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Pharmaceutical Biotechnology

By: Pharma Tips | Views: 8156 | Date: 28-Jun-2010

“Biotechnology” is a fast emerging thrust area with unprecedented opportunity for understanding of fundamental life processes as well as physical well being & improvement in standard of living.

Pharmaceutical Biotechnology

         
                             

 1. INTRODUCTION

“Biotechnology” is a fast emerging thrust area with unprecedented opportunity for understanding of fundamental life processes as well as physical well being & improvement in standard of living. Biotechnology is the integrated use of biochemistry, microbiology and chemical engineering in order to achieve the technological applications of the capacities of microbes and cultured cells.      Biotechnology is the application of the basic concepts of molecular biology to produce useful products for pharmaceutical, veterinary, clinical biochemistry and agricultural uses.
The British biotechnologists define biotechnology as “the application of biological organisms, systems, or processes to manufacturing and service industries”.
European Federation of Biotechnology defines the term biotechnology as “the integration of natural sciences in order to achieve the applications of organisms, cells, parts thereof and molecular analogues for products and services.”
The development of recombinant DNA technology during 1970s laid a foundation for the development of modern biotechnology. Plants are the sources for phytopharmaceuticals, flavors, fragrances, colours, pesticides and food. The availability of these plants became scarce because of ruthless exploitation and depletion of forest. Many of the pharmaceuticals are chemically complex in nature and there is no economically viable method of synthesis. The alternatives for the production of phytopharmaceuticals and allied products are met through the plant tissue culture. Thus biotechnology requires the knowledge of chemical engineering, life sciences and pharmaceutical sciences. Medicinal plant biotechnology has been successfully employed for the production of phytopharmaceuticals, perfumes, colours, flavours and biopesticides. Medicinal plants are the most important source of life saving drug for the majority of world’s population. The biotechnological tools are important to select, multiply and conserve the critical genotypes of medicinal plants. In-vitro regeneration holds tremendous potential for the production of high quality plant based medicine. Cryopreservation is long term conservation method in liquid nitrogen and provides an opportunity for the conservation of endangered medicinal plants. In-vitro production of secondary metabolites in plant cell suspension cultures has been reported from various medicinal plants.                                                                                                                             
Bioreactors are the key step towards commercial production of secondary metabolites by plant biotechnology. Genetic transformation may be a powerful tool for enhancing the productivity of novel secondary metabolites especially by agrobacterium rhizogenes induced hairy roots. 
           Most crops developed through biotechnology that are on the market today provide farmers with increased convenience and product quality while requiring fewer chemical inputs. According to the USFDA economic research service, herbicide and insect resistant biotech varities accounted for about 85% of soybean acreage &45%of corn acreages in 2003. Plant breeders are concentrating on enhancing grains or protein sources to produce feed stuffs that will improve feed utilization, performance, product quality, health of livestock while reducing production costs and environmental impacts.(2)

2.LABORATORY FACILITIES REQUIRED(2)
 
 
3. HISTORY OF PLANT CELL CULTURES
            The technique of plant tissue and cell culture has evolved over several decades.this technique combined with recent  advances in developmental,  cellular,  molecular,  genetics,  metabolic engineering,  genetic transformation and using conventional plant breeding have turned plant biotechnology into an exciting research field with a significant impact on pharmaceutical industries,  agriculture, horticulture and forestry.

Gottlieb haberlandt accomplished the first successful plant tissue culture at the turn of the 20th century when he reported the culture of leaf mesophyll tissue and hair cells.(22)
            Schwan (1839) expressed the view that each living cell of multicellular organism should be capable of independent development if provided with proper external conditions(22). More ever, Haberlandt  pressed the view that each living cell of multicellular organism should be capable of independent development if provided with proper external conditions(22). Hannig (1904) cultured nearly matured embryo excised from seeds of several species of crucifers.(23). More ever, haberlandts lack of success was not invain because one of his students, Kotte (1922) from Germany reported the growth of isolated root tips on a medium consisting of inorganic salts(19).At the same time Robbins (1922) reported the similar success with root and stem tips and white (1934) reported that,  not only could be cultured tomato root tips to grow, but they could be repeatedly sub cultured to fresh medium of inorganic salts supplemented with yeast extract,  which is a good source of vitamin B. White (1939) reported the growth promoting effects of thiamine isolated from tomato root tips(13).Went and Thiemann (1937) discovered the Indole Acetic Acid (IAA) , a natural auxin(10). Duhamet (1939) reported the stimulation of growth of excised roots by IAA(14). 
            The avenue was now open for rapid progress in the successful culture of plant tissue cultures during 1930 with refined media,  La Rue (1936) achieved better success at culturing embryo compared to Haning (1904) Johannes Van Overbeek and his co workers (1942) reported that they were able to obtain seedlings from heart shaped embryos by enriching culture media with coconut milk in addition to the usual salts, vitamins and other nutrients(23). At the same time, Panchanan Maheshwari and co-workers from India were very active in angiosperm embryology research. Braun (1943) reported tumour induction related crown gall disease. Folke Skoog (1944) described the organ formation in cultured tissue and organs of tobacco.(19). Camus (1943) from Europe was the first to report grafting experiments in plant tissue cultures.haberlandt and his associates (1946) made improvements on the medium for the growth of tobacco and sunflower tissue and Morel (1948) was well into applying tissue technique to study of parasites associated with plant tissues(19). Street (1950) and his associates began a series of extensive studies on the nutrition of excised tomato root tips. Skoog and coworkers (1951) worked on investigations on the nutritional requirements of tobacco cells and they discovered a compound (kinetin) which promotes cells division(19). Stove and Yamaki (1957) discovered gibberellins another plant growth regulator(31). Crocker et al (1935) were the first to propose that ethylene was involved in fruit ripening.(10)    
           Murashige and Skoog (1962) developed a culture medium for the rapid growth of tobacco callus.(19) Morel (1965) reported the micropropagation of orchids.(19) Guha and Maheshwari (1966) reported the first successful culturing of haploid cells of datura.Cocking (1960) isolated the plant protoplast and began to be cultured in the media(16). This led to somatic hybridization. In early 1970’s discovery of endonuclease enzymes, which led to rapid development of gene transformation.the prospects of success with the genetics of plants have created considerable public interest. Melchers, Sacristan and Holder (1978) produced somatic hybrid plants from fusion of potato and tomato protoplasts.Several research  groups have produced transformed tobacco plants following single cell transformation (i.e. gene insertion, Chilton 1983)(13). The use of agrobacterium mediated plant transformation, and the use of the so called gene gun to shoot DNA into plant cells,  has led to the development of novel plants.The Calgene of UA is the first tocommerceialize the technique of production of transformed plants(19).


4. NUTRITIONAL REQUIREMENTS OF PLANT  CELL CULTURES. 

Introduction
          There are three essential sources of nutrition for plants growing in nature. The mineral nutrients are obtained, along with water, from the root system .atmospheric carbon dioxide is used in the process of photosynthesis to provide carbon as a source of basic energy. Lastly,   the plant, particularly its meristematic regions and young organs such as leaves, using fixed carbon and minerals, synthesizes all of the vitamins and various plant growth substances that are critical and essential for normal growth and development of the plant. 

          The requirements of plant tissues grown in vitro are similar in general to those of infact plants growing in nature .the nature of explant and the composition of the nutrient medium  generally determine the successful establishment and growth of plant cells in vitro. In the first culturing attempts, either media that were known from nutrition experiments with intact plants (knoop solution), or media consisting of juices and extracts of biological origin were used.

Today, the only that is still commonly used is coconut milk, however only for monocotyledonous cultures. Mainly media of purely chemical composition are used. Media containing nutrients of plant origin that are chemically not precisely characterized are called complex or highly enriched media ,while those containing exclusively chemically defined compounds are called synthetic or regular media. To maintain the vital functions of a culture, the basic medium consisting of inorganic salts,organic compounds (amino acids, vitamins),growth regulators (phytohormones) and carbon sources recognized as essential.(2)

Inorganic Components

           Basic media are solutions salts in differents concentrations, called macro and micro nutrients. Cultured plant tissues required a continuous supply of certain inorganic chemicals. The macro nutrients are the compounds containing N, S, P, K, Mg, Cl  and Na added in concentrations of more than 30 ppm.in contrasts the elements added in the concentrations of less called micronutrients. The micronutrients are necessary as cofactor or enzyme synthesis e.g. nickel is essential for urease synthesis. 

