BTG 402 - BIOTECHNOLOGY - BHU2022-2023 - Notes

PCT406 (2Cr) – PHARMACEUTICAL BIOTECHNOLOGY_ Pharm. (Prof.) Y.B.
Ngwai
DEPARTMENT OF PHARMACEUTICAL MICROBIOLOGY AND BIOTECHNOLOGY

FACULTY OF PHARMACEUTICAL SCIENCES
BINGHAM UNIVERSITY, KARU
BIOTECHNOLOGY
By
Pharm (Prof.) Y.B. NGWAI, PhD
COURSE OUTLINE
(BTG 402: Biotechnology [3 Credit Unit]
1) PROF. NGWAI: Basic techniques in biotechnology – cutting and joining of DNA molecules, cloning techniques and gene
manipulation. Polymerase Chain Reaction (PCR). Clinical importance of recombinant proteins. Plant Biotechnology.
2) PROF. ONANUGA: Identification of potential biotechnological products. Gene Therapy.
Biotechnology in vaccines development. Plants and transgenic animals as potential sources of recombinant biotechnological
products.
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PCT406 (2Cr) – PHARMACEUTICAL BIOTECHNOLOGY_ Pharm. (Prof.) Y.B. Ngwai
OVERVIEW OF BIOTECHNOLOGY
WHAT IS BIOTECHNOLOGY?
 The term “Biotechnology” has been defined in many ways:

1) First by the Hungarian engineer Károly Ereky (in 1919), as the production of products from raw
materials with the aid of living organisms.
2) The use of an organism, or a component of an organism or other biological system, to make a
product or process for a specific use.
3) The scientific manipulation of living organisms or their products to produce new products or new
forms of organisms (so-called GMO- genetically modified organisms or GEO- genetically enhanced
organisms).
4) The manipulation of living organisms or their products for useful purposes which may be medical,
agricultural, and/or industrial.
5) The procedures for modifying living organisms according to human purposes.
6) An amalgamation of biological science with engineering whereby living organisms or cells or parts
are used for production of products and services.
7) As the application of biological organisms, systems, or processes by various industries to learning
about the science of life and the improvement of the value of materials and organisms such as
pharmaceuticals, crops, and livestock.
 Forms of Biotechnology
(i) “Organismic Biotechnology”- when manipulation uses intact organisms, cells or tissues and does
not alter genetic material.
(ii) “Molecular Biotechnology”- when manipulation alters genetic makeup to achieve specific goals.
This is achieved by creating Recombinant DNA (rDNA) or “Chimera” - DNA that has been
created artificially by the incorporation of DNA from two or more sources into a single molecule.
 Scope of Biotechnology
(i) Conventional activities- baking bread, brewing alcoholic beverages, breeding of animals;
cultivation of the plants, and their improvements through artificial selection and hybridization, etc
(ii) Modern activities- cell and tissue culture technologies, cell fusion, molecular biology, and in
particular, genetic engineering or rDNA technology to generate unique organisms with new traits or
organisms that have the potential to produce specific products.
 Biotechnology is thus a diverse field which involves either working with living cells or using molecules
derived from them for applications oriented toward human welfare using varied types of tools and
technologies.
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HISTORICAL DEVELOPMENT OF BIOTECHNOLOGY
 Biotechnology has been practiced by human society since the beginning of recorded history in such
activities as baking bread, brewing alcoholic beverages, or breeding food crops or domestic animals.
 The history of the development of biotechnology consists of three stages, namely:
(i) Ancient Biotechnology- early history as related to food and shelter; includes domestication.
o Paleolithic peoples began to settle and develop agrarian societies about 10,000 years ago.
o Early farmers in the Near East cultivated wheat, barley, and possibly rye
o Archaeologists have found ancient farming sites in the Americas, the Far East, and Europe.
o People collected the seeds of wild plants for cultivation and domesticated some species of
wild animals living around them, performing selective breeding.
(ii) Classical Biotechnology- built on ancient biotechnology; fermentation-promoted food production,

and medicine.
o Fermented food- bread; wine (Egypt ~5000 B.C made wine from grapes); beer (began as
early as 6000-5000 B.C.).
o Medicine- antibiotics; steroids from cholesterol (in 1950s); amino acids, enzymes and
vitamins (by mid-1950s); proteins in 1960s); biomass (e.g. single-cell protein); chemicals,
hormones, etc.
(iii) Modern Biotechnology

o Involves the use of cell and tissue culture, cell fusion and molecular biology to generate
unique organisms with new traits or organisms that have the potential to produce specific
products.
(iv)Molecular Biotechnology
o This is a type of modern biotechnology that manipulates genetic information in organisms to
achieve specific goals.
o In molecular biotechnology, donor DNA is taken from one organism (or type of cell) and
placed into new location in same organism or the DNA of another organism (or type of cell)
resulting to GMOs:
 GMOs can be cultured in fermenters and are modified to produce large quantities of
desirable products, which are extracted and purified.
o Molecular biotechnology has been used to increase the amount and purity of enzymes, to
improve an enzyme’s function, and to provide a more cost-efficient method to produce
enzymes. E.g. of GMO product: chymosin (used in cheese production)
o The foundation of molecular biotechnology was laid down after the discovery of structure of
DNA (the hereditary material which contains all the information that dictates each and every
step of an individual’s life) in the early 1950s.
o History of Molecular biotechnology

 1953: elucidation of the structure of DNA by Watson and Crick.
 1973: Cohen and Boyer develop genetic engineering techniques to "cut and paste" DNA and
to amplify the new DNA in bacteria.
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 1977: The first human protein (somatostatin) is produced in a bacterium (E. coli).
 1982: The first recombinant protein (human insulin) appears on the market.
 1983: Polymerase chain reaction (PCR) technique conceived.
 1990: Launch of the Human Genome Project (HGP), an international effort to sequence the
human genome.
 1995: The first genome sequence of an organism (Haemophilus influenzae) is determined.
 2000: A first draft of the human genome sequence is completed.
APPLICATIONS AND BRANCHES OF BIOTECHNOLOGY
 Biotechnology has applications in several fields like production of medicines; diagnostics; therapeutics like
monoclonal antibodies, stem cells, and gene therapy; agricultural biotechnology; pollution control
(bioremediation); industrial and marine biotechnology, amongst others.
 The main subfields of biotechnology, based on applications in various industries, include the following:
(i) Medical (RED) biotechnology- use of biotechnology in the medical and pharmaceutical industries,
and health preservation. This branch involves:
o Production of vaccines and antibiotics
o Regenerative therapies
o Creation of artificial organs
o Creation of new diagnostics of diseases
o Development of hormones, stem cells, antibodies, siRNA and diagnostic tests
(ii) Agricultural (GREEN) biotechnology- as applied to crop production and agricultural processes,
and include:
o Selection and domestication of plants via micropropagation
o Designing of environmentally friendly “transgenic” plants that are either more fertile or can
grow under specific environments in the presence (or absence) of biotic and abiotic
(chemicals) stress. For example, Bt corn that was engineered to express a pesticide, thereby
ending the need of external application of pesticides.
o Microorganisms to clean and reduce waste
(iii) Industrial (WHITE) biotechnology- applied to industrial processes, and include:
o Designing of an organism to produce a useful chemical
o Using of enzymes as industrial catalysts to either produce valuable chemicals or destroy
hazardous/polluting chemicals
(iv)Marine (BLUE) biotechnology- relates to the exploitation of sea resources to create products and
industrial applications. For example:
o Production of bio-oils with photosynthetic micro-algae.
(v) Food (YELLOW) biotechnology- as applied to:
o Food production (food industry), such as making wine (winemaking), cheese
(cheesemaking), and beer (brewing) by fermentation.
o Insects- biotechnology-based control of harmful insects; the characterization and utilization
of active ingredients or genes of insects for research, or application in agriculture and
medicine and various other approaches.
(vi)Environmental (GRAY) biotechnology- focused on the maintenance of biodiversity and the
removal of pollutants.
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Major applications of biotechnology in different areas and some of their important products
(vii) BROWN biotechnology- focused on the management of arid lands and deserts, such as:
o Creation of enhanced seeds that resist extreme environmental conditions of arid regions,
which is related to the innovation, creation of agriculture techniques and management of
resources.
(viii) VIOLET biotechnology- is related to law, ethical and philosophical issues around
biotechnology. It deals with ethical and safety issues associated with some of the products.
(ix)DARK biotechnology- associated with bioterrorism or biological weapons and biowarfare which
use microorganisms, and toxins to cause diseases and death in humans, livestock and crops.
(x) Bioinformatics (GOLD) biotechnology/Computational biology- an interdisciplinary field that
addresses biological problems using computational techniques, and makes the rapid organization as
well as analysis of biological data possible. Bioinformatics plays a key role in:
o Functional genomics- attempts to use current knowledge on gene function to link genotype
(DNA sequences) to phenotype (physical expression of gene).
o Structural genomics/proteomics- seeks to describe the 3-dimensional structure of every
protein encoded by a given “genome” (genome is the total genetic material of an organism
and includes both the genes and the noncoding sequences).
o Proteomics- the large-scale study of proteomes (a proteome is a set of proteins produced in
an organism, system, or biological context).
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BIOTECHNOLOGY ETHICAL PROBLEMS
 Like other technologies, biotechnology has potential benefits to people and societies, and poses different
types of dilemmas as well.
 Some concerns include:
1) Privacy and non-discrimination. For instance should your health insurance company be able to
charge you more if you have a gene variant that makes you likely to develop a disease? How would
you feel if your school or employer had access to your genome?
2) Safety and health effects
3) Ecological impacts of biotechnologies. For example, crops genetically engineered to make their own
insecticide reduce the need for chemical spraying, but also raise concerns about plants escaping into
the wild or interbreeding with local populations (potentially causing unintended ecological
consequences).
4) Biotechnology may provide knowledge that creates hard dilemmas for individuals. For example, a
couple may learn via prenatal testing that their fetus has a genetic disorder. Similarly, a person who
has her genome sequenced for the sake of curiosity may learn that she is going to develop an
incurable, late-onset genetic disease, such as Huntington’s.
BASIC TECHNIQUES IN BIOTECHNOLOGY

