechnology…..
21

 Pharmaceutical biotechnology; use of living organisms

such as microorganisms, to create new pharmaceutical

products, or safer & more effective versions of conventionally

produced pharmaceuticals, more cost-effectively.

 Use of biological systems to produce drug substances.

 Exploitation of modern technologies to produce drug

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substances from biological systems.
 Pharmaceutical biotechnology……
22

 Fermentation technology was first used in World War I to

ferment corn starch and produce acetone for manufacturing

explosives.

 with the help of Clostridium acetobutylicum.

 In the 1950’s, cholesterol was converted to cortisol and sex

hormones by reactions such as microbial hydroxylation.

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 Pharmaceutical biotechnology……
23

 In 1953- Watson and Crick;

 Determined the structure of DNA,

 DNA replication model &

 DNA bases made up of purine & pyrimidine.

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 Pharmaceutical biotechnology……..
24

 In 1975, scientists developed monoclonal antibody technology.

 Used as a tool to characterize and purify proteins that would

selectively bind to respective antibodies with high specificity.

 These tools for preparation and characterization of recombinant

products are essential for developing macromolecules into

therapeutic products.
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 BIOTECHNOLOGY TIMELINE

25

 1982: Humulin, the first recombinant insulin.

 1983: Amplification of DNA with PCR technique.

 1986: Recombivax; the first recombinant vaccine for human

use, Roferon A; the first recombinant anticancer interferon, &

OKT-3; the first recombinant immunosuppressant monoclonal

antibody for therapeutic use.

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 Biotechnology Timeline…
26

 1988: Patent awarded for first transgenic mouse.

 1990: First clinical trial for gene therapy, launch of

human genome project.

 1994: ReoPro; the first chimeric human-murine

monoclonal antibody, BRCA-1; the first breast cancer
susceptibility gene is discovered.

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 Biotechnology Timeline…
27

 1995: Popularization of combinatorial chemistry.

 1996: Commercialization of gene-chip technology.

 2000: First draft of human genome completed.

 2002: Development of first commercial protein chip.

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 Introduction to Genetic Engineering or (rDNA)
technology:
28

 The process of manipulating genes outside of a cell’s/

organism’s normal reproductive process.
 It generally involves the isolation, manipulation and
subsequent reintroduction of stretches of DNA into cells.

 The collection of techniques for manipulating DNA and

making recombinant molecules is known as rDNA
technology /genetic engineering.

 The deliberate modification of an organism’s genetic

information by directly changing its nucleic acid genome is
called genetic engineering or recombinant DNA technology or
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gene cloning.
 Genetic Engineering or (rDNA) technology……
29

 ‘rDNA technology’ is a term used interchangeably with

‘genetic engineering’.
 rDNA is a piece of DNA artificially created in vitro which
contains DNA (natural or synthetic) obtained from two or
more sources.
 It usually undertaken in order to confer on the recipient cell
the ability to produce a specific protein, such as a
biopharmaceutical.
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 BASIC PRINCIPLE

30

 Select the desired gene (or genes) to be inserted into the

organism.
 Cut DNA molecules into fragments with special (restriction)
enzymes.
 Splice(join) the fragments together in the desired combination.

 Introduce the new DNA into a living cell for replication.

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 FUNDAMENTAL TECHNIQUES
31

 The fundamental techniques involved in working with rDNA involves;

 Isolating or copying a gene.
 An efficient method for cleaving and rejoining phosphodiester bonds on
fragments of DNA;
 Inserting the precise gene into a transmissible vector that can be
transcribed, amplified, and propagated by a host cell's biochemical
machinery.
 Transferring it to that host cell; and facilitating the transcription into

mRNA and translation into proteins.

