@Nabin Bhusal

BIT 401

Introductory Biotechnology and Biodiversity

S.N Topics No of
Lectures
1 Introduction to Biotechnology (Definition, History and Fields of Biotechnology) 1
Current activities and Future scope of biotechnology in the context of Nepal 1
2 Plant Biotechnology (Definition, types, relationship to other disciplines, Future scope of plant biotechnology in the context of 1
Nepal)
3 Genetic engineering and gene cloning (Definition, History, basic steps involved and methods of genetic engineering 1
4 Restriction enzymes and its types 1
5 Gene cloning vectors 1
6 Methods of Gene cloning 1
7 Applications of genetic engineering in the field of crop improvement 1
8 Plant cell and tissue culture (Definition, History , basic steps/techniques and types of tissue culture 1
9 Culture (Callus, Cell suspension and Protoplast) 1
10 Anther/Pollen culture and its application 1
11 Meristem and embryo culture and its application 1
12 Applications of tissue culture for crop improvement (Haploid and triploid production, invitro pollination and fertilization 1
13 Somatic hybridization and cybridization 1
14 Genetic transformation and wide hybridization 1
15 Soma-clonal/gametoclonal variants selection, production of pathogen free plants 1
16 Polymerase chain reaction and gel electrophoresis 1
17 Molecular Markers 1
18 Application of molecular Markers/MAS 1
19 Marker assisted selection (MAS) and Mapping 1
20 Introduction to Biodiversity (Basic concepts and aim of biodiversity; (Alfa, beta, gamma, and endemic diversity) 1
21 Scope and factors affecting biodiversity 1
22 Biological hierarchy of biodiversity (genes-populations-species-communities –ecosystems-landscapes-biosphere) 1
23 Biodiversity database 1
24 Biodiversity indexing 1
25 Species and genetic diversity; wild genetic diversity of some important crops 1
26 Centres of diversity of crops 1
27 Germplasm collection, conservation and utilization (concept of conservation; Ex-situ conservation and In-situ conservation) 1
28 Risk of extinction and Recovery Program 1
29 National legislation and intellectual property rights, conflict and nature of policies 1
 1. Introduction to Biotechnology (Definition,
History and Fields of Biotechnology)
• Biotechnology is the product of interaction between science of biology and
technology which deals with the controlled use of biological agents such as
microorganism or cellular components for the beneficial use.

• However, it can also defined as the science of using living organisms, or the
products of living organisms, for human benefit.

• It creates novel products for agricultural, pharmaceutical, medical and

environmental applications.

• The term “Biotechnology” was first coined by a Hungarian agricultural engineer

Károly Ereky in 1919.
 Introduction and prospective of biotechnology
Periods of biotechnology history
• Ancient biotechnology: (Pre-1800 AD) Early applications and speculation

• Classical biotechnology: (1800–1950 AD): Significant advances in the basic

understanding of genetics

• Modern biotechnology: (1950 AD onward): Discovery of DNA, Recombinant DNA

technology, genetically modified organisms, animal cloning, and stem cell research
Introduction and prospective of biotechnology
Ancient biotechnology

❑Mainly based on the common observations of nature (Pre-1800 AD).

❑Domestication of plants: as a reliable source of food.

❑Selective domestication and breeding of wild animals: (mule, which is an

offspring of a female horse and a male donkey).

❑Food processing: making of cheese and curd, bread, alcohol and vinegar.
 Introduction and prospective of biotechnology
Classical biotechnology
• Based on the scientific background to many of the

common observations (1800–1950 AD): .

• Microscope (Antonie van Leeuwenhoek (1632–1723).

• Cells (Robert Hooke (1635–1703)

• Nucleus in the cell. (Robert Brown)

• Fermentation biology, vaccination, and pasteurization (Louis

Pasteur (1822–95)

• Theory of Inheritance (Gregor John Mendel (1822–84)

• Nucleic acids (Fredrich Miescher, In 1868)

Post-Mendelian
History of Biotechnology
Genetics

Frederick Griffith

1928

He was working with two strains of bacterium Streptococcus pneumoniae

Colonies of rough (R (type-IIR, the small colonies) and smooth (S (Type-IIIS), the
large colonies) and proposed that some unknown "principle" had transformed
the harmless R strain of Diplococcus to the virulent S strain.
History of Biotechnology

Avery, MacLeod, and McCarty’s experiment revealed

the nature of the transforming principle
Post-Mendelian
History of Biotechnology
Genetics
Modern biotechnology
• Based on the Discovery of DNA (1950 AD onward)
Post-Mendelian
History of Biotechnology
Genetics
 The phase consists of DNA within a protein
coat. The virus attaches to the bacterial host
cell and injects its genes (the DNA) through
the bacterial cell wall into the host cell
cytoplasm. Inside the host cell, these genes
direct the formation of new phage DNA and
proteins, which assemble into progeny
T2 bacteriophage particles either with 32P-labeled DNA or phages that are released into the
with 35S-labeled proteins were used to infect bacterial cells. environment when the cell bursts.
After a short incubation, they shook the cultures in a Waring 35S-labeled proteins remained with
blender and spun the samples in a centrifuge to separate the Most of the
the ghosts, while most of the 32P-labeled DNA
empty viral ghosts from the heavier infected cells.
was found in the sediment with the T2 gene-
containing infected cells
Post-Mendelian
History of Biotechnology
Genetics
Post-Mendelian
History of Biotechnology
Genetics
Post-Mendelian
History of Biotechnology
Genetics
Post-Mendelian
History of Biotechnology
Genetics
Post-Mendelian
History of Biotechnology
Genetics
Post-Mendelian
History of Biotechnology
Genetics
 Post-Mendelian
History of Biotechnology
Genetics

1972
History of Biotechnology
Post-Mendelian Genetics
Post-Mendelian
History of Biotechnology
Genetics
Fields of Biotechnology

Blue
Biotechnology

White Bioinformatics
Biotechnology

Biotechnology
Animal Red
Biotechnology Biotechnology

Environmental Green
Biotechnology Biotechnology
 Plant Biotechnology
• Plant Biotechnology is a technique used for development of
desired varieties of plants by manipulating its genetics.

• Plant biotechnology assist in developing new varieties and traits

using the techniques of genetics and genomics, marker-assisted
selection (MAS) and transgenic (genetic engineered) in crops.