 Many examples of modern biotechnology depend on the ability to analyze, manipulate, and cut and paste
pieces of DNA using tools referred to as DNA technology.
 Some DNA technology tools are hereby presented.
1. Cutting DNA and Joining DNA fragments
 Cutting DNA and joining DNA fragments together are among the first techniques learned in the
molecular biology lab and are fundamental to all recombinant DNA work.
 Such manipulations of DNA are conducted by a toolkit of enzymes collectively called “DNA
modifying enzymes” as follows:
1) Restriction endonucleases found in bacteria that cleave the deoxyribose-phosphate

backbone of DNA. They are used as molecular scissors. E.g. EcoRI was isolated from
Escherichia coli (strain RY13), Hind II and Hind III from Haemophilus influenzae, and XhoI
from Xanthomonas holcicola.
2) DNA ligase which functions to bond pieces of DNA together. It catalyzes formation of a
phosphodiester bond between the 5' phosphate of one strand of DNA and the 3'
hydroxyl of another. Example: T4 DNA ligase derived from the T4 bacteriophage, a DNA
ligase from E. coli.
3) DNA polymerases synthesize complementary strands during DNA replication. Examples:
Thermostable DNA Polymerases (Taq, Pfu, Vent, etc.) isolated from the hot springs
bacterium Thermus aquaticus, Reverse Transcriptases (RT) - an RNA-dependent DNA
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polymerases, Bacteriophage RNA Polymerases (T7, T3 and SP6 RNA polymerases) - Phage-
encoded DNA-dependent RNA polymerases, E. coli DNA Polymerase I, Klenow Fragment of
E. coli DNA Polymerase I, T4 DNA Polymerase derived from T4, a bacteriophage of E. coli,
T7 DNA Polymerase derived from T7 bacteriophage, Terminal Transferase catalyzes the
addition of nucleotides to the 3' terminus of DNA,
4) Others- Nucleases- e,g. DNase (deoxyribonuclease) I and RNase (ribonuclease) A;
Polynucleotide Kinase (PNK) produced from the T4 bacteriophage that catalyzes the
transfer of a phosphate from ATP to the 5' end of either DNA or RNA.
2. Making a Recombinant DNA (rDNA)
 Remember an rDNA is a DNA that has been created artificially by the incorporation of DNA from
two or more sources into a single molecule.
 To make rDNA, treat DNA from both sources with the same restriction endonuclease (e.g. BamHI).
Then, DNA ligase is added to covalently link the two into a molecule of recombinant DNA.
3. DNA Cloning
 “Cloning” is the technique of producing many identical copies of the same DNA fragment of interest
(e.g. gene) or recombinant molecule.
 Cloning can be done in vitro (by a process called the polymerase chain reaction (PCR) or in vivo.
 In vivo cloning can be done in:
(i) Unicellular prokaryotes like E. coli;
(ii) Unicellular eukaryotes like yeast; or
(iii) Mammalian cells grown in tissue culture.
 In every case of in vivo cloning:
(i) Section of DNA that contains target gene is identified and extracted from source/donor
chromosome.
(ii) Isolated gene is inserted into a vector genome to form rDNA that can be replicated and
expressed in the host cell. A vector is just a piece of DNA which carries our wanted gene. In
many cases, the vector used is a circular DNA molecule called a plasmid. Remember
bacterial cells naturally exchange plasmids so they will readily take up any plasmids they are
exposed to. When they do this, they are unintentionally also making many copies of gene of
interest. The plasmid can be replicated in bacteria, making many copies of the target gene. In
some cases, the gene is also expressed in the bacteria, making a protein (such as the insulin
used by diabetics).
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(iii) Growing the transformed cells in the host to produce a GM organism. Given the right
conditions, the transformed host bacteria cells will then go on to divide just like any other
bacteria cells. As they do so, once again they will be making many copies of our wanted
gene as well as even begin expressing the gene too.
Cloning of Gene
4. Polymerase Chain Reaction (PCR)
 Polymerase chain reaction is another widely used DNA manipulation technique, one with
applications in almost every area of modern biology.
 PCR can be used to make many copies of DNA that is present in trace amounts (e.g., in a droplet of
blood at a crime scene). Larger amounts of DNA mean more accurate and reliable results for your
later techniques.
 PCR reactions produce many copies of a target DNA sequence starting from a piece of template
DNA.
 PCR was developed by Nobel laureate biochemist Kary Mullis in 1984 (while working at the Cetus
Corporation in Emeryville, CA) and is based on the discovery of the biological activity at high
temperatures of DNA polymerases found in thermophiles (bacteria that live in hot springs).
 Requirements for PCR
(i) Target sample. This is the biological sample you want to amplify DNA from.
(ii) A primer. Short strands of DNA that adhere to the target segment; they identify the portion
of DNA to be multiplied and provide a starting place for replication.
(iii) Taq polymerase. This is the enzyme that is in charge of replicating DNA. This is the
polymerase part of the name polymerase chain reaction.
(iv)Nucleotides. You'll need to add nucleotides (dNTPs) so the DNA polymerase has building
blocks to work with.
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 Steps in PCR- three steps that are repeated automatically, usually 25 to 75 times
(i) Your target sample is heated. This denatures the DNA, unwinding it and breaking the
bonds that hold together the two strands of the DNA molecule, leaving you with single
stranded DNA (ssDNA).
(ii) Temperature is reduced and the primer is added. The primer molecules now have the
opportunity to bind (anneal) to the pieces of ssDNA. This labels the portions of DNA to be
amplified and provides a starting place for replication.
(iii) New pieces of ssDNA are made. Taq polymerase catalyzes the generation of new pieces
of ssDNA that are complimentary to the portions marked by the primers. The job of Taq
polymerase is to move along the strand of DNA and use it as a template for assembling a new
stand that is complimentary to the template. This is the chain reaction in the name
polymerase chain reaction.
Steps in PCR
5. Agarose Gel Electrophoresis

 Agarose Gel electrophoresis is a technique used to visualize (directly see) DNA fragments on an
agarose gel.
 Typical use for AGE is to analyze the results of a PCR reaction by examining the DNA fragments it
produces on the agarose gel.
 Gel electrophoresis separates DNA fragments based on their size, and the fragments are stained with
a dye so the researcher can see them.
 In AGE, an electric current is used to move the DNA molecules across an agarose gel, which is a
polysaccharide matrix that functions as a sort of sieve to help “catch” the molecules as they are
transported by the electric current.
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6. DNA Sequencing
 DNA sequencing involves determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a
DNA molecule.
 In some cases, just one piece of DNA is sequenced at a time, while in other cases, a large collection
of DNA fragments (such as those from an entire genome) may be sequenced as a group.
 One of the major methods of DNA sequencing is known as “chain termination” sequencing,
“dideoxy” sequencing, or “Sanger” sequencing, named after its inventor biochemist Frederick
Sanger.
 Automated sequencing exist where an automated sequencer runs on the same principle as the Sanger
method (dideoxynucleotide chain termination) except that where the manual/Sanger method uses
radioactive labeling, automated sequencing uses fluorescent tags on the ddNTPs (a different dye for
each nucleotide).
Agarose gel Electrophoresis
7. Polyacrylamide gel electrophoresis (PAGE)
 In non-denaturing conditions, PAG separates protein both size and charge of the proteins.
o However, a denaturing PAGE called sodium dodecyl (lauryl) sulfate-PAGE (SDS-PAGE)
separates proteins according to size of the proteins.
 In PAGE, an electric current is used to move the protein molecules across a polyacrylamide gel.
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 The polyacrylamide gel is a cross-linked matrix that functions as a sort of three-dimensional

mesh/sieve or screen to help “catch” the molecules as they are transported by the electric current.
CLINICAL IMPORTANCE OF RECOMBINANT PROTEINS

