Plant Tissue Culture

enjoy in this websitePlant Tissue Culture
Presenter: Lydiane (Ann) Kyte
Host: Kathy Liu
Discussion
Did you ever have a plant that was so unique or so beautiful that you wished you had hundreds or thousands of them to enjoy or to sell? Plant tissue culture (micropropagation) is a technique which will do just that for us. We are going to discuss this tool which is used so extensively in the nursery business and in plant biotechnology. It is a fascinating and useful tool which allows the rapid production of many genetically identical plants using relatively small amounts of space, supplies and time.

Basically the technique consists of taking a piece of a plant (such as a stem tip, node, meristem, embryo, or even a seed) and placing it in a sterile, (usually gel-based) nutrient medium where it multiplies. The formulation of the growth medium is changed depending upon whether you are trying to get the plant to produce undifferentiated callus tissue, multiply the number of plantlets, grow roots, or multiply embryos for “artificial seed”.

For many who become superficially aware of the technique it seems shrouded in mystery and is shrugged off as too technical to be of concern. Actually, it is no more of a mystery than taking a cutting of your favorite house plant and growing it to share with a friend. As for being technical, you can begin plant tissue culture with as little as a cookbook approach and a feeling for sterile technique.

Some people have visions of scientists doing plant tissue cultures in white gowns and masks in hospital-clean environments. Such conditions are excessive. While it is true that mold spores, bacteria, and other contaminants will grow and overrun a culture, air that is not moving has a minimum of contaminants. In addition, disinfection of implements, work surface and nearby areas helps eliminate contaminants.

The guidelines for preparation and the laboratory protocol provided here are given as a place to begin. Included with is a limited discussion of some of the many options you have as you explore micropropagation. We can discuss these in more depth if you have questions, concerns or related experiences to share. I would be particularly interested in success and challenges you may have had or are currently having in your classroom.

Some suggestions are given for the following
(a) Selecting plant sources. Some species, or even clones are easier to grow in culture than others. Some respond reluctantly to culture, some do not respond at all, and many plants have never been tried.

(b) Choosing a growth medium (price, convenience, type of plant and purpose of the micropropagation all enter into this decision.) How important are the kinds of hormones used? On limited scale, media ingredients are available at the grocery and health food stores.

(c) Suggestions for media preparation and sterilization. There are alternatives to sterilization in a pressure cooker or an autoclave.

(d) Methods for cleaning, storing and manipulating explants (plant pieces to be cultured).

Given certain basics there are many options for procedure, equipment and supplies for plant tissue culture. Some of your decisions will be based upon the amount of time, money and space you have. Other decisions will be based upon why you are doing plant tissue culture and what you expect as a result (more plants?) . Catalogs, such as Sigma, Carolina Biological, or Edmund Scientific are good reference and they are for purchasing needed materials..

I look forward to sharing tissue culture experiences with you.

References:
Debergh, P.C. and R.H. Zimmerman, eds. 1991. Micropropagation, Technology and Application. Kluwer Academic Publishers. $61.50. Lab design, info on labs worldwide, in depth discussions of problems. Not for the beginner.

Donnelly, D.J., and W.E.Vidaver, 1988. Glossary of Plant Tissue Culture, Portland, OR. Timber Press, $22.95. Good definitions of tissue culture terms.

Kyte, Lydiane and J. Kleyn, 1996. Plants from Test Tubes: An Introduction to Micropropagation, 3rd ed., Timber Press, 1996 $29.95. Good basics for the beginning amateur or grower.

Smith, Roberta H., 1992. Plant Tissue Culture-Techniques and Experiments. Academic Press. $35.00. Good introduction and broad base for college course.

Trigiano, Robert N, and Dennis J. Gray, eds.1996,Plant Tissue Culture Concepts and Laboratory Exercises. CRC Press. $65.00. For the advanced student.

Sources of supplies:

Carolina Biological

Edmond Scientific

PhytoTechnology Laboratories
This company specializes in plant tissue culture supplies. Downloadable documents (choose MS Word or PDF format) on Media Preparation, Setting Up a Tissue Culture Lab, Basic Laboratory Procedures, and more are available in the “Technical” section of the web site.

Sigma, 1996. “Plant Tissue Culture Catalog”.
In addition to media ingredients, premixes, equipment and supplies, this catalog contains a media comparison chart, procedures for media preparations.references and other valuable data.

REPEREN :http://www.accessexcellence.org/LC/ST/st2bgplant.php Readmore »»
Posted by panciamis@gmail.com at 10:51 PM 0 comments
Plant Tissue Culture Media
Plant Tissue Culture Media

Plant Tissue Culture refers to the technique of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium. Culture media used for in vitro cultivation of plant cells are composed of following basic components:

Complex Mixture of Salts: Essential elements, or mineral ions

Organic Supplement: vitamins and/or amino acids

Carbon Source: usually sugar sucrose

Gelling Agents

Plant Growth Regulators

Antibiotics
Complex Mixture of Salts
These include essential elements or mineral ions important for plant nutrition and their physiological function. The essential elements can further be divided into the following categories:

Macroelements (or macronutrients)
Microelements (or micronutrients)
Iron source

Macroelements :- These elements are required in large amounts for plant growth and development. Nitrogen, phosphorus, potassium, magnesium, calcium and sulphur (and carbon, which is added separately) are regarded as macroelements. These elements comprise at least 0.1% of the dry weight of plants.

Microelements :- These elements are required in trace amounts for plant growth and development. Manganese, iodine, copper, cobalt, boron, molybdenum, iron and zinc are regarded as microelements, although other elements like aluminium and nickel are frequently found in some formulations.

