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 Post subject: Tissu culture
PostPosted: Thu Apr 17, 2008 2:41 am 
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The practice of plant tissue culture has changed the way some nurserymen approach plant propagation. In the recent past, the applicability of this technology to the propagation of trees and shrubs has been documented. Some firms have established tissue culture facilities and commercial scale operations are presently in operation for the mass propagation of apples, crabapples, rhododendrons, and a few other selected woody species. The intent of this research update is to briefly examine "what is being done" and to explore "what can be done" with regard to the tissue culture of ornamental plants. Such a consideration necessarily includes an overview of tissue culture as a propagation tool. The major impact of plant tissue culture will not be felt in the area of micropropagation, however, but in the area of controlled manipulations of plants at the cellular level in ways which have not been possible prior to the introduction of tissue culture techniques.

Tissue culture refers to the growth of tissues and/or cells separate from the organism. In 1907 the American zoologist Ross Granville Harrison demonstrated the growth of frog nerve cell processes in a medium of clotted lymph. This term usually is used in the context of animal tissue culture, while the more specific term plant tissue culture is used for plants.


In modern usage, "tissue culture" often refers to the growth of animal or plant cells in vitro. In particular, the term is often used interchangeably with cell culture to specifically describe the in vitro culturing of mammalian cells.

Plant tissue culture is the aseptic (free from microorganism) culture of any plant part in vitro. Tissue culture is utilized in the field of Biotechnology. Micro-propagation is the rapid vegetative propagation of plants via tissue culture techniques. Micro-propagation permits the manipulation of physical and chemical conditions in the production of large numbers of high quality plant material within a short period of time.


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 Post subject: Re: Tissu culture
PostPosted: Thu Apr 17, 2008 2:42 am 
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Adantages of Propagation by Tissue Culture
The elimination of diseases and the production of disease free plantlets
The rapid production of large numbers of genetically identical plantlets
Introduction of new varieties and or genotypes
Preservation of germplasm
Production of haploid plants which can be used for plant breeding
Production of plantlets from species in which plant development from seed is difficult


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 Post subject: Re: Tissu culture
PostPosted: Thu Apr 17, 2008 2:42 am 
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Infrastructure Requirements for Plant Tissue Culture
a washing area (vessels and planting material are cleaned, plantlets may be weaned)
a media preparation room ( preparation of media, storage and sterilization)
a aseptic transfer area (initiation and sub-culturing of plantlets)
an incubator or a culture room (provide plantlets in culture with temperature and light
requirement)


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 Post subject: Re: Tissu culture
PostPosted: Thu Apr 17, 2008 2:42 am 
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Equipment Used in Tissue Culture Laboratory
Analytical balance (for weighing nutrients for media)
Graduated cylinders and pipettes (for measuring stock solutions)
pH meter (to regulate pH of media)
Hot plate or stove (to heat and dissolve gelling agent)
Glass containers (for heating and dissolving media)
Dispensing devices (to dispense equal quantities of media)
A Still or de-ionizer (water needed for media)
Pressure steam sterilizer (for sterilizing instruments and media)
Transfer instruments (forceps, scalpels spatulas, blades)
Refrigerator (storage of chemicals and stock solutions)
Stereo-microscope (use for meristem culture)
Laminar Flow Hood (provide a sterile area for transfers during initiation and sub-culturing)


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 Post subject: Re: Tissu culture
PostPosted: Thu Apr 17, 2008 2:43 am 
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Media and Media Components
The composition of the cultural media is one of the most important factors in determining the growth and morphology of in vitro plants. Plants grown in vitro require similar nutrients to plants grown in the soil. The basic components of any cultural medium are: The composition of the cultural media is one of the most important factors in determining the growth and morphology of in vitro plants. Plants grown in vitro require similar nutrients to plants grown in the soil. The basic components of any cultural medium are: The composition of the cultural media is one of the most important factors in determining the growth and morphology of in vitro plants. Plants grown in vitro require similar nutrients to plants grown in the soil. The basic components of any cultural medium are:

Macronutrients: There are six macronutrients, nitrogen, potassium, phosphorous, calcium,
magnesium and sulphur.
Micronutrients: They include, iron, manganese, zinc, boron, copper, molybdenum, cobalt and
iodine.
A Carbon source: The carbohydrates usually used are sucrose and glucose.
Vitamins: They are thiamin (B1) nicotinic acid, pyridoxine (B6) and myo-inositol
Growth Regulators: Both auxins (IAA, NAA) and cytokinins ( BA).
Solidifying or Gelling Agent: The two commonly used are agar and phytogel.

Several media formulations are sold prepackaged. Some of these are Murashige and Skoog Based Media, Gamborg’s Based Media, and Orchid Tissue Culture Media. They may contain all components of the media except the carbon source and the solidifying agent, and some may lack vitamins and or growth regulators.


However, "tissue culture" can also be used to refer to the culturing of tissue pieces, i.e. explant culture or whole organs, i.e. organ culture.


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 Post subject: Re: Tissu culture
PostPosted: Thu Apr 17, 2008 2:45 am 
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CELL CULTURE
Cell culture is the process by which either prokaryotic or eukaryotic 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.

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 an animal or person 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 (e.g. collagen or fibronectin) to increase its adhesion properties and provide other signals needed for growth.


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:

Nutrient depletion in the growth media

Accumulation of apoptotic/necrotic (dead) cells
Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing
Cell-to-cell contact can stimulate promicuous and unwanted cellular differentiation
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 biproducts 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
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 was historically done with a mixture of trypsin-EDTA, 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.