(i) Improved efficacy of Medicines
(ii) Lowering the cost of medicines (i.e. Insulin)
(iii) Increased access to medicines
(iv)Safer Medicines (i.e. Insulin)
PLANT BIOTECHNOLOGY
 Plant Biotechnology (PBT) is the application of technology to develop useful and beneficial plants or
plant products.
 It can involve the use of tissue culture or genetic engineering (changing or adding genes from another
organism) techniques to produce plants/products that are new or have improved desirable characteristics.
HISTORY OF PLANT BIOTECHNOLOGY
1) Early Plant breeding (12,000 years ago)

a) Humans domesticate crops; breed plants to further improve desirable characteristics.
b) Bread-making wheat grown in Egypt, rice cultivated in China (8,000-4,000 BC)
c) All major food crops being cultivated in Eurasia and in Americas. i.e. Potato, Wheat, Pea,
Sunflower, Millet (4,000-1,000 BC).
d) Babylonians use selective breeding techniques with date palm (1,000 – 0 BC) (870 BC)
o Traditional plant breeding selects mutants for best yield and quality (e.g., tomatoes).
o Artificial selection of kernel oil content
2) Classical Plant breeding

o The founding of the science of genetics;
o Cross-breeding to strengthen traits
a) 1735- Linnaeus publishes ‘Species Plantarum’, effectively beginning the science of plant taxonomy.
b) 1859- Charles Darwin publishes the theory of evolution by natural selection.
c) 1865- Gregor Mendel discovers the laws of inheritance by studying flowers in his garden. The
science of genetic begins.
3) Hybrid breeding
o Two parental lines of normally out-breeding species are inbred through several self-pollinations.
o When crossing such lines the first generation has hybrid vigor.
o The vigor gradually disappears over the next generations so new sowing seeds have to be
purchased every year.
o Selection operates on desirable traits, not on survival in the wild.
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o Examples:
 Hybrid breeding to strengthen traits- variety in genetic structure enhances viability
against disease, pests, climate variability.
 1923- Russet Burbank hybrid potato launched
 1933- First hybrid maize variety launched in USA
 Selection is the process that plants with desirable characteristics are chosen from a
population and then reproduced.
4) Modern Plant breeding

o A basic type of modern plant breeding:
a) Mutation breeding
- Seeds are treated with either radiation or mutagenic chemicals to induce larger or smaller
lesions in the genes.
- The mutations are at random over the genome.
- Usually mutation results in a loss of function of genes
- Examples:
 1928- Stadler produced mutations in barley
b) Green revolution (1960-1970)
- Green revolution’ leads to greatly increased crop yields
- Based on the incorporation of dwarfing genes discovered by Norman Borlaug and the
widespread use of agrochemicals.
c) Plant tissue culture (PTC) breeding
- The process of selectively mating plants in aseptic culture.
- PTC involves:
 Embryo rescue;
 Somaclonal variation selection;
 Somatic hybrid (i.e. fusion protoplast);
 Generation of haploid (i.e. anther/microspore culture)
- PTC History:
 1902- Gottlieb Haberlandt proposed that all cells are totipotent.
 1904- Hanning isolated nearly mature zygotic embryos from seeds of Crucifers
and successfully grew them to maturity in a defined medium.
 1925- Laibach isolated and grew embryos of interspecific cross Linum perenne and
L. austriacum that aborted in vivo.
 1948- Folke Skoog discovered that kinetin could induce organogenesis in callus
culture of tobacco.
 1957- Skoog and Miller demonstrated the effects and interaction of phytohormones:
 Auxin : cytokin > 1 = root formation
 Auxin : cytokin <1 = shoot formation
 Auxin : cytokin = 1 = callus formation
 1962- Development of MS medium (Murashige T. and Skoog F., Physiol. Plant., 15:
473-497).
 1964- Haploid plants derived from cultured Datura anthers
 1972- First interspecific hybridization of Nicotiana sp. by fusing protoplast
 1974- Haploid plants derived from cultured tobacco microspores
 1977- Successful integration of T-DNA in plants.
SCOPE OF PLANT BIOTECHNOLOGY
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 Major areas of PBT are:
1) Plant Tissue Culture (PTC)/ Plant Micropropagation
2) Recombinant DNA Technology (Gene Cloning)/Plant Genetic Engineering

o New hybrid DNA (recombinant DNA) is created artificially by the incorporation of DNA
from two or more sources into a single molecule.
o Recombinant DNA is then introduced into plant cells/plant to transform the plant with new
traits.
3) Plant Mutation Cloning
4) Plant Cells Technology
APPLICATIONS OF PLANT BIOTECHNOLOGY
1) Environmental applications
(i) To develop plants that are more environmentally friendly
(ii) Agricultural Biotechnology allows farmers can grow more food on less land using farming
practices that are environmentally sustainable.
2) Agricultural applications
(i) Higher yields of food crops per acre to help feed the growing world population
(ii) Reduced production costs.
(iii) Improve photosynthetic efficiency of plants by:
o Transferring abiotic stress tolerance into plants or crops- tomato plants that are resistant
to salinity; genes from marine plants (halophytes) to grain and vegetable crops; gene,
which encodes a protein from a flounder fish, has been transformed to plants to protect
them against freezing damage; and/or
o Development of nitrogen fixing capacity in non-leguminous crops by transferring
nitrogen fixing genes (nif genes) from leguminous to non-leguminous crops, to provide a
natural source of nitrogen within the plant, which can offer a cost-effective alternative to
the expensive fertilizers.
(iv)Improved nutritional (protein, vitamin, carbohydrate, etc) content of foods with- e.g.
o High beta carotene (pro-vitamin A) engineered into “golden rice”, which the body
converts to vitamin A;
o High protein introduced into potato by engineering potato with a gene from amaranth;
o Addition of gluten proteins in wheat to improve its elasticity to dough
o Enhanced starch content of starchy foods or altered properties of plant starches.
(v) Improved functional quality of foods- e.g.
o Improved flavor and texture of fruits and vegetables by manipulating their maturing
process
o Longer shelf-life engineered into “Flavr-Savr” tomato by slowing its ripening process
(vi)Eliminating/reducing undesirable food components- e.g.
o allergens in rice, peanuts or soybeans;
o caffeine from coffee;
o mycotoxins in corn,
o protease inhibitors in legumes
o hemaglutinins in legumes, etc.
(vii) Herbicide resistance-
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o Herbicide resistance genes transferred into plants offer protection for the manipulated
plants against herbicides by detoxifying (converting it to an inactive form) the herbicide.
(viii) Disease resistance- e.g.
o Transferring genes for farnase (a serochemical- chemical which alter the insect behavior)
into susceptible potato crop confers aphid resistance on it. Serochemicals are usually
produced by certain insect and plant species.
o Virus resistance- expression of viral coat protein gene in plants has conferred virus
resistance; sense and antisense expression of parts of the viral genome can be protective
against viral infection
o Nematode resistance- transfer of nematode resistance genes into plants confers nematode
resistance on the transgenic plants.
(ix)Insect resistance ensures safe storage- e.g.
o Protease inhibitors transferred into plants prevent the digestion of proteins by insects-
hence protect plants against insect attack.
o Avidin introduced into transgenic corn makes the corn highly resistant to insects during
storage. Avidin blocks the availability of biotin, a vitamin required by insects to grow.
o Bacillus thuringiensis (Bt) genes inserted into a plant/crop produces a Bt toxin that kills
certain harmful insects and their larvae- a kind of a built-in defense.
(x) Production of Neutraceuticals (“Functional Foods”, “Made-up Foods”, “Designer Foods”)
o vitamin-enriched breakfast cereals
o margarine spread (e.g. Benecol), a that actually lowers LDL cholesterol
(xi)Plant micropropagation
(xii) Somatic embryogenesis
(xiii) Plant development (complex process of development of a plant, which involves the role of
light receptors like phytochrome, chloroplast gene expression, mitochondrial gene expression in
relation to male sterility, storage product accumulation, and storage organ (fruits) development.
o Early flowering genes have been reported to alter the properties of the late maturing
varieties.
o Genes responsible for color formation can be transferred to plants bearing colorless
flowers.
o Genes that control flowering and pollen formation can generate transgenic plants with
altered fertility.
3) Industrial applications
(i) Production of food additives
(ii) Improved quality and flavor of beer due to manipulation of the barley grain used in beer
production
(iii) Alteration of properties of plant starches- e.g.
o proportion and quality of amylase and amylopectin in the starch
(iv)Improved sucrose content of sugar crops like sugar cane and sugar beet.
(v) Increased levels of fructans (a form of glucose) are already being produced using a levansucrase
from bacteria.
(vi)In vitro production of fine chemicals using plant cell/organ cultures in bioreactors- e.g.
o Enzymes, shikonin, berberine, ginseng, sanguinarine, rosmarinic acid, digoxin, geraniol,
immunologically active polysaccharides, etc
(vii) fuel- increasing biomass for biofuel production
(viii) raw materials- pigments, resins, perfumes, etc
4) Pharmaceutical applications
(i) Pharmaceutically useful compounds (chemical drugs, enzymes, hormones, etc) have been
developed and grown in whole plant or part of plants;
(ii) Vaccine production in plants
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o Genes for Hepatitis B virus antigens have been successfully expressed in tobacco plants
and used to immunize mice.
o Genes for P-sub unit of E. coli enterotoxin LT-B has been expressed in potatoes and used
to immunize mice, thus protecting the mice against the bacterial toxin.
o Oral vaccines against cholera have already been expressed in plants.
PLANT TRANSGENESIS (PLANT GENE TRANSFER)
 Trangenesis is generally the process of transferring genes of interest from one source to a recipient.
 Plant Transgenesis is thus the process of transferring transgenes (genes of interest) to plants directly
resulting in change in the genetic makeup of the recipient plant.
 The new plant is called TRANSGENIC, since it contains a gene or genes that have been artificially
inserted.
 The aim of transgenesis is to introduce a new trait to the plant which does not occur naturally in the
species.
 The inserted transgene may come from an unrelated plant or from a completely different species. The
inserted genes can come from species within the same kingdom (plant to plant) or between kingdoms
(bacteria to plant). In many cases, the inserted DNA has to be modified slightly in order to correctly and
efficiently express in the host organism.
 Plant transgenesis where a combination of genes are inserted in a plant confer several advantages as
follows:
1) improving shelf life
2) higher yield
3) improved quality
4) pest resistance
5) stress resistance (tolerance to heat, cold, drought and a variety of biotic and abiotic stresses)
6) expression of foreign proteins with industrial and pharmaceutical value (proteins, antibodies,
enzymes, etc)
 The first transgenic plant to be produced was a tobacco plant that expressed antibiotic resistance,
performed way back in 1982. Herbicide resistant tobacco plants underwent the first field trials in 1986 in
the USA and France.
 Since then, many recombinant proteins have been expressed in several important agronomic species of
plants including tobacco, corn, tomato, potato, banana, alfalfa and canola. Tobacco plants were
generally used, however potatoes and bananas are also considered, for the purpose of vaccines for
human beings.
Methods used in Plant Transgenesis

