Tissue Culture

History of Tissue Culture Techniques

The in vitro techniques were developed initially to demonstrate the totipotency of plant cells predicted by Haberlandt in 1902. Totipotency is the ability of a plant cell to perform all the functions of development, which are characteristic of zygote, i.e., ability to develop into a complete plant. In 1902, Haberlandt reported culture of isolated single palisade cells from leaves in Knop’s salt solution enriched with sucrose.

Plant Tissue Culture

Plant Tissue Culture

The cells remained alive for up to 1 month, increased in size, accumulated starch but failed to divide. Efforts to demonstrate totipotency led to the development of techniques for cultivation of plant cells under defined conditions.

This was made possible by the brilliant contributions from RJ. Gautheret in France and P.R. White in U.S.A. during the third and the fourth decades of 20th century. Most of the modern tissue culture media derive from the work of Skoog and coworkers during 1950s and 1960s.
The first embryo culture, although crude, was done by Hanning in 1904; he cultured nearly mature embryos of certain crucifers and grew them to maturity. The technique was utilised by Laibach in 1925 to recover hybrid progeny from an interspecific cross in Linum. Subsequently, contributions from several workers led to the refinement of this technigue.

Haploid plants from pollen grains were first produced by Maheshwari and Guha in 1964 by culturing anthers of Datura. This marked the beginning of anther culture or pollen culture for the production of haploid plants.

The technique was further developed by many workers, more notably by JP. Nitch, C. Nitch and coworkers. These workers showed that isolated microspores of tobacco produce complete plants.

Plant protoplasts are naked cells from which cell wall has been removed. In 1960, Cocking produced large quantities of protoplasts by using cell wall degrading enzymes.

The techniques of protoplast production have now been considerably refined. It is now possible to regenerate whole plants from protoplasts and also to fuse protoplasts of different plant species. In 1972, Carlson and coworkers produced the first somatic hybrid plant by fusing the protoplasts of Nicotiana glauca and N. langsdorfii. Since then many divergent somatic hybrids have been produced.
A successful establishment of callus cultures depended on the discovery during mid-thirties of IAA (idole-3-acetic acid), the endogenous auxin, and of the role of B vitamins in plant growth and in root cultures.

The first continuously growing callus cultures were established from cambium tissue in 1939 independently by Gautheret, White and Nobecourt. The subsequent discovery of kinetin by Miller and coworkers in 1955 enabled the initiation of callus cultures from differentiated tissues. Shoot bud differentiation from tobacco pith tissues cultured in vitro was reported by Skoog in 1944, and in 1957 Skoog and Miller proposed that root-shoot differentiation in this system was regulated by auxin-cytokinin ratio.

The first plant from a mature plant cell was regenerated by Braun in 1959. Development of somatic embryos was first reported in 1958- 1959 from carrot tissues independently by Reinert and Steward.

Thus within a brief period, the tissue culture techniques have made a great progress. From the sole objective of demonstrating the totipotency of differentiated plant -cells, the technique now finds application in both basic and applied researches in a number of-fields of enquiry.

Cell Culture

Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. In practice the term “cell culture” has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.

Animal cell culture became a routine laboratory technique in the 1950s,[1] but the concept of maintaining live cell lines separated from their original tissue source was discovered in the 19th century.[2]

The 19th-century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body.[1] In 1885 Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the principle of tissue culture.[3] Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907-1910, establishing the methodology of tissue culture.[4]

Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in virology. Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. The Salk polio vaccine was one of the first products mass-produced using cell culture techniques. This vaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins, who were awarded a Nobel Prize for their discovery of a method of growing the virus in monkey kidney cell cultures.

Concepts in mammalian cell culture

Isolation of cells

Cells can be isolated from tissues for ex vivo culture in several ways. Cells can be easily purified from blood, however only the white cells are capable of growth in culture. Mononuclear cells can be released from soft tissues by enzymatic digestion with enzymes such as collagenase, trypsin, or pronase, which break down the extracellular matrix. Alternatively, pieces of tissue can be placed in growth media, and the cells that grow out are available for culture. This method is known as explant culture.

Cells that are cultured directly from a subject are known as primary cells. With the exception of some derived from tumours, most primary cell cultures have limited lifespan. After a certain number of population doublings cells undergo the process of senescence and stop dividing, while generally retaining viability.