• These biotechnologies allow researchers to detect and map

genes, discover their functions, select for specific genes, and
transfer genes for specific traits into plants where they are
needed.
 Fields of plant Biotechnology
• Plant Tissue culture : Plant Tissue Culture is a process that uses plant
material in a growing medium to grow new platelets.

• Plant Genome sequencing: Genome sequencing involves revealing the order

of bases present in the entire genome of an organism.

• Genetic structures and mechanisms: Genes that code for amino acid
sequences are known as 'structural genes'.

• Genetic engineering (Methods for transgenic biotechnology)

• Molecular markers/ Marker assisted selection and bioinformatics

• Gene Editing/Genome Editing: These technologies allow genetic material to

be added, removed, or altered at particular locations in the genome.

• Synthetic Biology: Design and construction of new biological parts, devices,

and systems, and the re-design of existing, natural biological systems for
useful purposes.
 Genetic engineering and gene cloning
• The process of altering an organism’s DNA

• Involves in the direct manipulation of genetic material of an

organism, using molecular biology techniques.

• Genetically engineered organisms have enhance certain desired

traits like disease resistance, nutrient quality, increased crop
yield, and delayed ripening.

• Bt cotton is a genetically modified cotton variety, which has

been genetically engineered to produce an insecticide for boll-
worm.
 Steps of Genetic engineering
Whole genome of an organism
Plasmid isolation (Vectors)

Isolation of Gene of Interest and its Multiplication using

PCR
Linearization and compatible sticky end
generation using Restriction Enzyme Compatible Sticky end generation using Restriction
Enzyme

Recombinant DNA and Transformation into bacterial host

Screening of True transformants

Transformation of Clones in desired host

Selection for the expression of desired gene

Expression of the traits in next generation

 Restriction enzymes
• Microbiologists in the 1960s discovered that a bacteriophage λ which

can grow well in one strain of bacteria (E coli K).

• When it allowed to grown in another strain (E. coli C), its yields can

drop significantly.

• The host cell, (E. coli C), is called as the restricting host and have the

capability to decrease the phage activity. The ability of that phage to

grow in the other strains also becomes restricted.

• The restriction was instigated by an enzymatic break -down of the

phage λ DNA by cleaving the phosphodiester bond (in the sugar-

phosphate back -bone) that joins adjacent nucleotides in a DNA

strand

• The enzyme involved in the breakdown of phage DNA was coined as a

Restriction enzymes.

• Restriction enzymes are sophisticated “scissors” that molecular

biologists use to manipulate DNA.

 DNA Palindromes
• The palindrome in which the same forward and backwards
are on a single strand of DNA strand, as in GTAATG.

• The Inverted repeat palindrome is also a sequence that

reads the same forward and backwards, but the forward and
backward sequences are found in complementary DNA
strands (GTATAC being complementary to CATATG).

• Inverted repeat palindromes are more common and have

greater biological importance than mirror like palindromes.
 Restriction enzymes Mode of Action
• Restriction enzymes recognize a specific sequence of
nucleotides, and produce a double-stranded cut in the
DNA. these cuts are of two types:

BLUNT STICKY
END END
These blunt ended DNA fragments with
fragments can be complimentary sticky
joined to any other ends can be combined to
DNA fragment with create new molecules
blunt ends.

Enzymes useful for Which allows the

certain types of DNA creation and
cloning experiments. manipulation of DNA
sequences from different
sources.
 Restriction enzymes types
The restriction endonucleases are divided into three main groups (types I, II, and III).

Type I restriction enzymes

➢ Type I restriction enzymes have recognition sequence of 15 bp.
➢ They cleave the DNA about 1000 bp away from the 5` end of the sequence “TCA” located
within the recognition site. Eg:- EcoK-12,EcoB,etc

Type II restriction enzymes

➢ Cleaves DNA at defined positions close to or within their recognition sequences.
➢ The type II Restriction Enzyme work with the help of only single co factor (Mg2+)
➢ They produce discrete restriction fragments and distinct gel banding patterns, and they are
the only class used in the laboratory for routine DNA analysis and gene cloning.
➢ The Ist type II Enzyme to be isolated was Hind II in 1970.
➢ Mostly Type II endonucleases have recognition sites of 4, 5 or 6 bp, predominantly GC rich.

Type III restriction enzymes

➢ Type III restriction enzymes are intermediate between type I and type II endonucleases .
➢ large combination restriction-and-modification enzymes. They cut the DNA about 20–30 bp
away from the recognition site. They cleave outside of their recognition sequences

However, type IV and type V are also reported

 Restriction enzymes Nomenclature

• The name of the EcoRI restriction enzyme was derived as

• E= from the first letter of genus Escherichia,

• Co=from first two letters of species coli,

• R= the strain RY13,

• I= first identified (order of identification in bacteria).

 Features of Cloning Vectors
• Vectors are DNA (or viruses) that can be used to carry and replicate other
pieces of DNA in molecular biology experiments

• Size- small enough to be easily separated from the chromosomal DNA of

the host bacteria.

• Origin of replication (ori)-Site for DNA replication that allows plasmids to

replicate separately from the host cell’s chromosome.

• Multiple cloning site (MCS)-The MCS is a segment of DNA with

recognition sites for different common restriction enzymes.

• Selectable marker genes-These genes allow for the selection and

identification of bacteria that have been transformed with a recombinant
plasmid. (antibiotic resistance, b-galactosidase, Green Flourescent
Proteins (GFP))

• RNA polymerase promoter sequences-These sequences are used for the

transcription of RNA in vivo and in vitro by RNA polymerase.
 Cloning Vectors

• Plasmid vectors

• lambda phage

• Cosmid

• Yeast artificial chromosomes (YACs)

• Bacterial artificial chromosomes (BACs)

 Methods of Transformation
• Transformation is the process by which genetic makeup of
an organism is altered by the insertion of new gene into its
genome.

• Methods of transformation was divided into two parts

• Direct Method
• Particle gun/biolistic method of DNA Delivery
• Chemical Method; Polyethylene Glycol (PEG)/ protoplast fusion
• Microinjections
• Electroporation

• Indirect Method
• Agrobacterium mediated gene transfer
• In-planta method of transformation
 Direct Method of Transformation

Micro Injection
• The DNA can be introduced into cells or protoplast
with the help of fine needles or glass micropipettes
having diameter of 0.5 to 10 µm.

• Computerized control of holding pipette, needle,

microscope stage and video technology has
improved the efficiency of this technique.

