Showing posts with label Biotechnology. Show all posts
Showing posts with label Biotechnology. Show all posts

The Bioreactors

 Bioreactors

A bioreactor is generally defined as a system capable of providing the cells the intake of nutrients and removal of waste products of cell metabolism. Normally bioreactors are used in industry for the cultivation of bacteria, yeasts, fungi, algae, plant cells and animal cells on a large scale, thanks to the advantage of being able to optimize the control of vital parameters with respect to culture performed on the plate. The environment in which they grow the cells is tightly controlled from the point of view of the chemical-physical parameters: thanks to the presence of special sensors can monitor specific parameters of the crop, such as the temperature, the concentration of gases dissolved in the culture medium, the pH, concentration of inorganic ions and carbohydrates. In case be detected alterations of these components, control systems ensure their appropriate correction via the administration of liquid or gas.
The cells can grow in suspension or adherent to a substrate; the homogeneous distribution of the cell suspension through the substrate is ensured by a perfusion system that uses a flow of liquid through the substrate. Even the flow of culture medium within the bioreactor can increase the viability and cellular activity. The use of the bioreactors was thus proposed for the construction of artificial tissues in a three-dimensional system, where also the mechanical stress influence the development and tissue remodeling. In this regard, the bioreactor can be used to generate specific physical stimuli, for example compression and tension, which can stimulate the growth and maturation of the tissues during their development in vitro . Numerous studies have been made ​​for the building of tissues in the bioreactor such as cartilage, tendon and blood vessels, as well, although still at the theoretical level, for the generation of organs such as liver, pancreas and kidney.

The applications of cell theraphy

applications of cell theraphy

The history of the use of artificial tissues as a substitute for compromised tissue is born in the eighties, when they were put in place the conditions to expand the keratinocytes, the cells of the epidermis that is, to reconstruct in the laboratory flaps of tissue to be transplanted into patients who had suffered burns. From a small skin biopsy of the patient are isolated keratinocytes, cultivated in a test tube until the formation of a package that can be transplanted on the surface burned. Other interesting applications have focused on the reconstruction of the corneal epithelium of patients who had undergone thermal or chemical burns to the eye. More recently the interest of scientists turned to the regeneration and repair of skeletal tissues, bone and cartilage, through the use of cells isolated from the bone marrow, respectively, or from a small biopsy of cartilage. In the case of large bone defects, the regenerative ability of the bone is insufficient to ensure the repair of the lesion and the current therapeutic techniques of transplantation of autologous bone or from donor have limitations. Stromal cells derived from bone marrow can in these cases represent a suitable cell type in the regeneration of bone tissue, given their osteogenic potential. There are also numerous studies related to the use of cartilage cells, chondrocytes for the repair of cartilage lesions. The progress achieved by encouraging scientific research have opened new possibilities in the use of tissue engineering for the treatment of diabetes, the regeneration of cardiac muscle tissue when the heart is damaged by a heart attack and the use of endothelial cells for the coating of implants vascular. Even the nerve cells destroyed by degenerative diseases such as Parkinson's disease and Alzheimer's disease could be replaced by the use of cell therapy.

Construction of complex tissues

Construction of complex tissues

The development of knowledge of cell biology integrated with those of bioengineering has recently opened new perspectives in the field of tissue engineering has established itself, which proposes the use of the cells in the laboratory for the construction of artificial biological tissues to be used as a substitute for damaged ones in result of disease or trauma. This would help solve the problems of reduced availability of organs for transplantation and the risk of graft rejection derived from the donor. The association of cells with biocompatible and biodegradable materials is leading to the production of engineered tissues transplantable. The actors involved in the scenario biotech tissue regeneration are basically three: the cells, signaling molecules and biomaterials .
To ensure the maintenance of the function of the implanted cells using a material or scaffold that acts as a scaffold to guide the three-dimensional organization of the cells in the final construction. The scaffold , synthetic or natural, must be able to support the growth and differentiation of cells, ie it must recreate the more possible the microenvironment characteristic of the fabric that you want to replace. Once transplanted, it will perform the function of guide in tissue growth, before being completely resorbed. For the preparation of the plant to be transplanted, the cells are derived from a fragment of the healthy tissue of the patient to which you want to reconstruct the damaged tissue; follows the combination between the cells and biomaterial appropriate, in the presence or absence of signal molecules necessary for proper cell differentiation. Recently aroused great excitement in the scientific community as a resource in the stem cell regenerative therapy due to their ability to give rise to different cell types depending on how they are stimulated.

