Plant Tissue Culture And its
Applications in Crop
Improvements
Arun patel
M.Sc- Agriculture Biotechnolgy
Learning Objectives
 Introduction to Plant Tissue Culture
 History of Tissue Culture
 Media For Tissue Culture
 Various Techniques for Tissue Culture
 Germplasm Preservation
 Applications
 Limitations
Introduction to Tissue Culture
 Tissue Culture (also known as Micropropagation or
In vitro culture) is:
 The growing of plant cells, tissues, organs, seeds or
other plant parts in a sterile environment on a
nutrient medium.
HISTORY OF PLANT TISSUE CULTURE
1838-39
cellular theory (Cell is
autonom and totipotent)
SchleidenSchwann
1902
First attempt of plant tissue
culture
Continuously growing callus
culture
Harberlandt
1946
Whole plant developed from
shoot tip
Ball
1950
Organs regenerated on callus
Ball
1954
Plant from single cell
Muir
1960
Protoplast isolation
Cocking
1939
White
1962
MS media
Murashige Skoog
1964
Clonal propagation of orchids
Morel
1964
Haploids from pollen
Guha
1970
Fusion of protoplasts
Power
1971
Plants from protoplasts
Takebe
1981
Somaclonal variation
Larkin
1967
Anther Culture
Maheshwari
Landmark Achievers of PTC
G. Haberladnt
Skoog
White
Gautheret
Murashige
Maheshwai
Nutrient medium and the role of growth
hormones?
 The nutrient medium commonly contains
 Macronutrient, micronutrient, vitamins, Ferron &
carban source
 The optimum culture medium may vary with the
species, the genotype within the species, and the
origin and age of the cultured tissue.
 The preferred physical state of the culture medium,
whether a liquid medium or a solid agar gel, may
vary with the species and the culture environment.
 pH- 5.8
Hormones in the agar
 Two Hormones Affect Plant Differentiation:
 Auxin: Stimulates Root Development
 Cytokinin: Stimulates Shoot Development
 Generally, the ratio of these two hormones can
determine plant development:



 Auxin ↓Cytokinin = Root Development
 Cytokinin ↓Auxin = Shoot Development
Auxin = Cytokinin = Callus Development
Basic Procedure of Tissue Culture
Types of In Vitro Culture
 Culture of intact plants (seed and seedling culture)
 Embryo culture (immature embryo culture)
 Organ culture
Callus culture
 Cell suspension culture
 Protoplast culture
 Somatic Embryogenesis
 Micropropagation
 Somaclonal variation
Micropropagation
 Embryogenesis
 Direct embryogenesis
 Indirect embryogenesis
 Organogenesis
 Organogenesis via callus formation
 Direct adventitious organ formation
 Microcutting
 Meristem and shoot tip culture
 Bud culture
Steps of Micropropagation
 Stage 0 – Selection & preparation of the mother plant
 sterilization of the plant tissue takes place
 Stage I - Initiation of culture
 explant placed into growth media
 Stage II - Multiplication
 explant transferred to shoot media; shoots can be constantly divided
 Stage III - Rooting
 explant transferred to root media
 Stage IV - Transfer to soil
 explant returned to soil; hardened off
Features of Micropropagation
 Clonal reproduction.
 Way of maintaining heterozygosity.
 Multiplication Stage can be recycled many times to
produce an unlimited number of clones.

Routinely used commercially for many ornamental species,
some vegetatively propagated crops.
 Easy to manipulate production cycles
 Not limited by field seasons/environmental influences.
 Disease-free plants can be produced
 Has been used to eliminate viruses from donor plants.
Types of Plant Tissue Culture
What is Callus development
 A callus is a blob of tissue – (mostly undifferentiated
cells)
 A callus is naturally developed on a plant as a result
of a wound
 This callus can be left to develop or can be further
divided
Callus Culture
 Equimolar amounts of auxin and cytokinin stimulate




cell division. Leads to a mass proliferation of an
unorganised mass of cells called a callus.
Requirement for support ensures that scale-up is
limited.
Callus Suspension Culture
When callus pieces are agitated in a liquid medium,
they tend to break up.
Suspensions are much easier to bulk up than callus
since there is no manual transfer or solid support.
Protoplast Isolation
 Created by degrading the cell wall using enzymes.
 Very fragile, can’t pipette.
 The membranes are made to fuse.
 osmotic shock, electrical current, virus
 Regenerate the hybrid fusion product.
 Contain genome from both organisms.
 Very, very difficult .
Use of enzymes results
in a high yield of
uniform protoplasts
after removal of cellular
debris Protoplasts can
originate from different
sources: greenhouse or
field material,
micropropagated
plants, calli,
Protoplast Fusion Techniques
 Protoplast fuse spontaneously during isolation process
mainly due to physical contact.
 Induced Fusion.
 Chemofusion- fusion induced by chemicals.
 Types of fusogens
PEG
 NaNo3
 Ca 2+ ions
 Polyvinyl alcohal