An established or immortalised cell line has acquired the ability to proliferate indefinitely either through random mutation or deliberate modification, such as artificial expression of the telomerase gene. There are numerous well established cell lines representative of particular cell types.

Maintaining cells in culture

Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO2) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.

Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrient components. The growth factors used to supplement media are often derived from animal blood, such as calf serum. These blood-derived ingredients pose the potential for contamination of derived pharmaceutical products with viruses or prions. Current practice is to minimize or eliminate the use of these ingredients where possible.

Some cells naturally live without attaching to a surface, such as cells that exist in the bloodstream. Others require a surface, such as most cells derived from solid tissues. Cells grown unattached to a surface are referred to as suspension cultures. Other adherent cultures cells can be grown on tissue culture plastic, which may be coated with extracellular matrix components to increase its adhesion properties and provide other signals needed for growth. Organotypic cultures involves growing cells in a 3-dimensional environment as opposed to 2-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but is technically challenging to maintain.

Manipulation of cultured cells

As cells generally continue to divide in culture, they generally grow to fill the available area or volume. This can generate several issues:

These issues can be dealt with using tissue culture methods that rely on sterile technique. These methods aim to avoid contamination with bacteria or yeast that will compete with mammalian cells for nutrients and/or cause cell infection and cell death. Manipulations are typically carried out in a biosafety hood or laminar flow cabinet to exclude contaminating micro-organisms. Antibiotics can also be added to the growth media.

Amongst the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells.

Media changes

The purpose of media changes is to replenish nutrients and avoid the build up of potentially harmful metabolic byproducts and dead cells. In the case of suspension cultures, cells can be separated from the media by centrifugation and resuspended in fresh media. In the case of adherent cultures, the media can be removed directly by aspiration and replaced.

Passaging cells

Main article: Passaging

Passaging or splitting cells involves transferring a small number of cells into a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached; this is commonly done with a mixture of trypsinEDTA, however other enzyme mixes are now available for this purpose. A small number of detached cells can then be used to seed a new culture.

Transfection and transduction

Main article: transfection

Main article: transformation (genetics)

Another common method for manipulating cells involves the introduction of foreign DNA by transfection. This is often performed to cause cells to express a protein of interest. More recently, the transfection of RNAi constructs have been realized as a convenient mechanism for suppressing the expression of a particular gene/protein.

DNA can also be inserted into cells using viruses, in methods referred to as transduction, infection or transformation. Viruses, as parasitic agents, are well suited to introducing DNA into cells, as this is a part of their normal course of reproduction. see concentration

Established human cell lines

One of the earliest human cell lines, descended from Henrietta Lacks, who died of the cancer that those cells originated from, the cultured HeLa cells shown here have been stained with Hoechst turning their nuclei blue.

Cell lines that originate with humans have been somewhat controversial in bioethics, as they may outlive their parent organism and later be used in the discovery of lucrative medical treatments. In the pioneering decision in this area, the Supreme Court of California held in 1990 that human patients have no property rights in cell lines derived from organs removed with their consent. [5] It is estimated that about 20% of human cell lines are not the kind of cells they were generally assumed to be.[6] The reason for this is that some cell lines exhibit vigorous growth and thus can cross-contaminate cultures of other cell lines, in time overgrowing and displacing the original cells. The most common contaminant is the HeLa cell line. While this may not be of significance when general properties such as cell metabolism are researched, it is highly relevant e.g. in medical research focusing on a specific type of cell. Results of such research will be at least flawed, if not outright wrong in their conclusion, with possible consequences if therapeutic approaches are developed based on it. [7]

Generation of hybridomas

For more details on this topic, see Hybridoma.

It is possible to fuse normal cells with an immortalised cell line. This method is used to produce monoclonal antibodies. In brief, lymphocytes isolated from the spleen (or possibly blood) of an immunised animal are combined with an immortal myeloma cell line (B cell lineage) to produce a hybridoma which has the antibody specifity of the primary lymphoctye and the immortality of the myleoma. Selective growth medium (HA or HAT) is used to select against unfused myleoma cells; primary lymphoctyes die quickly in culture and only the fused cells survive. These are screened for production of the required antibody, generally in pools to start with and then after single cloning.