Cell differentiation

Cell differentiation

The process by which a relatively non-specialized cell becomes a very specialized is called 'differentiation' and affects both the embryo is an individual adult. During embryonic development, many cell types are generated from the fertilized egg cell, to get the range of cells that characterize the adult subject. The route followed by each embryonic cell differentiation is fundamentally dependent on the signals it receives from the environment; the latter, in turn, depend on the position of the cell in the embryo. As a result of the differentiation of different cell types acquire different shapes and express specific proteins: for example, the skeletal muscle cells contain contractile proteins particular, plasma cells are specialized for the production of antibodies, etc.. The differentiation processes continue in the adult body in those tissues that are subject to continuous renewal (eg., Hematopoietic cells).
Each fabric is renewed with its characteristic rhythm, thanks to the presence of stem cells undifferentiated which help keep constant the number of cells in the tissue. Stem cells are defined as undifferentiated cells capable of self-renewal, that is, to produce cells similar to themselves and to generate cells destined to differentiate. They therefore represent a reservoir of cells which the body draws to ensure the renewal of cell tissue death. An example of a stem cell is provided by the hematopoietic stem cells of the bone marrow, capable of giving rise to all cell types of the blood. The adult contains different types of stem cells, which give rise to the cells of the organs in which they are located. Recent studies have however shown that certain adult stem cells have plasticity, ie they are able to differentiate in cell types other than those of the tissue of origin.

Cell division

Cell division

Most cell undergoes division, ie, the formation of two daughter cells identical to the parent cell. This event occurs at the end of the cell cycle, which represents the set of modifications that must be met from the moment in which a cell is formed at the moment in which it divides into two daughter cells. The division is preceded by stages in which the cell doubles its intracellular content. Based on the ability to divide, cells are divided into three categories: ( a ) cells in continuous division, as the skin cells, which have a short life and must be replaced quickly; ( b ) stable cells, ie cells that after differentiation entering a phase of rest, from which they can return to the cycle if properly stimulated (eg., hepatocytes); ( c ) perennial cells, ie cells that after differentiation out permanently from the cycle (eg., neurons).
The rate of cell division is controlled by mechanisms partially known, that allow a cell to divide only if you need more cells. Numerous specific protein factors regulate the cell cycle, such as hormones and growth factors. The cell acts as a target, thanks to the presence of recognition systems of the stimulus to the division. Normally the number of cycles of replication is related inversely to the age of the animal from which the tissue was taken. For this reason, the cells derived from embryonic tissues can replicate in vitro for a longer time compared to cells isolated from tissues of adult organisms. The cells often lose the ability to divide after a certain period of time; this period is variable for the different cell types and to human cells is approximately 50 divisions. This was demonstrated in 1961 by Leonard Hayflick and Paul S. Moorhead, who observed that human fibroblasts died after a finite number of divisions in culture.

From healthy cell to the transformed cell

Some cells derived from multicellular organisms have a capacity of unlimited division in culture and can be used for the production of cell lines. The cell line is composed of transformed cells derived from tumor tissues or cells in primary culture have been changed as a result of spontaneous mutations or induced by exposure to viruses, chemical mutagens or radiation. Keep in mind that the transformed cells have many unusual characteristics that differentiate them from healthy cells. The alterations that occur as a result of processing related to the presence of a genetic aberrant, the reduction and the alteration of the cellular skeleton ( cytoskeleton ), modifications on the cell surface about the expression of antigens, the lower adhesiveness to the substrate and then the ' increased ability to proliferate in suspension and form multiple layers in the plate. Furthermore, the transformed cells have a lower dependence on the presence of serum in the culture medium and in some cases can produce tumors if they are injected into a suitable host organism.
In 1951, George O. Gey and his colleagues cultured the first human cell line, stabilized by a biopsy of the tumor tissue of the uterine cervix taken from a patient named Henrietta Lacks. The patient died shortly afterwards but his cancer cells were maintained in culture until today. These cells were called HeLa in their name and are currently used in many laboratories. We can obtain continuous cell lines through the introduction in normal cells of specific genes ( oncogenes ) by viral infection. If the tumor virus is added to the culture medium appear in a short time, small colonies of transformed cells that proliferate abnormally.