 Mechanical Fusion- Physical fusion of protoplasts under
microscope by using micromanipulator and perfusion
micropipette.
Somatic & Cybridization
Somatic & Cybridization
Uses for Protoplast Fusion
 Combine two complete genomes
 Another way to create allopolyploids
 Partial genome transfer
 Exchange single or few traits between species
 May or may not require ionizing radiation
 Genetic engineering
 Micro-injection, electroporation, Agrobacterium
 Transfer of organelles
 Unique to protoplast fusion
 The transfer of mitochondria and/or chloroplasts between
species
Somaclonal Variation
 Variation found in somatic cells dividing mitotically in culture
 A general phenomenon of all plant regeneration systems that involve a
callus phase
Some mechanisms:
 Karyotipic alteration
 Sequence variation
 Variation in DNA Methylation
Two general types of Somaclonal Variation:
 Heritable, genetic changes (alter the DNA)
 Stable, but non-heritable changes (alter gene expression, epigenetic)
Somaclonal Breeding Procedures
 Use plant cultures as starting material
 Idea is to target single cells in multi-cellular culture.
 Usually suspension culture, but callus culture can work (want as much
contact with selective agent as possible).
 Optional: apply physical or chemical mutagen.
 Apply selection pressure to culture.
 Target: very high kill rate, you want very few cells to survive, so long as
selection is effective.
 Regenerate whole plants from surviving cells.
Advantages of somatic hybridization
 Production of novel interspecific and intergenic hybrid
 Pomato (Hybrid of potato and tomato).
 Transfer gene for disease resistance, abiotic stress
resistance, herbicide resistance and many other quality
characters.
 Production of heterozygous lines in the single species
which cannot be propagated by vegetative means.
 Production of unique hybrids of nucleus and cytoplasm.
Plant germplasm preservation
 In situ : Conservation in ‘normal’ habitat
 rain forests, gardens, farms
 Ex Situ :
 Field collection, Botanical gardens
 Seed collections
 In vitro collection: Extension of micropropagation techniques
Normal growth (short term storage)
 Slow growth (medium term storage)
 Cryopreservation (long term storage

 DNA Banks
Cryopreservation


Storage of living tissues at ultra-low temperatures (-196°C)
Conservation of plant germplasm


Vegetatively propagated species (root and tubers, ornamental,
fruit trees).
Conservation of tissue with specific characteristics
Medicinal and alcohol producing cell lines
 Genetically transformed tissues.
 Transformation/Mutagenesis competent tissues (ECSs).


Conservation of plant pathogens (fungi, nematodes)
Applications:
 Study of Biochemical & Physiological activities.
 The effect of various hormones.
 Production of Secondary Metabolites.
 To preserve the plant species which are on red-line.
 Improve crop yield with regard to molecular
breeding & Genetic Engineering.
 To make transgenic & cis-genic plants.
Commercial Applications of Clonal Propagation
 Clonal propagation has the potential for propagation
of thousands of plantlets from a single genetic stock.
 Examples:





orchids,
potato,
asparagus,
strawberry, and
various flowers or herbaceous ornamentals that set seed
poorly.
 This may not be suitable for seeding field crops.
Problems in Tissue Culture
 Application of protoplast technology requires efficient






plant regeneration system.
The lack of an efficient selection method for fused
product is sometimes a major problem.
The end-product after somatic hybridization is often
unbalanced.
Regeneration products after somatic hybridization are
often variable.
It is never certain that a particular characteristic will be
expressed.
Genetic stability.
Sexual reproduction of somatic hybrids.
Conclusion
 PTC is the technique by which plant cells can be
grown in vitro sexually & asexually. By the help of this
we can study biochemical, physiological and
hormones activity.
 High yield, good quality of crops can be obtained.
 PTC , G.E. and Molecular breeding these techniques
are used to transfer the gene of same species or from
different species.
References
 Plant Tissue Culture, ELESIVISER Publishers
,Bhojwani & Rajdhan
 H.S. Chawla
 M. S. Shekhawat
 Images from google search engine