Macro Elements
Sulfur
           It is primarily supplied as sulphate. Usually it is utilized for protein synthesis via sulphate respiration as soluble cysteine (99.9%) and a smaller proportion as soluble methionine .the sulphate supply is directly incorporated only beneath a minimal concentration threshold. In a medium lacking sulfur or in the presence of growth limiting factors, e.g. the nonprotein Amino acid djenkolic acid (H-COOH (NH2) -CH2-s-CH2-s-CH2-CCH(NH2) -COOH), this becomes a apparent due to a five to ten fold increase in the level of ATP-dependent sulphurylase .The sulphur requirements of a culture vary depending on the object (0.5 to 10 mm) .Inorganic sulphur may be replaced by organically bound sulphur (dl-cysteine,  dl-methionine,  dl-homocysteine and glutathione) .

Phosphorus
It is commonly added as phosphate at concentrations of 1.1-1.25mm. Due to rapid uptake and interactions with other components (Fe, k, sucrose), deficiencies may rapidly arise in a medium. In addition, its uptake is influenced by the supply of other elements. For example, boron deficiency induces in daucus carota cultures a reduction in the phosphorus uptake capacity.
Nitrogen
            Most standard media offer nitrogen NH4+ and NO3-.Individual cultures (cannabis sativa, ipomoea, daucus carota) prefer NH4+ under certain conditions. Utilization of NO 3- requires functioning nitrate reductase, the presence of which has by now been described in numerous callus and suspension culture.(2)

Magnesium, Potassium, Calcium
These cations play an essential role in cell metabolism. For e.g., Mg2+ is one of the essential  factors in translation. It acts as a cofactor (e.g. Glutamine synthase, gs) and activator of various enzymes. Therefore, not least in photoautotrophic cultures,  it is of central significance+,  and especially Ca+2, inhibit enzymes such as the glycolysis enzyme pyruvate kinase, while others requires Ca+2to maintain their activity (NAD-kinase, Protein-kinase,  α-amylase) or stability (α-amylase). The  Ca+2 triggered binding of pectic acid, polyerization product of galacturonic acid, to calcium pectate is an elementary step in cell wall formation. Ca+2 are also required for deposition of phospholipids and proteins on within plasma membranes. Its importance is further demonstrated by the efforts of cells to maintain their intracellular concentration 10-6 to 10-8 mm even against a concentration gradient using specific Ca+2  pumps and Ca+2 binding proteins (calmodulin) located in the cytoplasm and/or individuals organelles. The concentration increase to a value of 10-5mm  induced by the sesquiterpenoid phytohormone  abscisic acid (ABA) , an apo-carotenoid and by light is only  temporary  and  is the basis of its signaling  effect in its function  as a second  messenger. In Nicotiana tabacum culture deficiency in nitrate reductase, an increased level of Ca+2 induces increased ammonium utilization.  Chlorine plays a role by binding to positively charged histidine residues of proteins like the enzymes of the photosystem 2 and atpases of the tonoplast and by influencing osmoregulation.(2)

Micronutrients
            The Fe, Mn, Zn, Cu, Mo,  B,  Co,  and Ni act as cofactors and as inducers of enzyme synthesis,  as for example nickel in urease synthesis in tobacco,  rice and soybean cell suspension cultures. Boron is essential for membrane function, permeability and integrity. Therefore, membrane fixed processes like ATP-ase; membrane potential and iron flow and phytohormone metabolism are influenced. Lack of iron results in increased contents of DNA and free amino acids, as well as a reduced RNA content. In order to maintain a minimum supply of Fe it is therefore usually added in complexes with EDTA or sequestrin. This also facilitates uptake over a broad pH range, which varies depending on the content of phosphate, NO3- and NH4+ in the medium. Although iodine in the form of KI is a constituent of several media, the necessity of this element remains questionable. A trace of cobalt is found in several media and yet this element is not known to have any function in higher plants.(2)

Organic   Components

Amino acids
           Amino acids are added for substitution or augmentation of the nitrogen supply. It is to be noted that threonine, glycine and valine reduce ammonium utilization by inactivating glutamate synthase located in chlorophasts and cytoplasm. Arginine is usually able to compensate this inactivation.(2)


Vitamins
           Plant cells are usually autotrophic with respect to vitamins. However, in most cases, the amount of vitamins synthesized even in photosynthetically active cells and tissues is insufficient to guarantee a sufficient supply. Thiamine (vitamin B1) may be the only essential vitamin for nearly all plant tissue cultures, whereas nicotinic acid (Niacin) and Pyridoxine (vitamin B6) may stimulate growth. Thiamine is added as thiamine hydrochloride in amounts ranging from 0.1-10mg/l.
           Some other vitamins that have been used in tissue culture media include p-amino benzoic acid (PABA), ascorbic acid (vitamin C), Tocopherol (vitamin E),  Biotin (vitamin H) Choline chloride,  Cyanocobalamine (vitamin b12) ,  Folic acid,  Riboflavin (vitamin B2) and Calcium pantothenate.

Carbon source
All plant tissue culture media requires the presence of a carbon source and is added in the form of carbohydrates. Sucrose is usually added in concentration of 20-30g/l. often, myoinositol is also used. Occasionally less common carbon sources such as lactose, galactose, glycerin and unrefined natural carbon sources like coffea Arabica and daucus carota are also employed. The various carbon sources used in tissue culture medium are pentoses, uronic acid, molasses, whey, potato starch, grain starch.


5. TECHNIQUES OF BIOTECHNOLOGY FOR MEDICINAL PLANTS

1. Biotransformation Using Plant Cell Culture.

Introduction
Biotransformation is a process through which the functional groups of organic compounds are modified by living cells. This process can be done by using micro organism or plant cell suspension, hairy root culture and immobilized cell. Microbial systems provide a vast range of advantages for biotransformation process. The number and diversity of microbial species with their associated wide range of potential for enzymatic catalysis ,  the relative ease with which they can be  grown in large volumes ,  and high growth  and metabolism are factors which shows microbes are efficient  agents for biotransformation reactions . This potential of microorganism was exploited by human beings long ago. For  e.g.  Acidification of wine into vinegar in 6th  millennium BC.

In the past two decades significant achievements were accomplished in the field of plant biotechnology. There are hundreds of plantpecies in culture in different centers of the world especially Japan, Germany, USA and India. The cell cultures of Catharanthus roseus have been extensively studied in terms of their growth, primary and secondary metabolism the potential of plant tissue cultures for biotransformation process was realized soon after their establishment, and they are used as tools for biosynthetic studies.

Because of factors such as difficulty of maintaining culture sterility, slow growth rate etc. plant cell cultures cannot compete with microbial system for same biotransformation reaction. However, the plants are genetically very diverse and possess a rich repetory of enzymes. The industrial utility of biotransformation using plant cell culture system will be possible if the biotransformation reaction is unique to plant cells and the product has a high market value e.g. paclitaxel. 
            A major disadvantage of plant tissue culture compared with the whole plant in connection with secondary metabolism is that the biosynthetic potential of the species in question is very often not expressed in culture.  
Conditions For Biotransformation 
            The substrate of biotransformation reaction must be easily assimilated by the cell and reach the appropriate cellular compartment or organelle without significant degradation.