How to prepare a cell culture

How to prepare a cell culture

The first stage in the preparation of a cell culture consists in isolating the desired population of cells from a tissue fragment; in an initial mechanical disaggregation of the tissue is followed by enzymatic digestion, to degrade the extracellular matrix that surrounds and holds the cells together. The resulting cell population is heterogeneous, but using selective media or by separating the cells based on the molecules expressed on the cytoplasmic membrane (antigens), it is possible to isolate specific cell populations. The isolated cells are then grown in an appropriate culture medium; these proliferate and reached confluence, can be detached and moved to another container to keep them in constant division. The cells can be grown to high density (mass culture) or low density (clonal culture), giving rise to the formation of single colonies.

The culture of plant cells

The culture of plant cells

The techniques for the cultivation of plant cells were developed in the fifties of the twentieth century., When he began to realize that the crops have the potential to produce a wide range of molecules useful in various areas, and currently many types of plant cells can be grown in test tubes. Plant cells are surrounded by a cell wall rather rigid, consisting mainly of cellulose, which gives them a mechanical support, the shape and a permeability barrier. To them we must cultivate the cells treated with an enzyme called 'cellulase', which degrades the cellulose wall of the cell releasing 'naked', also called 'protoplast'. Protoplasts are grown in a culture medium with defined chemical composition, which supports the growth and division. The soil must contain between ingredients also plant hormones, such as auxins, essential for cell division . The cultured cells multiply and form a mass of undifferentiated cells said 'callus', from which, by appropriate stimulation, can originate roots, stalks, leaves and even a whole plant. The pharmaceutical industry uses vegetable crops in the production of anti-cancer drugs, anti-inflammatory, antibacterial; the agricultural sector for the production of pesticides; the food industry with regard to the synthesis of additives (eg., dyes).

How to feed the cells in Tissue culture

 feed the cells in Tissue culture

In the living cells remain viable thanks to the contribution of nutrients, guaranteed by the vascular system and, via the capillary network, nourishes the tissue at the cellular level and removes waste products derived from cellular metabolism . In vitro functions vascular vicariate are from the culture medium, a liquid medium highly nutritious. It consists of basic substances, such as glucose, amino acids, vitamins, minerals and trace elements present, necessary for the normal physiological functions of the cell, and from animal serum (usually fetal bovine serum), which supports the growth and proliferation phone. The whey is in most cases used at a concentration of 5 to 20% and contains growth factors, such as platelet growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factors (IGF) , hormones (eg., insulin), lipids (fatty acids, phospholipids, lecithin and cholesterol) are important as a source of energy and for the synthesis of the plasma membrane and adhesive factors, such as fibronectin and vitronectin, transferrin, important for the metabolism of iron, and albumin, capable of transporting vitamins and lipids. The cells should be fed regularly under aseptic conditions to ensure its viability. Normal cells adhere to surfaces of glass or plastic treated and proliferate to form a confluent monolayer that completely covers the surface of the container (petri dish or flask). To ensure the maintenance of the cells in a microenvironment similar to native cells in the laboratory are kept in incubator at 37 ° C with controlled atmosphere (95% air, 5% CO 2 ), which enables it to maintain the proper pH phone. Depending on the tissue of origin, the cells may require the addition to the culture medium of specific factors, in order to maintain the same degree of proliferation and differentiation.