Applications of cell culture

Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biological products produced by recombinant DNA (rDNA) technology in animal cell cultures include enzymes, hormones, immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticancer agents. Although many simpler proteins can be produced using rDNA in bacterial cultures, more complex proteins that are glycosylated (carbohydrate-modified), currently must be made in animal cells. An important example of such a complex protein is the hormone erythropoietin. The cost of growing mammalian cell cultures is high, so research is underway to produce such complex proteins in insect cells or in higher plants.

Tissue culture and engineering

Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells ex vivo.

Vaccines

Vaccines for polio, measles, mumps, rubella, and chickenpox are currently made in cell cultures. Due to the H5N1 pandemic threat, research into using cell culture for influenza vaccines is being funded by the United States government. Novel ideas in the field include recombinant DNA-based vaccines, such as one made using human adenovirus (a common cold virus) as a vector,[8] [9] or the use of adjuvants. [10]

Culture of non-mammalian cells

Plant cell culture methods

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

Bacterial/Yeast culture methods

Main article: microbiological culture

For bacteria and yeast, small quantities of cells are usually grown on a solid support that contains nutrients embedded in it, usually a gel such as agar, while large-scale cultures are grown with the cells suspended in a nutrient broth.

Viral culture methods

The culture of viruses requires the culture of cells of mammalian, plant, fungal or bacterial origin as hosts for the growth and replication of the virus. Whole wild type viruses, recombinant viruses or viral products may be generated in cell types other than their natural hosts under the right conditions. Depending on the species of the virus, infection and viral replication may result in host cell lysis and formation of a viral plaque.

Plant Tissue Culture

Introduction

Plant tissue culture involves the growing of plant tissue from plant material taken from a source plant.It has been found that plants can reproduce whole plants from fragments of plant material when given a nutrient media capable of supporting growth and appropriate hormone control.

The nutrient media used in plant tissue culture is an agar media with macro and micro nutrients dissolved in it.Unlike plants growing from a seed, tissue cultures require a supply of  carbon in an organic form such as sugars.They also require amino acids,B vitamins and growth hormones .The constituents of the media will vary with the plant material being cultured.

Plant tissue culture can be used to clone plants and produce many identical plants for a particular market.This can be used when a new variety is grown and other methods of cultivation are too slow for the desired market.It can also be used if a stock plant has been infected and material taken from the plant that is not infected.The excised plant material can be grown on and any disease free plants grown on for propagation.Plant tissue culture is also of use in research for biochemists,geniticists,plant breeders and plant pathologists.Plant tissue culture has also proved more efficient in the production of secondary metabolites than the use of the parent plants in various instances and has been used in the commercial production of the napthoquinone pigment Shikonin.Plant tissue caulture has also been used in the production of flavours,sweeteners,natural colourants and pharmaceuticals.With the advent of gene insertion plant cells with gene material inserted can be regenerated using tissue culture to produce a whole new plant.

Some plants are better suited to plant tissue culture than others but most plants can be cultivated with time and practise.

Methods and Materials

When taking plant material to grow on using plant tissue culture it is important to get the most appropriate material for the end product you are aiming for.Plant tissue has been shown to be totipotent,but different tissues will require different treatment to produce whole plants if that is the aim.Tissue that is dividing such as at the nodes and leaf axils,leaf peiole material are often used.

Sharp cuts will decrease the amount of decaying material present,and decrease the possibility of infection.The use of a sharp scalpel is advised.Forceps are necessary to move the plant material to the growing media.The cutting should take place in a sterile environment and the growing media only exposed when the plant material is placed in it,after which it should be sealed.

Forceps

Scalpel

Disinfectants-for surfaces,implements and plant material.

Laminar Flow Cabinet

Bunsen burner

Containers-petri dishes,small clear plastic containers,glass jars.

