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1. 2024
Agricultural Biotechnology

2. 2
How human civilization transformed Agriculture?
▪From food gathering to digital Agro-economy

3. 1
Contents
6.1. Plant cell and tissue culture
6.2. Protoplast culture
6.3. Microspore/anther culture and
haploid production
6.4. In vitro fertilization and embryo
rescue methods
6.5. Genetic transformation
6.6. Embryo transfer and transgenic
animal production
6.7. Transgenic plants
6.8. Bio-fertilizers

4. 4
6.1. Plant cell and tissue culture
▪Plants are key to life on earth. Why?
✓Supply 90% of human calorie intake
✓80% of the protein intake
▪Plants are characterized by the ability to regenerate from a piece of tissue
into a complete plant in in vitro conditions, a phenomenon called totipotency
and autonomy.
▪Over the past few years, a number of methodologies have been developed in
advancing research in plant science on:
1.Growth of cells, tissues, organs, protoplasts, etc. by tissue culturing method
2.Plant transformation by genetic engineering (rDNA technology)

5. 5
Terms
Explant - Tissue taken from its original site and transferred to an artificial
medium for growth or maintenance.
Hormones-Growth regulators, generally synthetic in occurrence, that
strongly affects tissue growth and development.
Medium - A nutritive solution (solid or liquid) for culturing cells.
Micropropagation - In vitro clonal propagation of plants from shoot tip
or nodal explants in artificial medium
Differentiation - Modifications of new cells to form tissues or organs
with a specific function.
Culture - plant growing in formulated media.
Callus- unorganized, proliferated mass of plant cells.
Aseptic Technique---Procedures used to prevent the introduction of
fungi, bacteria, viruses,,,, into the culture.

6. 6
Terms cont …
Agar-a complex polysaccharide powder derived from algae used to gel a
medium.
Regeneration---In plant cultures, a morphogenetic response to a
stimulus that results in the products of organs embryos or whole plants
results in the products of organs, embryos, or whole plants.
Somaclonal Variation- Phenotypic variation, either genetic or
epigenetic in origin, displayed among somaclones.
Somaclones- Plants derived from any form of cell culture involving the
use of somatic plant cells. „
 Sterile - Without life or a culture that is free of viable microorganisms
Subculture - the process by which the tissue or explants is transferred
into fresh culture medium. „

7. 7
Tissue culture
▪It is the practice of growing plant cells or tissues on artificial nutrient
media using small pieces of plant part as a starting material under
controlled environmental conditions.
✓Produce clones of plants with the same genotype, except few cases.
✓Widely used for large scale plant multiplication.
▪Used as important tool for research and development activities.
▪Plant tissue culture is important in the agro-industry area of:
✓Clonal plant propagation
✓Plant disease elimination
✓Plant variety improvement
✓Production of secondary metabolites, etc.

8. 8
Tissue culture cont …
▪The plant part that is used as a starting material for tissue culture is called
explant.
▪Explant is a any piece of plant part (root ,shoots …) used as a
starting material for culturing.
▪In vitro cultivation of plants by cell/tissue culture is possible due to
totipotency, autonomy and plasticity of plant cells.
▪Totipotency – it is the capacity of plant cells to develop into a new
complete plant from small explant.
▪Plasticity – ability of a plant to endure severe conditions by changing
growth and development of their organs.

9. 9
Tissue culture cont.
Dedifferentiation - is the capacity of mature cells to return to
meristematic condition and development of a new growing point.
Redifferentiation - is the ability of meristematic tissues to
organize into new tissue and organ after dedifferentiation.
Phases of explant development into a complete plant:
Mature plant → Explant → Dedifferentiation → Callus →
Redifferentiation → Complete plant

10. 10
Brief History of tissue culture
The first tissue culture technique was initiated by Gottlieb Haberlandt in
1902.
▪He used single cells isolated from:-)
▪ palisade tissue of leaves
▪ pith parenchyma
▪ epidermal layer of various plants
▪He grew them on Knop’s salt solution with sucrose. He observed
growth in the cultured palisade cells
▪Composition of Knops solution:-)
-Ca(NO3)2, 3 g - Sucrose, 50 g (optional) - MgSO4, 1 g
-KNO3, 1 g - K2HPO4, 1 g - Deionized Water

11. 11
What conditions do plant cells need to multiply in vitro?
▪Tissue culture has several critical requirements. These are:
✓Appropriate starting material (explant)
✓A suitable growth medium containing energy sources and inorganic salts.
➢The medium can be liquid or semisolid.
✓Aseptic (sterile) conditions:- as microorganisms grow much more
quickly than plant and animal tissue and can overrun a culture.
✓Growth regulators – Hormones like auxins and cytokinins.
✓Frequent sub-culturing to ensure supply of adequate nutrition and to
avoid the build-up of waste metabolites.

