Tuesday, October 2, 2012

Tissue Culture























 Tissue culture is the growth of tissues or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as *broth   or **agar .

* Broth :   ( a liquid containing nutrients for culturing microorganisms- microbiology - a liquid medium containing proteins and other nutrients for the culture of bacteria: in vitro cultures -broth cultures of intestinal tissue -a liquid mixture for the preservation of tissue )

** Agar :  Agar or agar-agar is a gelatinous substance derived by boiling a polysaccharide in red algae, where it accumulates in the cell walls of agarophyte and serves as the primary structural support for the algae's cell walls. Agar is a mixture of two components: the linear polysaccharide agarose, and a heterogeneous mixture of smaller molecules called agaropectin.

Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium for microbiological work. Agar (agar-agar) can be used as a laxative, an appetite suppressant, vegetarian gelatin substitute, a thickener for soups, in fruit preserves, ice cream, and other desserts, as a clarifying agent in brewing, and for sizing paper and fabrics.

The gelling agent is an unbranched polysaccharide obtained from the cell walls of some species of red algae, primarily from the genera Gelidium and Gracilaria, or seaweed (Sphaerococcus euchema). For commercial purposes, it is derived primarily from Gelidium amansii. In chemical terms, agar is a polymer made up of subunits of the sugar galactose.


 Tissue culture commonly refers to the culture of animal cells and tissues, while the more specific term plant tissue culture is being named for the plants.

What are Plant Hormones

Plant hormones, also know and plant growth regulator or PGRs, are signal molecules produced within the plant at extremely low concentrations. Plant hormones regulate cellular processes and growth of the plant in various ways. In tissue culture, plant hormones are added to the media to promote a certain type of growth (division, root formation, etc.) depending on what is desired by the propagator at the time.

What is a callus

A callus of cells is a mass of undifferentiated plant cells. It often starts as a lumpy growth on existing tissue and can develop into odd-looking, perhaps "hairy" masses as many tiny plantlets begin to emerge from the callus.

Plant cell culture methods



Plant cell cultures are typically grown as cell suspension cultures in a liquid medium or as callus cultures on a solid medium. The culturing of undifferentiated plant cells and calli requires the proper balance of the plant growth hormones auxin and cytokinin.

Historical usage

In 1885 Wilhelm Roux removed a section of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the basic principle of tissue culture. In 1907 the zoologist Ross Granville Harrison demonstrated the growth of frog nerve cell processes in a medium of clotted lymph.

 
In 1913, E. Steinhardt, C. Israeli, and R. A. Lambert grew vaccinia virus in fragments of guinea pig corneal tissue. In 1996, the first use of regenerative tissue was used to replace a small distance of a urethra, which led to the understanding that the technique of obtaining samples of tissue, growing it outside the body without a scaffold, and reapplying it, can be used for only small distances of less than 1 cm.

Modern usage


In modern usage, tissue culture generally refers to the growth of cells from a tissue from a multicellular organism in vitro. These cells may be cells isolated from a donor organism, primary cells, or an immortalised cell line. The term tissue culture is often used interchangeably with cell culture

The literal meaning of tissue culture refers to the culturing of tissue pieces, i.e. explant culture.

Tissue culture is an important tool for the study of the biology of cells from multicellular organisms. It provides an in vitro model of the tissue in a well defined environment which can be easily manipulated and analysed.

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation, including:

The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits.

 

To quickly produce mature plants.

The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.
The regeneration of whole plants from plant cells that have been genetically modified.

The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens.

The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and nepenthes.

To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture.

Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). Single cells, plant cells without cell walls (protoplasts), pieces of leaves, or (less commonly) roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.

Choice of explant

The tissue obtained from the plant to culture is called an explant. Based on work with certain model systems, particularly tobacco, it has often been claimed that a totipotent explant can be grown from any part of the plant. However, this concept has been vitiated in practice. In many species explants of various organs vary in their rates of growth and regeneration, while some do not grow at all. The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased with inappropriate explants. Thus it is very important that an appropriate choice of explant be made prior to tissue culture.

The specific differences in the regeneration potential of different organs and explants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle, the availability of or ability to transport endogenous growth regulators, and the metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic ends of the plants like the stem tip, auxiliary bud tip and root tip. These tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins.