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 RECOMBINANT DNA TECHNOLOGY TOOLS
32

 DNA to be cloned
 Enzyme
 Polymerases - produce new strands of DNA.
 Endonucleases -they can cut DNA at specific locations
 Ligase - it can attach segments of double stranded DNA
 Vector
 Plasmids, Cosmids etc
 Host
 Bacteria, Yeast, etc
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 Overview/Steps:
33

Isolate / select DNA

Cut with restriction enzymes(cleavage)

Ligate into cloning vector

Transform rDNA molecule into host cell

Each transformed cell will divide many, many times to form a

colony of millions of cells, each of which carries the rDNA
molecule (DNA clone).
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01/31/202433333333331131
 A)Isolating donor and plasmid DNA
1) Isolating donor DNA
35

 Crude isolation of donor (foreign) DNA is accomplished by

isolating cells disrupting lipid membranes with detergent
destroying proteins with phenol or protease degrading
RNAs with RNase leaving DNA at the end.
 Plants and some microorganisms, initial disruption of the cell
wall may require application of physical or other vigorous
disruptive influences.

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 Isolating donor DNA…...
36

 The gentlest method of cell lysis usually involves incubation

with cell-wall-degrading enzymes, and detergent.
 Following cellular disruption,
 Solvent-based extraction/precipitation.
 E.g, Shaking in the presence of phenol results in
extraction of;
 denatured proteins into the phenol phase,
 nucleic acids remaining in the aqueous phase.
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 Isolating donor DNA…...
37

 Further purification may be achieved by selective

precipitation of the nucleic acids using ethanol or isopropanol

as precipitant.

 If DNA is required, then the RNA may be removed by the

enzyme ribonuclease, which selectively degrades RNA.

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 2) Isolating plasmid DNA
38

 Density gradient centrifugation is the method employed for the

isolation of bacterial plasmid.
 During this technique, a tube containing solution of Cesium
Chloride and Ethidium Bromide is spun at high speed in
centrifuge.
 This distributes the Cesium Chloride in a continuous concentration
gradient, with the top segment containing the least concentration
of Cesium Chloride and the bottom high concentration.
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 Isolating plasmid DNA…
39

 Because plasmid DNA and chromosomal DNA have different

densities in the solution of Cesium Chloride and Ethidium

Bromide, each DNA will form a separate band.

 The band containing plasmid can be collected and used for

genetic engineering

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40 01/31/2024
 B) Cutting of donor DNA and plasmid
41

 Both the plasmid DNA and donor DNA must be cleaved in such a way that the
sticky ended fragments are formed.
 DNA can be cut into large fragments by;
 mechanical shearing or Enzymes
 Restriction endonucleases
 are bacterial enzymes that cleave internal phosphodiester bonds of a DNA
molecule.
 are the scissors of molecular genetics
 more than 500 restriction endonucleases have been discovered, and these
react with more than 100 different cleavage sites. 01/31/2024
 Cutting of donor DNA and plasmid……
42

 There are three major classes of restriction endonucleases.

 Their grouping is based on:

 the types of sequences recognized,

 the nature of the cut made in the DNA, and

the enzyme structure.

 Accordingly, the enzymes are grouped as Type I, II and III
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43

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 Cutting of donor DNA and plasmid……
44

 Type I and III restriction endonucleases are not useful for

gene cloning.
 They cleave DNA at sites other than the recognition sites
and thus cause random cleavage patterns.
 Type II endonucleases are widely used for mapping and
reconstructing DNA in vitro.
 they recognize specific sites and cleave just at these sites.

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 Cutting of donor DNA and plasmid……
45

 Restriction endonucleases are named for the organism in

which they were discovered, using a system of letters and

numbers.

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 Cutting of donor DNA and plasmid……
46

 Type II restriction endonuclease recognize short symmetric

DNA sequences of 4–8 bp.
 Six base pair cutters are the most commonly used in
molecular biology research.
 Restriction endonucleases catalyze the hydrolysis of
phosphodiester bonds in palindromic DNA sequences.
 formation of 5′-PO4− and 3′-OH termini

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 Cutting of donor DNA and plasmid……
47

 Some enzymes, such as EcoR1, generate a staggered cut, in which the

single-stranded complementary tails are called “sticky” or cohesive ends

because they can form hydrogen bond to the single stranded

complementary tails of other DNA fragments.