12. 12
Major steps in tissue culture
i.Preparation of suitable nutrient media.
ii.Selection of appropriate explants.
iii.Surface sterilization of the explants by disinfectants (eg. by Sodium
hypochlorite solution)
iv.Transferring explants onto the nutrient medium in culture vessels
under aseptic conditions in laminar air flow hood
v.Growing the cultures in the growth chamber or plant tissue culture
room at optimum conditions of light, temp and humidity.
vi.Hardening - it is the gradual exposure of plantlets for acclimatization
to environmental conditions in the green house
vii.Transfer of plants to the field conditions following the acclimatization.

13. 13
Major steps in tissue culture

14. 14
Spacial requirements of a tissue culture work in the lab
▪Media preparation area
▪Aseptic transfer area
▪Culture area
▪Acclimatization area (green house)

15. 15
Applications of plant tissue culture
▪In forestry industry
▪secondary metabolite production (eg. Pharmaceuticals)
▪Selection of crops with advantageous characters(e.g. herbicide
resistance/tolerance)
▪to cross distantly related species
▪Production of diploids from haploids (eg. protoplast fusion)
▪Produce large numbers of identical clones (Micropropagation)
▪Germplasm preservation
▪Production of variability by somaclonal variation, etc.

16. 16
Sterilization methods
▪Aseptic condition is maintained by sterilization.
▪Different sterilization techniques
1. Chemical sterilization - using alcohol and bleaching agents
✓Surface-sterilization
2. Autoclaving – sterilization by heating to 121˚C at 103.5kPa for 15-20
min.
✓eliminate bacterial and fungal contaminants
3. UV radiation- for 15 minutes or slightly more
4. Filter Sterilization - for heat labile compounds.
✓Certain media components are susceptible to heat denaturation.
✓filter such components using a 0.22µm pore size filter.
✓add to the media after autoclaving.

17. 17
Plant tissue culture techniques
There are two major techniques in plant tissue culture:
a.Static/solid culture - using solid medium
✓the explant grows on solid agarmedium andgives rise to aggregated
mass of tissue called a callus.
b.Suspension cultures – growth on liquid medium
✓Keep cells in suspension.
✓Has higher growth rate than cells in solid-agar medium.
Suspension cultures are two types:
1.Batch suspension cell culture - cells or tissues are grown in a
fixedvolumeofnutrientmedium.
✓ When the cells reach exponential phase, the entire culture is
replaced with new medium.
✓It is a closed type of culture.

18. 18
2. Continuous suspension culture- cells grow by continuous addition
ofnutrients to the growing media.
✓to maintain the dilution rate, an equivalent volume of media is
removed out proportional to the in flow from top.
✓the cells are always kept in exponential growth phase.
✓It is open type of culture.

19. 19
Advantages of tissue culture
➢Large numbers of plants can be produced from a single plant in relatively
small space in a short period of time.
✓ This reduces growing space, labor and plant maintenance
requirements
➢Viruses and other systemic diseases can be eliminated by propagating
meristematic cells.
➢Avoid seasonal restrictions for seed germination
➢Produce exact copies (clones) of plants. Eg, produce good quality flowers,
fruits or have other desirable traits.
➢Produce plants that otherwise have very low chances of germinating (Eg.
Orchids)
Orchid italica

20. 20
Overview on the basic requirements of tissue culture
1. Starting tissue or explant
➢Cell, tissue or organ of a plant can be used to start in vitro cultures.
✓Axillary buds and meristems are most commonly used
➢Many plants have polyphenolic compounds.
✓Phenolic products inhibit enzyme activities and may kill the explants.
✓During tissue dissection, the injury may cause oxidation of phenolics
by polyphenol oxidases (PPO).
✓ PPO turn tissues to brown.
➢Browing can be avoided by the following methods:
✓adding antioxidants such as ascorbic acid, citric acid,
polyvinylpyrrolidone (PVP)
✓Activated charcoal - absorb toxic pigments and stabilize pH.
✓Frequent transfer into fresh medium.

21. 21
2. Nutrient medium
➢When an explant is isolated from the mother plant, it is no longer able to
get nutrients or hormones from the parent plant.
➢they must be provided with artificial nutrient medium to allow growth in
vitro.
➢Contain all the nutrients required for the normal growth and development
of plants.
➢Composed of macronutrients, micronutrients, vitamins, other organic
components, plant growth regulators, carbon source and gelling agents in
case of solid medium.
➢Waste products of cell metabolism are eliminated by replenishing the
culture media periodically.

