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 CultureVarious Techniques for Tissue Culture Germplasm PreservationApplications 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 CULTURE1838-39 cellular theory (Cell is

autonom and totipotent)Schleiden-Schwann

1902 First attempt of plant tissue culture

Harberlandt

1939 Continuously growing callus culture

White

1946 Whole plant developed from shoot tip

Ball

1950 Organs regenerated on callus

Ball

1954 Plant from single cell Muir

1960 Protoplast isolation Cocking

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 White

Murashige

Skoog Gautheret Maheshwai

Nutrient medium and the role of growth hormones?

The nutrient medium commonly contains Macronutrient, micronutrient, vitamins, Ferron

& carban sourceThe 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 CultureWhen 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. ChawlaM. S. ShekhawatImages from google search engine

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