Cultured cells and tissues

Cultured cells and tissues

The cell culture techniques have allowed to study the behavior of cells outside of the living organism in artificial conditions which reproduce, as faithfully as possible, the microenvironment of the tissue or organ from which they derive. The experiments performed on cultured cells are defined in vitro to distinguish them from those in vivo that are executed on the living organism. The tissue culture and cell phones affect many areas of scientific research. Among the possible applications are: the study of the regulation of cell life and response to external stimuli under controlled conditions; the verification of the effect of various chemical compounds and drugs on specific cell types (eg,., tumor cells); the study of the use of cells for the generation of tissue in the test tube (eg., artificial skin); the synthesis of organic products on a large scale (eg,., therapeutic proteins). There have been numerous milestones in the history of the development of cell cultures and tissues.
The first experiment in tissue culture dates back to 1907, when Ross Harrison (1870-1959) of Yale University withdrew a small piece of tissue from the spinal cord of a frog embryo and laid him in a clot of sap. The observation of the tissue under a microscope for several days allowed Harrison to find out that the nerve cells were maintained viable in the medium used. A short time later, Alexis Carrel (1873-1944), Nobel Prize in 1912 for medicine and physiology and considered one of the pioneers in the history of the cell cultures, showed that they could keep the cells outside the body under sterile conditions . In the fifties Harry Eagle gave a major boost to this area of ​​research by studying the necessary nutrients to the cells in culture. Proved fact that animal cells could grow in a cocktail of substances in the chemical composition defined in the presence of serum. Cell cultures have been and still are a very useful tool for the development of vaccines. In 1949 it was shown that the poliovirus could grow in cultures of human cells. The polio vaccine virus deactivated became one of the first commercial products of crops animals. Another milestone in the technology of the cell cultures was placed in 1975 with the development of a technique for the production of hybrid cells, including cells capable of producing antibodies, macromolecules that have an important value for both diagnostic for therapy.
The knowledge and techniques in the field of cell cultures accumulated over time allow us today to cultivate in the laboratory many cell types. From a small fragment of tissue is possible to isolate, through specific procedures, individual cells kept viable thanks to the contribution of nutrients provided by the culture medium. Thanks to the process of division, in culture the cells are able to replicate rise to new cells identical to the parent cell for a defined number of times. In addition, under the influence of appropriate stimuli, may undergo differentiation, namely the assumption of a specific shape and function. Because of this property it is possible today to propose cell cultures as a tool for regeneration of artificial tissues that can be used in the context of new therapeutic strategies. In appropriate experimental conditions the cells may undergo a transformation process that involves a series of changes at the expense of the nucleus and the cytoplasm, altering their normal properties and in some cases making them look like cancer cells.

Difference Between Micro and Nano Technology

Nano and micro technology

Our scientific campaigns haven't simply taken us to the domain of huge spatial figures additionally to the field of the minutest. Micro and Nano engineering, manage scaled down items offering smaller and exceedingly proficient results. There is as of now no incredible distinction between these two fields as both of them have kind of a comparative objective; to prepare innovative units of the minutest sizes. The main major distinction is in the scale. Nano scale is three times more modest than the microscale. Nanotech typically worries about the nuclear or atomic scale while Microtechnology manages electrical and mechanical gadgets that are close to one millionth of a metre in size. Operations identified with both of them notwithstanding, require a comparable setup which is free of dust and earth. Extra steps, for example extraordinary clothing standards and so on are taken to guarantee that no dust particles connect with the moment items. The accompanying segment of the article, tries to carry out a percentage of the paramount distinctions in these two fields with a perspective to carry out a clearer picture. 

Scale 

This is one of the clearer refinements between micro and nano technology. A micrometer is 106 m while a nanometer is 109 m. Researchers have watched that a few diverse phenomena appear as the size diminishes. Consequently the pertinent hypotheses in regards to the two fields are additionally sort of diverse. A greater amount of quantum mechanics plays a hand in nanotech. In the course of the most recent not many years, numerous items that were under the extent of Microtechnology have further scaled down and are constantly treated as nanotech items. 

Application

Different between micro and nano innovation can likewise be seen in the diverse provisions of the two fields. Microelectromechanical System or Mems is presumably the most ubiquitous provision of Microtechnology. Mems units hold mechanical segments and additionally electronic circuits installed onto a little chip. Nanotechnology has appropriated more consideration in the later years has different requisitions in numerous territories, for example human services, It, auto, material and biochemical commercial ventures. 