1. The substance must be non-toxic to the cell culture.
2. The rate of product formation must significantly exceed the rate of its further metabolism.
The advantage of biotransformation reactions by plant cells and microbial system over non-biochemical reaction system include.
1. Selectivity,  stereo-selectivity,  regio-selectivity
2. Mild reaction conditions
3. Environmentally friendly; avoiding extremes of PH  and metal  catalysts
4. Specificity; reaction on particular molecules
5. Wide range of catalytic activities available

              Biotransformation by plant cell cultures yield a wide range of reactions, such as Glycosylation,  Glucosylesterification,  Hydroxylation,  Oxido-reductions between alcohols and caronyl compounds,  Reduction of carbon double bonds,  Hydrolysis, Epoxidation, Isomerization, Methylation, Demethylation and Dehydrogenation etc.

        Glucose conjugations of exogenously added substances are considered tobe detoxificaton reactions. The terpenoids and cholesterol (hydrophobic) may disturb the membranes of cells and organelles, when the molecules are taken into the cells, or phenolics may cause generation of active oxygen in the cell. However if the substrate are non-toxic to cells,  the cells may not respond to them for e.g. Glycyrrhiza  cultured cells are sensitive to papaverine above 250mg and yielded several biotransformation products. Even normal metabolites of plant cells can be toxic for plants if these are supplied exogenously. For e.g. Cinnamic acid is usually metabolized to coumarin, flavone, and lignin and related compounds in plant cells, however, even cinnamic acid when added exogenously, was toxic at 100mg/l to cell cultures of several species. The reaction may depend on the concentration cinnamic acid added; it may be metabolized normally when supplied exogenously at a lower concentration.(2)

6. FACTORS INFLUENCING BIOTRANSFORMATIONS.

1. Improvements cell viability
            Many substrates are harmful to cultured cells. So it is necessary to decrease the toxicity in order to increase the yield of the products.
            Yokoyama et al. (1990) proposed that sugar could increase cell viability during glycosylation of phenolic compounds. They reported that sucrose (6%w/v) improved the cell viability to a large extent when hydroquinone was biotransformed to arbutin by Catharanthus roseus. Arbutin production was enhanced by 2-3 folds, with the increase being directly dependent on the concentration of sucrose. The exogenously added sucrose was not metabolized and the concentration remained unchanged. The reason for the effect is probably related to the effect of these sugars to act as the scavenger of hydroxyl radicals presumed to be generated on the surface or within the cell. A sugar is known to be specific scavengers of hydroxyl radical and not reacts with superoxide. These assumption leads to the conclusion that antioxidants can improve cell viability and increase the product formation in the biotransformation of phenolics. Yokoyama (1991) reported that antioxidants such as Gallic acid, Ascorbic acid, Cystein and Tannins at 200mg/l could increase the production of arbutin when hydroquinone was added to the cell cultures of Chatharanthus roseus.

2. Selection of Plant Species
           The capacity for biotransformation is diverse among plant species. Tabatas et al. (1988) reported that among seven species of plant cell cultures, the datura had only capacity to biotransform coumarins, flavonoids, anthraquinones and phenolic acids. Datura had the greatest capability to glycosylate coumarins, where as Mallotas japonica did not biotransform coumarins. Different biotransformation can be found among strains of the same species in culture. Courtois et al (1988) showed 3.5  fold difference in the biotransformation rate of tryptamine to serotonin by Peganum harmala cell cultures.
      
            There are several reports on effect of culture age on glucosylation biotransformation. The culture age yielding glucosylation is variously reported. The lag phase or exponential phase,  when pentachlorophenol was glucosylated using soybean cell cultures or late exponential phase when umbelliferone was glucosylated by Datura innoxia or exponential phase when salicyl alcohol was glucosylated by gardenia jasminoide. However these results do not directly reflect the biotransformation ability of the cells.(15)

3. Immobilized Plant Cells
            Immobilization of plant cells has some distinct advantages e.g. reuse of the expensive biocatalyst, continuous process and process control is simplified. polyurethane foam immobilized cells of Pyrethrum somniferum biotransformed codeinone to codeine with increased efficiency (57-79%) as compared to suspension cultures. Using alginate immobilized cells of the same species, the biotransformation efficiency was 70.4% compared with cell cultures of 60.8%. It was interesting to note that 88% of the converted codeine was excreted into the medium in both experiments. It was observed that the immobilization does not effect rather decreases biotransformation ability. 

4. Root Culture
            Cell suspension cultures are generally believed to have excellent biotransformation capacity, especially for glucosylation. However, furuya et al (1989) has found that the root cultures showed higher glycosylation activity than cell culture. Panax ginseng root cultures are able to covert (R,S) -2 phenyl propionic acid into four kinds of glycosyl conjugates where as the cell cultures biotransformed 2-phenyl propionic acid into only (R,S) -2-phenyl propionyl-β-d-glucopyranoside.(16) 


7. IMMOBILIZATION OF PLANT CELLS

Introduction 
Plant cell culture has been for sometime considered as an alternative method for the production of flavours, colors and pharmaceuticals to their extraction from plants. Characteristics of plant cell cultures, such as slow growth, the compounds produced should be of high value ($ 500-1000kg-1) and low volume. One of the major limiting factors in the development of a commercial production system using plant cell culture has been the production cost of phytopharmaceuticals. The use of high biomass levels for extended periods would be one method of increasing productivity and hence reducing the costs. This can be achieved by the immobilization of plant cells. 
           The immobilization of enzymes and cells has received increasing attention, and used to produce amino acids and carbohydrates. The immobilization of microbial cells is not new,  it has known for sometime that cells will adhere to many surface in nature i.e. polymers.
           Immobilization is the newest culture technology of plant cell, and considered as to be the most “natural”. It has been defined as a technique, which confines to a catalytically active enzyme or to a cell within a reactor system and prevents its entry into the mobile phase, which carries the substrate and product. The first successful immobilization of plant cells was reported by Brodellus et al. (1979) and they entrapped Catharathus roseus and daucus carota cells in alginate beds. Following success with enzymatic and microbial process, immobilization has been suggested as a strategy to enhance the overall productivity of secondary metabolite in plant cell culture. The ability to immobilize plant cells has been reported for a large number of plant cells and protoplasts by using a variety of polymers. Immobilization of plant cells has been used for a wide range of reactions, which can be divided into three groups. (1) Biotransformation or bioconversion, (2) synthesis from precursors and (3) the De- Novo synthesis of compounds.(16)


Advantages of Plant Cell Immobilization:
 
           Retention of biomass enables its continuous reutilization as a production system, a definite advantage with slow growing plant cells e.g. Papaver somniferum have remained stable and active for up to six months.
          High biomass levels: The immobilization of cells allows the use of a higher biomass level compared to cell suspension culture,  because of the limitation of mass transfer and settling,  e.g. bead densities of 110 g dry weight/L have been obtained with calcium alginate  entrapped cells when 30 g dry weight/L in suspension cultures. The high cell density allows a reduction in contact in packed bed catalyst leading to an increased volumetric productivity. 
          Separation of cells from medium: the immobilization separates cells from medium and the product is extra cellular, which will simplify down stream processing  compared to extract from tissue. 
          Continuous process: Immobilization allows a continuous process, which increase volumetric productivity and allows the removal of metabolic inhibitors.
          Decoupling of growth and product formation: Immobilization is compatible with non-growth associated product formation.
          Reduces problems such as aggregate, growth and foaming: the immobilization reduces some of the physical problems associated with the cultivation of plant cells such as the formation of aggregates, and susceptibility to mechanical damage (shear stress) are problems which do not affect immobilized system compared to cell culture.

Disadvantage of Plant Cell Immobilization: 

          Secretion of secondary metabolites requires cellular transport or artificially altered membrane permeability.
          The efficiency of the production process depends on the rate of release of products rather than actual rate of biosynthesis. The immobilization process may reduce biosynthetic capacity. Products must be released from the cell into medium. Release of single cells from cell aggregate may make processing of the product more difficult.  The microenvironment avouring optimal production can be unfavorable for released secondary metabolites and cause their degradation or metabolization.The prerequisites for a successful immobilization of plant cells are as follows:
1. Non-growing cells must produce products.
2. Products must be released from the cell into the medium.