• The major limitations of microinjection are that it is

slow, expensive, and has to be performed by
trained and skilled personnel.
 Electroporation
• Suspension of protoplast with desired DNA are prepared passes
from high electric shock for few seconds cause temporarily pores
to open allows DNA to enter the cell.

• Preferably protoplasts used largely

Low voltage-long pulses method (300-400 V cm-1 for 10-15 ms (millisecond)
High voltage-short pulses method (1000-1500 V cm-1 for 10 µs (microsecond)
Protoplast fusion
Particle gun/biolistic method of DNA
Delivery
 Agrobacterium mediated gene transfer
• The A. tumefaciens-mediated plant genetic transformation process requires
the presence of two genetic components located on the bacterial Ti-plasmid.

• The first essential component is the T-DNA, defined by conserved 25-base pair
imperfect repeats at the ends of the T-region called border sequences.

• The second is the virulence (vir) region, which is composed of at least seven
major loci (virA, virB, virC, virD, virE, virF, and virG) encoding components of
the bacterial protein machinery mediating T-DNA processing and transfer
 Agrobacterium mediated gene transfer
• Recognition and attachment of the Agrobacterium to the host cells.
• The sensing of specific plant signals by the Agrobacterium VirA/VirG
• Generation and transport of T-DNA and virulence proteins from the bacterial cells into plant cells.
• Nuclear import of T-DNA and effector proteins in the plant cells.
• T-DNA integration and expression in the plant genome.
 Applications of genetic engineering in the field
of crop improvement
• Herbicide resistance: Herbicides normally affect processes like photosynthesis or biosynthesis of
essential amino acids. Transformation of cereal crops with Glyphosate resistant gene (Glyphosate =
herbicide). Herbicide tolerant (HT) soybean and canola are released for commercial cultivation.

• Insect resistance: Insect-resistant crops contain genes from the soil bacterium Bacillus thuringiensis
(Bt). The genes which responsible for the production of delta-endotoxin in Bacillus thuringiensis is used
as biological insecticide. The transgene has been transferred to many crops for example looper
resistance in soybean, pod borer resistance in groundnut, head borer resistance in sunflower, semi-
looper resistance in castor etc.,

• Resistance against viral infection: coat protein gene from Tobacco Mosaic Virus (TMV) was transferred
to develop resistant varieties of crop plants. The resistant varieties developed in crop plants like
soybean for resistant to yellow mosaic virus, groundnut for resistant to bud and stem necrosis, clump
and stripe virus resistance, whereas in sunflower, resistance developed for bud necrosis.

• Resistance against bacterial and fungal pathogens: Chitinase genes was transferred to crops like
Brassica, Soybean, Sunflower, Sesame etc for alternaria leaf spot disease, where as in case of groundnut
which was introduced against leaf spot and alternaria blight.
 Applications of genetic engineering in the field
of crop improvement

• Improvement of the nutritional qualities in crop plants: The carotene gene has been transferred from
daphoddils to rice grains (Golden Rice) for increasing Beta-carotene content in grains and for solving
the blindness in childerns. Antisense Fae 1 gene transferred to Brassica napus and Brassica juncea for
low erucic acid content and also for low linolenic acid content in case of linseed.

• Improvement of crop plants against abiotic stresses: transcription factor genes, structural genes,
regulatory genes were introduced into the groundnut, soybean, Brassica juncea, B. napus to develop
drought and salinity tolerant types.

• Development of transgenic male sterile lines: transgenic male sterile lines of safflower Brassica juncea
were developed through the transfer of Barnase gene from Bacteria (Bacillus amyloliquefaciens). A long
term goal in agriculture is to introduce the genes (Nif genes) for nitrogen fixation in crop plants
 Plant tissue culture

➢Tissue culture is the culture and maintenance of plant cells or

organs in sterile, nutritionally and environmentally supportive
conditions (in vitro).

➢The basic principle of tissue culture is the totipotency of cells

➢Totipotency: The ability of undifferentiated plant tissues to

differentiate into functional plants when cultured in vitro
 Plant tissue culture requirements
➢The main aspect of a tissue culture laboratory is the maintenance of aseptic
working environment so as to avoid the contamination of growing cultures with
microorganisms like bacteria, fungi, and their spores.

1. Washing and storage of glassware, plastic ware and other lab

wares

2. Preparation, sterilization and storage of nutrient media

3. Aseptic manipulation of plant material

4. Maintenance of cultures under controlled conditions of

temperature, light and humidity.

5. Hardening of in vitro developed plants.

 Steps of plant tissue culture
Selection of Explants

Preparation of Nutrient Media

Sterilization of explants

Inoculation in a nutrient media

Callus induction (culturing)

Sub culturing

Shooting media

Rooting media

Plant regeneration and transfer to sterile soil (green house)

Plant regeneration and transfer to non sterile soil

 Sterilization
 The sterilization of materials, e.g., vessels, instruments,
medium, plant material, etc used in culture work can be
achieved by:

1) Dry heat treatment (150°C for 3-4 hrs

2) Flame sterilization
3) Autoclaving (121°C and 15 p.s.i, 15-20 min)
4) Filter sterilization
5) Wiping with 70% ethanol
6) Surface sterilization
 Surface Sterilization
 The plant material should be surface sterilized to remove the
surface borne micro-organisms.

 For surface sterilization chromic acid, Hgcl2(0.11%),calcium

hypochlorite, sodium hypochlorite(1-2%),alcohal(70%) are used.
Process depends on the type of explant.


absolute ethyl calcium sterile water
SEED bromine water
alcohol hypochlorite
Surface Sterilization
FRUIT ethyl alcohol sodium hypochlorite sterile water

LEAF surface clean Hgcl2 sterile water dried

STEM Running water sodium hypochlorite sterile water

Culture media

 Optimal growth and morphogenesis of tissue may very

from different plants according to their nutritional
requirement.

 The type of tissue culture medium depends on:

 The Species to be cultured
 Age of the plant
 Part of the plant used in culture

 Growth and development of an ex-plant in vitro is a

product of its genetics, surrounding environment and
component of tissue culture medium.
 Culture media

 Approximately 20 different components are needed in tissue culture

medium i.e. Macro-nutrients, Micro-nutrients, vitamins, amino acids,
Source of Carbon, undefined organic supplements, growth regulators and
Solidifying agents.