 Other type II enzymes, such as SmaI, cut both strands of the DNA at the

same position and generate blunt ends with no unpaired nucleotides

when they cleave the DNA.

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 C) Joining DNA
48

 If two different kinds of DNA are treated with same restriction

endonucleases(RE), then each DNA will have same sticky
ends.
 DNA ligase is the glue of molecular genetics that holds the
ends of the DNAs together.
 DNA ligase creates a phosphodiester bond between two DNA
ends. This joining of linear DNA fragments together with
covalent bonds is called ligation.
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 Joining DNA…….
49

 If restriction-digested fragments of DNA are placed together

under appropriate conditions, the DNA fragments from two
sources can anneal.
 hydrogen bonding between the complementary base pairs
of the sticky ends covalently bonded by phosphodiester
bonds.
 DNA ligase is required to seal the gaps, covalently bonding
the two strands and regenerating a circular molecule.
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 Joining DNA…….
50

 Recombinant DNA molecules can be formed from DNA cut

with restriction endonucleases that leave blunt ends, such as
SmaI.
 Without end modification, blunt end ligation is of low
efficiency. The efficiency is increased through using the
enzyme terminal deoxynucleotidyl transferase to create
complementary tails by the addition of poly(dA) and poly(dT)
to the cleaved fragments.
 These tails allow DNA fragments from two different
sources to anneal.
 The two DNA sources are then covalently linked by treatment
with DNA ligase to create a recombinant DNA molecule.
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 D) Transforming recombinant plasmid and
Amplifying the recombinant DNA
51

 To recover large amounts of the recombinant DNA molecule, it

must be amplified.
 There are different ways that amplify (clone) rDNA products

I) In vitro cloning: This is accomplished via Polymerase Chain

Reaction (PCR)

II) In vivo cloning : using cellular prokaryotes (e.g E.coli) ,

unicellular eukaryotes (e.g yeast, or mammalian tissue culture
cells).
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 Transforming recombinant plasmid…….
52

 In vivo: transforming the recombinant DNA into a bacterial or other host

strain.

1) To incubate the cells in a concentrated Ca salt solution

 Make host membrane leaky(make in a hole)

2) Using Electroporation
 Drive DNA into cells by a strong electric current
 Once in a cell, the recombinant DNA will be replicated. When the cell
divides, the replicated recombinant molecules go to both daughter cells
which themselves will divide later. Thus, the DNA is amplified.
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 Vector DNA
53

 Vectors are genetic elements that have been engineered, so that

they can accept fragments of foreign DNA and used for the

insertion of genetic material into a cell.

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 Vector DNA…..
54

Four important features of all cloning vectors are:

1. Can independently replicate themselves and the foreign DNA
segments they carry.
2. Contain a number of unique restriction endonuclease
cleavage sites
3. Carry a selectable marker, such as, antibiotic resistance
genes.
 Their very purpose is to distinguish host cells that carry
vectors from host cells that do not contain a vector.
4. Are relatively easy to recover from the host cell.

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 Vector DNA…..
55

 There are many possible choices of vector depending on;

 Insert size and application

 The classic cloning vectors are plasmids, phages, and

cosmids, which are limited to the size insert they can

accommodate, taking up to 10, 20, and 45 kb, respectively.

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56

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 Plasmid

57

 Plasmids are small extra chromosomal molecules of DNA that

replicate independently of the bacterial chromosomes.
 They are normally covalently closed, circular, super coiled
molecules.
 Plasmids do not normally contain the genetic information for
the essential metabolic activities of microorganism, but they
generally contain genetic information for specialized features,
such as resistance to heavy metals and antibiotics.
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 Plasmid……
58

 E.g. the R (resistance) plasmids carry genes that code for

antibiotic resistance.