22. 22
Nutrient medium cont ….
➢The composition of the medium, particularly the plant hormones and the
nitrogen source has profound effects on the response of the initial explant.
➢Both the solid and liquid medium can be used for culturing.
➢Murashige and Skoog medium (MS medium) is most extensively used
for the vegetative propagation of many plant species in vitro.
➢The pH of the media is important that affects both the growth of plants and
activity of plant growth regulators.
▪The pH is adjusted between 5.4 - 5.8.

23. 23
Carbon sources and vitamins
➢Sucrose, glucose or fructose at a concentration of 2-5%
➢Myoinositol - improves cell growth
➢Vitamin B1 (thiamine) - is an absolute requirement
➢Tissue growth is also improved by the addition of nicotinic acid and
vitamin B6 (pyridoxine)
➢Some media contain pantothenic acid, biotin, folic acid, choline chloride,
riboflavin and ascorbic acid
Plant growth regulators
➢Plant growth regulators play an essential role in determining the
development pathway of plant cells and tissues in culture medium.
➢Auxins , cytokinin and gibberellins are most commonly used as plant
growth regulators.

24. Murashige and Skoog Basal Medium composition (Sigma-Aldrich)
24
Components mg/L
Ammonium nitrate 1,650.0
Boric acid 6.20
 CaCl (anhydrous) 332.20
 Cobalt chloride hexahydrate 0.0250
 Cupric sulfate pentahydrate 0.0250
Disodium EDTA dihydrate 37.260
 Ferrous sulfate heptahydrate 27.80
Glycine 2.0
 Magnesium sulfate (anhydrous) 180.70
Components mg/L
▪Manganese sulfate monohydrate 16.90
▪myo-Inositol 100.0
▪Nicotinic acid 0.50
▪ Potassium iodide 0.830
▪Potassium nitrate 1,900.0
▪Potassium phosphate monobasic 170.0
▪Pyridoxine hydrochloride 0.50
▪Sodium molybdate dihydrate 0.250
▪Thiamine hydrochloride 0.10
▪Zinc sulfate heptahydrate 8.60

25. 25
Plant growth regulators ……
Auxins:
▪high auxin to cytokinin ratio enhances root formation.
▪Induces cell division, cell elongation, swelling of tissues, formation of
callus, formation of adventitious roots.
▪Inhibits adventitious and auxiliary shoot formation
✓ Eg. 2,4-D, NAA, IAA, IBA ..
Cytokinin:
▪Induce shoot formation and cell division, Eg, BAP, Kinetin, zeatin
▪High cytokinin to auxin ratio promotes shoot formation
▪A balance of both auxin and cytokinin leads to the development of mass of
undifferentiated cells known as callus.

26. 26
Plant growth regulators …..
Gibberellins:
▪Used for plant regeneration, elongation of internodes, Eg. GA3
Abscisic acid:
▪Induce embryogenesis , Eg. ABA
Organic supplements
▪N containing amino acids (glutamine, asparagine) and nucleotides
(adenine)
▪Organic acids: TCA cycle acids (citrate, malate, succinate, fumarate),
pyruvate
▪Complex substances -Yeast extract, malt extract, coconut milk ..

27. 27
Mineral salts
▪Macroelements - are essential elements required in concentration
above 0.5 mM/L. These are N, K, P, Ca, S, Mg, Cl
▪Microelements - are elements required in conc. < 0.5 mmol/L.
✓These include Fe, Mn, B, Cu, Zn, I, Mo, Co ……..
Different Techniques of Plant Tissue Culture:
➢Micropropagation
➢Callus and Cell culture
➢Somatic embryogenesis
➢Haploid culture
➢Protoplast culture
➢Organogenesis
➢Somaclonal variation ,,,,,,

28. 28
Types of tissue culture
Micropropagation
In vitro clonal or mass multiplication and propagation of agricultural
and horticultural plants
To generate large number of identical plantlets rapidly
Explants are usually derived from meristematic tissues
Steps of Micropropagation
Stage 0 -Selection and preparation of the mother plant
✓Sterilization of the plant tissue
✓Sterilization of explants to remove microbial contaminants.
 usually done by chemical surface sterilization
Stage I - Initiation of culture
✓Explant placed into growth media