Nanotech as the Heir to Microtechnology 

Micro and nano technologt are at present the most prominent regions of experimental study. Nanotechnology is required to be the following major upset and more consideration and subsidizing is presently being redirected towards this field. Nanotech has assumed control over numerous requisitions that were formerly under the extent of Microtechnology. Governments, instructive establishments and major organizations all around the globe are currently contributing for the most part on nanotechnology research and development.

Laboratory Safety

Laboratory Safety 


Students must wear lab coat in laboratory every time.

Students must wear good shoes (no slippers allowed).

Students are expected to strictly follow instruction of each experiments.

Do not work alone in laboratory.

Eating, drinking and smoking is strictly prohibited in the laboratory.

Unauthorized experiments and experiments not related to food microbiology course will not be allowed.

Students who are not taking the food microbiology course are not expected to be in 
   lab.

All experiments must be conducted under supervision of relevant lecturer or 
  demonstrators.

After conducting experiments,

Switch off power on all equipment used.

Keep all the chemicals in place ensuring the containers are tightly covered.

Clean the working table and equipment s eg. PH probe, weighing balance etc.

Switch off light and lock the door.
 

Safety


This is a prime important and all students are expected to cooperate in this respect.

•Chemicals used in laboratory could be flammable, toxic, and cause external or internal injury.

Microbes used in laboratory could be pathogenic.

•So, everyone work in laboratory must be well prepared with various safety measures.

Laboratory should be equipped with various safety equipments.

Fire extinguishers

Eye washer

Safety showers

Fume hood

Chemical disposal

First aid supplies

Special aids for particular experiments ( Glasses, shield, UV protectors, etc.)        


Hygiene

•Must keep tables clean.
Must use gloves and wash them after use with detergents.
•Glassware should keep clean and dry.
All glassware and utensils in use must be properly labeled.

Rules for biotechnology practical


Do not eat, drink, apply cosmetics or lip balm, or store food in laboratory.

•Wear a protective laboratory coat or apron when you are working with cultures, and avoid 
  wearing long, full sleeves if possible.

Tie long hair back or put it up.

•Carry and store cultures of microorganisms in racks or baskets. Do not leave cultures on the 
  table or unmarked areas after laboratory session is completed.

Cultures to be discarded should be clearly labeled. All those cultures should be autoclaved 
  before discarding.

•Decontaminate work space after spills at the beginning and at the end of experiments using 
  disinfectant.

Mix liquid cultures gently.

•Never pipette cultures by mouth.

Wash hands using soap after any contamination and after laboratory session and dry hands 
  after washing.

•Open cuts should be properly covered.

Students with special health problems should inform the laboratory staff.

•Shake broth cultures in a manner that avoid wetting the plug or cap.

All contaminated liquid or solid waste must be decontaminated before disposal.


Emergency procedure while doing biotechnology practical


•Spills

     Flood the infected area with disinfectant and dry with paper towels.

•Ingestion

Do not swallow.
Empty content of mouth into sink.
Rinse mouth with copious amount of water.
Report to lab staff.

•Exposure

Eye
     Immediately walk to eye wash station and hold eyelids open and wash at least two 
     min. Inform instructor.  

Cuts
    do not put mouth over cut or breath on the cut. Wash cut with water, follow with 
    disinfectant. Place a clean material over cut.

Needle injection
     Report immediately to instructor and contact medical Center

Equipments used in biotechnology.





Gel electrophoresis apparatus
Culture and media storing refrigerators




















Stomacher Blender
 Top Loading Balance
Laminar flow
Digital microscope

  Sterilization oven



          






                                                        
Analytical Balance    




                                                      






 Autoclaves
Vortex Mixture



                
            
            
      






 Test tubes and Screawcap tubes

 PCR machine   





                            













                                                                      
                                                
 Petridishes,Conical flasks & Measuring cylinders for preparation of media










                      



                                               

        







Tissue Culture

Tissue Culture Techniques: An Introduction
Tissue Culture

      Tissue culture is the culture and maintenance of plant cells or organs in sterile, nutritionally and environmentally supportive conditions (in vitro). Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture). It has applications in research and commerce. In commercial settings, tissue culture is primarily used for plant propagation and is often referred to as micro propagation.


