plant tissue culture

Biotechnology

Biotechnology is any technique that uses living organisms or substances derived from these organisms to make or modify a product, improve plants or animals or develop microorganisms for specific uses” It ranges from traditional biotechnology to most modern biotechnology

History

n  The person who should be mentioned: Haberlandt, a plant physiologist, is the first person performing the real plant tissue culture experiment, and the time was 1902.

Theory

n  Theory for plant cell and tissue culture: the totipotency, which states that cells are autonomic, and in principle, are capable of regenerating to give a complete plant.

Plant tissue culture

n  Plant tissue culture is defined as culture under sterile conditions, of plants, seeds, embryos, organs, tissues, cells and protoplasts.

Characteristics of Plant Tissue Culture Techniques

• Occur on a micro-scale.
•  Environmental conditions optimized                    (nutrition, light, temperature, phytochemistry).
•  All microbes (fungi, bacteria, viruses, and nematodes are excluded).
• The normal pattern of plant development breaks down giving rise to callus, adventitious roots, shoots, and/or embryos.
• Ability to grow as single cells (protoplasts, microspores, suspension cultures)
• Plant cells are totipotent (e.g.: able to regenerate a whole plant).

Uses of Plant Cell and Tissue Culture

n  Micropropagation

n  Production of pathogen free plants 

n  Meristem Culture

n  Somatic embryogenesis

n  Organ Culture Callus Culture

n  Anther and pollen grain Culture (Haploid production)

n  Protoplast culture

n  Germplasm preservation

Main Objectives

n  Elimination of viruses from infected plants 

n  Rapid multiplication of clones

n  Vegetative propagation of difficult to propagate species

n  All the year round propagation of clones

n  Rapid multiplication of seedlings (in cases where seed is hard to get

n  Virus Eradication

Tissue Culture Methodology

n  Disinfest plant material and culture buds; 

n  Discard contaminated field taken material /cultures and rear aseptic cultures until they are small plants; or

n  Expose these field taken material/aseptic plants to elevated temperatures (34-37 ^oC) during incubation for various times.

n  Excise small stem tips from these incubated plants and incubate in culture at 25°C;

n  Incubate until each plant is quite large and test for presence of viruses; 

n  If free of detectable viruses, re-culture from this (or these) plant(s) and multiply to obtain a population of virus-free clonal material.

Meristem Tip Culture

n  Meristem tip refers to the region of shoot apex lying distal to the youngest leaf primordial, whereas shoot apex includes Meristem tips plus a few sub adjacent leaf primordial.

Micropropagation

n  Enhanced axillary shoot proliferation

n  Node culture,

n  de novo formation of adventitious shoot through shoot organogenesis,

n  Nonzygotic embryogenesis.

Advantages of Micropropagation

n  Plant material require less attention between subcultures and there is no labour or material requirement for watering, weeding, spraying, etc. Micropropagation is most advantageous where costs is less than traditional methods of multiplication.

n  Certified virus free plants can be produced in large number.

n  It is possible to produce clones of same kinds of plants that are otherwise slow and difficult to propagate vegetative.

n  Only limited space is required to maintain and produce large number of plants.

n  Plants may acquire a temporary vigor through micropropagation which makes them more desirable to the grower than conventionally raised stock.

n  Production can be continued all the year round and is independent of seasonal changes.

n  Plants may acquire a temporary vigor through micropropagation which makes them more desirable to the grower than conventionally raised stock.

n  Production can be continued all the year round and is independent of seasonal changes.

n  Propagation is ideally carried out under aseptic conditions, free from pathogens. Once culture have been started there should be no loss through disease and the plantlets finally produced should be free from bacteria, fungi and other microorganisms.

Somatic Embryogenesis

n  Somatic embryogenesis is the production of embryos from somatic or “non-germ” cells.

n  Not a common micropropagation technique but is currently being used to produce superior spruce seedlings here in B.C.

n  Usually involves a callus intermediate stage which can result in variation among seedlings

Organogenesis

n  This is the production of roots, shoots or leaves.

n  These organs may arise out of pre-existing meristems or out of differentiated cells.

n  This, like embryogenesis, may involve a callus intermediate but often occurs without callus.

Somatic Embryogenesis and Organogenesis

n  Both of these technologies can be used as methods of micropropagation.

n  Not always desirable because they may not always result in populations of identical plants.

n  The most beneficial use of somatic embryogenesis and organogenesis is in the production of whole plants from a single cell (or a few cells).

Callus Culture

n  Callus is an unorganized mass of cell which can be induce to re-differentiate on appropriate medium to develop embryoids which directly develop into plantlets, eventually giving rise to a whole viable plant.

Anther and Pollen Grain Culture

n  The culture of immature anthers and pollen grains is done so as to induce the pollen grains to develop into multicellular forms, particularly into embryos, with   half the normal number of chromosomes for the species.

n  When such haploid  embryos are treated with chromosome doubling agents, e.g. colchicines, their  normal chromosome number is restored (and thus their fertility) and, in addition, the plants are pure lines.

Protoplast Culture

n  Enzymatic removal of the mainly cellulose wall surrounding a plant cell results in a protoplast. When these are cultured, a new wall is synthesized thus restoring the original state of the plant cell. However, during the period that the protoplast is wall-less, there is the possibility:-

n  Of adding foreign genetic material to the protoplast,

n  Of fusing the protoplasts of two cultivars or even species to form hybrid cells.

Germplasm Conservation

n  The preservation of Germplasm in vitro by tissue culture is an extension of micropropagation techniques.

n  Simply it is that by the micropropagation techniques scientists  get plant materials to be conserved. Then by the same techniques they make these materials grow slowly in the culture room and keep their genetic characteristics stable

Banana

In Pakistan, experimental banana cultivation was started after Independence, and soon after its success in Sindh the banana emerged as an important fruit crop.  It is now grown on 30.3,000 ha with a total annual production of 139.5,000 metric tons.

Overview

Area (Pakistan)                                 = 30,300 hectare

 Production                                         = 1,39,500 tones

 Distribution                                       = Sindh (86.8%), Punjab (8.6%),

                                                                Balochistan(3.0%), NWFP (2.0%)

VARIETY

 Locally called Bombay

 Dwarf Cavendish                                                             (95%)

 William hybrid                                                                  (3%)

Setting up of Tissue Culture Laboratory

n  Media Preparation Area/Room

n  Aseptic transfer Chamber Area

n  Environmentally Controlled Culture Room

n  Analytical Room and Acclimatization Room

Conclusion

The application of tissue culture techniques has helped breeders to improve varieties more efficiently, and the breeders are able to handle the crops in such a new way that they were unable to do with conventional methods only. For many agronomic and horticultural crops, tissue culture can accelerate growth of seeds and seedlings which contributes directly to an improved agricultural production.