Need for immobilization:
Plant cells are characterized by large size, sensitivity to shear and oxygen and need of a cell to cell contact for metabolic production. The secondary metabolites are triggered by short periods of stress in cultures. Immobilization can overcome many of the limiting factors of suspension cultures with the distinct advantages of easier operation of biocatalyst from the product and also being amenable for biotransformation of low value compounds to high value products.

Different types of immobilization:
1. Direct intracellular binding due to natural affinity (adsorption, adhesion and agglutination).
2.   Covalent coupling on otherwise inert matrices.
3.   Intracellular connection via bi or poly functional reagent (cross-linking).
4. Mixing with suitable materials, changing their consistency with temperature (embedding).
5.  Physical retention within the framework of diverse pore size and permeability (entrapment, micro encapsulation).

Selection of immobilization system:
The choice of a suitable immobilization system is determined by the following requirements.
1. The polymer material used for immobilization must be available in large quantities; itmust be inert, non-toxic and cheap.
2. It must be able to carry large quantities of biomass and its fixing potential must be high.
3. The immobilization process must not diminish enzymatic activity of biological catalyst.
4. Manipulation of the biological catalyst must be as simple as possible (hall et al 1988).

Methods For Immobilization

Gel entrapment by polymerization:
            A monomer or a mixture of monomers is polymerized in the presence of a cell suspension, which is entrapped inside the lattice of the polymer. The most common example is polyacrylamide. The method is based on the free radical polymerization of acrylamide in an aqueous solution. As the linear polymers are soluble in water, they have to be insolubilized with bifunctional compounds such as N, N, - Ethylene bisacrylamide. The free radical polymerization of acrylamide is conducted in an aqueous solution containing the cells and the cross-linking agent. Polymerization is commonly carried out in the absence of oxygen and at lower temperature (100C) to avoid damage to the cell during the operation. An initiator N,N,’ Tetramethylethylene diamine (TEMED) is used. Both the initiation and the cross-linking agents are toxic to the cells and therefore, their viability can be lost e.g. Chatharanthus. roseus and Silybum marianum.

Gel entrapment by ionic net work formation:
In this method, polymerization of polyelectrolyte is achieved by addition of multivalent ions. The most common method is the entrapment in calcium alginate. This is a non-toxic process in which sodium alginate solution containing the cell suspension is dropped into a mixture of counter ion solution such as calcium chloride. A uniform, spherical and highly microporus structure results, which retains the cell.

The method may be inconvenient over long run in media containing calcium chelating agents,  such as phosphates and certain cations such as Mg+2 that may accelerate disruption of gel by solubilizing bound Ca+2.Carrageenan is also a suitable matrix for the immobilization of plant cells. The gels of calcium and potassium carrageenates are very stable at room temperature and PH above 4.5.

Gel entrapment formation by precipitation:
Gels may be formed by precipitation of some natural and synthetic polymers by changing one or more parameters in the solution, such as temperature, salinity or PH of solvent. Several materials can be used for entrapment. The examples include methods involving thermal treatment. Some disruption of viability can occur naturally.

Entrapment in preformed structures
Hollow fiber reactors can be used to immobilize plant cells by entrapment. The cells are placed on the shall side of the reactor and nutrient medium is rapidly re-circulated through the fibers. This may have important applications in large-scale.
In other examples, the cells are added to preform polymerized structures such as polyurethane foam. When cells and suspansion are mixed with these materials, they are rapidly incorporated into the net work and subsequently grown into the cavities of the mesh and are entrapped by physical restriction and attachment to the matrix material. The mechanism of this involvement is at first a mechanical entrapment and later, the fixation of the cells due to mechanisms of adsorption and adhesion or even due to their natural tendency for aggregation. These methods of immobilization have several advantages over other methods in that they are simple, cheap, gentle and rapid and maintain cellular functions.

Surface immobilization:
Surface immobilization may occur on both natural and other matrices. Examples of natural matrices are deeper callus layers and cellulose, while synthetic one includes nets of steel and nylon. For e.g. cells of Solanum aviculare were covalently linked to beds of polyphenylene oxide, which had been achieved using glutaraldehyde. Archambault et al (1986) described the spontaneous and rapid binding of Chatharanthus. roseus cells to a man made material.(16)


Immobilization by embedding:
The temperature dependent solubility of macromolecules like agarose,  agar and carrageenan or the differing solubility of the sodium and calcium salts in the case of alginate are utilized to form polymeric gels or gel combination. Insoluble are formed under cold conditions (Agar) or in aqueous CaCl2 solutions (Alginate). Their structure is non-uniform, with differing pore diameters at the surface and in deeper layers. The size and form of the beds can be determined in part by stirring speed and using alginate, by the viscosity of the solution and dropping aperature

Types of bioreactors used for immobilization of plant cells:
            The following types of reactors are generally used for immobilized plant cell.

Packed bed reactors
In this reactor, cells can be immobilized either on the surface or through the support and fluid containing S flows past the support particles. When the cells are immobilized through the support, the packed bed can accommodate a large number of cells per reactor volume.

Disadvantages:
1.  Low degree of mixing causes difficulties in transfers of oxygen, CO2 etc, when it is necessary.
2. The pressure required to pump fluid through the packed bed is inversely proportional to the support particle size, and thus reduces the energy requirements for flow, large incompressible support particles are needed.
3.  The packed bed reactors are having filters. When particulate matters are included in the media or when the support particles fragment during operation, they will be trapped in the column and block the pathways for fluid flow.


Well mixed reactor
The advantages of a well-mixed reactor for immobilized cells are that gaseous substrates can be directly sparged, the temperature and PH can be carefully controlled, and mass transfer rates can be improved over packed bed reactors. The suspension culture with complete cell recycle is essentially a well-mixed “immobilized” cell reactor.

Disadvantages:
The disadvantages of well-mixed reactor for immobilized cell systems are the possibility of fragmenting the support due to particle collision and shear. Using gentle pneumatic agitation can reduce this problem.

Fluidized bed reactors
Typical fluidized bed reactors utilize the energy of the following fluid (liquid and/or gas) to suspend the particles. Because the energy required for fluidization increases with increased particle size, small immobilized particles are often employed. The mass transfer advantages of these small particles is one of the major benefits of the fluid must often be retained in the reactor for an extended period. This may be incompatible with the requirement that the fluid flow rate be sufficient to suspend the particles. To satisfy these two requirements, large gas volumes can be used to suspend the immobilized cells while maintaining low liquid flow rates, or the liquid can be rapidly recirculated through the bed. Both these conditions lead to a large degree of fluid mixing. Large recirculation rates were in the fluidized bed reactors.

Disadvantages:
The disadvantages of the fluidized bed reactor are that shear and particle collisions may damage the immobilized supports and the complex fluid dynamics of such reactors make scale-up difficult.

Membrane reactors
The most commonly considered membrane reactors are the hollow fiber and spiral wound reactors. In the hollow fiber reactor, the cells are retained either within the tubes or in the outer region. Preliminary studies to evaluate the use of these reactors have been reported by shuler et al., (1983); Jose et al., (1983) and prenosil and Pederson (1983). The spiral wound membrane reactor is essentially a flat plate reactor rolled into a cylindrical shape and although no reports have appeared on the use of spiral wound reactor. Hallsby and shuler (1986) have studied plate reactor with tobacco cells. The studies have described that in addition to the membrane, the thickness of the cell layer is also important. The inner portions of thick cell layers are generally characterized by substrate deficiencies and product accumulation and these conditions may prove beneficial for the morphological and chemical differentiation of plant cells.
Advantages:
The advantages of membrane reactors are the possibilities that these membranes can be reused. Thus, despite the high capital costs, the reusability may make this form of immobilization more economical. Also because plant cells are metabolically less active than microbes, thicker cell layers can be employed. Since reactor inoculation, or cell loading is easier with larger cell layer thicknesses it may be possible to develop plant cell reactors,  which can be easily inoculated.