29. 29
Stage II - Multiplication/ shoot formation
✓Explants transferred to shoot media
✓shoots can be constantly divided
Stage III - Rooting
✓Explant transferred to rooting media
Stage IV – acclimatization (transfer to soil in the green
house)
✓Growing explants returned to soil
(hardening)

30. 30

31. 31
Organ Culture
the culture of isolated organs under laboratory conditions.
different names are given depending upon the type of organ used for the
culture.
E.g. the culture of roots, endosperm, ovary, and ovule are called as
root culture, endosperm culture, ovary culture, etc respectively.
Skoog (1944) suggested that organogenesis could be chemically
controlled for the first time.
Organogenesis
▪It refers to the process of differentiation by which plant organs are
formed (roots, shoot, buds, stem etc.) either de novo (from callus) or
adventitious (from the explants) in origin.

32. 32
Callus culture
▪Callus refers to an unorganized mass of parenchymatous cells.
▪It has biological potential to develop tissues (root, shoots and
embryoids) and then to complete plant.
▪Callus cultures can be maintained indefinitely by repeated sub-
culturing.
✓Naturally, callus is formed by the infection of plant with MOs at
wounded tissues due to stimulation by endogenous growth hormones;
▪the auxins and cytokinins.
✓ it is now possible to artificially develop callus by using tissue culture
techniques.
✓Callus production is maintained when auxin and cytokinins are present
at equal proportion in nutrient medium

33. 33
Callus culture …..
▪Nutrient medium composition is an important factor for callus
formation.
▪Callus culture can be compact or friable.
✓Compact callus - strong aggregations of cells
✓Friable callus – loose collection of cells
✓Friable cells can be broken into single cell culture grown in
suspension
▪Callus cultures are used for:
✓ plant regeneration,
✓ preparation of single cell suspensions and protoplasts,
✓ genetic transformation studies.

34. 34
Three stages of callus culture:
1.Induction – cells begin to differentiate and begin
to divide
2.Proliferative stage – rapid cell division
3.Morphogenesis stage – Differentiate and
formation of organized structures differentiation
may lead to:
✓Organogenesis
✓Somatic embryogenesis – embryo initiation and
development from somatic cells

35. 35

36. 36
6.2. Protoplast culture
Protoplasts are plant cells without cell wall.
The term protoplast was first used by Manstein in 1880.
Protoplast is isolated by using enzymes like cellulases and pectinases
from leaf mesophyll, seedling, callus, …
Protoplasts can undergo cell division, regenerate cell wall and form
callus.
Examples of plants regenerated from protoplasts :
Cucumis sativusCapsicum annum
Ipomoea batata
Glycine max

37. 37
Protoplast cont …..
▪Protoplast cultures have the following applications:
✓biochemical and metabolic studies
✓fusion of two somatic cells to create somatic hybrids
✓for transformation or fusion of protoplasts from different cell lines
✓fusion of enucleated and nucleated protoplasts to create Cybrids
(cytoplasmic hybrids)
✓Genetic manipulation
✓Drug sensitivity test

38. 38
6.3. Microspore/anther culture and Haploid production
Anther/pollen culture
▪Anther is part of the stamen where pollen is produced
▪Anther culture is a method of producing haploid plants in which a mature
plant is formed from a single microspore.
▪The existence of haploid plants was 1
st
reported by Bergner (1921) in
Datura stramonium.
▪Haploid embryos and plantlets can develop from microspores of excised
anthers.
Datura stramonium
Stamen

39. 39
▪Haploids have single set of chromosomes
▪Haploid plants are useful in breeding for several reasons:
✓Haploids carry only one allele of each gene, so recessive characters
and mutations are expressed
✓Plants with lethal or undesired traits are eliminated from the gene
pool
✓Can produce homozygous diploids and polyploids which are valuable
in breeding.
✓Shorten the period of production of superior hybrids in genotype
Haploid production

40. 40
Cont …..
✓Development of homozygous lines in a short span of time.
✓The generation of exclusive male plants by the process of androgenesis to
double the chromosome numbers.
✓Production of disease resistant plants by introducing disease resistant
genes.

41. 41
Somatic hybridization
▪Somatic hybrids are made by fusing protoplasts isolated from two
distantly related plants.
➢produce hybrids or cybrids with desirable characteristics.
▪It is of particular interest for characters related to the chloroplast or
mitochondrion.
➢These genes are inherited maternally in sexual crossings.
▪In the fusion process, the nucleus and cytoplasm of both parents are
mixed in the hybrid cell (heterokaryon).
➢results in various nucleo-cytoplasmic combinations.
▪Sometimes interactions in the plastome and genome contribute to the
formation of cybrids (cytoplasmid hybrids).