 Macronutrients (>0.5mM): Essential elements C, H, O

 Macro-molecules; N, P, K, Ca, Mg, S for satisfactory growth and morphogenesis
 At least 25-60mM of inorganic nitrogen for satisfactory cell growth
 K ranging from 20-30mM
 P, Mg, S, and Ca range from 1-3mM

 Micronutrients: Iron (Fe)-0.1µM, Manganese (Mn)-20-90µM, zinc (Zn)-5-

30µM, boron (B)-25-100µM, Copper (Cu)-0.1µM and Molybdenum (Mo)-
1µM
 Culture media

 Source of Carbon: A concentration of 20-60g/L sucrose is the most

often used carbon and energy source. Besides sucrose 2-5%, other
carbohydrates are also used i.e. Lactose, galactose, maltose. It was
frequently demonstrated that autoclaved sucrose was better for growth
than filter sterilized sucrose.

 Vitamins and myo-inositol: The vitamins most used in the cell and
tissue culture media includes: Thiamin (B1), nicotinic acid and Pyrodoxine
(B6). Thiamin and nicotinic acid is used in ranging from 0.1 to 10 mg. Myo-
inositol is play very important role in cell growth.

 Amino acids: The amino acids used for enhancement of cell growth in
culture media included; Glycine (2mg), glutamine (8mM), asparagine
(100mg), L-arginine and Cysteine (10mg) and L-tyrosine (10mg).
 Culture media

 Undefined organic supplements: natural substances or extracts such as

protein hydrolysates, coconut milk, yeast extract, malt extract, ground
banana, orange juice and tomato juice to test their effect on growth and
enhancement.

 Solidifying agents: Hardness of the culture medium greatly influences the

growth of cultivated tissue.

 Growth Regulators: These are Auxins, Cytokinins, Gibberellins and

Abscisic acid (0.001-10µM)

 PH: 5.5
 Culture media
 White’s medium - is one of the earliest plant tissue culture media

 MS medium - formulated by Murashige and Skoog (MS) is most

widely used for many types of culture systems

 B5 medium - developed by Gamborg for cell suspension and callus

cultures and at present it’s modified form used for protoplast culture

 N6 medium - formulated by Chu and used for cereal anther

culture

 Nitsch’s medium- developed by Nitsch and Nitsch and used for

anther culture
 Types of plant tissue culture

1. Type of in vitro growth: Callus & Suspension cultures.

2. Type of Explant:
1. Single cell culture
2. Shoot & root culture
3. Somatic embryo culture
4. Meristem culture
5. Anther culture & haploid production
6. Protoplast culture & somatic hybridization
7. Embryo culture, Ovule culture, Ovary culture
 Callus and Suspension Culture

• Callus Culture means an Callus Culture Suspension Culture

In this culture, cell division in It consists of single cells and small
unorganised proliferative explants forms a callus. groups of cells suspended in a liquid
mass of cells produced medium

from isolated plant cells,

tissues or organs The culture is maintained on agar The culture is maintained in liquid
medium medium

• Suspension Culture is a The medium contains growth The Medium contains growth
regulators auxin such as 2, 4-D and regulators auxin such as 2, 4-D only
type of culture in which cytokinins like BAP
single cells or small
Callus is obtained within 2-3 weeks Suspension culture grows much faster
aggregates of cells than callus culture.
multiply while
It does not need to be agitated. It must be constantly agitated at 10-
suspended in agitated 250 rpm (revolutions per minute).
liquid medium.
 Meristem culture
• Morel and Martin (1952) developed
the technique of meristem culture
for in vitro virus eradication of
Dahlia.

• The apical meristem is usually tissue

located at the extreme tip of a shoot
and measures approx. 0.1 mm in
diameter and 0.25 to 0.3 mm in
length.

• Meristem or shoot tip is isolated

from stem by applying a V-shaped
cut
 Application of Meristem Culture

• Virus elimination

• Micro Propagation

• Storage of Genetic Resources (Cryopreservation or in-vitro

conservation of germplasm)

• Use in Plant Breeding

• Quarantine

• Facilitation of exchange between locations

 Anther/Pollen culture

• Anther: A part of stamen containing pollen.

• Pollen: A fertilising powder discharged from flowers anther.

• It is the process of formation of haploid plants from microspores

(pollen) cultured individually or anthers, reported in about 250
species.
 Pathways to Androgenesis
• Pathways1: Microspores divide by equal division and
two daughter cells contribute to sporophyte. Vegetative
and generative cells are not distinct.

• Pathway 2: Unequal division of uninucleate microscopre

resulting in formation off vegetative and generative cell.
Sporophyte rise due to division of vegetative cell.
Generative cells dives slow or does not divide before
degenerates.

• Pathway 3: The uninucleate microspore undergoes a

normal unequal division but pollen embryo
predominantly formed from generative cell.

• Pathway 4: The division of microspore in asymmetrical

as in pathway II. Both generative and vegetative cell
divide and contribute to sporophyte
 Factors influencing Anther culture
• Genotype of donor plants
• Culture medium and culture density:
Without hormones - mostly dicots (Most success with solanaceous species)
With hormones - most non-solanaceous species.
Many monocots require hormones or complex organics such as coconut
milk.
Sucrose - ranges from 2% (Nicotiana) to 10% (Brassica)

• Stage of microspore or pollen development: Best time either just prior to

division of the microspore or after microspore mitosis (forms generative and
vegetative cells)
• Effect of temperature and/or light:
• Physiological status of donor plant: Anthers removed from field-grown plants gave a
better response than those picked from greenhouse-grown plants.
• anthers from the first flush of flowers in the season were found to be more
responsive
 Somatic Hybridization
• The production of hybrid plants through fusion of two different plant
protoplasts (wall less naked cells) is known as Somatic hybridization
and such hybrids are called somatic hybrids.

• Somatic hybridization involves the following steps:

1. Isolation of protoplasts.

2. Fusion of the protoplasts of desired species.

3. Selection of somatic hybrid cells.

4. Culture of the hybrid cells and regeneration of hybrid plants from them
 Isolation of protoplasts
• Protoplast are plant cell with the plasma membrane but without the cell
wall. Protoplast allow the fusion of similar or different species and the
fused product can generate into the whole plant.

• Isolation of protoplast can be done by following methods:

1. Mechanical method
2. Enzymatic method
 Mechanical Method
• Cut the tissue which are first
plasmolysed with a sharp knife into
small pieces.