What conditions do plant cells need to multiply in vitro?

Tissue culture has several critical requirements:


·  Appropriate tissue (some tissues culture better than others)
· A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs.     This can be liquid or semisolid.
· Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and  animal tissue and can overrun a culture.
·  Growth regulators - in plants, both auxins & cytokinins.

· Frequent subculturing to ensure adequate nutrition and to avoid the build-up of waste metabolites

         Appropriate tissue (Explant)

·  Cell, tissue or organ of a plant that is used to start in vitro cultures.
·   Axillary buds, meristems Leaves, stems, roots, hypocotyl…
·  Many different explants can be used for tissue culture, but  axillary buds and meristems are most commonly used.
· The explants must be sterilized to remove microbial  contaminants. This is usually done by chemical surface          sterilization of the explants.
·  With an agent such as bleach at a concentration and for a duration that will kill or remove pathogens without injuring the plant cells.
·   Many plants are rich in polyphenolics
·  After tissue injury during dissection, such compounds will be oxidized by polyphenol oxidases
            → tissue turn brown/black

· Phenolic products inhibit enzyme activities and may kill the explants

Methods to overcome browning:
  Adding antioxidants [ascorbic acid, citric acid, PVP (polyvinylpyrrolidone), dithiothreitol], activated charcoal or presoaking explants in antioxidant Incubating the initial period of culturing in reduced light/darkness Frequently transfer into fresh medium.

Nutrition medium

·  When an explant is isolated, it is no longer able to receive nutrients or hormones from the plant, and these must be provided to allow growth in vitro.
·  The composition of the nutrient medium is for the most part  similar, although the exact components and quantities will    vary for different species and purpose of culture.
·   Types and amounts of hormones vary greatly.
·  In addition, the culture must be provided with the ability to excrete the waste products of cell metabolism.
·  This is accomplished by culturing on or in a defined culture  medium which is periodically replenished.
·  A nutrient medium is defined by its mineral salt composition, carbon source, vitamins, plant growth regulators and other organic supplements.

· pH determines many important aspects of the structure and  activity of biological macromolecules. Optimum pH of 5.0-  6.0 tends to fall during autoclaving and growth.

Mineral salt

§ NH4NO3 Ammonium nitrate
§ KNO3 Potassium nitrate
§ CaCl2 -2 H2O Calcium chloride (Anhydrous)
§ MgSO4 -7 H2O Magnesium sulfide (Epsom Salts)
§ KH2PO4 Potassium hypophosphate
§ FeNaEDTA Fe/Na ethylene-diamine-tetra acetate
§ H3BO3 Boric Acid
§ MnSO4 - 4 H2O Manganese sulfate
§ ZnSO4 - 7 H2O Zinc sulfate
§ KI Potassium iodide
§ Na2MoO4 - 2 H2O Sodium molybdate
§ CuSO4 - 5 H2O Cupric sulfate
§ CoCl2 - H2O Cobaltous sulfide


Mineral salt composition

v Macroelements: The elements required in concentration > 0.5 mmol/l
v  The essential macroelements: N, K, P, Ca, S, Mg, Cl
v  Microelements: The elements required in conc. < 0.5 mmol/l
v  The essential microelements: Fe, Mn, B, Cu, Zn, I, Mo, Co
v  The optimum concentration → maximum growth rate

Carbon sources and vitamins

·  Sucrose or glucose (sometimes fructose), concentration 2-5%
·  Most media contain myo-inositol, which improves cell growth
·  An absolute requirement for vitamin B1 (thiamine)
· Growth is also improved by the addition of nicotinic acid and vitamin B6 (pyridoxine)
· Some media contain pantothenic acid, biotin, folic acid, p-amino benzoic acid, choline chloride, riboflavine and ascorbic acid (C-vitamin)