Application of plant cell immobilization:
Biotransformation:
          Hydroxylation of cardiac glycosides has proved an interesting application of immobilized plant cells. Alfermann et al. (1982) used Digitalis lanata in alginate beds in a process aimed at converting beta- methyldigitoxin into clinically more beta-methyl-digoxin.(15) In batch process, the fluid bathing of the cells was changed every second day over a period of 59 days and resulted in a higher cell productivity than that of a free cell system. Brodelius (1981) successfully used a column of similar beds for upto 70 days.(16) Using alginate immobilized cells of Daucus carota;  jones and Veliky (1981) achieved 5-Hydroxylation of digitoxigenin into periplogenin over a period of 12 days, but noted deterioration in biocatalytic activity due to accumulation of inhibitory levels of toxic substrate in the cells.(16) 

8. MICRO PROPAGATION
Introduction:
Micro propagation is a field dealing with the ability to regenerate plants directly from explants. It is defined as “true-to-type propagation of selected genotypes using in vitro culture techniques”. Unlike animals where differentiation is generally irreversible, in plants (due to an intact membrane system and a variable nucleus) even highly mature and differentiated cells retain the ability to regress to a meristematic state. The phenomenon of a mature cell reverting to the meristematic state and forming undifferentiated callus tissue is termed ‘dedifferentiation’. The degree of regression a cell can undergo would depend on the cytological and physiological state it had reached in situ. The ability of plant cells to be cultured indefinitely on fully defined medium and their capacity to regenerate (cellular totipotency) into the whole plant via organogenesis or embryogenesis (redifferentiation) have helped to study some of the problems haunting plant scientists.
The vegetative method of propagation plants is termed as micropropagation or cloning tissue culture or growing in vitro. Morel (1965) used the technique for the first time for orchids.(26) This propagation is now commonly accepted and adopted widely with lot of significance in commercial horticulture. It has important agro economic applications in medicinal plant preservation, floriculture and forestry.
Hundreds of commercial laboratories in different parts of the world are currently involved in micropropagation   some of them producing over 20 million plants a year.(1)

Advantages:
      The various advantages of micropropagation are:
1. Useful for plants that are difficult to propagate by conventional methods (e.g. plants producing little viable seeds ) 
2. In relatively short time (due to increased multiplication rate ) and space,  a large number of plants that is
(a) Genetically stable and true-to-type progeny (by rapid clonal propagation ) 
(b) Virus-free.
(c) Insect-resistant, disease-resistant, herbicide-resistant. With special phenotypic characteristic (i.e.) changed genotypes (tetraploids, haploids, and hybrids) can be produced from single individual.
1. Conservation of genetic resources of species and threatened medicinal plants (by axillary bud proliferation) 
2. Plant improvement by regeneration technique in conjuction with in vitro cell manipulation and
3. To solve some theoretical problems connected with the pathway of biogenesis of chemical compounds in in plants and the relation between organogenesis and production of metabolites.

Disadvantages:
Though, there are several advantages, there are also certain disadvantages with   
the system. They are:
1. Micropropagation methods through use of tissue culture involve capital intensive expensive materials like autoclave, laminar airflow bench, controlled culture rooms etc.
2. This is a technically skilled work, knowledge about material, techniques and decisions making (during subculture and multiplication of propagule) are required in the personnel.
3. Contamination is a serious threat and cause severe damage to material and adds substantively lot to the cost of production, affects time schedule delivery of the material.
4. Specific conditions of micropropagation, rooting and hardening may be required. Therefore, each material requires separate research methods.
5. Small delicate plantlets are produced, which take longer initial time to grow.
6. Genetic stability is doubtful in certain methods.
7. It is a capital-intensive industry, if plants are produced in small number, they cost too much.
8. Otherwise also cost is a major factor for the production and sale of tissue culture raised plants.

Factors that influence micro propagation:
 The factors that important roles in the degree of success achieved in a given        micropropagation or plant regeneration system include
1. The genotype of the donor, 
2. The physiological conditions of the donor material, 
3. The explants source, 
4. The orientation and size of explants in culture, 
5. The culture medium composition (s), 
6. Interactions of endogenous hormones with exogenously supplied growth regulators, 
7. The incubation conditions (including light quality and Intensity, Temperature,  Relative humidity and Air quality) and
8. The timing of the subculture interval/changes in medium / incubation treatment.
      Cells must be  physiologically receptive to the hormone induction  signals before they can be induced to regenerate a plant or organ .thus ,  the timing of the application of induction signals can be critical . This physiology receptiveness to be induced is termed ‘competence’ (graham and wearing 1984, Christianson and warnic 1987). Komalavalli and Rao (2000) reported that the nature of the explant ,  seedling age ,  medium type, plant growth regulators, complex extracts (casein hydrolysate ,  coconut milk, malt extract and yeast extract ) and antioxidants (activated charcoal ,  ascorbic acid, citric acid and polyvinylepyrrolidone ) markedly influence in- vitro propagation of gymnema sylvestre  . 
The other problems commonly encountered in the micropropagation industry are 
1. Vitrification: in repeated cycles of in-vitro shoot multiplication of culture show water –soaked (almost translucent) leaves and may eventually die.
2. High price of propagated plants: plant propagation by tissue culture techniques is encumbered by the intensive labour requirement for the multiplication  process; thus scaling-up systems and automation of unit operations (i.e.) increased mechanization step or by placing them in ‘low vage ’ countries are necessary to cut down the production coasts (Aitken- Christiie 1991 , Vasil 1991) .
3. Somaclonal variation; it is defined, as the variation, which occurs in culture of cells and tissues that may be either genetic or epigenetic. By selecting somaclonal variants, disease resistant as well as herbicide resistant plants could be achieved. This phenomenon has not yet been used to improve medicinal plants but several examples of changes in the colour of flowers of ornamental plants indicate that secondary metabolism can be affected. The factors that frequency of somaclonal variation among regenerated plants are 
1. Length of time in culture.
2. Culture stresses (Improper media components or mutagens, certain growth regulator treatments, delayed subculture intervals leadings to nutrient stress, or extreme or highly variable incubation conditions) and 
3. Explant source (Non-Meristematic explants that do not orderly mitoses, used in adventitious or de novo regeneration systems).(18)

General technique of micro propagation:

Stage 1:
In stage 1, suitable media (MS/modified MS media), plant growth regulator levels and their combinations are selected in order to promote explant establishment and shoot growth. Physiological stabilization may require (3-24) months and (4-6) sub cultures on stage 1 medium. Failure to do so before transfer to stage 2 medium containing higher cytokinin level (to disrupt apical dominance of shoot tip ) may result in diminished shoot multiplication rates or production of undesirable basal callus and adventitious shoots. In many commercial labs, stabilized cultures verified as having specific pathogen tested and free of cultivable contaminants, are often maintained on media that limit shoot production to maintain genetic stability. These cultures called “mother blocks” serve as sources of shoot tips or nodal segments for initiation of new stage 2 cultures. The following factors may affect successful stage 1 establishment of meristem explants:
Explantation time: (beginning of growing seasons generally gives best results), 
Position of explant on the stem, 
Explant size:
Polyphenol oxidation (tissue/medium browning). Excision of explant promotes release of polyphenol, which stimulates the activity of polyphenol oxidase. Tissue browning can be reduced by the use of liquid medium with frequent transfer, the addition of antioxidants (ascorbic acid, PVP) or culturing in reduced light intensity/darkness.
Cytokinins and/or Auxins are most frequently added to stage 1 media to enhance explant survival and shoot development. BA, NAA and IBA are most widely used plant growth regulators. 2, 4-D is more effective in somatic embryogenesis.