 Plasmids are excellent vector for small DNA fragment and

hence are important tool for genetic engineering.

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 Bacteriophages

59

 The most popular bacteriophage is the Escherichia coli

(lambda) bacteriophage, which is made of approximately 50kb
of DNA
 Out of the 50kb that make the lamda bacteriophage less than
half are essential for the propagation of the phage and around
20kb can be replaced for recombinant DNA, hence their name
lamda replacement vectors.
 Therefore, these vectors are very useful for the generation of
genomic libraries. 01/31/2024
 Cosmids

60

 In essence, they are plasmids that have been engineered with

the addition of cos ends. This allows them to be packed into
lamda phage particles using in vitro packaging.
 Once inside the bacterial host the DNA from the cosmid is
circularized by the joining of the cos ends and thereafter
behaves as a normal plasmid.

 Cloning vectors that can carry up to 40kb of cloned DNA and

can also be maintained in E. coli.
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 PCR (Polymerase Chain Reaction)
61

 It is a fast and inexpensive technique used to amplify small

and targeted segments of DNA to produce million of copies,
sometimes called "molecular photocopying" of a specific gene
fragment.
 It is a technique used to study the molecular pathogenesis and
diagnosis of a variety of acquired, inherited, viral and
bacterial diseases

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 PCR……
62

 Wide varieties of living organism have been tried for the

production of recombinant proteins.

 The most commonly used hosts for the production of usable

quantities of the desired rDNA products are either

 Prokaryotic (bacteria) or

 Eukaryotic (yeast and mammalian cell culture)

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 PCR…..
63

 Most of the rDNA products approved by FDA are being

produced using the following hosts:

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 PCR…..
64

 Currently, the most important expression systems used in

the industry are the following:
 Bacteria (Escherichia coli [E. coli]
 Yeast (Saccharomyces cerevisiae and Pichia pastoris ]
 Mammalian cell (Chinese hamster ovary (CHO) cells)
 Transgenic animals.

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 PCR…..
65

 The two most important recombinant biomolecules,

insulin and erythropoietin were produced using E.
coli and mammalian (CHO) cells respectively.
 Most of the recombinant biomolecules are produced
industrially using these two organism followed by
yeast.

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 PCR…..
66

 The choice of production systems depends on;

1. The expression level

2. Quality of biomolecules, safety, manipulation of genetic

system

3. The cost of production.

4. Requirement of some proteins to be modified, or glycosylated,

in order to retain bioactivity, which can only be attained in
certain expression systems.
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 Commonly used hosts cells in rDNA technology
67

1. Prokaryotic
 Prokaryotic cells are known to be the earliest on earth.
 A prokaryotic is a single cell organism, whose cell lacks a
nucleus and other membrane bound organelles.
 They are unicellular and includes; Bacteria & Archaea.
 Bacteria such as Escherichia coli are widely used for the
expression of rDNA products.

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 Prokaryotic…..
68

 Advantages;
 high level of recombinant protein expression,
 rapid growth of cell and
 simple media requirement.
 Disadvantage;
 intracellular accumulation of heterologous proteins,
 the potential for product degradation due to trace of
protease impurities and
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
 2. Eukaryotic
69

 Eukaryotes are organisms whose cells contain a nucleus &

other membrane bound organelles.
 There is a wide range of eukaryotic organisms, including all
animals, plants, fungi and protein, as well as most algae.
 Eukaryotes may be either unicellular or multicellular.

I. Yeast
 Yeasts such as; Saccharomyces cerevisiae, Hansenulla
polymorpha and Pichia pastoris are among the simplest
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eukaryotic organisms.
 Eukaryotic…..
70
 Advantage;
 they grow relatively quickly and
 are highly adaptable to large-scale production.
 these organisms do not produce endotoxin.