• Then these pieces are deplasmolysed by

using dilute solution to release the
protoplasts.

• Generally protoplasts were isolated

from highly vacuolated cells of storage
tissues (onion bulbs, scales, radish root,
beet root).
 Enzymatic method

• Protoplast using enzymes may be isolated by sequential

method or mixed enzyme method.

• In the first process two enzymes-pectinase and cellulase are

used sequentially, while in the second process two enzymes
are used simultaneously.

• The enzyme mixture macerates the cells and simultaneously

destroys their walls.
 Enzymatic method

Leaf Sterilization, removal

of epidermis

Plasmolysed Plasmolysed
cells cells

Pectinase+
Cellulase Pectinase

Protoplasm Release of
released isolated cells

Protoplasm
Protoplasm
released
isolation
 Protoplast purification
• Enzyme solutions are filtered with nylon mesh to remove insoluble
impurities.

• Filtrate is centrifuged for 5 minutes at 700 rpm.

• The protoplast forms pellet and goes at the bottom of centrifuge tube.

• Supernatant is removed with Pasteur pipette.

• The pellet at the base is suspended in 10 ml of MS medium plus

mannitol and the process is repeated thrice.

• The resultant protoplast is pure

 Induced fusion
• Mechanical fusion: Physical fusion of protoplasts under microscope by
using micro manipulator and perfusion micropipette.

• Electrofusion: Fusion induced by electrical stimulation Fusion of

protoplasts of pearl chain is induced by the application of high strength
electric field (100kv m-1) for few microsecond.

• Chemofusion: fusion induced by chemicals

– PEG (Polyethylene Glycol)
– NaNo3
– Ca 2+ ions
– Polyvinyl alcohal
PEG (Polyethylene Glycol)
Agglutination and addition
 Identification and Selection

• Following fusion of protoplasts, identification of protoplast

fusion product is necessary to determine fusion frequency
and to monitor the fusion products.

• The preliminary identification of fusion product is done

under microscope.

• The microscopic identification is based on differences

between the parental cells with respect to pigmentation,
presence of chloroplast, nuclear staining, cytoplasmic
marker etc.
 Protoplast culture and regeneration
• Hybrid cells are cultured on nutrient medium in petri dishes.
• The plates are incubated at 25°C in a dim white light.

• The protoplasts regenerate a cell wall, undergo cell division and form
callus. The callus can also be sub-cultured.

• Embryogenesis begins from callus when it is placed on nutrient

medium lacking mannitol and auxin.

• The embryo develops into seedlings and finally mature plants.

• These hybrid plants must be at least partially fertile, in addition to

having some useful property, to be of any use in breeding schemes.
 Advantages of somatic hybridization
• Production of novel interspecific and intergenic hybrid e.g. Pomato
(Hybrid of potato and tomato)

• Production of fertile diploids and polypoids from sexually sterile

haploids, triploids and aneuploids

• Transfer gene for disease resistance, abiotic stress resistance,

herbicide resistance and many other quality characters

• Production of heterozygous lines in the single species

• Studies on the fate of plasma genes

• Production of unique hybrids of nucleus and cytoplasm

 Limitations of Somatic hybridization
• Poor regeneration of hybrid plants

• Non-viability of fused products

• Not successful in all plants

• Production of unfavourable hybrids

• Lack of an efficient method for selection of hybrids

• No confirmation of expression of particular trait in somatic hybrids

 Somaclonal and gametoclonal variation selection

• Somaclonal variation is defined as genetic variation observed among

progeny plants obtained after somatic tissue culture in vitro.
• The variations might occur in number of progeny which are known as
somaclones and they are genetically variable from their explant.
• The initiating explant for a tissue culture cycle may come virtually from
any plant organ or cell type including embryos, microspores, roots, leaves
and protoplasts. So, all somatic tissue culture can result in somaclonal
variation.
• Somaclonal variation is a phenotypic changes as a result of chromosomal
rearrangement during tissue culture.
Somaclonal and gametoclonal variation selection
Somaclonal and gametoclonal variation selection

Following are some of the basis of chromosomal rearrangement which results

in soma clonal variation
1. Karyotype changes
2. Changes in chromosome structure
3. Single gene mutations
4. Cytoplasmic genetic changes
5. Mitotic crossing over
6. Gene amplification and nuclear changes
7. Transposable elements
8. DNA methylation
Applications of Somaclonal Variation:

i. Production of Novel variants:

ii. Production of disease resistance variety:
iii. Production of abiotic stress resistance variety:
iv. Production of Cold tolerance:
v. Production of Salt tolerance:
vi. Production of Aluminium tolerance:
vii. Production of Drought tolerance:
viii. Production of Herbicide resistance:
ix. Production of Insect resistance:
x. Seed quality improvement:
xi. Introgression of Alien gene:
 Application of plant tissue culture
• Micro-propagation

• Germplasm conservation

• Somaclonal variation

• Haploid and dihaploid production

• In-vitro hybridization- protoplast fusion

• Embryo rescue

• Synthetic seed production

Polymerase chain reaction and gel electrophoresis

• This system for DNA replication that allows a "target" DNA sequence
to be selectively amplified, several million-fold in just a few hours
under in vitro condition.

• It targets and amplifies a special region of DNA strand.

• Two methods currently exist for amplifying the DNA or making copies.

• Cloning: Takes a long time for enough clones to reach maturity

• PCR: Works on even a single molecule quickly

 Requirements of PCR

• DNA template (contains the sequence

of DNA you wanted to amplify. The
DNA can be from animals, plants,
viruses, or bacteria.)

• Primers

• Taq polymerase

• Deoxyribonuleoside triphosphates
(dNTPs)

• Buffer solution is a salt-solution that

helps to stabilize the DNA and other
components of the reaction.
Buffer Solution
 Tris-HCl 10mM (10-50mM) PH 8.3 (PH 8.3-8.8 at
20°C)
 KCl 50mM
 MgCL2 1.5mM (0.5-10mM)
 Gelatin or Bovine Serum Albumin 100µg/ml
 Application of PCR
• Disease diagnosis
• Cloning
• Mutation analysis
• Detection of gene expression
• Mapping
• Site directed mutagenesis
• Sequencing
• Forensic medicine
• Pre-natal detection
• Scientific research
 Gel Electrophoresis
• Gel electrophoresis is a laboratory method used to separate mixtures of
DNA (or other macromolecules, such as RNA and proteins) based on
their size and charge..

