CONCEPTS OF BIOTECHNOLOGY

TOPIC: Culture media in plant tissue culture BTC 012
SUBMITTED BY: TO: ANCHAL GUPTA MR.HARSH BTB-A ROLLNO: 17 REGD.NO: 1040070080 SUBMITTED

Acknowledgement

With profound reverence express my deepest sense of gratitude to my sir Mr. Harsh from biotech department of lovely professional university, who helped me on my topic “Culture

media for plant tissue culture”,

Special thanks goes to my elder sister puja sivastava for her consistent moral support and guidance without which i would not have completed my term paper. Uniting help rendered by my rum mates which were at most important for my term paper.

Above all with deepest sense of regards i bow to my esteemed parents for their blessings on me to complete my task successfully

Index
S.no Contents 1 Introduction 2 Plant tissue culture media 3 Preparation of plant tissue culture media 4 Application 5 Conclusion 6 Reference Page no. 4-4 5-8 9 9 10 11

Introduction

Plant Tissue Culture
Plant tissue culture is a practice used to propagate plants under sterile conditions, often to produce clones of a plant. 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 plant 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

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. As different plants required different media like murashige and skoog’s(m s) media, white media and gamborg media etc .culture media. But generally all the 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. :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.

Microelements

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.

Organic Supplements

These include vitamins and amino acids. Two vitamins, i.e., thiamine (vitamin B1) and myoinositol (a Vitamins 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.

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.

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.

Growth Plant 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-3acetic 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.

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 embryo genesis. 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.

Antibiotics

Antibiotics are substances produced by certain microorganisms that suppress the growth of other microorganisms and eventually destroy them. Their applications include:
A. B. C. Suppresses bacterial infections in plant cell and tissue culture. Suppresses mould and yeast infections in cell cultures. 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

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, Neomycin,, Tobramycin, Streptomycin,

Gentamicin,

Kanamycin,

• •

Inhibitors of Nucleic Acid Metabolism: e.g. Rifampicin, Mitomycin C and Nalidixic acid Antimetabolites: Antibiotics, which block specific metabolic steps that are essential to microorganism’s e.g. Metronidazole, Miconazole, Nitrofurantoin, Trimethoprim and Sulphomethoxazole. Nucleic Acid Analogs, which inhibit enzymes essential for DNA synthesis. e.g. 5-Fluorouracil, Mercaptopurine

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.

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

Micro propagation 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 inside bioreactors as a source of secondary products, like 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 this would otherwise normally die (Embryo Rescue).

For production of doubled monoploid 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.

Conclusion

By studying the whole topic I have found that different growth media is required for to grow different plant cells like m.s media, white and gambards etc all the media which is used in plant tissue culture slightly different with each other and each constituent has its own importance in media like nitrogen is one of the main component to the growth of plant in vitro and in vivo, phosphorus for cell division, sulfure which is present in some protein. The amino acid like cystine which is used in as antioxidant and prevent blacking of tissue sucrose is widely used in carbon source. in addition to nutrients growth regulator also play an important like auxin for root initiation, cytokinins for cell division with auxin and in shoot formation,gibberellin for somatic embriogeninses.One of the important point in preparation of media is its ph which varies with different stages of preparation and culture .

Reference
1. http://www.planttissueculturemedia.com/ 2. en.wikipedia.org/wiki/Plant_tissue_culture - 32k

3. S.S Bhojwani & M.K Razdan.1996.Plant Tissue Culture.Theory and practice, a revised edition.pp.39-52. 4. K.G ramabhwat. Plant Tissue Culture.pp.41-43.

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