EDIBLE VACCINE

1. PLANT TISSUE
CULTURE AND ITS
APPLICATIONS

2. Contents:
• History
• Introduction
• Tissue culture
• Nutrient medium
• Fundamental Principles of
plant tissue culture
• Plant tissue culture Techniques
• Micropropagation
• Somatic cell Genesis
• Transgenic Plants
• Major steps of Tissue Culture
• Initiation phase
• Multiplication phase
• Root formation phase
• Types of Tissue culture
• Cell culture
• Callus culture
• Protoplast culture
• Organ culture
• Seed culture
• Embryo culture
• Meristem culture
• Anther culture
• Applications of Tissue
Culture

3. History
• Haberlandt, Father of plant tissue culture. cultured single
cells on Knop’s salt solution.
• Hanning(1904) Embryo culture of selected
crucifers.
• Robbins(1922), in vitro culture of root tips.
• Muir(1953) isolation and culture of single plant cells.
• Skoog and Miller(1957) hormonal control in tissue culture.
• Reinert and Steward(1958) report somatic embryogenesis.

4. History
• E C Cocking (1960) Isolation of
enzymatic degradation method.
• Murashige and Skoog (1962)
media.
protoplasts by
Development of MS
• Guha and Maheshwari (1964) Production of First
haploid plant by anther culture

5. Plant Tissue Culture
• The growth or regeneration of plant cells,
tissues, organs or whole plants in artificial
medium under aseptic conditions.
• In this technique use
called Explant.
plant parts or cells
INTRODUCTION:
• The most widely used artificial nutrient
medium is MS medium.

6. Fundamental Principles of Plant Tissue Culture
• Totipotency, ability of plant cells to regenerate
into whole new plant
• Plasticity, ability of plants to alter their
metabolism, growth and development to best
suit their environment
Plant cell
Plant
tissue
culture
depends
upon;
Tissue
Callus
Plant organ Embryo
New plant

7. Plant Tissue Culture Techniques:
1. Micropropagation:
• Developing high- quality clonal plants.
• Provide rapid and large scale propagation of new
genotypes.
• Steps:
• Sterilization and introduction
• Shoot production
• Root production
• Transfer to soil

8. 2. Somatic cell genetics:
• It is used mostly in terms of haploid production
and somatic hybridization.
• Steps:
• Isolation of protoplast
• Fusion by using chemicals
• Plating of fused protoplast
• Selection

9. 3. Transgenic plants:
• Transgenic plants are plants that have been
genetically engineered, uses recombinant DNA
techniques to create plants with new
characteristics.
•They are identified as a class of genetically
modified organism (GMO).
•The complete process is
described as:

10. Major Steps of Tissue Culture:
• Initiation Phase (Stage 1)
• The tissue of interest is obtained,
and introduced.
• Multiplication Phase (Stage 2)
• The in vitro plant material is introduced in to
the medium.
• Proliferation of the tissue and the production
of multiple shoots.
sterilized

11. Major Steps of Tissue Culture:
• Root formation (Stage 3)
• Roots are formed.
• Here, hormones are required in order to
induce rooting such as “Auxin”.
• Consequently complete plantlets.

12. Types of
Tissue
Culture
Seed
culture
Embryo
culture
Organ
culture
Meristem
culture
Protoplast
culture
Callus
culture
Cell
culture
Anther
culture

13. Cell culture:
• The culture of isolated individual cells.
• Carried out in dispersion medium.
Callus culture:
• The growth of callus from explant by
providing appropriate conditions.
• Darkness and solid medium gelled by agar
stimulate callus formation.

14. Protoplast culture:
• The cell in which the cell wall has been removed.
• In this, isolated protoplasts are cultured on a
suitable medium under the aseptic condition.
• The protoplast culture is aimed to develop
genetically transformed plant.

15. Organ culture:
• Isolated plant organs (Shoot, root, leaf, and flower) are
used as explant.
• The organ culture may be organized or unorganized.
• New growth (differentiated structures)
continues given that the organ retains its:
• Physiological features,
• Provide information on patterns of growth,
• As well as development.

16. Seed culture:
• For this method, explants are obtained from an in-vitro
derived plant.
• Introduced into an artificial environment.
• Where they get to proliferate.
• Seed culture is primarily used for plants such as orchids

17. Embryo Culture:
• Embryo culture is the isolation and growth of
mature or immature embryo in-vitro.
• For embryo culture, the ovule, seed or fruit
from which the embryo is to be obtained is
sterilized.
• Culturing them in nutrient media, help in
developing viable seedlings.

18. Meristem culture:
• A meristem is a localized group of cells, which
are actively dividing and give rise to
permanent tissue.
• The apical meristem of
cultured.
shoots is
• To get the disease-free plants.

19. Anther Culture:
• Anthers of some plants are cultured on a
suitable medium to produce haploid plants,
it is called anther culture.
• Separated anthers from flowers.
• Cultured on a suitable medium.
• This technique was first used in India to
produce haploids of Datura.

