Sanjeet
The living cells spontaneously propagate and differentiate themselves in via condition. But now-a-days it is possible to multiply the cells, tissues or plant organs outsides is artificially in a controlled vitro condition to obtain the all parts of plants or for other biotechnological use is called tissue culture or cell culture or organ culture. The term tissue culture is commonly used in a very wide sense to include in vitro culture of plant cells, tissues as well as organs. But in strict sense tissue culture denotes the in vitro cultivation of plant cells in our unorganized mass. Tissue culture is used for in vitro culture of single or relatively small groups of plant cells i.e. suspension cultures plant tissue, culture is sued in its broad sense to denote aseptic in vitro culture of plant cells, tissues and organs. The beginning of plant tissue culture was made as early as 1898, when the German Botanist G. Haborland successfully cultured fully differentiated individual plant cells isolated from different living tissue in several plant species. During 1934-39, due to discovery of auxins, B-Vitamins laid the foundation of plant tissue, culture by workers like Gautherot, while and Nobecourt. Thus, the method of plant cell, tissue and organ culture in vitro was regionally well developed.
The technique of in vitro cultivation of plant cells or organs is primarily devoted to solve two basic problems i.e. first, to keep the plant ells and organs free from microbes i.e. bacteria and fungi. Second, to ensure the desired development in the cells and organs by providing suitable nutrient media and other environmental conditions. The first problem can be eliminated by using modern equipment and careful handling during various equipments. The second problem remains the area of active research and is likely to do so for quite same time in the future.
(1) Laboratory space
The organization of a tissue culture laboratory depends mainly on the nature of scale of activity. There are following needed i.e.
(a) Washing, drying and storage of vessels.
(b) Preparation, sterilization and storage of media.
(c) Aseptic handling of explants and cultures.
(d) Maintenance of cultures.
(e) Observation of culture.
Laboratory requirements:
(1) Laminar air flow hood.
(2) Autoclave/pressure cookers.
(3) Refrigerator
(4) Freezer
(5) Balance, preferably electronic
(6) PH meter
(7) Water distillation unit
(8) Magnetic stirrer
(9) Ovens
(10) Water bathes with temp. Control.
(11) Hot plate/gas plate
(12) Microwave oven for heating
(13) Centrifuge table top
(14) Vortex
(15) Shaker, gyratory with platform and clips for different size flasks.
(16) Dissecting microscope.
(17) Lab carts and trays.
(2) Culture Room:
The culture room should have the following facilities i.e.
(i) Controlled temperature (usually at 250 + 20C) with the help of air conditioners and room locators’
(ii) Culture racks fitted with light (1000 flux).
(iii) A shaker for agitation of liquid cultures.
(3) Culture vessels and their washing:
Culture vessels made of clear plastic are available for a variety of purpose; vessels are generally presterilized and disposable. But certain types are autoclave and for reusable. Tissues are generally cultured in culture tuber, flasks and petriplates. Culture tubes and flasks are usually stopped with cotton plugs.
(4) Sterilization Techniques:
Sterilization is a procedure used for elimination of microorganisms. The maintenances of aseptic or sterile conditions is essential for successful tissue culture procedures. All operations are carried out in laminar our flow sterile cabinets. Sterilization is made by several methods. Such as :-
(i) Dry heat
(ii) Flank sterilization
(iii) Autoclave
(iv) Filter sterilization
(v) Wiping with 70% ethanol
(vi) Surface sterilization
(vii) Steam sterilization
(viii) Ultraviolet sterilization
(ix) Alcohol sterilization
(x) Chemical sterilization
(5) Nutrient medium:
The greatest progress towards the development of nutrition medium for plant calls grown in culture took place in 1960s and 1970s. No single medium is advisable for all types of plant tissue culture, so that several culture media with their nutrients in due preparation have been prepared by different workers. But of which MS medium is the most important one. There are a list of media constituent i.e. MS, BS, White’s, Nitsche and Nitsche, Gamborg’s and else medium.
Media constituents:
The major constituents of a culture medium include inorganic minerals, organic nutrients, phytohormones, vitamin, Amino acids, Agar in specific proportion.
(a) inorganic minerals:- N. P. K, Ca, Mg, B, Mo, Cu, Zn, Mn, cl.
(b) Organic nutrients:- Sucrose, glucose, peptones, Glyurol, Cacsnut water etc.
(c) Growth Hormones:- Auzin, Cytokinins, Gibbere thins.
(d) Vitamins: - Vitamin B2, C, B12, Nicotinic acid.
(e) Amino acids:- L-Aspartic acid, glutauric acid, Aspargin and L.glutamiue.