The pathways through which whole plants are regenerated from cells and tissues or explants such as meristems broadly fall into three types:

The method in which explants that include a meristem (viz. the shoot tips or nodes) are grown on appropriate media supplemented with plant growth regulators to induce proliferation of multiple shoots, followed by rooting of the excised shoots to regenerate whole plants,

The method in which totipotency of cells is realized in the form of de novo organogenesis, either directly in the form of induction of shoot meristems on the explants or indirectly via a callus ( unorganised mass of cells resulting from proliferation of cells of the explant) and plants are regenerated through induction of roots on the resultant shoots,

Somatic embryogenesis, in which asexual adventive embryos( comparable to zygotic embryos in their structure and development) are induced directly on explants or indirectly through a callus phase.

The first method involving the meristems and induction of multiple shoots is the preferred method for the micropropagation industry since the risks of somaclonal variation (genetic variation induced in tissue culture) are minimal when compared to the other two methods. Somatic embryogenesis is a method that has the potential to be several times higher in multiplication rates and is amenable to handling in liquid culture systems like bioreactors.

Some explants, like the root tip, are hard to isolate and are contaminated with soil microflora that become problematic during the tissue culture process. Certain soil microflora can form tight associations with the root systems, or even grow within the root. Soil particles bound to roots are difficult to remove without injury to the roots that then allows microbial attack. These associated microflora will generally overgrow the tissue culture medium before there is significant growth of plant tissue.

 

Aerial (above soil) explants are also rich in undesirable microflora. However, they are more easily removed from the explant by gentle rinsing, and the remainder usually can be killed by surface sterilization. Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic, de-colorization or localized necrosis on the surface of the explant.

An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to penetration of harsh surface sterilizing agents, such as hypochlorite, so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues.

Tissue cultured plants are clones, if the original mother plant used to produce the first explants is susceptible to a pathogen or environmental condition, the entire crop would be susceptible to the same problem, conversely any positive traits would remain within the line also.

Applications

Plant tissue culture is used widely in plant science; it also has a number of commercial applications. Applications include:

Micropropagation is widely used in forestry and in floriculture. Micropropagation can also be used to conserve rare or endangered plant species.

A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance.

Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds, like plant-derived secondary metabolites and recombinant proteins used as biopharmaceuticals.

To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.
To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die (Embryo Rescue).

For production of doubled monoploid (dihaploid) plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes, usually by treatment with colchicine which causes doubling of the chromosome number.

As a tissue for transformation, followed by either short-term testing of genetic constructs or regeneration of transgenic plants.

Certain techniques such as meristem tip culture can be used to produce clean plant material from virused stock, such as potatoes and many species of soft fruit.

Micropropagation using meristem and shoot culture to produce large numbers of identical individuals.
Production of identical sterile hybrid species can be obtained.

Laboratories

Although some growers and nurseries have their own labs for propagating plants by the technique of tissue culture, a number of independent laboratories provide custom propagation services. The Plant
 
Tissue Culture Information Exchange lists many commercial tissue culture labs. Since plant tissue culture is a very labour intensive process, this would be an important factor in determining which plants would be commercially viable to propagate in a laboratory.

The following plants can be produced by using tissue culture method :

Ornamental Plants
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MARANTHA
SYNGONIUM
CALLALILY
LIMONIUM
FERNS
CORDYLINE
DRACENA
SPATHIPHYLLUM
FICUS
PHILODENDRON
VENUS FLY TRAP
ORCHIDS
ROSE - Miniature
CALADIUM
DIFFENBACHIA
YUCCA
MARIGOLD
CALATHEA
ALPINIA
GENTIANA
CARNATION
BEAUCARNIA
CACTUS
LEGULARIA
AGLOENEMA
POMEGRANATE
PINEAPPLE
WAX FLOWER
Commercial Crops

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SUGARCANE
TURMERIC
POTATO
ASPARAGUS
BITTER GUARD
TAPIOCA



Tree Crops
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CASUARINA
BAMBOO
PAULOWNIA
NEEM
EUCALYPTUS



Fruit Crops
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BANANA
PRUNUS

TOMATO
STRAWBERRY
WATERMELON


Medicinal Plants
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PATCHOULI
MUSLI
ROSEMARY
THYME
VANILA
GLORIOSA
TULSI - Ocimum
CURRY LEAF
NAGABASHANAM
GYMNEMA SYLVESTRES
NAGATHALI
ALOE VERA
AROKYAPATCHAI
GERANIUM
STEVIA


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Starting of plant tissue culture - various steps

Plant Tissue Culture Media Preparation

Plant Tissue Culture owes its origin to the ideas of the German Scientist, Haberlandt, in the beginning of the 20th century. This was just the beginning of the tissue culture; thereafter in 70’s began the commercialization of the technology. Currently the world is revolving around it due to predicated future grain shortage; green house effect and total environmental disbalance.

