2. What is tissue culture?
Tissue culture is the term used for “the process of growing
cells artificially in the laboratory”.
Tissue culture involves both plant and animal cells.
Tissue culture produces clones in which all product cells
have the same genotype.
3. Plant Tissue Culture
Plant tissue culture refers to the techniques of
growing plant cells, tissues, organs, seeds or
other plant parts in a sterile environment on a
nutrient medium.
5. A glance in History
In 1902, Haberlandt; father of plant tissue culture ( He proposed
that plant cells could be cultured.
In 1930- White cultured tomato root tip and subculutred to fresh
medium containing salts, yeast extract and sucrose and vit B
During this period, some plant growth regulators, additives and
vitamins was discovered for the plant micro- propagation.
discovery of first PGR ---IndoleAcetic Acid , called IAA, in 1937
In 1962, Murashige and Skoog published a recipe for MS media.
In 1972, protoplast fusion has been done in toabacco.
6.
7. Micropropagation is rapid clonal in vitro
propagation of plants from cells, tissues or organs
cultured aseptically on defined media contained in
culture vessels maintained under controlled
conditions of light and temperature.
Briefly, it is the art and science of multiplying plants
in- vitro.
8. Explants
Explant is an excised piece of tissue or organ taken
from the plant to initiate a culture.
They can be :
• shoot meristem, tip, bud
• leaf or stem (internode)
• root
• anther / microspore
• ovule
• embryo associated seed parts
21. Steps of micro-propagation
Stage 0- Selection and preparation of the mother
plant(Sterilization of the explant tissues)
Stage I- Initiation of culture ( Explant placed into growth
media)
Stage II- Multiplication ( Explant transferred to shoot
media and shoot can be constantly divided)
Stage III- Rooting ( Explant transferred to rooting media)
Stage IV- Transfer to soil ( Hardening off)
22.
23. Why do micro-propagation
A single explant can be multiplied into several thousand
plants in less than a year.
Once established, it can give a continuous supply of
young plants throughout the year i.e. irrespective of
season.
Taking an explant does not usually destroy the mother
plant, so rare and endangered plants can be saved
somehow.
Clones through micro-propagation are ‘true to type’ as
compared with seedlings, which show greater variability
24. Why do micro-propagation?
This allows fast selection for crop improvement - explants
are chosen from superior plants, then cloned.
Plant ‘tissue banks’ can be frozen, then regenerated
through micro-propagation.
Disease and virus free plants can be produced through
this technique.
Greater credibility in international market as plantlets are
produced through micro-propagation( Phyto-sanitary
perspective)
25. How possible is this ??
Micro-propagation of almost all vegetables and fruit
crops is possible.
Some examples,mustard, corn, soybean, azalea,
dwarfing sweet cherry, strawberry, mango, banana,
rose, orchid, nutraceutical plants, rhododendron,
citrus, potato, tomato,legumes etc.
26. Applications of micro-propagation
Somaclonal Variation
Germplasm Conservation
Mutation Breeding
Inducing mutation
Embryo culture
Haploid and Dihaploid production
In Vitro Hybridization- protoplast fusion
Production of Disease free plants
Molecular farming
Genetic engineering
Production of secondary metabolites
27. Applications ..
Somaclonal variation
Somaclonal variation is a general phenomenon of all
plant regeneration systems that involve a callus phase.
There are two types of Somaclonal variation:
Heritable genetic variation
Non- Heritable genetic variation
28. Applications..
Advantages of somaclonal variation
It helps in crop improvement
Creates additional genetic variants
Plants with resistant and tolerant to toxins, herbicides,
high salt and even mineral toxicity
Suitable for breeding purposes
Increased and improved production of secondary
metabolites
29. Applications..
Germplasm conservation
Micropropgation is utilized in conserving genetic
resources.
Depending upon the crop species and method of
preservation, tissue culture can help in the
preservation of genetic resources from 1 to 15 years.
Cyopreservation – the preservation of germplasm in a
dormant state at ultra-low temperatures, usually in
liquid nitrogen (-196 °C) – is a type of tissue culture
which can be used to preserve seeds.
30. Applications..
Mutation Breeding
1927: Muller produced mutations in fruit flies using x-
rays
1928: Stadler produced mutations in barley
Basically, there are three groups of breeders
1) Mutation breeding is useless, we can accomplish the
same thing with conventional methods
2) Mutation breeding will produce a breakthrough
given enough effort
3) Mutation breeding is a tool, useful to meet specific
objectives
31. Applications..
Types of mutation
Spontaneous (natural mutation)
Some type of spontaneous mutation have played an
outstanding role in development of valuable crop
cultivars and hybrids.
But limitation of this is that, it can not form the basis
of modern plant breeding
Induced mutation
Any mutation found in nature can be induced by
mutation breeding.