Applications of cell culture
Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and many products of biotechnology. Biologicals 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.

PLANT IMPROVEMENT THROUGH TISSUE CULTURE

In introducing this research update, it was mentioned that the major impact of tissue culture technology would not be in the area of micropropagation, but rather in the area of controlled manipulations of plant germplasm at the cellular level. The ability to unorganize, rearrange, and reorganize the constituents of higher plants has been demonstrated with a few model systems to date, but such basic research is already being conducted on ornamental trees and shrubs with the intent of obtaining new and better landscape plants.

SELECTION OF PLANTS WITH ENHANCED STRESS OR PEST RESISTANCE
Perhaps the most heavily researched area of tissue culture today is the concept of selecting disease, insect, or stress resistant plants through tissue culture. Just as significant gains in the adaptability of many species have been obtained by selecting and propagating superior individuals, so the search for these superior individuals can be tremendously accelerated using in vitro systems. Such systems can attempt to exploit the natural variability known to occur in plants or variability can be induced by chemical or physical agents known to cause mutations.
All who are familiar with bud sports, variegated foliage and other types of chimeras have an appreciation for the natural variability in the genetic makeup or expression in plants. Chimeras are the altered cellular expressions which are visible, but for each of these which are observed many more differences probably exist but are masked by the overall organization of the plant as a whole. For example, even in frost-tender species, certain cells or groups of cells may be frost hardy. However, because most of the organism is killed by frost, the tolerant cells eventually die because they are unable to support themselves without the remainder of the organized plant. Plant tissues grown in vitro can be released from the organization of the whole plant through callus formation. If these groups of cells are then subjected to a selection agent such as freezing, then those tolerant ones can survive while all those which are susceptible will be killed. This concept can be applied to many types of stress as well as resistance to fungal and bacterial pathogens and various types of phytotoxic chemical agents. Current research in this area extends across many interests including attempts to select salt tolerant lines of tomato, freezing resistant tobacco plants, herbicide resistant agronomic crops, and various species of plants with enhanced pathogen resistance. Imagine, if you will, the impact of a fireblight-resistant Bartlett pear, a clone of pin oak for alkaline soils, or a selection of southern magnolia hardy to zone 4!

TISSUE CULTURE AND PATHOGEN FREE PLANTS
Another purpose for which plant tissue culture is uniquely suited is in the obtaining, maintaining, and mass propagating of specific pathogen-free plants. The concept behind indexing plants free of pests is closely allied to the concept of using tissue culture as a selection system. Plant tissues known to be free of the pathogen under consideration (viral, bacterial, or fungal) are physically selected as the explant for tissue culture. Cultures which reveal the presence of the pathogen are destroyed, while those which are indexed free of pathogen are maintained as a stock of pathogen-free material. Procedures similar to these have been used successfully to obtain virus-free plants of a number of species and bacteria-free plants of species known to have certain leaf spot diseases. The impact of obtaining pathogen-free nursery stock can only be speculative, since little research documenting viral,
ing nitrogen-fixing corn plants on the one extreme to discovering a yellow-flowered African violet on the other extreme.

Culture of non-mammalian cells

Bacterial/Yeast culture methods

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 as hosts for the growth and replication of the virus. Viruses infect cells, causing them to lyse and form a viral plaque.

MICROBIOLOGICAL CULTURE

A microbiological culture, or microbial culture, is a method of growing a microbial organism to determine what it is, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology. A tool is often used to determine the cause of infectious disease by letting the agent multiply (reproduce) in predetermined media in laboratory.

The most common method of microbiological culture uses Petri dishes with a layer of agar-based growth medium in them to grow bacterial cultures. This is generally done inside of an incubator. Another method is liquid culture, where the bacteria are grown suspended in a liquid nutrient medium. Bottles of liquid culture are often placed in shakers in order to introduce oxygen to the liquid and maintaining the uniformity of the culture.

The term culture can also, though infrequently and informally, be used as a synonym for tissue culture, which involves the growth of cells or tissues explanted from a multi-cellular organism.

TYPES OF CULTURE

Blood culture
Sputum culture
Tissue culture
Culture of various fluids such as pleural fluid and peritoneal fluid
Urine culture


ORGAN CULTURE


Organ culture is a development from tissue culture methods of research, the organ culture is able to accurately model functions of an organ in various states and conditions by the use of the actual in vitro organ itself.

Parts of an organ or a whole organ can be cultured in vitro. The main objective is to maintain the architecture of the tissue and direct it towards normal development. In this technique, it is essential that the tissue is never be disrupted or damaged. It thus requires careful handling. The media used for a growing organ culture are generally the same as those used for tissue culture. The techniques for organ culture can be classified into (i) those employing a solid medium and (ii) those employing liquid medium.

SUMMARY
Plant tissue culture research is multi-dimensional. While most nurserymen have been introduced to the techniques and advantages of micropropagation, few have ventured to use it as a propagation tool. The applicability of micropropagation for woody trees has been demonstrated as feasible since all aspects of the technology have confirmed the fact that trees produced by this method look like and grow like their counterparts produced by traditional methods of cloning.
Other dimensions of tissue culture research have been less well publicized. The potential for selecting pathogen free plants, for selecting stress-tolerant and pathogen-resistant clones of plants, and the novel genetic combinations to be achieved through somatic hybridization are all lines of research which can have a profound impact on the nursery industry.


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