42. 42
Somatic Embryogenesis
▪It is the process of developing embryos from vegetative cells
▪Embryo can be developed from starting material by direct or indirect
methods.
➢Direct method – use plant cells (explants), no callus
➢Indirect method – use of callus made from explants
▪It depend on plant regeneration through organogenesis.
▪Organogenesis can lead to genetic variation (somaclonal variation)
Embryogenesis is the production of embryos from
somatic or “non-germ” cells.

43. 43
Somaclonal variation
▪It is variation generated by plants produced by asexual means of
propagation, a phenomenon mostly associated with callus culture.
➢The variation (mutation) is caused by genetic changes
➢The mutation can be stable and heritable.
➢The plants produced from somatic tissue with new characteristics are
called somaclones.
➢Mutants may arise spontaneously or induced with chemicals, DNA-
altering mutagenic agents.
➢It allows the generation of plants with new and useful traits.
Eg. Herbicide resistance, stress tolerance, disease resistance.
➢Long-term cell-suspension cultures are prone to somaclonal variation.

44. 44
6.4. In vitro fertilization and embryo rescue methods
▪There is a reproductive barrier between distantly related organisms.
▪Hybridization between distantly related organisms result in premature
death of embryo and production of sterile seed.
▪Interspecific and intergentic reproductive barrier can be overcome by in
vitro fertilization on suitable media
▪This method of protecting embryo and its viability in vitro is termed as
embryo rescue methods.

45. 45
Embryo Culture
▪Embryo culture is developed due to the need to rescue embryos from
wide crosses where fertilization occurred, but embryo development
did not occur.
▪Embryo rescue techniques enable to overcome embryo inviability.
▪Embryo rescue and culture is a particularly attractive technique for
recovering plants from sexual crosses where the majority of embryos
cannot survive in vivo.
▪These techniques have been further developed for the production of
plants from embryos developed by non-sexual methods, e.g., haploid
production.

46. 46

47. 47
6.5. Genetic transformation in plants
➢It is the means to transfer genes with desirable trait into plants and
recovery of transgenic plants
▪It is the most recent aspect of tissue culture
➢It has a great potential of genetic improvement of various crop plants by
integrating plant biotechnology to plant breeding programs.
➢Used to introduce agronomically important traits that :
▪enhance resistance to pests, drought, salt and diseases
▪Increase quality and quantity of yield

48. 48
Gene transfer mechanisms
➢Genetic transformation in plants can be achieved by:
❑Vector mediated
✓Agrobacterium based vector
✓Virus-based vectors
❑Direct gene transfer (with out vector)
A) Vector mediated
1.Agrobacterium mediated gene transfer
➢It is widely used for the expression of foreign genes in plant cells.
➢Discovered by Marc Van Montagn and Jeff Schell
Introduction of agronomic traits into plants

49. Gene transfer mechanisms ….
49
➢Agrobacterium tumefaciens – is a gram –ve soil bacteria that cause the
development of a tumor called gall located on the crown of the plant.
❑Naturally invades plants through wounds and induces crown galls
(tumors)
▪It is called a “crown gall disease” affecting broad leaf plants, E.g. Grape.
▪A. tumefaciens contain a special large plasmid called the Ti-plasmid
▪Part of the Ti- plasmid called T-DNA is transferred to a plant cell
when A. tumefaciens infects a plant.
In the plant, part of the Ti plasmid DNA integrates into one of the plant
chromosomes, and it is transcribed and translated to produce several
enzymes that help support the bacterium

50. 50
Gene transfer mechanisms ….

51. 51
The Ti plasmid can be used to transfer genes into plants.

52. 52
Flanking sequences LB and RB are required for the transfer of the DNA
segment from bacteria to the plant cell

53. 53
Gene transfer mechanisms ….
Tumor inducing (Ti)-Plasmid

54. Tumor inducing (Ti)-Plasmid
54
Ti-plasmid is a circular DNA found in Agrobacterium
Ti-plasmid contains genes for the synthesis of:
Opium-amino acids - food for the bacterium
Plant hormones – auxin and cytokinin
Virulence (vir) genes
 all these genes are expressed at high level
Thus, Ti-Plasmid has three main regions:
T-DNA region
Vir genes
Opine catabolism region

55. Transferred(T)-DNA
55
T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid
found in A. tumefaciens.
T-DNA is flanked by left (LB) and right(RB) borders
LB and RB are excision points
Excision starts at RB and follows by nick at LB as a single strand
Coated by vir E proteins
Transportation of T-DNA is via membrane channel formed by vir B
protein