• The molecules are separated by electrical field through a gel that

contains small pores.

• Agarose gels
• Polyacrylamide gel electrophoresis (PAGE)

• That is based on the mobility of ions in an electric field.

• Positively charged ions migrate towards a negative electrode and

negatively-charged ions migrate toward a positive electrode

• Agarose is a polysaccharide, generally extracted from certain red

seaweed.
Polyacrylamide gel electrophoresis
(PAGE):Polyacrylamide gels are formed by
copolymerization of acrylamide and bis-acrylamide
DNA and Electrical Current

+
 Requirements
• Box to hold the gel

• Comb to create small wells

in the agarose gel to put the
DNA sample into at the
beginning of the gel

• Positive and negative

electrodes to create the
electrical current

• Power supply

• Buffers

• Gel photo imaging system

 Types of Buffers

• Running/Electrophoresis buffer
➢Usually Tris-acetate-EDTA (TAE) or Tris-
borate-EDTA (TBE).

• Loading buffer/sample buffer

➢ It contains something dense (glycerol
or sucrose) to allow the sample to
"fall" into the sample wells

➢It also contains one or two tracking

dyes, which migrate in the gel to allow
visualize how far the electrophoresis
run. E.g., bromophenol blue & xylene
cyanol are used.
Procedure of Gel electrophoresis
• Prepare sufficient electrophoresis buffer (1X TAE or TBE) to
fill the electrophoresis tank and to cast the gel

• Prepare a solution of agarose in electrophoresis buffer at an

appropriate concentration
• E. g. For 2% (2gm of agarose in 100ml of electrophoresis
buffer)
Procedure of Gel electrophoresis
• Heat in a microwave oven until the agarose dissolves.

• Use insulated gloves to transfer the flask/bottle into water

0
bath at 55 C. When the molten gen has cooled, at 0.5ug/ml of
ethidium bromide. Mix the gel solution thoroughly by gentle
swirling.
Procedure of Gel electrophoresis
• Pour the warm agarose solution into the mold and allow the
gel to set completely (30-45 min at room temperature) , then
pour a small amount of gel electrophoresis buffer on the top
of the gel and carefully remove the comb.

• Pour enough 1X TAE electrophoresis buffer to cover the gel in

the chamber (prior to loading samples
Procedure of Gel electrophoresis
• Mix the DNA with a loading dye and slowly load the sample
mixture into the slots of the submerged gel using automatic
micropipette. Load size standards into slots on both the right
and left.

• Close the lid of the gel tank and attach the electrical leads so
that the DNA will migrate towards the positive anode. Apply a
voltage of 1-5V/cm)
• Run the gel until the bromophenol blue and xylene-cyanol
have migrated an appropriate distance through the gel
 Interpretation

• The sizes of the various

fragments can be identified by
including a “ladder” in the gel.

• A ladder is a mixture of DNA

fragments of known size.

• It is usually run beside the

unknown sample so that the
sizes of various DNA
fragments in the sample can
be identified
 Factors affecting mobility of molecules in gel

• Charge

• Size of DNA fragment

• Shape of DNA fragment

• Buffer conditions

• Gel concentration

• Voltage
 Applications of gel electrophoresis

• Analytical purposes : after amplification of DNA via PCR.

• preparative purposes : prior to use of other methods such as

southern blotting , cloning, PCR for further characterization.

• Check the quality and quantity of genomic DNA after DNA

extraction
Molecular Markers
• Marker: constituents that determines the function of a
construction.

• Genetic marker:
➢Any stable and inherited variation
➢That can be measured or detected by the suitable method
➢Can be subsequently used to detect the presence of a specific
genotype or phenotype other than itself, which otherwise is not
measurable or difficult to detect
➢It is an indirect way of predicting variation

• Molecular Markers are classified as:

➢Morphological Markers
➢Protein Based Markers/ Biochemical Markers
➢DNA Based Markers
Phenotypic Markers
• Phenotype for which the variation observed in the population
of interest is entirely explained by mendelian factor. E.g.
height, color, shape…etc

➢Limitations:
➢Show low degree of polymorphism
➢Reflect variability in the coding sequence only
 DNA Based Markers

• On the basis of ability to discriminate between same or different

species
– Co-dominant: discriminate between homo and heterozygotes

– Dominant: which do not discriminate between homo and

heterozygotes
DNA markers
▪ DNA markers are defined as a fragment of DNA which can be used to
detect polymorphism between different genotypes or alleles of a gene for
a particular sequence of DNA in a population or gene pool.

▪ These fragments are associated with a certain location within the genome
and may be detected by means of certain molecular technology.

▪ Reflect heritable differences in homologous DNA sequences among

individuals.
▪ That may be due to:
1. Base pair changes.
2. Rearrangements (translocation or inversion).
3. Insertions or deletions.
4. Variation in the number of tandem repeats.
Restriction Fragment Length Polymorphism (RFLP)

The Restriction Fragment Length

Polymorphism (RFLP) is based on
the Southern method for the
detection of DNA polymorphisms.

high-quality DNA is isolated from

the samples and digested with one
or more restriction enzymes.

The fragments are segregated on

AGE, transferred to a nitrocellulose
or nylon membrane and hybridized
with a probe.

The DNA bands are detected by

autoradiography,
chemiluminescence or fluorescence
PCR based markers
Randomly Amplified Polymorphic DNA (RAPD)
RAPD refers to polymorphism found in the randomly amplified fragments of DNA
generated by using single DNA primer of arbitrary sequence.
Polymorphism result from change in the primer-binding site in the DNA sequence

In variety A there are 4 primer binding sites

resulting in two RAPD products, variety B lacks
one of the binding sites resulting in only one
RAPD marker being produced
PCR based markers Randomly Amplified Polymorphic DNA (RAPD)

Primers point in the same Primers too far apart, so Primers are just the
direction, so amplification won’t right distance apart,
amplification happen so fragment is
won’t happen amplified
Disadvantages
• RAPD markers are dominant.

• The quality and concentration of template DNA

influence the outcome.

• Mismatches between the primer and the template may

decreased amount of the product.

• Problems with reproducibility.

Single-Nucleotide Polymorphism (SNP)

The Single-Nucleotide Polymorphism

(SNP) is a molecular marker based on
the detection of the mutations of
individual nucleotide bases on the
DNA

The DNA is isolated and then

subjected to a reaction to call the DNA
bases.