Plant growth regulators

Auxins:   
· Induces cell division, cell elongation, swelling of tissues, formation of callus, formation of  adventitious roots.
·  Inhibits adventitious and axillary shoot formation
                - 2,4-D, NAA, IAA, IBA, pCPA…

Cytokinins:
·  Shoot induction, cell division
·  BAP, Kinetin, zeatin, 2iP…

Gibberellins:
·  Plant regeneration, elongation of internodes
        GA3…

Abscisic acid:
·  Induction of embryogenesis
           - ABA

Organic supplements

· N in the form of amino acids (glutamine, asparagine) and nucleotides (adenine)
· Organic acids: TCA cycle acids (citrate, malate, succinate, fumarate), pyruvate
· Complex substances: yeast extract, malt extract, coconut milk, protein hydrolysate
·  Activated charcoal is used where phenol-like compounds are a problem, absorbing  toxic pigments and stabilizing pH. Also, to prevent oxidation of phenols PVP          (polyvinylpyrrolidone), citric acid, ascorbic acid, thiourea and L-cysteine are used.

Different Techniques of Plant Tissue Culture:

• Callus and Cell culture
• Somatic embryogenesis
• Haploid culture
• Protoplast culture
• Micro propagation
• Organogenesis
• Soma clonal variation
• In vitro Mutagenesis


This includes:

ØCell culture
ØTissue culture
ØOrgan culture

Cellular totipotency and plant regeneration

§ The capacity of a cell (or a group of cells) to give rise to an entire organism.
§ Unlike an animal cell, a plant cell, even one that highly maturated and differentiated, retains the ability to change a meristematic state and differentiate into a whole plant if it has retained an intact membrane system and a viable nucleus.
§ An excised piece of differentiated tissue or organ (Explant) → dedifferentiation → callus (heterogenous) → re differentiation (whole plant) = cellular totipotency.
§  Two hormones affect explants’ differentiation:
§  ↑ Auxin ↓Cytokinin = Root development
§  ↑ Cytokinin ↓Auxin = Shoot development
§  Auxin = Cytokinin = Callus development

Callus culture

§ Un-differentiated mass of cells.
§ Callus is formed at the peripheral surfaces as a result of wounding and hormones.

§ Genotype, composition of nutrient medium, and physical  growth factors are important for callus formation.

Organogenesis

§  Process of differentiation by which plant organs are formed (roots, shoot, buds, stem etc.).

§  Plant development through organogenesis is the formation of organs either de novo (from callus) or adventitious (from the explants) in origin.

Why we need plant tissue culture?

§  Create a large number of clones
§  New genes can be introduced in to the plants which boost crop yield resistance to pests and infections
§ Time required is much shortened & Decrease the amount of space required for field trials
§  Rapid propagation is possible.
§  Overcomes  seasonal restrictions for seed germination
§  Preserve  pollen and cell collections
§  Helps to eliminate plant diseases
§  Reduces  the  storage  space
§  The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits
   E.g.: Tea crop plantation
                    Rubber crop plantation
§  The production of plants from seeds that other wise have very low chances of germinating.
      E.g. : Orchids, Nepenthes

§  If there is plant with partially infected tissue, it is possible to  produce a new plant without infection.    


Applications

· Widely used in forestry
· Pharmaceuticals
          e.g.Ginseng and taxol
· Selection  of crops with advantageous characters e.g. herbicide resistance/tolerance
· To cross distantly related species
· Production of dihaploids from haploids
· Produce large numbers of identical individuals

Basic requirements of a tissue culture laboratory

ü  Preparation area
ü  Transfer area
ü  Culture area
ü  Acclimatization area


Preparation area

ü  Running water facility
ü  Drainage system
ü  Equipments for sterilization (Autoclave etc.)
ü  Distilled water supply
ü  Necessary chemicals and glass-ware

Transfer area

ü  Laminar air flow bench
ü  Disinfectants
ü  Other sanitary aids (glows, masks etc.)

Culture area

ü  Incubators
ü  Shakers
ü  Controlled light, temperature RH etc.


Acclimatization area

ü  Facility to provide different shade levels