1) Conventional Selective Breeding and Hybridization
2) Cloning
o Protoplast Fusion
o Leaf Fragment Technique (Agrobacterium tumafaciens mediated transformation)
o “Gene Gun” (or Micro-Projectile Bombardment” or “Bolistic”)
o Chloroplast Engineering
o Antisense Technology
1) Conventional Selective Breeding and Hybridization

- Sexual cross between two lines and repeated backcrossing between hybrid offspring and parent
o Can take years
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- Polyploid plants (multiple chromosome sets greater than normal)

o Increases desirable traits, especially size
o Whole chromosomes can be transferred rather than single genes
2) Cloning
- Growing plants from a single cell.
(a) Protoplast Fusion is the fusion of two protoplast cells from different species
o Protoplast cell = a callus cell whose cell wall has been dissolved by the enzyme
cellulase.
o Fusion of the two protoplast cells creates a cell that can grow into a hybrid plant.
o Examples: broccoflower
Cloning by Protoplast Fusion Technique
(b) Leaf Fragment Technique (“Agrobacterium”) Method

o Small discs are cut from leaf;
o Leaf discs are cultured in a medium containing genetically modified Agrobacter
(Agrobacterium tumefaciens);
- A soil bacterium that infects plants
- Bacterium contains a plasmid, the TI plasmid, that can be genetically modified
16
- DNA from the TI plasmid integrates with DNA of the host cell
o Engineered leaf discs are transferred to filter paper over feeder cells embedded in agar
and cultured for 2-3 days;
o Leaf discs are transferred to shoot-stimulating medium containing appropriate plant
hormones and cultured for 2-4 weeks;
o Growing shoots are transferred to root-stimulating medium with appropriate plant
hormones and cultured for 3 weeks;
o Regenerated engineered plant is transferred to the soil.
Cloning by Leaf Fragment Technique (“Agrobacterium”) Method
(c) “Gene Gun” (“Bolistic”) Method

o Used to blast tiny metal beads coated with DNA into an embryonic plant cell.
o Aimed at the nucleus or the chloroplast.
o Use marker genes to distinguish genetically transformed cells.
- Antibiotic resistance marker
17
o Technique is useful in plants that are resistant to Agrobacterium.
(d) Chloroplast Engineering Method

o A chloroplast is an organelle within the cells of plants and certain algae that is the site
of photosynthesis (process of synthesis and storage of foodstuffs).
o DNA in chloroplast can accept several new genes at once.
o High percentage of genes will remain active.
o DNA in chloroplast is completely separate from DNA released in pollen - no chance that
transformed genes will be carried on wind to distant crops.
(e) Antisense Technology

o Process of inserting a complementary copy of a gene into a cell.
o Gene encodes an mRNA molecule called an antisense molecule.
o Antisense molecule binds to normal mRNA (sense molecule) and inactivates it
o Example is Flavr Savr tomato
Cloning by Antisense Technique
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EXAMPLES OF PLANT-DERIVED THERAPEUTIC PROTEINS
Plant-derived pharmaceutical proteins close to commercialization for the treatment of human disease
Product Indication Company/ Crop Expressio Status Reference
organization n System
E. coli heat Diarrhoea Prodigene Inc. Maize & Transgenic Phase I Tacket et
labile toxin Potato al., 1998,
2004
Hepatitis B Hepatitis B Arntzen group Potato & Transgenic Phase I Kapusta et

virus surface Thomas Jefferson Lettuce al., 1999
antigen University/ and
Polish Academy of Thanavala
Sciences et al.,
2005
Norwald virus Norwald Arntzen group Potato Transgenic Phase I

capsid protein virus
infection
Norovirus Norovirus Potato Transgenic Phase I Tacket et
Capsid protein infection al., 2000
Rabies Rabies Yusibov et al. 2002 Spinach Viral Phase I Yusibov
glycoprotein vectors et al. 2002
and
nucleoprotein
Influenza Influenza Cummings et al, 2014 Nicotina Launch Phase I Cumming
virus (H5N1; virus H5N1 benthamiana vector s et al,
2009 2014
pandemic)
HA antigen
Cholera Cholera Nochi et al., 2009 and Rice Transgenic Phase I Nochi et
Cholera Toxin infection Yuki et al., 2013 al., 2009
B antigen and Yuki
et al.,
2013
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SOMATIC EMBRYOGENESIS AND ORGANOGENESIS
 Embryogenesis and organogenesis are two important processes in the development of an organism.
 Definitions
o Embryogenesis- is the process that forms an embryo from a zygote developed from the syngamy
(fusion of two gametes).
o Somatic embryogenesis- the artificial formation of an embryo from somatic cells of plants.
 Somatic embryogenesis possible in plant cells since they are totipotent.
 Two forms exist: DIRECT (no callus is formed; embryos can be developed directly from
the explant) and INDIRECT (embryo formation proceeds via callus phase).
o Organogenesis- the formation of organs (e.g. leaves, shoots, roots) of an organism from the
three germ layers (ectoderm, endoderm, mesoderm) of the embryo.
Somatic Embryogenesis
 Steps in Somatic Embryogenesis
o Induction of somatic cells- a single somatic cell is induced to become mature.
 Induction can be done by supplying nutrients and plant hormones.
 The plant hormone auxin is used in the early stage of somatic embryogenesis. Once the
auxin is applied, cells will start to grow and divide rapidly.
 After that, the second hormone gibberellin is supplied. Then cells differentiate into an
undifferentiated cell mass called callus.
o Maturation of Callus
 Callus has the capability to mature into a plant.
 Hence, it is transferred into a fresh nutrient medium to develop into an embryo.
o Development into embryo
 Embryo development has different stages such as globular, heart shaped, and small
plant.
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Callus formed during somatic embryogenesis
 Advantages of Somatic embryogenesis

o When the plant is infected, a mature plant can be made from a single unaffected cell using this
process.
o Artificial seed can also be prepared by somatic embryogenesis.
 Disadvantages
o Cannot be applied to all the plants- limited for certain plant species due to induction difficulties.
o Time-consuming process
o Requires expertise
o High probability of mutations
o The method is usually rather difficult.
o Losing regenerative capacity become greater with repeated subculture
o A deep dormancy often occurs with somatic embryogenesis.
Organogenesis
 Inducing a vegetative tissue to form organs (shoot or root) which eventually develop into a complete
plantlet (small but whole plant).
 The piece of tissue from original plant part used to initiate the culture is known as explant.
 Explant can be:
o Pieces of organs- leaves, roots, stems, cotyledons, embryos, etc
o Special cell types- leaf tissue, embryo, pollen, endosperm, nucellus, etc
 Organogenesis may be:
o Direct- when explant develops shoot and root directly through the manipulation of plant growth
regulators and culture conditions.
o Indirect- if the formation of shoot and root need to go through a callus phase.
 Induction of organogenesis is by use of plant growth regulators (PGRs), usually the cytokinins i.e. 6-
benzylaminopurine (BAP) alone in different concentrations or in combination with an auxin for
example, Naphtalene Acetic Acid (NAA) or 2,4-Dichlorophenoxyacetic acid (2,4-D) also at different
concentrations.
Organogenesis Vs Somatic Embryogenesis
S/N Feature Organogenesis Somatic Embryogenesis