56. Vir genes
56
Vir genes – located at 35kb region of the Ti-plasmid
Mediate the transfer of T DNA and its integration into the plant
genome.
Vir gene is organized into 8 operons (Vir A to H) and has
approximately 25 genes.
Needed for the production of trans acting proteins that are essential
for the transformation of plant cells
Switched on by chemicals called acetosyringone
Acetosyringone is produced by wounded plant cells
Activation of vir genes excise T-DNA from the plasmid

57. 57
Transportation of T-DNA to plant cells mediated by cascades of vir gene
expressions
➢It is a binary vector system
▪Essential transfer functions are supplied separately

58. 58
Unique Biological characteristics of Ti-plasmid:
➢It contain certain genes that cut and insert into the plant genome
➢The genes expressed in the host and produce protein hormones that
induce uncontrolled cell division causing the formation of tumor
➢T-DNA has genes responsible in synthesizing metabolites that is used by
the bacteria living in the plant tissue.
➢The borders of T-DNA in the plasmid has recognition sequences
➢The natural gene transfer system is exploited for the transfer of valuable
traits into plant hosts.
Agrobacterium rhizogenes – is another species of Agrobacterium
It induce extensive hairy root formation in infected plants
Harbour root-inducing plasmid (Ri-plasmid)

59. 59
Infection cycle of Agrobacterium tumefaciens

60. 60
Different crown gall tumors

61. 61
Application of Agrobacterium genetic engineering?
T-DNA can be excised out and replaced by gene of interest (eg,
herbicide tolerant gene)
Agrobacterium can transfer the gene of interest to the host plant
The gene can be expressed in the host plant to produce new traits that
enable the plant to acquire the desired traits, for example herbicide
resistance, longer shelf life, disease resistances ….
Eg, flavor-savor tomato- the 1
st
GM food

62. 62
Ti plasmid mediated genetic transformation of plants

63. 63
Limitations :
Host range is limited: not all plants are susceptible to Agrobacterium
✓Monocotyledons are not very susceptible to Agrobacterium infection
Major challenges of Agrobacterium gene transfer are host specificity by the
infectious agent and difficulty in plant regeneration after transformation
Plant viruses as vectors
Caulimoviruses – ds-DNA CaMV
Geminiviruses – 2ss DNA – maize streak virus
RNA plant viruses - TMV

64. 64
2. Direct gene transfer methods
Physical methods of gene transfer, not biological
Overcome host specificity and regeneration problems
Some of the direct gene transfer methods are:
✓Microinjection
✓Electroporation
✓Chemical – mediated
✓Biolistic method or particle gun method

65. 65
Direct gene
transfer methods
Mechanism of gene transfer Draw backs
Microinjection ➢Direct injection of the gene or plasmid
into the protoplast using micro-needles
➢Laborious, and
produce few
transgenics
Electroporation ➢Transfer of DNA through the membrane
under high voltage direct current
➢Require
formation of
protoplast like
microinjection
Chemical mediated ➢Cell wall removed by enzymes
➢Activate membrane by chemical agents
like polyethylene glycol and dextran
sulfate
➢Transfer of DNA into the protoplast
➢Depend on the
development of
protoplast
Biolistic or particle-
gun method
➢Small particles of tunguston or gold
coated with DNA accelerated into cell
interior using external pressure
➢In the cell, the DNA dissolve and integrate
to the genome
➢Can be applied to
intact cell
➢This method does
not require
protoplast

66. 66
Particle gun
The Helium Gas gun

67. 67
Transgenic plants
➢Transgenic plants are plants which express the traits coded by the
transgenes
What is the basic difference between transgenics and traditional breeding in
plants?
▪Traditional breeding allow movement of genes only between members
of a particular genus or close species of plants.
▪Transgenic technology can transform genes with out species barriers
from any source (Eg: from animals, bacteria, virus …)
Seedlings Mature plants
Wild-type Wild-type
Drought
tolerant
Drought
tolerant

68. Transgenic plants …..
68
➢What are the essences of gene transfer in plant?
✓Crop improvement
✓Diseases resistance
✓Stress tolerance
✓Improved performance
✓Value-added traits
✓Longer shelf life
✓edible vaccines and antibodies, etc.
Transgenic plants
74% of soybeans
71% of cotton
32% of corn