For instance, fluorescent dideoxy

Nucleoside Tri Phosphates (ddNTP;
ddATP, ddCTP, ddGTP & ddTTP) can be
used as with sequencing so the
polymerization reaction is stopped
when any of such terminators is
incorporated in the growing chain.

The products are then subjected to

PAGE electrophoresis and the
fluorescence is detected
Applications of Molecular markers
Applications of Molecular markers
Applications of Molecular markers
GMM: Genic molecular marker
FM: Functional markers
RDM: Random DNA markers
Applications of Molecular markers
Applications of Molecular markers
Applications of Molecular markers
Applications of Molecular markers
Applications of Molecular markers
Applications of Molecular markers

Haplotypes are groups of SNPs that are

inherited as a single block. An example of
SNPs (A or G, G or T, and A or T) found in
close proximity in the genome. During
reproduction, these three SNPs are inherited
as a group. Therefore, an individual can
inherit one of three distinct haplotypes (AGT,
GTA, AGA) depending on the SNP profile of
their parents.
Applications of Molecular markers
Applications of Molecular markers
MOLECULAR MARKERS AND MAPPING OF QTLS
MAPPING
▪ The order and relative distance of
genetic features that are associated with
genetic variation or polymorphisms can
be determined by genetic mapping.

▪ Genetic maps constructed using

molecular markers can also be used to
locate major genes which can then also
be used as genetic markers.

▪ Genetic Maps are Generally Three Types

A. Linkage maps: frequency of recombination between pairs of markers

B. Cytogenetic or cytological maps: characteristic banding pattern.
C. Physical maps: markers are depicted in terms of base pairs
MAPPING OF QUANTITATIVE TRAIT LOCI
▪ Quantitative genetics study the inheritance of quantitative of
polygenic characters in the experimental population.

▪ Quantitative characters are traits which show continuous variation

and governed by a large number of genes called multiple genes
 Mapping of Quantitative Trait Loci
• The genomic regions associated with the expression of a quantitative trait;
such a genomic region is referred to as quantitative trait locus (QTL).

• A QTL may contain one or more genes affecting the concerned quantitative
trait.

• QTLs have been grouped into different categories on the basis of their effect
size, effect of the environment on their expression, the type of effect
produced by them, and the manner of their action.

• Main effect QTLs produce direct effect on the expression of the concerned
traits.

• Epistatic QTLs interact with the main effect QTLs to influence the trait
phenotype

• Minor QTLs with a smaller effect size (explains <10 % of the phenotypic
variance
 Mapping Population

• A population that is suitable for linkage

mapping of genetic markers is known
as mapping population.

• Mapping populations are generated by

crossing two or more genetically
diverse lines and handling the progeny
in a definite fashion.

• Mapping populations are used for

determining genetic distances between
pairs of loci/genes and to map them to
specific locations in the genome
RIL Population
 Quantitative Trait Loci Methods
• Single-Marker Analysis: simplest and the earliest used method
of QTL detection. each marker is separately tested for its
association with the target trait.

• Simple Interval Mapping: requires a marker linkage map for

QTL search as it uses neighboring marker pairs to define
marker intervals and searches QTLs within these intervals.

• Composite Interval Mapping: controls the effects of QTLs

present in other marker intervals of the same chromosome,
and in other chromosomes as well
MOLECULAR MARKERS AND MAPPING OF QTLS
Mapping QTL
 Marker Assisted Selection

• Selection for the desirable allele of a gene/quantitative trait locus

(QTL) on the basis of molecular marker(s) linked to it in the place
of phenotype generated by this allele.

• Marker-Assisted Characterization of Germplasm and Genetic Purity

• Marker-Assisted Backcrossing:
 Marker-Assisted Backcrossing
MABC aims to transfer one or a few genes/QTLs of Interest from one
genetic source into a superior cultivar or elite breeding line to
improve the targeted trait.

Select backcross progeny carrying the target gene which tightly-linked to

flanking markers (foreground selection)
 Marker-Assisted Backcrossing

Select backcross progeny with background markers (background

selection) to accelerate the recovery of the recurrent parent genome
 Marker-Assisted Backcrossing

This method of MABC approach

is used to reduce the number of
deleterious genes (linkage drag)
that are transferred from the
donor parent.
BIODIVERSITY
 BIODIVERSITY-LEVELS
Genetic Diversity : Genetic variation
within species, both (within
individuals of single population and
geographically separated population).
 BIODIVERSITY-LEVELS
Species Diversity : Variety of different
types (species) of organisms: Includes
all the species, microbes, viruses,
bacteria to animals and plants
BIODIVERSITY-LEVELS
➢ Ecosystem Diversity: Biodiversity includes
variation in the geographical community.
➢ Variations in the community in which the
species live
➢ The ecosystem were the community exists
➢ Interaction within and between biotic and
abiotic components.
BIODIVERSITY-MEASURE
Alpha Diversity
➢Numbers of species in a unit area (single community)
➢This diversity closest to the popular concept of species richness
and can be used to compare the number of species in different
ecosystem types or communities
➢Richness and of evenness of the individuals within a habitat unit
can me measured as

Alpha Diversity of site-A= 7, Site-B=5, Site-C=7

A vs B= 8 species
Site-C
B vs C=4 species
Site-B
Site-A
C vs A=10 species
BIODIVERSITY-MEASURE
Beta Diversity
➢ The degree to which species composition changes along an
environment gradient
➢ The Beta diversity is high e.g. if the species composition of moss
communities changes at successively higher elevations on a mountain
slope, but it is low if the same species occupy the whole mountain
side.
➢ it express the diversity between habitats

Beta Diversity observed between site A and C with 10 species that differ
between them and only 2 species in common

A vs B= 8 species
Site-C
B vs C=4 species
Site-B
Site-A
C vs A=10 species
BIODIVERSITY-MEASURE
Gamma Diversity
➢Applies to larger geographical scale
➢The rate at which additional species are encountered as
geographical replacements within a habitat type in different
localities
➢it a species turnover rate with distance between sites of similar
habitat or with expanding geographical areas.
Gamma Diversity: landscape diversity or diversity of habitats within a landscape
region. The gamma diversity is 3 habitats with 12 species total diversity.