Stage 2:
           This is characterized by repeated enhanced formation of axillary shoots from shoot tips or lateral buds cultured on medium supplemented with a relatively higher cytokinin level to disrupt apical dominance of the shoot tip. Subcultures inoculated with explants that had been shoot apices in the previous subculture often exhibit higher multiplication rates than lateral bud explants. Inverting shoot explants in the medium can double/triple the number of axillary shoots produced on vertically oriented explants per culture period in some species. Selecting only terminal shoots of axillary origin for subculture, instead of shoot bases, decrease the frequency of off-types including the preclinal chimeras. Addition of auxin, often mitigates the inhibitory effect of cytokinin on shoot elongation, (but May from callus) thus increase the number of usable shoots of sufficient length for rooting. Selection of stage 2 cytokinin type and concentration is based on shoot multiplication rate; shoot length, frequency of genetic variation and possibility of adverse carry over effects on the survivability and rooting of plantlets in stage 4. Using liquid shake cultures has the special advantage that the shoots broke apart as they multiplied and the manual cutting of shoot cultures is not required. Media with reduced salt levels are used if necessary. The nature of organogenetic differentiation is determined by the relative concentration of Auxins and Cytokinins.(19)

Stage 3:
            This may involve elongation of shoot prior to rooting (GA3 may be added), rooting of individual shoots/shoot clumps, fulfilling dormancy requirements of storage organs by cold treatment and prehardening cultures to increase survival. Where possible, commercial labs have developed procedures to transfer stage 2 micro cuttings to soil, thus by-passing stage 3 rooting. A low salt medium with optimum auxin concentration is determined based upon percentage of rooting, root number and root length (upto max. 5 mm, approx. 15 days) to prevent root damage during transplanting.(19) 

9. GENE CLONNING AND DNA ANALYSIS IN AGRICULTURE:
1.  Current uses of biotechnology in agriculture 
Some of the most important commercial applications of biotechnology are discussed below.

Engineered Crops:
The most widespread application of genetic engineering in agriculture by far is in engineered crops. Thousands of such products have been field tested and over a dozen have been approved for commercial use. The traits most commonly introduced into crops are herbicide tolerance, insect tolerance, and virus tolerance.

Herbicide Tolerance: 
Herbicide tolerance allows crops to withstand otherwise lethal doses of herbicides, which are chemicals that kill plants. Some herbicides kill virtually all plants and cannot be used on crops. By offering crops tolerant to herbicides, chemical companies can expand the market for their products. Indeed, the major developers of herbicide tolerant plants are companies that sell herbicides. The current set of commercially available herbicide-tolerant crops is tolerant to three herbicides based on three active ingredients: bromoxynil, glyphosate, and glufosinate.

Insect Tolerance:
All of the commercially available insect-tolerant plants contain a version of the toxin Bacillus thuringiensis, which is found in nature in soil bacteria. Bacillus thuringiensis toxins are highly effective for many pest organisms, like beetles and moth larva, but not toxic to mammals and most other nontarget organisms. A major concern among farmers and environmentalists is that wide use of Bacillus thuringiensis crops will lead to the rapid development (over the course of perhaps as few as three to five years) of resistance to the toxin. If resistance develops, the Bacillus thuringiensis toxin will be useless as a pesticide. In this case, the environmental benefits of the product will be short lived.

Loss of Bacillus thuringiensis efficacy will affect those who currently use the engineered Bacillus thuringiensis crops, but also many other farmers who use Bacillus thuringiensis in its natural bacterial form, usually as a spray. These other farmers include those who grow food organically and those who use Bacillus thuringiensis  as part of integrated pest management (IPM) plans. Natural  Bacillus thuringiensis sprays are a valuable mode of pest control for these farmers. Organic farmers and others who rely on Bacillus thuringiensis question whether the companies who sell the Bt crops have the right to use up this resource guided only by commercial calculations. UCS considers  to be a public good that should be reserved for everyone.

Virus Tolerance:
The third major application of biotechnology to crops is virus tolerance. These crops contain a gene taken from a virus. By a process that is not well understood, plants that produce certain viral proteins are able to fend off infections by the viruses from which the proteins were taken. Two virus-tolerant crops are currently approved for commercial use, papaya and squash. The squash, which is resistant to two viruses, is currently off the market. Although it is difficult to get information on why products are not on the market, it is possible that the squash did not perform well enough in the field to capture market share.

Resistance to environmental stress :
In addition to the biological challenges to plant growth and development just described, crops plant must contained with a biotic stresses nature dispenses regularly drought , cold, heat and soils that are too acidic or salty to support plant growth .while plant readers successfully incorporated genetic resistance to biotic stresses into many crop plants through crossbreeding, their success at creating crops resistance to a biotic stresses has been more limited, largely because few crops have close relatives with genes for resistance to these stresses. The crossbreeding limitation posed by reproductive compatibility does not impede crop biotechnology; genes found in any organism can be used to improve crop production. As a result, scientists are making great strides in developing crops that can tolerate difficult growing conditions for example; researchers have genetically modified tomato and canola plants that tolerate salt levels 300% greater than none genetically modified varieties. Other researchers have identified many genes involved in cold, heat and drought tolerance found naturally in some plants and bacteria .scientist in Mexico have produced maize and papaya that are tolerant to the high levels of aluminum that significantly impede crop plant productivity in many developing countries.

Increasing yields:
In addition to increasing crop productivity by using built in protection against diseases, pests , environmental stresses and weeds to minimize losses, scientists use biotechnology to improve crop yields directly. Researchers at Japan’s national institute of agro biological resources added maize photosynthesis genes to rise to increase its efficiency at a converting sunlight to plant starch and increased yields by 30 %. Other scientist are altering plant metabolism by blocking gene action in order to shunt nutrients to certain plant parts. Yields increase as starch accumulates in potato tubers and not leaves, or as oil-seed crops, such as canola, allocate most fatty acid to the seeds.
    
2. Transgenic plants:

A procedure used to make a transgenic plant.(9) (A) Outline of the process. A disc is cut out of a leaf and incubated in culture with Agro bacteria that carry a recombinant plasmid with both a selectable marker and a desired transgene. The wounded cells at the edge of the disc release substances that attract the Agro bacteria and cause them to inject DNA into these cells. Only those plant cells that take up the appropriate DNA and express the selectable marker gene survive to proliferate and form a callus. The manipulation of growth factors supplied to the callus induces it to form shoots that subsequently root and grow into adult plants carrying the transgene. 
(B) The preparation of the recombinant plasmid and its transfer to plant cells. An Agrobacterium plasmid that normally carries the T-DNA sequence is modified by substituting a selectable marker (such as the kanamycin-resistance gene) and a desired transgene between the 25-nucleotide-pair T-DNA repeats. When the Agrobacterium recognizes a plant cell, it efficiently passes a DNA strand that carries these sequences into the plant cell, using the special machinery that normally transfers the plasmid's T-DNA sequence(9).
 
10. ORGANOGENESIS AND REGENERATION IN VITRO:

         Organogenesis refers to the process whereby explants, tissues or cells can be induced to form root/and or shoot and even whole plantlets. In other words, the formation of organs is called organogenesis. It may be categorized as rhizogenesis and caulogenesis. The process of root formation is called rhizogenesis and the process of shoot initiation is known as caulogenesis.          
            The earliest reports on controlled organogenesis in vitro were by white (1939) who obtained shoots on callus of a Nicotiana Hybrid and Nobecourt (1937), who observed root formation in carrot callus. Skoog (1944), showed that auxin could stimulate rooting and inhibit shoot formation.(19) Further studies of Skoog and Miller (1957) established that a balanced combination of auxin and cytokinin controls the root and shoot formation.(19) They also reported that a high ratio of auxin to cytokinin in the medium favoured root formation, a reverse ratio having high cytokinin favours shoot formation and that intermediate ratio promotes callus formation. Thus root and shoot differentiation is a function of quantitative interaction between an auxin and cytokinin. Casein hydrolysate or tyrosine also induces kinetin type bud formation even in the presence of higher levels of   IAA in the medium.(19)
            Light intensity plays important role in organogenesis. High light intensity has been shown to be inhibitory for shoot bud formation in tobacco. The quality of light also influences organogenesis. Blue light promotes shoot bud differentiation in tobacco callus while red light stimulates rooting. In general, maintenance of callus under alternating light and dark period may prove satisfactory for differentiation of shoots. Temperature also affects differentiation. Increase in temperature upto 330C may be associated with increased growth of callus in tobacco while low temperature favor shoot bud differentiation (180C is optimal).(13)
            A medium solidified with agar favors bud formation although there are some reports about the development of leafy shoot buds on cultures grown as suspension.  