Growth media-appropriate to plant material being cultured

Murashige Skoog

Constituents                                              Media mg/Litre

Inorganic

Ammonium Nitrate NH4NO3                     1650

Potassium Nitrate KNO3                            1900

Calcium Chloride CaCl2.2H2O                     440

Magnesium Sulphate MgSO4.7H2O              370

KH2PO4                                                       170

Potassium Iodide                                           0.83

H3BO3                                                           6.2

Manganese Sulphate MnSO4.4H2O              22.3

Zinc Sulphate ZnSO4.7H2O                            8.6

Na2Mo4.H2O                                                 0.25

Copper Sulphate CuSO4.5H2O                       0.025

Iron Sulphate FeSO4.7H2O                            27.8

Na2EDTA.2H2O                                            37.3

Organic

Inositol                                                            100

Nicitinic Acid                                                      0.5

Pyridoxine HCl                                                   0.5

Thiamine HCl                                                      0.1

Glycine                                                                2

Sucrose                                                              3%

BVitamins

Nicitinic acid

Thiamine HCl

Pyridoxine HCl

Growth Hormones-Auxins,

Benzyl Amino Purine

Auxin-Indoleacetic acid

Inositol

Cytokinins

Kinetin

For growing plant tissue cultures on a suitable site is required which is clean,warm (20deg C) and there is adequate light.

The source of the plant material is important as some plant tissue is better suited to tissue culture than others,the ability of plant material to grow and divide in vitro is known as totipotency,but different plant material will need different control to form new plant material.The plant material may form a new embryo,callous tissue or a whole plant depending on how it is looked after.

History of Plant Tissue Culture

This ability has been known for many years and much information has been gathered on the best ways to look after plant material of different species and sources for different uses using plant tissue culture.Vochtung in 1878 obderved that cells along a stems length were capable of generating roots or shoots.Gottlieb Haberlandt a German botanist was the first to generate tissue from fully differentiated  tissue.He was interested in thios as he thought it would give:

“interesting insight into the properties and potentialities which the cell as an elementary organism possesses.”

Hannig grew nearly mature embryos of crucifers and grew them to maturity on mineral salts and sugar solution.The embryos would not grow to form plants however without the addition of growth compounds.Later work was succesful in growing on embryos with the asddition of coconut milk.Nutrient media then included mineral salts,vitamins,amino acids, and sugar.Laibach succesfully reared embryos that were otherwise unviable using tissue culture.Several new hybrids have since ‘evolved’using tissue culture that would otherwise have been unviable at the embryo stage.

White succesfully reared tomato root cultures using a medium containing three B vitamins;Pyrodoxine,thiamine and Nicotinic acid,also inorganic salts and sucrose.In the 1030’s identification of auxin as a natural growth regulator and the recognition of the importance of the B vitamins led to improvements in tissue culture practises.Proliferation of cells was achieved using a solution containing Glucose and Cysteine Hydrochloride.The first continually growing cultures were achieved using carrot root cambium.

Cell division in tissue had to await the discovery of kinetin.This was first discovered by autoclaving freshly isolated slurries of DNA from Herring sperm.The discovery of cytokinins gave much impetus to tissue culture.

Techniques developed to stimulate cell division in liquid culture.In 1965 whole plants were raised from isolated single cells by filtering suspension cultures and growing on the isolated cells on solid media containin 0.6% agar.These techniques are now widely used for cloning tissues.

Hormonal control of tissue culture was achieved using the regulation of cytokinin:auxin ratio,thus controlling root and shoot development in tobacco.I then became possible to manipulate tissues and to develop whole plantlets via shoot and root development.Cells could also be controlled to produce somatic embryos which can then be used to produce whole plants.Rapid propagation of tissues from callus was used though genetic variation in tissue culture tended to occur.Production from shoot tip and leaf primordia proved a more succesful technique.The technique was used to produce virus free plants fropm infected Dahlias.Orchids were also cloned on a wide scale and the technique was adapted for ferns,foliage plants and fruit plants.

Growing on cytokinin rich media can reduce apical dominance leading to more shoots and to quicker regeneration of numbers.Invitro fertilisation made it possible to cross varieties unable to cross in nature.Haploid plants of tobacco were raised from pollen grains.A more recent development in tissue culture is protoplast culture.This technique has allowed the crossing of plants by protoplast fusion,somatic hybrids have thus been produced of Nicotiana glauca x Nicotiana langsdorfii (Bhojwani et al,1983).

Tissue culture is thus proving to be useful in a variety of ways including plant propagation,raising and maintenace of high health status plants,germ plasm storage,and a valuable technique in plant improvement..In plant improvement tissue culture maybe used in the technique of gene insertion to improve plant stocks.

Plant tissue culture has also been used in the production of secondary metabolites in plants.The production of flavours,sweeteners, naturl colourants, aswell as pharmaceuticals has all achieved using tissue culture.

(from wikipedia)

Related Reading

http://www.accessexcellence.org/LC/ST/st2bgplant.php

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