Plant Tissue Culture Media

Iron Source :- Iron is usually added in the medium as iron sulphate, although iron citrate can also be used. Ethylenediaminetetraacetic acid (EDTA) is usually used in conjunction with the iron sulphate. The EDTA complexes with the iron so as to allow the slow and continuous release of iron into the medium. Uncomplexed iron can precipitate out of the medium as ferric oxide.

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Organic Supplements
These include vitamins and amino acids. Two vitamins, i.e., thiamine (vitamin B1) and myoinositol (a vitamin B) are essential for the culture of plant cells in vitro. However, other vitamins are often added to for historical reasons. The most commonly used amino acid is glycine. However, arginine, 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. Casein hydrolysate can be used as a source of a mixture of amino acids.

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Carbon Source
The most commonly used carbon source is sucrose. It is readily assimilated and relatively stable. Other carbohydrates like glucose, maltose, galactose and sorbitol can also be used and may prove better than sucrose in specialized circumstances.
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Gelling Agents
Plant tissue culture media can be used in either liquid or ‘solid’ forms, depending on the type of culture being grown. Agar, produced from seaweed, is the most common type of gelling agent, and is ideal for routine applications. For more demanding applications, a range of purer gelling agents are available. Purified agar or agarose can be used, as can a variety of gellan gums.

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Plant Growth Regulators
Specific media manipulations can be used to direct the development of plant cells in culture due to plasticity and totipotency. Plant growth regulators are the critical media components in determining the developmental pathway of the plant cells. There are five main classes of plant growth regulator used in plant cell culture, namely:

Auxins

Cytokinins

Gibberellins

Abscisic Acid

Ethylene

Auxins

:-

Auxins promote both cell division and cell growth. IAA (indole-3-acetic acid) is the most important naturally occurring auxin but its use in plant tissue culture media is limited because it is unstable to both heat and light. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances.

Cytokinins

:-

Cytokinins promote cell division. Of the naturally occurring cytokinins, only zeatin and 2iP (2-isopentyl adenine have some use in plant tissue culture media. The synthetic analogues, kinetin and BAP (benzylaminopurine), are used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant tissue culture media.

Auxins

:-

Auxins promote both cell division and cell growth. IAA (indole-3-acetic acid) is the most important naturally occurring auxin but its use in plant tissue culture media is limited because it is unstable to both heat and light. 2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin and is extremely effective in most circumstances.

Plant Tissue Culture Media

Cytokinins

:-

Cytokinins promote cell division. Of the naturally occurring cytokinins, only zeatin and 2iP (2-isopentyl adenine have some use in plant tissue culture media. The synthetic analogues, kinetin and BAP (benzylaminopurine), are used more frequently. Non-purine-based chemicals, such as substituted phenylureas, are also used as cytokinins in plant tissue culture media.

Gibberellins

:-

Gibberellins are involved in regulating cell elongation, in determining plant height and fruit-set. Only a few of the gibberellins like GA3 are used in plant tissue culture media.
Abscisic Acid

:-

It is used in plant tissue culture to promote distinct developmental pathways such as somatic embryogenesis. Abscisic acid (ABA) inhibits cell division.

Ethylene

:-

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.

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Antibiotics
Antibiotics are substances produced by certain microorganisms that suppress the growth of other microorganisms and eventually destroy them. Their applications include:
A.

Suppresses bacterial infections in plant cell and tissue culture.
B.

Suppresses mould and yeast infections in cell cultures.
C.

Eliminates Agrobacterium species after the transformation of plant tissue.
These antibiotics can be divided into different classes on the basis of chemical structure and their mechanism of action:

Inhibitors of Bacterial Cell Wall Synthesis

e.g. β-lactam antibiotics, Penicillins and Cephalosporins.

Antibiotics that affect Cell Membrane permeability.

• Antibacterial e.g. Colistin Sulphate, Polymixin B Sulphate, Gramicidin
• Antifungal e.g. Amphotericin B, Nystatin, Pimaricin

Bacteriostatic Inhibitors of Protein

Plant Tissue Culture Media

Antibiotics that affect the function of 30 S or 50 S ribosomal subunits to cause a reversible inhibition of protein synthesis. e.g. Chloramphenicol, Chlortetracycline HCl, Clindamycin HCl, Doxycycline HCl, Erythromycin, Lincomycin HCl, Oxytetracycline HCl, Spectinomycin sulphate, Tetracycline HCl, Tylosin tartrate, Lincomycin HCl

Bactericide Inhibitors of Protein Synthesis

Antibiotics that bind to the 30 S ribosomal subunit and alter protein synthesis which eventually leads to cell death. This group includes:

* Aminoglycosides: Apramycin, Butirosine, Gentamicin, Kanamycin, Neomycin, Streptomycin,
Tobramycin.
* Inhibitors of Nucleic Acid Metabolism: e.g. Rifampicin, Mitomycin C and Nalidixic acid
* Antimetabolites: Antibiotics, which block specific metabolic steps that are essential to microorganisms
e.g. Metronidazole, Miconazole, Nitrofurantoin, Trimethoprim and Sulphomethoxazole.
* Nucleic Acid Analogs, which inhibit enzymes essential for DNA synthesis. e.g. 5-Fluorouracil, Mercaptopurine.

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Preparation of Plant Tissue Culture Medium

Measure approximately 90% of the required volume of the deionized-distilled water in a flask/container of double the size of the required volume.

Add the dehydrated medium into the water and stir to dissolve the medium completely. Gentle heating of the solution may be required to bring powder into solution.

Add desired heat stable supplements to the medium solution.

Add additional deionized-distilled water to the medium solution to obtain the final required volume.

Set the desired pH with NaOH or HCl.

Dispense the medium into culture vessels.

Sterilize the medium by autoclaving at 15 psi (121οC) for appropriate time period. Higher temperature

may result in poor cell growth.

Add heat labile supplements after autoclaving.

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