32. Applications..
Inducing Mutations
Physical mutagens (Irradiation)
These mutagens helps in the chromosome aberrations
and point mutations.
Neutrons, Alpha rays
Gamma, Beta, X-rays
Chemical mutagens
By using different carcinogenic chemicals which are
highly toxic in nature and eventually result in point
mutations.
33. Applications..
Embryo Culture
Embryo culture is usually done from the need to
rescue embryo from wide crosses where fertilization
occurred, but not the embryo development.
Objectives:
Rescue F1 Hybrid from wide crosses
Overcome seed dormancy by addition of hormone to
media. For ex. GA
To overcome immaturity in seed
To rescue valuable genotype from dead or dying plant
To speed up the generations in a breeding program
34. Applications..
Embryo culture as a source of genetic variation
Hybridization
Can introduce new genetic combinations through
inter-specific crosses
Can transfer mutant alleles between species
Polyploidy
It combines embryo culture with chromosome
doubling to create new polyploid species
35. Application…
Haploid and dihaploid production
Plants produced through the anther culture are the
haploids.
Doubling the chromosomes without going into series
of backcrossing produce homozygous plants.
This technique shortens the time of breeding by half.
36. Application…
In Vitro Hybridization( Protoplast fusion)
Created by degrading the cell wall using enzymes.
Very fragile in nature.
Protoplasts can be induced to fuse with one another.
Methods:
Electrofusion
Poly Ethylene Glycol(PEG)
Addition of calcium ions at high PH Values
37. Applications..
Production of Disease free plant
Heat treatment
Plants grow faster than viruses at higher temperature.
Meristemming
Viruses are transported from cell to cell through
plasmodesmata and vascular tissue.
Apical meristem are virus free in nature. So, micro-
propagation of these cells gives virus free plantlets.
Even, not all the cells in the plant are infected. For
example, adventitious shoots formed from single cells
can give virus free shoots.
38. Applications..
Molecular farming
Where plants are treated as bioreactors for the production
of specific compounds.
Range from simple peptides to a thermoplastic.
Two types of products:
1)High value compounds with small scale production
requirements such as pharmaceutical products.
For ex. Malaria epitope.
2)Compounds needed on a bulk scale with low production
costs (plant biotechnology has greatest potential in this
area)
For ex. α-Amylase (food + detergent industries)-starch
manipulation
39. Applications…
Genetic Engineering
Genetic engineering would not be possible without the
development of plant tissue culture.
Genetic engineering requires the regeneration of
whole plants from single cells.
Efficient regeneration systems are required for
commercial success of genetically engineered
products.
40. Application..
Protoplast fusion between male sterile cabbage and
normal cabbage was done and cybrids were selected
that contained the radish mitochondria and cabbage
chloroplast.
41. Applications..
Production of secondary metabolites
Secondary metabolites are those cell constituents
which are not essential for survival.
For example, alkaloids, glycosides,
terpenoids,latex,tannins etc.
In vitro production of secondary metabolites is much
higher from differentiated tissues compared to non-
differentiated tissues.
Azadirachtin from Azadirachta indica Insecticidal
Berberine from Coptis japonica Antibacterial, anti
inflammatory
Capsaicin from Capsicum annum Cures Rheumatic
pain
42. Problems of micro-propagation
Expensive laboratory equipment and service
No possibility of using mechanization
Plants are not autotrophic
Poor Acclimatization to the field is a common
problem (hyperhydricity)
Risk of genetic changes if 'de novo' regeneration is
used
Mass propagation cannot be done with all crops to
date. In cereals much less success is achieved
Regeneration is often not possible, especially with
adult woody plant material.
44. Micro-propagation a tool for commercialization (Taiwan story)
According to Taiwan Today, Jan 1, 2016
The nation’s orchids are exported to 36 countries in
Northern America, Northern Europe and South Africa.
This country began the export with US $ 23 million in
2004 but in 2015 they have exported with worth US $
130 million.
They are selling a Phalaenopsis or moth orchid in
Dubai at the price of US $ 1,000.
Won the bid to host the 23rd World Orchid Conference
in 2020.
45. What may be the reason for this achievement ?
According to John Feng, CEO of SOGO TEAM CO
LTD., they are exploiting micro-propagation as a
technique in mass production as well as in variety
improvement.
In I-Hsin Biotechnology Corp’s Tissue Culture Lab
only, they have conducted 12,000 breeding
experiments which have yielded 2,300 Phalaenopsis
varieties.
This all is being possible through tissue culture.
46. ALBIFLORA ORCHID OF NEPAL
In Nepal a total of 377 species of orchids belonging to 100
genera reported.
47. Future of micro-propagation
……………………………………………………………………………………
Perhaps some of the greatest discoveries of plant tissue
culture are yet to come.
SO BE READY !