69. Beneficial traits of transgenic plants
69
1. Stress tolerance
✓Herbicide tolerance
✓Disease resistance
✓Abiotic stress tolerance – eg, drought, salinity, pH …
2. Delayed ripening of fruits and vegetables
3. Male sterility – prevent self pollination
4. Improved nutrient quality
5. Transgenic plants can serve as bioreactors – molecular farming
✓Diagnostic and therapeutic proteins
✓Edible vaccines
✓Biodegradable plastics
6. Metabolic engineering and secondary metabolites

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Disease
▪Ug99 - wheat stem rust
▪Ug99 - is found in Uganda, Kenya,
Ethiopia, Sudan
Some examples
Value added nutrients
▪Vitamin A–enriched rice
Long shelf life
▪Wild-type (top) and antioxidant-enriched
tomatoes

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Herbicide resistance
▪Herbicides are chemical agents that destroy weed plants or inhibits their
growth.
Eg: Glyphosate – common herbicide
-Sold by Monsanto under the trade name RoundUp
-Phosphate derivative of amino acid Glycine
-Environmental friendly
-Breaks down into non-toxic compounds
-Kill weed plants by blocking the synthetic pathway of aromatic amino
acids (phenylalanine, tyrosine and tryptophan)
▪Inhibits the enzyme EPSPS (5-enolpyruvoylshikimate-3-phosphate
syntheses)

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EPSPS is product of aroA gene in chloroplast
▪It is found in plants, fungi, Bacteria

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EPSPS
▪Found resistance gene to Glyphosate in bacteria
▪Bacterial terminators and sequences were replaced by plant
▪Chloroplast transit peptide was added
▪Transit peptide cleaved off
▪Only functional enzyme enters the chloroplast
▪This Glyphosate resistant gene transfer were carried our in
different crops
▪Canola and cotton plants – Ti plasmid

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Insecticides
Are chemicals used to kill insects
Very costly, hazardous procedures
More toxic to human
Naturally occuring insecticides are only harmful to insects
Eg; Bt toxin (Bacillus thuringienis)
➢These toxins are used to prevent cotton boll worm (destroy cotton) and
European corn borer destroy corn)
Insect larvae are killed by Bt Toxins

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▪Corn based edible vaccine
▪Transgenic corn kernel
▪Eliminate unwanted chemicals
▪E.g. Tearless less Onion
Nutritional and medicinal values

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6.6. Embryo transfer and transgenic animal production
▪Culturing eukaryotic cells has more complex media requirements
▪Animal cell cultures are susceptible to contamination too.
▪They have limited growth even in nutritive media.
▪Most animal cell dies or stop growth in culture after limited generations,
except immortal cell lines and cancer cells
▪Cells derived from kidney, liver, embryo and muscles stop growth when
they come in contact to the wall of the container, a condition called
contact inhibition.

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Transgenic animals:
Carry foreign gene(s) that has been deliberately inserted into their
genome
Foreign genes are inserted into the germ line of the animal, so it can
be transmitted to the progeny
Transgenic technology create animals with altered, but useful genetic
profiles
The first transgenic animal was a ‘supermouse’ created at university of
washington in 1982.
It was created by inserting human growth hormone gene into mouse
genome.
❑Giant mouse developed
from a fertilized egg
transformed with human
growth hormone gene

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Steps of producing transgenic animals:
1.Construction of a (recombinant) transgene.
2.Introduction of foreign gene into the animal cell.
3.Screening for transgenic positives.
✓Transgenic progenies can be screened by PCR to examine the site of
incorporation of the gene.
✓Some transgenes may not be expressed if integrated into a
transcriptionally inactive sites.
4.Further animal breeding is done to obtain maximal gene expression.
✓Heterozygous offsprings are mated to form homozygous strains

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Two common methods

80. 80
Methods of gene transfer in creating transgenic animals
1. Microinjection method
A female animal is superovulated and eggs are collected
The eggs are fertilized in vitro.
The transgene containing solution is injected into the male
pronucleus using a micropipette.
 fertilized eggs with the transgenes are kept overnight in an
incubator to develop to a two cell stage.
The fertilized eggs are then implanted into the uterus of a pseudo-
pregnant female
Microinjection of
DNA into
fertilized eggs

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2. Embryonic stem cell method
Transgenic animals can be created by transforming embryonic stem cells
with transgenes.
ES cells are obtained from the inner cell mass of a blastocyst.
➢Transgenic stem cells are grown in vitro. Then they are inserted into a
blastocyst and implanted into a surrogate uterus.
➢Transgene is incorporated into the ES cell by microinjection, retrovirus,
or by electroporation.
Retrovirus-mediated transgenics
✓Infect early stage embryo with replication-defective retrovirus.
✓Infecting mouse embryos with retroviruses before the embryos are
implanted
Size of transgene (transferred genetic material) is limited.