Various steps have been taken in this field and have been introduced an extensive range of Ready to use PTC Medium; PTC Medium Ingredients like

Plant Growth Regulators or Phytoharmones (Auxins; Cytokinins; Gibberellins; Abscisic and & Others),

Macro & Micro Nutrients (Nitrogen; Potasssium; Phosphorus; Sulphur; Magnesium; Calcium;
                                Boron; Zinc; Iron; Iodine; Vitamins; Amino acids; Carbohydrates & Others),
Gelling Agents (Gelrite; Agar Agar & Others); 

Adsorbing Agents; 
Buffering Agents;
Pure Antibiotics and range of products used in the process.

Plant Tissue Culture Media Preparation is based on the unique property of the cell-totipotency. The cell-totipotency is the ability of the plant cell to regenerate into whole plant. In this process the excised bud is transferred into a tube containing a sterile nutrient medium. The success of tissue culture depends very much on the stage of explant selected, the sterilization period and the type of culture media used; different types of plants require different sets of culture media. Plant tissues are grown in vitro on artificial media, which supply the nutrients necessary for growth. The success of plant culture as a means of plant propagation is greatly influenced by the nature of the culture medium used. The rich tissue culture media provides a good nutrient source for bacteria and fungi, therefore precautions against microbial contamination must be taken in all in vitro procedures.

Tissue culture media used for in vitro cultivation of plant cells are composed of following basic components:
  • Complex mixture or Inorganic nutrients: Essential elements, or Mineral ions
  • Organic supplement: Vitamins and/or Amino acids
  • Carbon source: Sugar Sucrose
  • Gelling agents or Solidifying agents
  • Plant Growth Regulators
  • Growth Promoters
  • Complex organics
  • Activated charcoal
  • pH
  • Antibiotics

Complex mixture of salts is Inorganic nutrients (both micro- and macro-elements : C, H, O, N, P, S, Ca, K, Mg, Fe, Mn, Cu, Zn, B, Mb). For healthy and vigorous growth, intact plants need to take up from soil: relatively large amounts of some inorganic elements (the major plant nutrients): ions of Nitrogen (N), Potassium (K), Calcium (Ca), Phosphorous (P), Magnesium (Mg) and Sulphur (S). And some other elements in small quantities (minor plant nutrients or trace elements): Iron (Fe), Nickel (Ni), Chlorine (Cl), Manganese (Mn), Zinc (Zn), Boron (B), Copper (Cu), and Molybdenum (Mo). Among the micronutrients, iron requirement is very critical. Chelated form of Iron and Copper are commonly used in culture media.

Organic supplement: Vitamins and/or Amino acids (e.g. Nicotinic acid, Thiamine, Pyridoxine and myo-Inositol (Viamin B), Amino acids (e.g. Arginine) are essential for the culture of plant cells in vitro. The plant cells in culture are able to synthesize vitamins just like natural plants, but in suboptimal quantities which does not support proper growth of cells in culture. Therefore the medium is supplemented with vitamins to achieve good growth of cells. Similarly amino acids are added to the cell cultures to stimulate the cell growth and estabilish the cell lines. The most commonly used amino acid is glycine. However, Asparagine, Aspartic acid, Alanine, Glutamic acid, Glutamine and Proline are also used. Amino acids provide a source of reduced nitrogen and, like ammonium ions, uptake causes acidification of the medium. Organic acids especially the intermediates of krebs cycle e.g. Citrate, Malate, Succinate, Pyruvate also enhances the growth of plant cells. Sometimes antibiotics (e.g. Streptomycin, Kanamycin) are also added to the medium to prevent the growth of the microorganisms.