As an outcome of this approach, several hundred-plant species have been reported to form shoot and/or root in vitro.
            The process of embryo development is called embryogenesis. Somatic embryogenesis is defined as “a non-sexual developmental process which produces a bipolar embryo from somatic tissue”.The earliest report on controlled somatic embryogenesis in vitro was with carrot reported by reinsert (1958). Somatic embryo can be formed from callus, cell cultures, protoplasts or organized structure such as stem segments or zygotic embryo.(18)

REGENERATION:
It is defined as the tendency shown by a developing organisms to restore any part of it which has been removed or physiologically isolated and thus to produce a complete individual. The regeneration through plant biotechnological methods using organogenesis or embryogenesis has several advantages compared to conventional method of propagation. These are
1.  Efficiency of process (reduction in labour cost and time, the formation of plantlet is fewer steps),
2. The potential for the production of much higher number of plantlets and the morphological and cytological uniformity of the plantlets.
The report on plantlet formation is an orchid through the tissue culture was reported by morel in (1960). They are several plants produced through tissue culture on commercial scale, which includes food crops, vegetables, spices, fruits, medicinal and aromatic plants.(17)

Factors affecting Regeneration:
Regeneration via de novo organogenesis is a complex multi-stage phenomenon. The factors affecting the process of regeneration are:

Sources of Explant:
The factors which influence the response of inoculum in the culture are:
1. Size of the explant
2. The season in which the explant is produced.
3. The physiological age of the organ
4. The organ that is to be served as tissue source
5. The overall quality of the plant from which explants are taken.
Regeneration from any plant part can be achieved proper combination of the factors. The explants like stem segments and apices, leaf species, flower petals, hypocotyls, root segments and seed embryo etc., can give rise to organs and embryo directly or indirectly via callus. Explants consisting or mersitamatic actively dividing cells have been useful in initiating the cultures and subsequent regeneration. Young explants are generally more responsive in producing somatic embryogenesis.

Nutrient media and constituents:
The following medium constituents can influence the regeneration:

(a) Inorganic salts: MS medium is the most widely used salt formation compared to white, Heller, B5 and SH medium. The major difference in composition of these media is in the amount and form of nitrogen, and level of calcium.

(b) Organic substances: sucrose is the best carbon source for regeneration media (2-4%) although sometimes glucose or fructose can also be used. The most commonly used vitamin are inositol, pyridoxine, thiamin and nicotinic acid. All these media contain a balanced combination of an auxin and cytokinin.

There are many aspects of culture environment that can influence growth and organized development.
Those include
1) The ph of the medium,
2) The light quality and quantity,
3) Temperature,
4) Humidity,
5) Presence or absence of agar.

Regulation of Regeneration:
By employing a suitable explant and proper medium, callus or explants can be induced to form organs or embryos. The organogenesis and embryogenesis may occur in the same cultures. The induction of shoot or root is formed depending upon the growth regulators.

Organogenesis:
Evan et. Al. (1984) found that for 75% of the species forming shoots either kinetin or (0.05-46micro M). Auxins  such as NAA/IAA (0.06-27 micro M). Usually a high cytokinin concentration favors shoot formation. 2,4-D promotes cell proliferation and suppress cellular and organ differentiation in dicots.

Embryogenesis:
The process of somatic embryogenesis has two stages
1. Induction of somatic embryogenic competence (achieved through high concentration of auxin)
2.   Development of embryogenic cells into embryos (achieved through auxins). 2, 4-D is the commonly used auxin in somatic embryogenesis, the others such as, dicamba and picloram etc. the commonly used media in somatic embryogenesis is MS medium. Increased concentration of carbohydrates (from 2 to 6%w/v) in the medium caused osmotic stress and it has been observed that it enhances somatic embryogenesis in several species. It has also been observed that a high nitrate to low ammonium nitrogen favors somatic embryogenesis.(18)

11. ISOLATION, CULTURE AND REGENERATION OF PROTOPLAST:

Introduction:
           One of the most striking characteristics of plant cells is the presence of a thick and relatively relatively rigid cell wall. This wall functions as the mechanical support of the cell and in defence against physical damage and attack by pathogens. It may also play more subtle roles in cellure communication. Protoplasts are plant cells with a plasma membrane but without cell wall. Because of this, the protoplasts provide the starting point for many of the techniques of genetic manipulation of plants, in particular the induction of somaclonal variation, somatic hybridization and genetic transformation. E.C. Cocking in 1960 from University of Nottingham, U.K. found that cellulose-degrading enzymes, isolated from wood-rotting fungi, were able to dissolve away the cell wall and yield the fragile but still viable protoplast, a cell surrounded only by its membrane.(11)

Applications of Protoplast Isolation and Culture: 
           Plant cells are normally connected to each via many plasmodesmata in multicellure tissues. Plant protoplast culture provides an excellent system to obtain single cells or higher organism and to study its functioning in a controlled environment. Protoplast isolation provides millions of cells; which is comparable to microbial system and can be used many ways.
1. To develop novel hybrid plant through protoplast fusion.
2. Isolation of mutants developed spontaneously or through mutagens, is easier in single cell derived colony.
3. Single cell cloning can be easily performed with protoplasts.
4. The protoplast in culture can be regenerated into an entire plant.
5. Genetic transformation through DNA or organelles uptake can be achieved with protoplasts.

Isolation of Protoplasts:
         Protoplasts are the living material of the cell where as an isolated protoplast is the cell from which the cell wall is removed. Protoplasts can be isolated from almost all plant i.e., roots, leaves, fruits, tubers, root nodules, endosperm, pollen mother cells, pollens, callus, and suspension culture. Protoplasts are isolated from cells by two methods.
Medicinal Plant Biotechnology
(1) Mechanical
(2) Enzymatic

(1) Mechanical Method:
         The cells were kept in a suitable plasmolyticum and cut with a fine knife. In this process some of the plasmolysed cells were cut only through the cell wall, releasing intact protoplast.
        This method of isolation is suitable for limited variety of higher plant tissues, such as leaf, bulb scale, fruit epidermis, or storage tissues. Disadvantages of the technique are that it yields a very small number of protoplast after a rather tedious procedure.

(2) Enzymatic Method:
Isolation of protoplasts from leaves:
This involves four steps:
1) Sterilization of leaves.
2) Peeling off the lower epidermis.
3) Incubation in enzyme solution and
4) Isolation and cleaning of the protoplasts.

 Healthy leaves are obtained from plants growing under green house or wild source and are surface sterilized by using alcohol (70%v/v) for 1 min. then treating them with sodium hypochlorite (2%) solution for 15-20 min. Then leaves are rinsed 3 times with sterile distilled water and these operations are done under laminar flow. The lower epidermis of the sterilized leaves is carefully peeled off and the stripped leaves are cut into small pieces. From these peeled leaf segments mesophyll protoplasts can be obtained. The protoplasts can be isolated using any of the following methods.

1) Direct (One Step Method): In this method, treatment with macerozyme (or pectinace) and cellulose is done simultaneously. The leaf segments are incubated overnight (14-18 hrs) with enzyme mixture (0.5% macerozyme + 2% cellulose in 13% sorbitol or mannitol at PH 5.4) at 250C and treated gently to liberate the protoplasts. The mixture is filtered with wire gauze to remove leaf debris, transferred to screw cap tubes (13-x 1000 mm) and centrifuged for 1 min. The protoplasts from a pellet and supernatant removed. The process is repeated for 3 times and protoplasts are washed with 13% sorbitol solution, which is later replaced by sucrose (20%) solution and centrifuged for 1 min, the cleaned protoplast, which will float can be pipetted out.
2) Sequential (two step) method: the leaf segments with mixture a (0.5% macerozyme + 0.3% potassium dextran sulphate in 13% mannitol at PH 5.8) are vaccum filtered for 5-7 min, transferred to a water bath at 25.C and subjected to slow shaking. After 20 min, the enzyme mixture is replaced by fresh “Enzyme mixture A” and leaf segments incubated for another one hour. The mixture is filtered using nylon mesh, centrifuged (100g) for 1 min. and washed 3 times with 13% mannitol to get a pure sample of isolated cells. These cells are then, incubated with “Enzyme mixture B” (2% cellulose in a 13% solution of mannitol at pH 5.4) for about 90 min at 30.C. after incubation, the mixture is centrifuged at 100g for 1 min so that protoplasts from a pellet, which is cleaned 3 times to get a cleaned protoplast.