N.B: Yeast has been the most widely used fungus for production
of proteins and vaccines (because of its easy growth).

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 Eukaryotic……
71

II. Mammalian systems

 Example; Chinese hamster ovary (CHO) cell and Baby
hamster kidney (BHK) cell.
 They are the choice for production of human therapeutic
proteins.
 Advantage;
 production of complex protein such as glycoprotein.

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 Eukaryotic……
72

 Disadvantage;
 Mammalian cell culture is complex process, time consuming,
 cost of production of the products using these systems is high
 slow growth and expensive nutrient media.

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 Eukaryotic……
73

 N.B: In spite of these disadvantages, in comparison to the

microbial system, mammalian cells have been most
extensively used for the production of glycoprotein and
monoclonal antibodies.
 Expression system for glycoprotein;
 CHO cell > Yeast> Insect cell > Plant based

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 3. Transgenic animals and plants
74

 Transgene is a gene, that has been transferred naturally or by

any of a number of genetic engineering techniques from one
organism to another.
 Recent advances have been made in producing therapeutic
proteins by using transgenic animals.
 transgenic milk production is currently most feasible.
 The use of genetically engineered plants to produce valuable
proteins is increasing slowly.
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 Transgenic animals and plants…..
75

 The system has potential advantages of economy and

scalability.
 Advantages;
 are high expression levels and volume output,
 low capital investment, low operational costs and
 reproducible production facility.

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 Transgenic animals and plants…..
76

 Disadvantage;
 variation in product yield,
 contamination with agrochemical and fertilizer, &
 variable cultivation conditions.

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 Impacts of rDNA technology
77

 Recombinant DNA technology had a fourfold positive impact

upon the production of pharmaceutically important proteins:

1. It overcomes the problem of source availability.

 Many proteins of therapeutic potential are produced
naturally in the body in minute quantities, Eg; interferons,
interleukins and colony-stimulating factors.
 Recombinant production allows the manufacture of any
protein in whatever quantity it is required.
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 Impacts of rDNA technology……
78

2. It overcomes problems of product safety

 Direct extraction of product from some native biological
sources has, in the past, led to the unwitting transmission of
disease.
 Examples include the transmission of blood-borne pathogens
such as;
 Hepatitis B and C and HIV via infected blood products.

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 Impacts of rDNA technology……
79

3. It provides an alternative to direct extraction from

inappropriate/dangerous source material.
 A number of therapeutic proteins have traditionally been
extracted from human urine.
 Follicle stimulating hormone (FSH); urine of
postmenopausal women.
 Human chorionic gonadotrophin (hCG); the urine of
pregnant women.
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 Impacts of rDNA technology……
80

4. It facilitates the generation of engineered therapeutic proteins

displaying some clinical advantage over the native protein

product.

 Site-directed mutagenesis facilitate the logical introduction of

predefined changes in a protein’s amino acid sequence.

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 APPLICATION OF GENETIC ENGINEERING
81

 Broadly speaking genetic engineering plays its major role in;

 Production of drugs and biochemicals

 Genetically modified organisms

 Analysis of genetic diseases

 Correction of genetic defects

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 Produce proteins to treat diseases and abnormalities:
82

 Insulin………………..diabetes
 Growth hormone…….dwarfism
 Clotting factors………hemophilia
 TPA (clot buster)/tissue plasminogen activator….heart attacks
 Sex hormones………..infertility
 Interferon…………….hepatitis C
 Interleukin-2…………kidney cancer
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 Agricultural Applications:

83

 Genetic engineering, including gene editing, can have

numerous benefits:

 faster & more precise breeding,

 higher crop yields,

 development of more nutritious food &

 decreased need for herbicides and pesticides.

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 Agricultural Applications….
84

 All of this are possible with genetic engineering in

agricultural crops, as scientists can change the genes
of crops to meet all of the above requirements.

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85

Thank you

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