20. Applications of Plant Tissue Culture:

21. Clonal Propagation:
• Useful tool to get a large population of
species having desirable traits.
plant
• This technique is very much used in horticulture
and silviculture—orchids and many fruit plants.
• production of plants of same
Large scale
genetic stock.

22. Secondary Metabolites:
• Secondary metabolites produced by
cultures are rather small in amount.
plant cell
• By clonal selection the particular high yielding
clone of cells can be isolated.
• Production of many useful compounds
can be done by plant cell culture.

23. Genetic Variability:
• The chromosomal instability in the cultured
cells play an important role in polyploidization
of cells and genetically variable plants can be
raised.
•
• Such kind of variations may show some useful
characters such as:
Different kinds of morphological variations in leaf
and flowering.

24. Somatic Embryogenesis and Synthetic Seed:
• Direct or indirect somatic embryogenesis may be
achieved from:
• Pro-embryonic cell of the direct explant
• The embryoids developed withinthe callus
tissue.
• The application of this technique is:
• the mass production of adventitious embryos
which ultimately develop into complete
plantlet.

25. Haploid Plants:
• Haploid plants can be obtained through anther
pollen culture (androgenesis).
• Colchicine treatment within a very short period.
or
• These haploids produced homozygous diploid or
polyploid lines.

26. Somatic Hybrids:
 hybridization successful.
• Obtain somatic hybrid plants between sexually compatible
and incompatible plants.
• Production of cybrid is also of immense
importance in the plant breeding program.
• Somatic hybridization, using isolated
somatic
protoplasts is a new tool to make
the wide

27. Transgenic Plants:
• The genetically modified (GM) plants, in which a
functional foreign gene has been incorporated by
biotechnological method.
• A number of transgenic plants
have been produced carrying genes for different
traits.
• The direct DNA uptake through protoplast is
the most ideal method.

28. Germplasm Conservation:
• Many of the important crop species produce recalcitrant
seeds.
• Mainly
needed
the plant species which are endangered,
to be conserved by ex-situ method of
germplasm conservation.
• Plant tissue culture may be applied for this purpose.

29. EDIBLE
VACCINES

30. VACCINE
• A biological preparation that improves immunity to a particular disease.
• contains an agent that resembles a disease-causing microorganism and is
often made from weakened or killed forms of the microbe
• stimulate the body's immune system to recognize the agent, destroy it, and
keep a record of it for later encounters
• reduced mortality rate caused by various organisms.
• one of the safe and effective measure to control various infectious diseases.
• A protein which acts as the vaccine, present in food and consumed as the
internal composition of food is known as EDIBLE VACCINE

31. EDIBLE VACCINE- WHY?
• Immunization through DNA vaccines is an alternative but is an expensive
approach
• Edible vaccine gives cost-effective, easy-to-administer, easy-to-store and
socio-culturally readily acceptable vaccines for their delivery systems.
• Oral vaccines provide “mucosal immunity” at various sites by secreting
antibodies.
• Don’t need to worry about re-use, misuse and lack of sterilization. Thus,
low risk of infection.

32. History of Edible vaccines
Mason et al 1992 Haq et al 1995 McGarvey et al 1995 Mason et al 1996
Hepatitis
Hepatitis B
surface antigen
(HBsAg)
Tobacco/leaf
Norwalk virus
(NV)
Gastroenteritis
Norwalk virus
capsid protein
(NVCP)
Potato/tuber
tobacco/leaf
Rabies virus
Rabies
Rabies virus
glycoprotein
(RVG)
Tomato/leaf,
fruit
V. Cholerae
Cholera
Cholera toxin
B subunit
(CTB)
Tobacco/leaf
Hein et al 1996 ….
E. Coli
Diarrhea
Heat labile
toxin B subunit
(LTB)
Potato/tuber,
tobacco/
leaf
5

33. IDEAL PROPERTIES
EDIBLE
VACCINES
Nontoxic or
Nonpathogenic
very low levels of
side effects
Not
problems
cause
in
individuals with
impaired
immune system
Long lasting humoral
and cellular immunities
Vaccination
should be
Simple
Not
contaminate
the
Environment
Sould be
effective in
affordable

34. Plants used for edible vaccine
• Tobacco
• Potato
• Banana
• Tomato
• Rice
• Lettuce
• Soybean
• Alfalfa
• Carrot
• Peanuts
• Wheat
• corn

35. Plant species
Banana
Advantages
Do not need cooking.
Protein not destroyed even after cooking.
Inexpensive .
Grown widely in developing countries.
Disadvantages
Trees take 2-3 to mature years. Spoils
rapidly after ripening.
Potato :
Advantage
Easily transformed. Easily
propagated.
Stored for long periods without
refrigeration.
Disadvantage
Need cooking which denature antigen.
Rice
Advantages
Commonly used in baby food. High
expression of antigen.
Disadvantages
Grows slowly.
Requires glasshouse condition.
Tomato
Advantage
Grow quickly. Cultivate broadly.
High content Vitamin-A may boost
immune response.
Disadvantages
Spoils readily.