1 Product Organs from embryonic cells Embryo from 1 or more somatic cells
2 Nature Natural, more or less Artificial
3 Occurrence Plants and Animals Plants
PLANT TISSUE CULTURE
Definitions of PTC
1) The culture and maintenance of plant cells and organs.
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2) The culture of plant seeds, organs, tissues, cells, or protoplasts on nutrient media under sterile
conditions.
3) The growth and development of plant seeds, organs, tissues, cells or protoplasts on nutrient media under
sterile (axenic) conditions.
4) The in vitro, aseptic plant culture for any purpose including genetic transformation and other plant
breeding objectives, secondary product production, pathogen elimination or for asexual
(micropropagation) or sexual propagation.
Basis for PTC

1) Totipotency
o Potential or inherent capacity of a plant cell to regenerate into whole plant if suitably stimulated.
o It implies that all the information necessary for growth and reproduction of the organism is
contained in the cell.
2) Dedifferentiation
o Capacity of mature cells to return to meristematic condition and development of a new growing
point, followed by redifferentiation which is the ability to reorganize into new organ
3) Competency
o The endogenous potential of a given cells or tissue to develop in a particular way.
4) Ability of plant cells to give rise to callus, embryos, adventitious roots and shoots.
5) Ability of plant cells to grow as single cells (protoplasts, microspores, suspension cultures)
6) Two Hormones Affect Plant Differentiation:
o Auxin: Stimulates Root Development
o Cytokinin: Stimulates Shoot Development
7) Generally, the ratio of these two hormones can determine plant development:
o Auxin ↓Cytokinin = Root Development
o Cytokinin ↓Auxin = Shoot Development
o Auxin = Cytokinin = Callus Development
Stages of PTC
A. Sourcing explants
B. Explant culture on agar medium
C. Callus production
D. Plantlet regeneration
E. Transplantation of plantlet into sterile soil
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Examples of Hormones used in PTC
Hormones Product Name Functions in PTC
Auxins Indole-3-Acetic Acid Adventitous root formation (high concen);

Indole-3-Butyric Acid Adventitious shoot formation (low concentration);
Indole-3-Butyric Acid, Potassium Salt Induction of somatic embryos
a-Naphthaleneacetic Acid Cell Division
2,4-Dichlorophenoxyacetic Acid Callus formation and growth
p-Chlorophenoxyacetic acid Inhibition of axillary buds
Picloram Inhibition of root elongation
Dicamba
Cytokinins 6-Benzylaminopurine Adventitious shoot formation
6-g,g-Dimethylallylaminopurine (2iP) Inhibition of root formation
Kinetin Promotes cell division
Thidiazuron (TDZ) Modulates callus initiation and growth;
N-(2-chloro-4-pyridyl)-N’Phenylurea Stimulation of axillary’s bud breaking and growth;
Zeatin Inhibition of shoot elongation;
Zeatin Riboside Inhibition of leaf senescence.
Gibberellins Gibberellic Acid Stimulates shoot elongation
Release seeds, embryos, and apical buds from
dormancy
Inhibits adventitious root formation
Paclobutrazol and ancymidol inhibit gibberellin
synthesis thus resulting in shorter shoots, and
promoting tuber, corm, and bulb formation.
Abscisic Abscisic Acid Stimulates bulb and tuber formation;
Acid Stimulates the maturation of embryos;
Promotes the start of dormancy.
Polyamines Putrescine Promotes adventitious root formation;
Spermidine Promotes somatic embryogenesis;
Promotes shoot formation.
Why is PTC important?

1) Have value in studies such as cell biology, genetics, biochemistry, and many other research areas.
2) Crop Improvement
3) Seed Production – Plant Propagation Technique
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4) Genetic material conservation
Important considerations in PTC

1) Growth Media
– Minerals, Growth factors, Carbon source, Hormones
2) Environmental Factors
– Light, Temperature, Photoperiod, Sterility, Media
3) Explant Source
– Usually, the younger, less differentiated explant, the better for tissue culture
– Different species show differences in amenability to tissue culture
– In many cases, different genotypes within a species will have variable responses to tissue culture;
response to somatic embryogenesis has been transferred between melon cultivars through sexual
hybridization
Types of in vitro PTC
Types of Plant Tissue Culture
1) Culture of intact plants (seed and seedling culture)

 Growing seed aseptically in vitro on artificial media
 Increasing efficiency of germination of seeds that are difficult to germinate in vivo
 Precocious germination by application of plant growth regulators
 Production of clean seedlings for explants or meristem culture
2) Embryo culture (immature embryo culture)
 Growing embryo aseptically in vitro on artificial nutrient media
 It is developed from the need to rescue embryos (embryo rescue) from wide crosses where
fertilization occurred, but embryo development did not occur
 It has been further developed for the production of plants from embryos developed by non-sexual
methods (haploid production discussed later)
 Overcoming embryo abortion due to incompatibility barriers
 Overcoming seed dormancy and self-sterility of seeds
 Shortening of breeding cycle
3) Organ culture
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 Any plant organ can serve as an explant to initiate cultures.

 Many types of organ culture exist, depending on the plant part:
o Shoot Tip Culture- shoot as explant
o Root Culture- root as explant
o Leaf Culture- leaf as explants
o Anther/Ovary Culture- flower as explant
4) Callus culture
 A culture of callus (an un-organized mass of cells)
 A tissue that develops in response to injury caused by physical or chemical means
 Most cells of which are differentiated, although may be and are often highly unorganized within
the tissue.
Callus Culture
5) Cell suspension culture

 When callus pieces are agitated in a liquid medium, they tend to break up.
 Suspensions are much easier to bulk up than callus since there is no manual transfer or solid
support.
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Cell Suspension Culture
6) Protoplast culture
 The living material of a plant or bacterial cell, including the protoplasm and plasma membrane
after the cell wall has been removed.
Protoplast Culture
Applications of PTC
26
 Plant cell culture has direct commercial applications as well as value in basic research into cell
biology, genetics, and biochemistry.
1) Breeding and Genetics
 Micropropagation – using meristem and shoot culture to produce large numbers of identical
individuals;
 Selection – screening of cells, rather than plants, for advantageous characters;
 Crossing distantly related species by protoplast fusion and regeneration of the novel hybrid;
 Production of dihaploid plants from haploid cultures to achieve homozygous lines more
rapidly in breeding programs;
 Transformation, followed by either short-term testing of genetic constructs or regeneration of
transgenic plants;
 Removal of viruses by propagation from meristematic tissues;
2) Model system for study of plant cell genetics, physiology, biochemistry, and pathology;
3) Production of secondary metabolites – growth in liquid culture as a source of products.
PRODUCTION OF SECONDARY METABOLITE IN PLANT CELL CULTURE
 Plants are natural sources of the over 80% of the approx. 30,000 known natural products which include
both primary and secondary metabolites.
 Plant secondary metabolites can be defined as compounds that have no recognized role in the
maintenance of fundamental life processes in plants, but they do have an important role in the
interaction of the plant with its environment.
o They mostly have an ecological role as attractants of pollinating insects or in defense
mechanisms against predators.
 These secondary metabolites can be used as food additives (flavors, colorants, fragrances, etc),
nutraceuticals, and pharmaceuticals.
 Distribution of secondary metabolites in plants is far more restricted than that of primary metabolites:
o A compound is often only found in a few species, or even within a few varieties within a species.
o The production of these compounds is often low (less than 1% DW), and it depends greatly on
plant species and plant’s physiological and developmental stage.
o Often accumulate in the plant in specialized cells or organs.
 Traditional agriculture methods were used as sources for these plant secondary metabolites.
o But the natural habitats for a large number of plants are rapidly destroyed leading to extinction of
many valuable and even endemic species.
o Also, many plants containing high-value compounds are difficult to cultivate.
o At the same time, the chemical synthesis of plant-derived compounds is often not economically
feasible because of their highly complex structures and specific stereo-chemical characteristics.
 But PTC use for the production of valuable secondary metabolites in plant cell cultures is now an
attractive alternative to the extraction of the whole plant material.
o PTC explores the biosynthetic capabilities of various cell cultures for industrial production of
bioactive plant metabolites
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 Examples of PTC-sourced bioactive secondary metabolites

o medicinally important compounds/pharmaceuticals (alkaloids, terpenoids, steroids, saponins,
phenolics, flavanoids, anticancer drugs)
o recombinant proteins
o food additives
Some secondary metabolites produced in Plant Tissue Culture

S/N Product Plant
1 Ajmalicine Catharanthus roseus
2 Anthraquinones Morinda citrifolia
3 Berberine Coptis japonica
4 Caffeic acid Vanilla planifolia
5 Ginsenoside Panax ginseng
6 Nicotine Nicotiana tabacum
7 Rosmarinic acid Coleus blumei
8 Shikonin Lithospermum erythrorhizon
9 Ubiquinone-10 Nicotiana tabacum
Plant Tissue Culture derived Pharmaceuticals