(f) Agar: - It is heteropolysacolaride, used to solidify the nutrient medium at a cone of 0.8 to 1.0%.
Callus and suspension cultures:
The plant from which the tissue is to be recovered is called stock plant. Remove plant partier tissues are excised and surface sterilized. Explants may be pieces of stem leaf, root, flower, fruit or from seed. Then the explants is cultured in different cultures are they are-
(a) Callus culture: Tissues and cells cultured agar-gelled medium form an unorganized mass of cells called callus. Callus cultures need to be sub cultured every 3-5 weeks in view of cell growth, nutrient depletion and medium drying. Therefore, cultures are easy to maintain and are the most widely used.
(b) Suspension culture: Tissue and cells cultured in a liquid medium produce a suspension of single cells and cells stumps of few many cells. These are called suspension culture. Suspension cultures grow faster than callus culture need to be sub cultured. The suspension cultured are broadly grouped as follows i.e.-
(i) Batch culture
(ii) Continuous cultures
(iii) Immobilized cultures
(1) Batch culture:
In a batch culture, the same medium and all the cells produced are retained in the culture vessel. The cell number of a batch culture exhibits a typical sigmoid curve. The cell no. or biomass remains unchanged. Batch cultures are maintained by sub culturing.
(2) Continuous cultures:
In a continuous culture, the cell population is maintained in a steady state by regularly replacing a portion of the sued or spent medium by fresh medium, such systems are either closed or open type.
(3) Immobilized cell cultures:
Plant cells and cell groups may be encapsulation in a suitable material i.e. agars or calcium alginate gale. Immobilization of cells changes their cellular physiology is comparison to suspension culture cells.
(c) Subcultures:
After a period of time, it becomes necessary to transfer organs and tissues to fresh media chiefly due to nutrient depletion and medium drying. This is particularly true of tissue and cell cultures where a portion of tissue is used to inoculate new cultures tubes or flasks, this is known as sub culturing.
Micro propagation
Tissue culture techniques are becoming increasingly popular by plant vegetative propagation. Plant tissues culture involves a sexual method of propagation and its primary goal is crop improvement. Clinical propagation in vitro is called micro propagation. The word ‘clone’ was first used by Webber for apply to cultivated plants that were propagated vegetative. The word is derived from Greek Cton means twig and propagulates means multiplication clinical propagation is the multiplication of genetically identical individuals by a sexual reproduction. There are 3 major steps involved in micro propagation. They are:-
(i) Selection of suitable explants, their sterilization and transfer to nutrient media for establishment.
(ii) Proliferation or multiplication of shoots from the explants or medium.
(iii) Transfer of shoots to a rooting medium followed by planting into sail.
Major steps:
Stage-I
(Establishment)
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Selection of an elite mother plant
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Explants
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Surface sterilization and washing
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Establishment on growth medium
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State-II (proliferation)
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Transfer to proliferation medium
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Shoot or embryonic formation
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Stage-III (Rooting and hardening)
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Transfer of shoots or plantlets to sterilized soil or artificial medium by various gradual weakening process.
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Stage-I (Establishment):
In this stage, when explants may develo9p either into single shoots or multiple shorts. It is unlikely that meristem, shoot tip or bud explants have sufficient endogenous cytoking to support growth and development. Thus at this stage media are supplemented with cytokiwin like BA, Kinestine, and ZiP. Auxin is another hormone required for shoot growth. Since the young short apex is an active site for auxin biosynthesis.
Stage-II Proliferation:
In this stage, the propagates are multiply and for this auxiliary shoot proliferation is followed as it maintains higher genetic stability and is more easily achieved by most plant species then organogenic or embryogenic path. In auxiliary short proliferation, high levels of cytokinin re utilized to overcome the special dominance of shoots and to enhance the breaching of lateral bud from leaf axils. In general BA is the most effective cytokinin followed by kinetin and 2iP. Effect of high cytokinin concentration on auxiliary shoot elongation and to restore normal short growth.
Stage-III (Rooting and Hardening):
This stage is regeneration of roots from the shoots. Usually in vitro produced shoots of sufficient length are cut on separated and transferred to a medium containing auxin. Root initiations depend on a low cytokinin to a high auxin ratio. IAA, 2, 4-D and other are used for induction of root regeneration. If the roots, are unable to initiate in such cases, high salt media concentration is reduced. Riboflavin is reported to improve the quality of root system in some cases. Optimum inculcation conditions vary but generally the cultures are kept in the range of 20-280c. Light intensity varies with species i.e. 1-10 Klux, but in tissue culture lower light intensity is better.
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| My Research Team (From Lefft, Dr. Padan Kumar Jena, Me and Shanti Prava Behera) |
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