A vs B= 8 species
Site-C
B vs C=4 species
Site-B
Site-A
C vs A=10 species
BIODIVERSITY-MEASURE
Endemic Diversity
➢Endemic species are those that are found in just one region and
nowhere else in the world.
 Benefits Of Biodiversity

• Consumptive value:
➢Food/Drink
➢Fuel
➢Medicine
➢Batter crop varieties
➢Industrial Material
• Non-Consumptive Value:
➢Recreation
➢Education and Research
➢Traditional value
 Benefits Of Biodiversity
• Ecological services:
➢ Balance of nature
➢ Biological productivity
➢ Regulation of climate
➢ Degradation of waste
➢ Cleaning of air and water
➢ Cycling of nutrients
➢ Control of potential pest and disease causing species
➢ Detoxification of soil and sediments
➢ Stabilization of land against erosion
➢ Carbon sequestration and global climate change
➢ Maintenance of Soil fertility
 Threats to biodiversity

• Natural causes:
• Narrow geographical area
• Low population
• Low breeding rate
• Natural disasters
• Anthropogenic causes:
• Habitat modification
• Overexploitation of selected species
• Innovation by exotic species
 Threats to biodiversity

• Pollution
• Hunting
• Global warming and climate change
• Agriculture
 Biodiversity Database
• "All Catfish Species Inventory".
• "Arctos".
• "AntWeb".
• "ASEAN Biodiversity Information Sharing Service".
• "CITES-listed species database".
• "FishBase : A Global Information System on Fishes".
• "FLOW: Fulgoromorpha Lists on The Web".
• "Freshwater Ecoregions of the World".
• "HerpNET".
• "Integrated Biodiversity Information System".
• "Integrated Taxonomic Information System".
• "Natural History Information System".
• "NatureServe Explorer: An online encyclopedia of life".
• "Wikispecies, free species directory".
• "A Pan-European Species-directories Infrastructure (PESI)".
• "Naturdata - Biodiversidade online".
• "Georgian Biodiversity Database".
• "ScaleNet: Scale insect web catalog".
 Biodiversity index

D=1–[∑(n/ N) 2 ]

D=diversity index
n = number of individuals
N = total number of individuals
 Species observed Percentage cover
Field A (n) Field B (n)
Cocksfoot grass 57 38
Timothy grass 32 16
Buttercup 3 14
Clover 3 22
Thistle 1 5
Dandelion 4 5
Total (N) 100 100
 Species observed Percentage cover

Field A (n) n/N

Cocksfoot grass 57 0.57

Timothy grass 32 0.32

Buttercup 3 0.03

Clover 3 0.03

Thistle 1 0.01

Dandelion 4 0.04

Total (N) 100

 Species observed Percentage cover
Field A (n) n/N (n/N)2
Cocksfoot grass 57 0.57 0.349

Timothy grass 32 0.32 0.1024

Buttercup 3 0.03 0.0009

Clover 3 0.03 0.0009
Thistle 1 0.01 0.0001
Dandelion 4 0.04 0.0016
Total (N) 100 ∑ = 0.4308

D = 1 – [ ∑ ( n / N)2 ]
D = 1 – 0.4308
D = 0.5692
 Species observed Percentage cover
Field B (n) n/N (n/N)2
Cocksfoot grass 38 0.38 0.1444

Timothy grass 16 0.16 0.0256

Buttercup 14 0.14 0.0196

Clover 22 0.22 0.0484
Thistle 5 0.05 0.0025
Dandelion 5 0.05 0.0016
Total (N) 100 ∑ = 0.243

D = 1 – [ ∑ ( n / N)2 ]
D = 1 – 0.243
D = 0.757
• D for Field A = 0.5692
• D for Field B = 0.757

• “Field B has the higher diversity index, so has more species

richness AND evenness. It would be more resistant to any
environmental damage or change
 Centers of diversity of crops
• A centre of origin (or centre of diversity) is a geographical area where a group of
organisms, wild species either domesticated or, first developed its distinctive
properties.

• Vavilov concluded that each crop has a characteristic primary centre of diversity
which is also its centre of origin.
 Risk of extinction and Recovery Program
International Union for Conservation of Nature (IUCN)

• The IUCN used to assess an increasingly diverse range

of taxa occurring in a wide variety of habitats and makes
Red List of the species based on their extinction risk.
 Risk of extinction and Recovery Program
• A taxon is Extinct when there is no reasonable doubt that the last individual has
died.
• A taxon is Extinct in the Wild when it is known only to survive in cultivation.
• Critically Endangered, Endangered and Vulnerable, are assigned to taxa on the
basis of quantitative criteria that are designed to reflect varying degrees of threat
of extinction; taxa in any of these three categories are collectively referred to as
‘threatened’.
• Near Threatened is applied to taxa that do not qualify as threatened now, but may
be close to qualifying as threatened.
• Least Concern is applied to taxa that do not qualify (and are not close to
qualifying) as threatened or Near Threatened.
• Data Deficient highlights taxa for which sufficient information is lacking to make
a sound status assessment.
• Not Evaluated applies to taxa that have not yet been evaluated against the Red
List Criteria .
Germplasm collection, conservation and utilization
• The sum total of all the genes present in a crop and its related
species constitutes its germplasm.

• Germplasm provides the raw materials in plant breeding

programme, without which breeding is impossible to conduct.

• The conservation of germplasm involves the preservation of the

genetic diversity of a particular plant or genetic stock.

• In-situ conservation: The germplasm is conserved in natural

environment by establishing biosphere reserves such as
national parks, sanctuaries.

• Ex-situ conservation: It is the process of protecting an

endangered species, variety or breed, of plant or animal
 TYPES OF CONSERVATION

• Short term (Working Collections)

(>3-5 years) at 10-15°C at10% moisture

• Medium Term (Active Collections)

10-15 year at temperature 15°C (often near 0°C) at 5%
moisture

• Long Term (Base Collections)

50 years more stored about -20 °C with 5% moisture
content
 UTILIZATION OF PLANT GERM PLASM RESOURCES
• Genetic enhancement
• Promoting sustainable agriculture through diversification of
crop production

• Promoting network for plant genetic resources for food and

agriculture.
• Developing monitoring and early warning system for loss of
plant genetic resource for food and agriculture.
• Promising public awareness of the value of plant genetic
resources for food and agriculture conservation and use
• Used as a variety, as a parent in the hybridization and also
used to transfer resistance to biotic and abiotic stresses.