S/N Product Use Plant species
1 Ajmalicine Antihypertensive Catharanthus roseus
2 Ajmaline Antimalarial Rauvolfia serpentine
3 Camptothecin Antitumor Camptotheca acuminata
4 Codeine Sedative Papaver somniferum
5 Colchicine Antitumor Colchium autumnale
6 Ellipticine Antitumor Orchrosia elliptica
7 Morphine Sedative Papaver somniferum
8 Shikonin Antibacterial Lithospermum erythrorhizon
9 Taxol Anticancer Taxus brevifolia
10 Vinblastine Antileukemic Catharanthus roseus
11 Vincristine Antileukemic Catharanthus roseus
PTC Techniques for secondary metabolite production
1) Callus cultures- frequently used for production of flavonoids.

o Non-embryogenic callus cultures, containing more or less homogenous clumps of
dedifferentiated cells, are used for secondary metabolite production.
2) Cell suspension cultures
3) Organ cultures - they are relatively more stable in production of secondary metabolites than cultures of
undifferentiated cells, such as cells in callus or suspension culture
o Root: e.g. tropane alkaloids (hyoscyamine and scopolamine)
4) Shoots (aerial parts)
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Advantages of plant cell cultures over the conventional production in intact plant
1) It is independent of geographical and seasonal variations and environmental factors – the synthesis
of bioactive secondary metabolites runs in controlled environments and the negative biological
influences that affect secondary metabolites production in nature are eliminated (microorganisms
and insects);
2) It offers a defined production system which ensures the continuous supply of products, uniform
quality, and yield;
3) It is possible to select cell lines with higher production of secondary metabolites;
4) It is possible to produce novel compounds that are not normally found in parent plant;
5) It allows the efficient downstream production;
6) Plant cell can perform stereo- and regio-specific biotransformations for the production of novel
compounds from cheap precursors;
7) With automatization of cell growth control and regulation of metabolic processes, cost price can
decrease and productivity increase.
Problems with plant cell cultures
Biological Operational
o slow growth rate o wall adhesion
o physiological heterogeneity o light requirement
o genetic instability o mixing
o low metabolite content o shear sensitivity
o product secretion o aseptic condition
PLANT MICROPROPAGATION
 Plant micropropagation (in vitro clonal propagation) is a plant tissue culture technique in which cells,
tissues or organs of selected plants are isolated, surface sterilized, and incubated in a growth-promoting
aseptic environment to produce many clone plantlets.
o It is the culture of aseptic small sections of tissues and organs in vessels with defined culture
medium and under controlled environmental conditions
 It can be employed in mass multiplication of plants in relatively shorter time.
Steps in Micropropagation
Stage 0: Selection & preparation of the mother/stock plants under hygienic conditions in greenhouse for
about 3 months under controlled conditions
- sterilization of the plant tissue takes place
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Stage I: Initiation of culture under axenic (aseptic/sterile) conditions

- selection of explants is done
- explants are disinfected
- cultivation of explants in growth media under axenic conditions
Stage II: Multiplication (shoot proliferation and multiple shoot production)

- rapid regeneration and multiplication of shoots or rapid embryo formation occurs
- explants are transferred to shoot media; shoots can be constantly divided
Stage III: Rooting

- elongation and root induction or development occurs
- explants are transferred to root media
- This phase is designed to induce the establishment of fully developed plantlets.
- It is the last period in vitro before transferring the plantlets to soil (ex vitro) conditions.
Stage IV: Transfer to soil (acclimatization)

- explants are returned to soil to acclimatize (i.e. to adapt to new climatic or environmental
conditions); hardened off
Micropropagation Techniques
1) Micropropagation via meristem culture (or axillary bud/shoot tip culture)

o Quiescent or actively dividing meristems are present at the axillary and apical shoots (shoot tips).
o The axillary buds located in the axils of leaves are capable of developing into shoots.
o By means of induced in vitro multiplication in micro propagation, it is possible to develop plants
from meristem and shoot tip cultures and from bud cultures.
o Micropropagation via meristem is best possible means of virus elimination.
o It produces a large numbers of plants in a short span of time.
o It is a powerful tool for large-scale propagation of plants.
o The term ‘meristem culture’ specifically means that a meristem with no leaf primordia or at most
1-2 leaf primordial are excised and cultured.
2) Multiplication by Adventitious Shoots

o The stem and leaf structures that are naturally formed on plant tissues located in sites other than
the normal leaf axil regions are regarded as adventitious shoots.
o There are many adventitious shoots which include stems, bulbs, tubers and rhizomes.
o The adventitious shoots are useful for in vivo and in vitro clonal propagation.
o The meristematic regions of adventitious shoots can be induced in a suitable medium to
regenerate to plants.
3) Micropropagation via somatic embryogenesis

o Somatic embryos germinate readily (from somatic cells, tissues or organs), similar to their
seedling counterpart, under controlled environmental conditions.
o Only somatic embryo germination rate as high as 80-85% has commercial application.
o Applications- Dendrathema grandiflorum; Bouteloua gracilis.
4) Multiplication by Organogenesis
o The formation of individual organs such as shoots, roots, directly from an explant (lacking
preformed meristem) or indirectly from the callus and cell culture induced from the explant.
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Factors affecting Micropropagation
1) Media
o Significant effect of media has been observed on plant regeneration from different parts of plant.
o Selection, strength and combination of media are important parameters for optimizing the
regeneration protocol.
o MS medium (Murashige and Skoog, 1962) is most widely used culture medium
 It contains all the nutrients essential for plant growth in vitro; hence most of the plants
respond favorably it.
o But other basal media like White medium, Nitsch and Nitsch medium, B5 medium and Gamborg
medium have also been employed for micropropagation.
o The standard plant tissue culture media are suitable for micro propagation during stage I and
stage II.
 But for stage III (Rooting), certain modifications are required- Addition of growth
regulators (auxins and cytokinins) and alterations in mineral composition (This is largely
dependent on the type of culture- meristem, bud etc.).
2) Type of Explant
o Significantly affects tissue culture process of plants; most likely due to the different level of
endogenous plant hormones present in the plants parts.
o Types of explants- leaf, petiole, cotyledonary leaf, hypocotyle, epicotyle, embryo, internode, root .
o Leaf is the most commonly used due to more surface area available.
o Leaf gave best regeneration efficiency than: root and shoot of Cajanus cajan; petiole and
hypocotyle of Jatropha curcas.
3) Genotype
o Genotype affects shoot regeneration and elongation in many species.
 Partly due to differences in the levels of endogenous hormones, particularly cytokinins
levels during the induction period although the precise mechanism remains unclear.
o In general, plants with vigorous germination and branching capacity are more suitable for micro-
propagation.
o Genotypic differences with respect to embryogenesis and regeneration result from quantitative or
qualitative genetic differences.
4) Source and Physiological status of the explant

o Source
 In vitro explants differ from in vivo explants in regeneration capacity.
 The difference may be due to the level of endogenous hormones present in the plant
explant.
 In vitro explant > in vivo explants in suitability for organogenesis.
 Seedling explant > mature plants in responsiveness to regeneration due to different level
of plant hormones present in the plants.
 Effect of the source of explants on regeneration was seen in Jatropha curcas.
o Physiological status
 Explants from more recently produced parts of plants are more effective than those from
older regions.
 Good knowledge of donor plants’ natural propagation process with special reference to
growth stage and seasonal influence will be useful in selecting explants.
5) Orientation of explant
o Orientation of explant in the culture medium also affects the regeneration efficiency.
31
o Horizontal > Vertical in regeneration efficiency

 Due to little contact of explant to medium in vertical position as compared to horizontal
position.
o Examples:
 Explant orientation altered the initiation site, polarity, and efficiency of bud regeneration
in Dionaea muscipula
 Cotyledons placed in abaxial (lower surface facing down) orientation > adaxial (upper
surface facing down) orientation in shoot regenerative response = produced greater
numbers and taller shoots.
 Orientation of explants affects regeneration in Jatropha curcas.
6) Mineral nutrition
o Minerals are important component of culture medium- combined as macro and micro-salts.
o MS medium (most widely used culture medium due to its rich content of all the elements that
have been shown to be essential for plant growth in vitro) is classified as a high salt medium in
comparison to many other formulations.
o Due to the high salt content, MS medium is not necessarily always optimal for growth and
development of plants in vitro- its high concentration of salts may inhibit root growth, even in
presence of auxins in the culture medium.
 Hence, dilute media formulations are used generally to promote better formation of roots
as in rose explants.
7) Carbon source
o Because photosynthesis is insufficient, addition of carbon source is essential for in vitro growth
and development of many plant species.
o Sucrose is by far the most used carbon source
 Cheap
 Available readily
 Stable to autoclaving relatively
 Assimilated by plants readily.
o Other carbohydrates (glucose, maltose, galactose, glycerol, sorbitol) can be also used. such as
(Fowler, 2000).
o The carbohydrates added to the culture medium supply energy for the metabolism of the green
tissues under in vitro conditions.
8) Growth regulators
o Higher plants produce certain organic compounds naturally (so-called Growth regulators), which
influence their growth and development.
o Synthetic chemicals with growth regulating activities have been developed which correspond to
the natural ones.
o Several classes of plant growth regulators- cytokinins, auxins, gibberellins, ethylene and
abscisic acid.
o Interaction and balance between the growth regulators supplied in the medium, and the growth
substances produced endogenously affects in vitro growth and morphogenesis.
o A balance between auxin (e.g. IAA, 2,4-D) and cytokinin (e.g. Kinetin, BA) is most often
required for the formation of adventitious shoots and roots.
 In vitro tobacco culture: higher auxin than cytokinin promotes root formation; but
higher cytokinin than auxin favors shoot formation.
o The balance of growth regulators depends on the:
 objective of the cultivation in vitro (as e.g. shoot, root, callus or suspension culture)
32
 micropropagation phase considered (initiation, multiplication or rooting).