Carbon sources: usually sugar Sucrose (usually sucrose) - However, plant cells and tissues in the culture medium are heterotrophic and therefore depend on the external carbon for energy. During the sterilization of the medium, Sucrose gets hydrolyzed to glucose and fructose and the plant cells utilize first the glucose and than the fructose. The other carbohydrates such as Lactose, Maltose, Galactose etc., have been used in culture media but with limited success.

Gelling or Solidifying agents like Agar. Plant tissue culture media can be used in either liquid or ‘solid’ forms, depending on the type of culture being grown. Generally, a gelling agent Agar (a polysaccharide obtained from red algae, Gelidium amansil) is added to the liquid medium for its solidification. The agar obtained from seaweeds provides solid surface for the growth of cells because in the liquid medium, the tissue will be submerged and die due to lack of oxygen. Cells are grown in suspension medium without agar but such cultures are aerated regularly either by bubbling sterile air or by gentle agitation. Some other less frequently used solidifying agents are Gelrite, Biogel (polyacrlyamide pellets), Phytagel, and purified Agarose, as can a variety of Gellan gums.

Growth regulators (e.g. Auxins, Cytokinins and Gibberellins) - Plant hormones play an important role in growth and differentiation of cultured cells and tissues. There are many classes of plant growth regulators used in culture media involves namely: Auxins, Cytokinins, Gibberellins, Abscisic acid, Ethylene, 6 BAP (6 Benzyladenine), I.A.A (Indole Acetic Acid), I.B.A (Indole - -3- Butyric Acid), Zeatin, Zeatin Riboside. 

  • The Auxins facilitate the cell division and root differentiation. Auxins induce cell division, cell elongation, and formation of callus in cultures. For eg., 2,4-dichlorophenoxy acetic acid is one of the most commonly added auxins in plant cell cultures.
  • The Cytokinins induce cell division and differentiation. Cytokinins, promotes RNA synthesis and stimulate protein and enzyme activities in tissues. Kinetin and benzyl-aminopurine are the most frequently used cytokinins in plant cell cultures.
  • The Gibberellins is mainly used to induce plantlet formation from adventive embryos formed in culture.
  • Abscisic acid is used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis. Abscisic acid (ABA) inhibits cell division.
  • Ethylene is associated with controlling fruit ripening in climacteric fruits, and its use in plant tissue culture is not widespread. Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and development of the culture.
As in Plant tissue culture media the ratio of auxins and cytokinins play an important role in the morphogenesis of culture syatems. When the ratio of auxins to cytokinins is high, embryogenesis, callus initiation, and root initiation occur. For axillary proliferation and shoot proliferation, the ratio of auxins to cytokinins is kept low. GA3 enhances the callus growth and induces dwarf plantlets to elongate. 

Plant growth promoters are α - Naphthalene acetic acid, Humic acid (granular/ powder) etc. Plant growth promoters (PGP) are those substances used for better management of nutrients and plant growth. Many times some crops fail to produce optimum yields in spite of proper nutrient supply. Physiological inefficiency in plants is responsible for such effects. Therefore, these PGA play a major role in seed germination, fruit ripening, enhances uptake of nutrients, boost protein synthesis, augment immunity and helps to withstand stress conditions, reduce flowering and fruiting drop and help in better plant growth.

Complex organics like Casein hydrolysate, Coconut milk, Malt extract, Yeast extract, Tomato juice, etc. may be added for specific purposes. Casein hydrolysate has given significant success in tissue culture and potato extract also has been found useful for anther culture. However, these natural extracts are avoided as their composition is unknown and vary from lot to lot and also vary with age affecting reproducibility of results.

Activated charcoal acts both in promotion and inhibition of culture growth depending upon plant species being cultured. It is reported to stimulate growth and differentiation in orchids, carrot, ivy and tomato whereas inhibits tobacco, soybean etc. It absorbs brown-black pigments and oxidized phenolics produced during culture and thus reduce toxicity. It also absorbs other organic compounds like PGRs, vitamins etc which may cause the inhibition of growth. Another feature of activated charcoal is that it causes darkening of medium and so helps root formation and growth.

pH - affects absorption of ions and also solidification of gelling agent. An optimum pH for culture media is 5.8 before sterilization. Values of pH lower than 4.5 or higher that 7.0 greatly inhibit growth and development in vitro. The pH of culture media generally drops by 0.3 to 0.5 units after autoclaving and keeps changing through the period of culture due to oxidation and also differential uptake and secretion of substances by growing tissue. At pH higher than 7.0 and lower than 4.5, the plant cells stop growing in the medium. 