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➢An animal that gains
new genetic
information from the
addition of foreign
DNA is described as
Transgenic while
the introduced DNA
is called the
transgene
Embryonic
stem cell
method

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Pronucleus microinjection Retrovirus mediated gene transfer

84. 84
NB: For a short time after fertilization, the male pronucleus and female
pronucleus exist separately.
Female pronucleus:
In the maturing of the ovum preparing to impregnation part of the
germinal vesicle becomes converted into a number of small vesicles
which aggregate themselves into a single clear nucleus which travels
towards the center of the egg and is called the female pronucleus.
Male pronucleus:
In impregnation, the spermatozoan which enters the egg soon loses its
tail, while the head forms a nucleus, called the male pronucleus which
gradually travels towards the female pronucleus and eventually fuses
with it, forming the first segmentation nucleus.
The male pronucleus is larger than the female’s and can be seen easily
under a light microscope.

85. Some examples of transgenic animals
1. Fishes:
▪Superfish – Eg, Salmon, a growth
hormone inserted into fertilized egg
▪Glo fish –a GM zebra fish (Danio rerio) produced
by integrating a fluorescent protein gene from
Jelly fish into embryo of fish.
2. Mouse – 1
st
transgenic animal
3. Transgenic pig – Enviro Pig, created by
introducing phytase gene of E.coli
4. Transgenic cows – are made to produce proteins
Lactoferrin and interferons in their milk.
5. Transgenic sheep – eg, for production of good
quality of wool
6. Transgenic Monkey – ANDi
✓ANDi - 1
st
transgenic monkey
85

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Importance of creating transgenic animals
1. Gene transfer improve the productivity of livestock
Introduce genes for faster growth rates or leaner growth patterns =
quicker to market and healthier meat
Chickens
Dairy industry
2. Transgenic Animals as Bioreactors
Whole animals can serve as bioreactors to produce proteins
Gene for a desired protein is introduced via transgenics to the target
cell
By using cloning techniques, cell is raised to become an adult animal
Produce milk, eggs …. that are rich in the desired protein

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3. Medical importances
✓Provide animal models for study of human disease
✓Bioreactors for pharmaceuticals
✓Xenotransplantation
✓Resistance to bacterial infections
✓In vivo immunization
4. Agricultural importances
✓Disease resistant animals
✓Quality and quantity of milk, eggs, meat, wool
✓Increasing casein content of milk, and cheese production
✓Lactose free milk (transgene lactase)
5. Industrial importances
✓Toxicity sensitive transgenic animals to test chemicals
✓Spider silk in milk of goat ?

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6.8. Bio-fertilizers
▪Are biological preparation of live microbial inoculants
▪Fertilizer preparations containing live microbes that helps in
enhancing soil fertility
▪Help crop plants uptake of nutrients by their interactions in the
rhizosphere to biological processes.
➢ Eg, nitrogen fixing bacteria inoculants
➢Not harmful to the plants and environmentally friendly
▪It also include organic fertilizers such as manure which render
available nutrients

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Cyanobacteria

90. Phosphobacteria
90
P is key for root formation and generally for plant growth.
 plants utilize only 10-15% of the phosphate applied .
The remaining 85-90% remains insoluble in soil.
Bacteria such as Bacillus megatorium can dissolve insoluble
phosphate by secreting organic acids.
➢This increase availability of P for plants.

91. Potash mobilizing bio-fertilizers
91
Bacteria such as Frateuria aurantia mobilize potassium in soil
 Potash bio-fertilizer increase yield by up to 20%
Enhance quality of crop product
 improve water and mineral uptake

92. Compost Biofertilizers
92
Compost biofertilizers are prepared from animal dung and plant remains
Enrich the soil with useful nutrients and microorganisms
Microorganisms break down the waste matter by biological process
Cellulostic fungal cultures and Azetobactor cultures can be used for the
compost biofertilizers.

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Summary of major types of bio-fertilizers
▪Nitrogen bio-fertilizers – avail nitrogen in usable forms
▪Phosphorus bio-fertilizers – improve phosphorus level in soil
▪Compost bio-fertilizers – enrich the soil with useful microbes and
nutrients.
Eg, Cellulytic fungal cultures and Azetobacter cultures
Advantages of bio-fertilizers
High yield of crops
Reduce environmental pressure of chemical fertilizers
Microbes may protect plants from diseases
Not expensive
Environmental friendly