– Multiplication phase: cytokinins should be normally higher than of auxins;
– Rooting phase: auxins should be normally higher as the use of cytokinin is, in
some cases, not even necessary.
o Effect of the plant growth regulators on regeneration is demonstrated in Jatropha curcas.
9) Gelling agents
o Culture media can be classified as liquid or solid.
 The liquid media have the advantage of faster (and cheaper) preparation than the solid
ones.
 Liquid media are more homogeneous; but gradients of nutrients may appear during tissue
growing in solid media.
 One serious disadvantage of using liquid media for shoot growth and multiplication is
that shoots, which are perpetually submerged in liquid cultures, may become hyperhydric
and will then be useless for micropropagation.
o Liquid > solid media in propagation ratio for some species.
o Agar has traditionally been used as the preferred gelling agent for tissue culture.
 Agar concentration in the culture medium can influence hyperhydration and propagation
efficiency- there is a strong connection between culture medium hardiness, proliferation
ratio and hyperhydration.
– Increase in the agar concentration reduces hyperhydration symptoms in plants and
propagation rate (i.e. the efficiency of micropropagation).
 The concentration of agar in the medium may also affect the formation of roots.
– E.g. rooting performance of rose decreased with increasing agar concentration
(from 6 to 15 g/L).
o Alternative to agar as a gelling agent is called gelrite. Gelrite is a gellan gum - a hetero-
polysaccharide produced by the bacterium Pseudomonas elodea (Kang et al., 1982).
 Gelrite cost less than agar, and it produces a clear gel which facilitates the proper
observation of cultures and their possible contamination.
 Gelrite showed better performance than agar for some Australian woody plants.
10) Physical Environment

o Gas exchange and relative air humidity inside the vessel
 Exchange of gases (e.g. Carbon dioxide, oxygen and ethylene) in the culture atmosphere
(in or adjacent to the culture vessel) influences in vitro plant tissue culture.
– Gas exchange occurs despite the “closed” nature of the culture vessel depending
upon the type of vessel, the closure and how tightly they are sealed together.
– The sealing of the vessels must allow sufficient ventilation to prevent significant
accumulation of ethylene and depletion of CO2.
– CO2 concentrations inside the vessels are higher in the dark due to respiration and
photosynthesis, but lower during the light period.
– Tightly closed vessels that reduce the gas exchange may affect negatively the
normal growth and development of plants during cultivation in vitro.
– Extent of sealing can be controlled by using closures with filters or vented
vessels, which allow gas exchange, increasing the photosynthetic capacity, the
multiplication rate, and the survival of plants after transfer to ex vitro conditions.
 Relative air humidity (i.e. Water status) inside the vessel is influenced by the:
– culture medium used
- gelling agent used (or its absence)
- osmotic pressure (determined by salt concentration, amount and type of
carbohydrate and quantity and type of other constituents)
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- changes in the medium with time

– culture vessel itself
– physical environment
– The plants that develop under higher relative humidity in vitro have more
transpiration and more anatomical abnormalities under ex vitro conditions, which
may result in high mortality rate during acclimatization.
- It is required to reduce the relative air humidity inside the vessel by:
opening of culture containers for some days before acclimatization, the
use of special closures that facilitates water loss or the cooling of
container bottoms, which increases the condensation of water vapor on
the gel surface.
o Light
 Light is an important environmental factor that controls plant growth and development,
since it is related to photosynthesis, phototropism and morphogenesis.
 Features of light, which influence in vitro growth, are: wavelength, flux density and the
duration of light exposure or photoperiod.
 Influence of light on in vitro growth varies among species:
– enhances root formation and shoot growth in several cases; whereas in others
darkness favored root formation
 The reduced rooting in presence of light is due to the degradation of the endogenous IAA.
 Higher light intensity are associated with greater growth in some species but not others.
o Temperature
 Temperature influences plant tissue culture just like other physiological processes.
 20°C - 27°C is most common culture temperature range;
 Optimal temperatures vary widely, depending on genotype.
Methods for Micropropagation
 All operations in plant micropropagation- involving the handling of explants and their culture are carried
out in an axenic (aseptic; sterile) environment under defined conditions, including a basal culture
medium of known composition with specific types and concentrations of plant growth regulators,
controlled light, temperature and relative humidity, in culture room(s) or growth cabinet(s).
1) Explants and their Surface Disinfection/Sterilization
1) Small pieces of plants (explants)

are used as source material to
establish cells and
34
2) tissues in vitro. All operations

involving the handling of explants
and their culture
3) are carried out in an axenic
(aseptic; sterile) environment under
defined conditions,
4) including a basal culture medium
of known composition with specific
types and
5) concentrations of plant growth
regulators, controlled light,
temperature and relative
6) humidity, in culture room(s) or
growth cabinet(s). The disinfection
of explants
35
7) before culture is essential to

remove surface contaminants such
as bacteria and
8) fungal spores. Surface disinfection
must be efficient to remove
contaminants, with
9) minimal damage to plant cells.
This chapter focuses on the general
procedures for
10) developing in vitro cultures,
illustrated by protocols for specific
plants and explants.
establish cells and
36

and their culture
types and
of explants
37

as bacteria and
18) fungal spores. Surface
disinfection must be efficient to
remove contaminants, with
procedures for
o Explants (small pieces of plants) are used as source material to establish cells and tissues in vitro.
o The most commonly used explants are organs, shoot tips and axillary buds.
o Their surfaces are disinfected before culture to remove surface contaminants such as bacteria and
fungal spores.
o Surface disinfection must be efficient to remove contaminants, with minimal damage to plant
cells.
o Typical disinfection protocols:
 70% (v/v) ethanol (for 2 min) + 5% (w/v) NaClO, containing 20 drops per liter of
Tween 20 (for 15–30 min).
 0.1% (w/v) HgCl2 (Mercuric chloride) () aqueous solution (1–3 min for leaves and stems
of herbaceous plants; 8–10 min for nodal and apical segments of woody plants; 10–20
min for seeds.
 96% (v/v) H2SO4 solution (4–5 min for seeds with a hard testa).
2) Culture media and their Preparation
38

establish cells and
and their culture
types and
39

of explants
as bacteria and
8) fungal spores. Surface disinfection
must be efficient to remove
contaminants, with
procedures for
40

establish cells and
and their culture
types and
41

of explants
as bacteria and
18) fungal spores. Surface
disinfection must be efficient to
remove contaminants, with
procedures for
o Culture media contain macroelements, microelements, vitamins, other organic components (e.g.
amino acids), plant growth regulators, gelling agents (if semisolid) and sucrose. Gelling agents
are omitted for liquid media.
o The composition of the culture medium depends upon the plant species, the explants, and the aim
of the experiments.
o Certain standard media are used for most plants, but some modifications may be required to
achieve genotype-specific and stage-dependent optimizations, by manipulating the
42
concentrations of growth regulators, or by the addition of specific components to the culture

medium.
o Commercially available ready-made powdered medium or stock solutions can be used for the
preparation of culture media.
o A range of culture media of different formulations, and plant growth regulators are supplied by
companies such as Duchefa and Sigma-Aldrich.
o Murashige and Skoog medium (MS) is used most extensively.
3) Initiation and establishment of axenic cultures (Stage I)