Antibiotics used as Antmicrobial agents in Plant tissue culture media. The combination of antibiotics and chemical biocides has been tested for their ability to inhibit microbial contamination in plant cultures.

Antibiotics help in suppressing the bacterial infections in plant cell and tissue culture. Also helps in suppressing mould and yeast infections in cell cultures. Whereas, helps in elimination of Agrobacterium species after the transformation of plant tissue. The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement. The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. An alternative method is agroinfiltration.
The elements listed above are - together with Carbon (C), Oxygen (O) and Hydrogen (H) - the 17 essential elements. Certain others, such as Cobalt (Co), Aluminium (Al), Sodium (Na) and Iodine (I), are essential or beneficial for some species but their widespread essentiality has still to be established.
According to Epstein (1971), elements can be considered to be essential for plant growth if :
 
  • A plant fails to complete its life cycle without it
  • Its action is specific and cannot be replaced completely by any other element
  • Its effect on the organism is direct, not indirect on the environment
  • It is a constituent of a molecule that is known to be essential.
  •  
PLANT TISSUE CULTURE MEDIUM PREPARATION

For 1liter of growth medium, this is enough to prepare about 100 growing tubes.
  • Take a required amount of Murashige and Skoog Basal Salts with minimal organics medium (MSMO).
  • For 1liter of Murashige and Skoog Basal Salts with minimal organics medium, use a 2 liter container (because the medium boils up).
  • Add the dehydrated medium to the water and stir for a minute to dissolve completely.
  • Add the desired heat stable supplements to the medium solution.
  • Weigh out 6gms of Agar.
  • Add it to the MSMO solution.
  • Heat the solution gently while stirring until all the agar has dissolved.
  • Add more distilled water to make the total volume up to 1liter.
  • Pour the still-warm medium into the polycarbonate tubes to a depth of about 15 mm.
  • Place the tubes (with lids sitting on the tubes but not tightened) in an autoclave and sterilise for 20 minutes. (Note: Higher temperature may result in poor cell growth).
  • Cool the autoclve, then remove the tubes and tighten the lids.
  • Add heat labile supplements after autoclaving.
  •  
Plant Tissue Culture Media - 
 
  • Banana Micropropagation Medium
  • Banana Multiplication Medium
  • Gamborg Medium
  • Knudson-C Orchid Medium
  • The most extensively used nutrient medium is MS medium (developed by Murashige and Skoog in 1962) and many more...
  •  
Plant Tissue Culture Media Ingredients -
Macro & Micro Nutrients
  • Ammonium chloride
  • Ammonium molybdate
  • Ammonium sulphate
  • Potassium nitrate
  • Potassium sulphate
  • Sodium nitrate
  • Urea
  • Zinc nitrate
  • and many more...
Carbohydrates
  • Dextrose
  • Fructose
  • Maltose
  • D (+) Mannose
  • D- Ribose
  • D- Sorbitol
  • Sucrose
  • Tomato powder
  • and many more...

Amino acids
  • Aspartic acid
  • Glycine
  • L- Lysine mono- HCl
  • L- Proline
  • L- Tyrosine
  • and many more...
Plant Growth Regulators
  • IAA
  • IBA
  • NAA
  • 2,4,5 -T
  • and many more...

Antibiotics
  • Amicillin Sodium
  • Ciprofloxacin
  • Kanamycin sulphate
  • Gentamycin
  • Polymixin B sulphate
  • Vancomycin
  • and many more...
Vitamins
  • L - Ascorbic acid
  • D (+) Biotin
  • Niacin
  • Riboflavin
  • Retinol acetate
  • Thiamine HCl
  • and many more...

Gelling Agents
  • Agar Agar
  • Agarose
  • Gelrite
  • Gellan Gum
  • and many more...
Organic acids
  • Citric acid
  • Casein enzymatic hydrolysate
  • Malt extract
  • Soya peptone
  • Yeast extract
  • and many more...

Buffering agents
  • ACES buffers
  • CHAPS buffers
  • HEPES buffers
  • MES buffers
  • and many more...

2 comments:

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    Pole Saw

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