36. TARGET PATHOGENS EXPRESSED IN MODE OF ADMINESTRATION
Enterotoxigenic Ecoli (humans) Potato, tobacco Immunogenic and protective when
administered orally.
Vibrio cholera(humans) Potato Immunogenic and protective when
administered orally.
Hepatitis B virus Tobacco Extracted proteins is Immunogenic
(humans) when administered by injection
Hepatitis B virus (humans) Potato Immunogenic and protective when
administered orally.
Norwalk virus(humans) Potato Virus like particles form and
Immunogenic when administered orally.
Rabies virus (humans) Tomato Intact glycoproteins
Foot and mouth disease (agricultural
domestic animals)
Arabidopsis Immunogenic and protective when
administered orally
Foot and mouth disease (agricultural
domestic animals)
Alfalfa Immunogenic and protective when
administered by injection or orally
Transmissible Maize Protective when administered
gastroenteritis corona virus oral

37. recombinant DNA technology
 A technique mainly used to change the phenotype of an
organism (host) when a genetically altered vector is
introduced and integrated into the genome of the
organism.
 So, basically, this process involves the introduction of a
foreign piece of DNA structure into the genome which
contains our gene of interest.
 This gene which is introduced is the recombinant gene
and the technique is called the recombinant DNA
technology.

38. Tumour inducing plasmid Agrobactrium tumefaciens

39. Plant cell in protoplast
tissue culture media

42. CONCEPT OF EDIBLE VACCINE
Developed by Arntzen in the 1990s.
Introduce genes of interest into plants (Transformation)
Genes expressed in the plant tissues edible parts (Transgenic plants)
Genes encode putatively protective vaccine antigens from viral, bacterial, and
parasitic pathogens that cause disease in humans and animals
Ingestion of the edible part of the transgenic plant (Oral delivery of vaccine)

44. FACTORS
AFFECTING
EDIBLE
VACCINES
Antigen selection
Efficacy in model systems
Choice of plant species
Delivery and dosing issues
Safety issues
Public perceptions and
attitudes to genetic
modification
Quality control and licensing

46. 1. MALARIA
Three antigens
development
are currently being investigated for the
of a plant-based malaria vaccine,
merozoite surface protein (MSP) 4 and MSP 5 from
Plasmodium falciparum, and MSP 4/5 from P. yoelli.
Wang et al have demonstrated that oral immunization
of mice with recombinant MSP 4, MSP 4/5 and MSP1,
a mucosal adjuvant,
effective against blood-
co-administered with CTB as
induced antibody responses
stage parasite.
2. MEASLES
Mice fed with tobacco expressing MV-H (measles
haemagglutinin from Edmonston strain) could
virus
attain
antibody titers five times the level considered protective for
humans and they also demonstrated secretory IgA in their
faeces.
Carrot, banana and rice are the potential candidates

47. 3. HEPATITIS B
significantly exceeded the protective level
humans..
of 10 mIU/mL in
potato-based vaccine against hepatitis B have reported The
amount of HBsAg needed for one dose could be achieved in a
single potato.
4. STOPPING AUTOIMMUNITY
The transgenic potato and tobacco plants when
fed to nonobese diabetic mice showed increased
IgG, an antibody associated with
that suppress harmful immune
levels of
cytokines
response.

48. 5. CHOLERA
plants were transformed with the gene
encoding B subunit of the E. coli heat liable
enterotoxin (LT-B). Transgenic potatoes
expressing LT-B were found to induce both
serum and secretory antibodies when fed to
mice; these antibodies were
protective in bacterial toxin assay
in vitro. This is the first “proof of
concept” for the edible vaccine.

49. Advantages of Edible vaccines
 DO not require administration by injection.
 Possible production of vaccines with low costs.
 Do not require separation and purification of
vaccines from plant materials.
 Necessary syringe & needles not required.
 Economical in mass production and
transportation.
 Heat stable, eliminating the need for
refrigeration.

50. DISADVANTAGE OF EDIBLE VACCINE
Development of immunotolerance to vaccine peptide or protein.
Consistency of dosage form fruit to fruit, plant-to-plant, and generation-
to-generation is not similar.
Stability of vaccine in fruit is not known.
Dosage of vaccines would be variable.
Selection of best plant is difficult.
Certain foods like potato are not eaten raw, and cooking the food might
weakens the medicine present in it.
Not convenient for infants.

51. Safety aspects
• Contamination through cross pollination.
• vaccine antigen may affect browsing animals.
• Vaccine contamination via plant debris spreading on surfaces and
ground waters.
• Affect on humans living in the area drinking vaccine polluted water or
breathing vaccine polluted dust.
• cultivation and production of pharmaceutical crops should be limited to
control the production facilities like greenhouse, or in plant tissue culture,
that prevent the environmental release of biopharmaceuticals.