o Surface disinfected explants are introduced into culture, followed by initiation of shoot growth.
o The objective of this stage is to place selected explants into culture, avoiding contamination and
providing an environment that promotes shoot production.
o Shoot formation may be initiated, depending on the type of explant, from:
 apical and axillary buds (pre-existing meristems);
 adventitious meristems that originate on excised shoots, leaves, bulb scales, flower stems
or cotyledons (direct organogenesis); or
 callus that develops at the cut surfaces of explants (indirect organogenesis)
o Usually 4–6 weeks are required to complete this stage and to generate explants that are ready to
be moved to next stage.
 Some woody plants may take up to 12 months to complete this stage, termed
‘stabilization’. A culture is stabilized when explants produce a constant number of
normal shoots after subculture.
4) Multiplication –(Stage II)
o Shoot proliferation and multiple shoot production- multiplication of shoots or rapid embryo
formation from the explant occurs
 At this stage, each explant has expanded into a cluster of small shoots.
o Multiple shoots are separated and transplanted to new culture medium.
o Shoots are sub-cultured every 2–8 weeks.
o Material may be sub-cultured several times to new medium to maximize the quantity of shoots
produced.
5) Root formation (Stage III)
o Shoot elongation and rooting
 Transfer of shoots to a medium for rapid development into shoots and rooting.
o The rooting stage prepares the regenerated plants for transplanting from in vitro to ex vitro
conditions in controlled environment rooms, in the glasshouse and, later, to their ultimate
location.
o This stage involves not only rooting of shoots, but also conditioning of the plants to increase
their potential for acclimatization and survival during transplanting.
o The induction of adventitious roots may be achieved either in vitro or ex vitro in the presence of
auxins.
 The main advantage of ex vitro compared to in vitro rooting is that root damage during
transfer to soil is less likely to occur.
 The rates of root production are often greater and root quality is optimized when rooting
occurs ex vitro.
6) Acclimatization (Stage IV)
o Transfer of plantlets (regenerated plants) to soil (ex vitro) under natural environmental
conditions.
o Transplantation of in vitro-derived plants to soil is often characterized by lower survival rates.
 So, before transfer of soil-rooted plants to their final environment, they must be
acclimatized in a controlled environment room or in the glasshouse.
43
o Plants transferred from in vitro to ex vitro conditions, undergo gradual modification of leaf
anatomy and morphology, and their stomata begin to function (the stomata are usually open
when the plants are in culture). Plants also form a protective epicuticular wax layer over the
surface of their leaves.
o Regenerated plants gradually become adapted to survival in their new environment.
Applications/Advantages of Plant Micropropagation
1) High Rate of Plant Propagation

o A large number of plants can be grown from a piece of plant tissue within a short period.
o Another advantage is that micro propagation can be carried out throughout the year, irrespective
of the seasonal variations.
o Furthermore, for many plants that are highly resistant to conventional propagation,
micropropagation is the suitable alternative.
o The small sized propagules obtained in micro propagation can be easily stored for many years
(germplasm storage), and transported across international boundaries.
2) Production of Disease-free Plants

o It is possible to produce disease-free plants through micropropagation.
o Meristem tip cultures are generally employed to develop pathogen-free plants.
o In fact, micropropagation is successfully used for the production of virus-free plants of sweet
potato (Ipomea batatus), cassava (Manihot esculenta) and yam (Discorea rotundata).
3) Production of Seeds in some Crops

o Micropropagation, through axillary bud proliferation method, is suitable for seed production in
some plants.
o This is required in certain plants where the limitation for’ seed production is high degree of
genetic conservation e.g. cauliflower, onion.
4) Cost-effective Process
o Micropropagation requires minimum growing space.
 Millions of plant species can be maintained inside culture vials in a small room in a
nursery.
o The production cost is relatively low particularly in developing countries where the manpower
and labor charges are low.
5) Automated Micropropagation
o It has now become possible to automate micropropagation at various stages.
o In fact, bioreactors have been set up for large scale multiplication of shoots and bulbs.
o Some workers employ robots (in place of laborers) for micropropagation, and this further
reduces production cost of plants.
Disadvantages of Micropropagation
1) Contamination of Cultures
o During the course of micropropagation, several slow-growing microorganisms (e.g. Eswinia
spp., Bacillus spp.) contaminate and grow in cultures.
o The microbial infection can be controlled by addition of antibiotics or fungicides.
 However, this will adversely influence propagation of plants.
44
2) Brewing of Medium
o Micropropagation of certain plants (e.g. woody perennials) is often associated with accumulation
of growth inhibitory substances in the medium.
 These substances are phenolic compounds, which can turn the medium into dark color.
 Phenolic compounds are toxic and can inhibit the growth of tissues.
o Brewing of the medium can be prevented by the addition of ascorbic acid or citric acid or
polyvinyl pyrrolidone to the medium.
3) Genetic Variability
o When micropropagation is carried out through shoot tip cultures, genetic variability is very low.
o However, use of adventitious shoots is often associated with pronounced genetic variability.
4) Vitrification
o During the course of repeated in vitro shoot multiplication, the cultures exhibit water soaked or
almost translucent leaves.
o Such shoots cannot grow and even may die.
o This phenomenon is referred to as vitrification.
 Vitrification may be prevented by increasing the agar concentration (from 0.6 to 1%) in
the medium.
 But increased agar concentration reduces the growth rate of tissues.
5) Cost Factor
o For some micropropagation techniques, expensive equipment, sophisticated facilities and trained
manpower are needed. This limits its use.
HEALTH AND ENVIRONMENTAL CONCERNS OF PLANT BIOTECHNOLOGY
1) Human Health
- Opponents fear the effects of foreign genes, bits of DNA not naturally found in plants
o Allergic reactions
o Antibiotic-resistance marker genes could spread to disease-causing bacteria in humans
o Cause cancer
- To date, science has not supported any of these concerns.
2) Environmental Concerns
- Genes for pest or herbicide resistance could spread to weeds

- Few experts predict this will happen; further studies are needed
3) Regulation
- Nigeria:
o NBMA (National Biosafety Management Agency) regulates GMOs in Nigeria.
45
o NABDA (National Biotechnology Development Agency) to promotes biotechnology

practices in Nigeria.
- USA:
o FDA regulates foods on the market.
o USDA oversees growing practices.
o EPA controls use of Bt proteins and other pesticides.
46
47

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PCT406 (2Cr) – PHARMACEUTICAL BIOTECHNOLOGY_ Pharm. (Prof.) Y.B.

DEPARTMENT OF PHARMACEUTICAL MICROBIOLOGY AND BIOTECHNOLOGY

Pharm (Prof.) Y.B. NGWAI, PhD

 The term “Biotechnology” has been defined in many ways:

HISTORICAL DEVELOPMENT OF BIOTECHNOLOGY

(ii) Classical Biotechnology- built on ancient biotechnology; fermentation-promoted food production,

(iii) Modern Biotechnology

o History of Molecular biotechnology

APPLICATIONS AND BRANCHES OF BIOTECHNOLOGY

BIOTECHNOLOGY ETHICAL PROBLEMS

BASIC TECHNIQUES IN BIOTECHNOLOGY

1. Cutting DNA and Joining DNA fragments

1) Restriction endonucleases found in bacteria that cleave the deoxyribose-phosphate

2. Making a Recombinant DNA (rDNA)

4. Polymerase Chain Reaction (PCR)

 Requirements for PCR

5. Agarose Gel Electrophoresis

Agarose gel Electrophoresis

7. Polyacrylamide gel electrophoresis (PAGE)

 The polyacrylamide gel is a cross-linked matrix that functions as a sort of three-dimensional

CLINICAL IMPORTANCE OF RECOMBINANT PROTEINS

HISTORY OF PLANT BIOTECHNOLOGY

1) Early Plant breeding (12,000 years ago)

2) Classical Plant breeding

4) Modern Plant breeding

SCOPE OF PLANT BIOTECHNOLOGY

 Major areas of PBT are:

1) Plant Tissue Culture (PTC)/ Plant Micropropagation

2) Recombinant DNA Technology (Gene Cloning)/Plant Genetic Engineering

3) Plant Mutation Cloning

4) Plant Cells Technology

APPLICATIONS OF PLANT BIOTECHNOLOGY

PLANT TRANSGENESIS (PLANT GENE TRANSFER)

Methods used in Plant Transgenesis

1) Conventional Selective Breeding and Hybridization

- Polyploid plants (multiple chromosome sets greater than normal)

Cloning by Protoplast Fusion Technique

(b) Leaf Fragment Technique (“Agrobacterium”) Method

Cloning by Leaf Fragment Technique (“Agrobacterium”) Method

(c) “Gene Gun” (“Bolistic”) Method

o Technique is useful in plants that are resistant to Agrobacterium.

(d) Chloroplast Engineering Method

(e) Antisense Technology

Cloning by Antisense Technique

EXAMPLES OF PLANT-DERIVED THERAPEUTIC PROTEINS

Hepatitis B Hepatitis B Arntzen group Potato & Transgenic Phase I Kapusta et

Norwald virus Norwald Arntzen group Potato Transgenic Phase I

SOMATIC EMBRYOGENESIS AND ORGANOGENESIS

Callus formed during somatic embryogenesis

 Advantages of Somatic embryogenesis

Organogenesis Vs Somatic Embryogenesis

S/N Feature Organogenesis Somatic Embryogenesis

PLANT TISSUE CULTURE

Basis for PTC

Examples of Hormones used in PTC

Hormones Product Name Functions in PTC

Auxins Indole-3-Acetic Acid Adventitous root formation (high concen);

Why is PTC important?

4) Genetic material conservation

Important considerations in PTC

Types of in vitro PTC

Types of Plant Tissue Culture

1) Culture of intact plants (seed and seedling culture)