Isolation of Protoplasts from Culture cells:
Actively dividing cells in suspension cultures are proved as most suitable material for isolation of non-germ protoplast in large amount. Protoplasts have been isolated from suspension culture of many plant species like, Rose, Atropa sp., Carrot; Sugarcane etc. protoplasts can be easily isolated from such cultures by treating the filtered suspension with 2-4% Onozuka cellulose in 0.6 M mannitol, for 4-6 hours at 32.C in a gently shaking water bath.(11)

FACTORS AFFECTING YIELD AND VIABILITY OF PROTOPLASTS:
Source of material:
The most convenient source of plant protoplasts is the leaf because it allows the isolation of large number of uniform cells without the necessity of killing the plants. Since the mesophyll cells are loosely arranged, the enzymes have an easy access to the cell wall. When the protoplasts are prepared from leaves the age of the plant and the conditions under which it has been grown may be critical. To achieve maximum control on the growth conditions of source plants, several researchers have used in vitro growing shoots. It is very difficult to isolate protoplasts from leaf cells of cereals and other species. The alternative is the cultured cells. The yield of protoplasts from cultured cells depends on the growth rate and growth phase of the cells. Frequently sub cultured (3 days) suspension cultures, and cells taken from early log phase are most suitable.

Pre-enzyme treatments:
With tissues taken from plants from wild source the first step is to surface sterilize them. Scott et al. (1978) observed that the most effective and efficient ways to surface sterilize cereal leaves was to rinse them in Zephiran (alkyl dimethylbenzyl ammonium chloride) (0.1%)-ethanol (10% solution) for 5 min. to facilitate the penetration of enzyme solution into the intracellular spaces of leaf is to peel the lower epidermis and float the stripped pieces of on the enzyme solution. Agitation of the incubation mixture during the enzyme treatment improves the yields of protoplasts from culture cells.

Enzyme treatment:
The release of protoplasts is depends on the nature and concentration of the enzymes used. The two essential enzymes used for isolation of protoplasts are cellulose and pectinase. The latter degrades the middle lamella and the former is required to digest the cellulytic cell wall. The commercially available enzymes are following:

EnzymeSource
Cellulases
Cellulose onozuka r-10Trichoderma viride
Cellulose onozuka rsT. viride
DriselaseIrpex lacteus
Meicelase p-1T. viride
Hemicellulases
HelicaseHelix pomatia
HemicellulaseAspergillus niger
Hemicellulase h-2125Rhizopus sp.
Rhozyme hp 150Aspergillus niger

Osmoticum:
A fundamental property of isolated protoplasts is their osmotic fragility and hence, the need for a suitable osmotic stabilizer in the enzyme solution, the protoplast-washing medium, and the protoplasts culture. Most commonly used osmotica are sorbitol and mannitol in the range of 450-800 mmol-1. Uchimiya and murashige (1974) reported that for isolating protoplasts from tobacco suspension cultures several soluble carbohydrates, including glucose, fructose, galactose, sorbitol & mannitol, were equally effective. When non-ionic substances are calcium chloride (50-100 Mmol-1), potassium chloride (335Mmol-1) and they improve the stability of the plasma membrane(19).

Viability of the protoplasts:
Viability of the freshly prepared protoplasts can be checked by a number of methods.
(a) Observation of cyclosis or cytoplasmic streaming as indication of active metabolism.
Oxygen uptake measured by an oxygen electrode which indicate respiratory metabolism.
(b)   Photosynthetic activity.
(c)   Exclusion of Evans blue dye by intact membrane 
(d)   Staining with fluoresicene.

Techniques of Protoplast fusion:
The protoplast fusion may be of 3   kinds.
1. Spontaneous fusion
2. Mechanical fusion
3. Induced fusion

1. Spontaneous fusion:
During isolation of protoplasts for culture, when enzymatic degradation of cell wall is affected, some of the adjacent protoplast fuses together forming homokaryons or homokaryocytes, each with 2-40 nuclei. The occurrence of multinucleate fusion bodies is more frequent, when protoplasts are prepared from actively dividing cells. This spontaneous fusion, however, is strictly intraspecific. Young leaves are more likely to undergo this fusion

2.Mechanical fusion:
The giant protoplasts of Acetablularia have been fused mechanically. This kind of fusion is not dependent upon the presence of fusion-inducing agent. However, in this method protoplasts are likely to get injury.

3.Induced fusion:
      So far as somatic hybridization is concerned spontaneous fusion is of no value; it requires the fusion of protoplasts of different origin. To achieve the induce fusion a suitable agent (fusogen) is necessary. the various fusogens used for induced fusion are NaNO3, artificial sea water, lysozyme, mechanically induced adhesion, virus, gelatin, high pH, Polyethylene glycol, Antibodies, plant lectin concanavalin A, polyvinyl alcohol and electric stimulation.

Sodium nitrate treatment:
The technique suffers from a low frequency of heterokaryon formation especially when highly vacuolated mesophyll protoplasts are involved. The method involves the following stages.
(1) The isolated protoplasts are suspended in fusion inducing mixture (5.5% sodium nitrate in 10% sucrose solution) and causes fusion on incubation on water bath at 350C. in order to obtain a high frequency of fused protoplasts, mixture may be centrifuged and the pellet resuspended and incubated for one or more additional cycles.
(2) Finally the mixture is replaced by a liquid medium and the protoplasts in this mixture are incubated again; the cycle may be repeated twice before plating the protoplasts on a solid medium. E.g. interspecific somatic hybrids in the genus Nicotiana(17).
   
Poly ethylene glycol treatment:
Poly ethylene glycol has achieved widespread acceptance as a fusogen of plant protoplasts of the reproducible high frequency heterokaryon formation with low cytotoxicity. Another advantage of PEG-induced fusion is that the formation of a high proportion of binucleate heterokaryons. PEG-induced fusion is a non-specific, e.g. soyabean-tobacco, soyabean-maize, soyabean-barley, animal cells with yeast.
          The freshly isolated protoplasts from the two selected parents are mixed in appropriate proportions and treated with 28-50% PEG (1500-1600 molecular weight) solution for 15-30 min. followed by gradual washing of protoplasts with the culture medium. Kao et al. (1974) reported that eluting PEG with a highly alkaline solution (PH 9-10) containing a high Ca2+ ion (50 mMol calcium chloride) led to higher frequency of fusion than washing with the culture medium. Some modifications are suggested to the PEG method is the addition of Concanavallin A to the PEG solution for the purpose of increasing the incidence of fusion.

Factors affecting protoplast fusion by PEG:
(a) PEG of molecular weight higher than 1000 induces tight adhesion and high frequency fusion of protoplasts.
(b)  Excessive dilution of the enzyme solution leads to poor fusion.
(c)  Protoplast from young leaves and fast growing calli give better fusions.
(d)  Prolonged incubation in PEG solution reduces heterokaryon formations.(11).   
    

12. CRYOPRESERVATION AND GERMPLASM:
Introduction:
In the recent years, with the tremendous increase in the population, pressure on the forest and land resources have increased, which in turn caused decrease in the population of medicinal and aromatic plant species. Even some of the plants species are at the verge of vanishing from the forest. The list of endangered species is growing day by day. The conventional methods of germplasm preservation are prone to possible catastrophic losses because of:
1. Attack by pests and pathogens
2. Climatic disorders
3. Natural disasters and
4. Political and economic causes.
In addition, the seeds of many important medicinal plants lose their viability in a short time under conventional storage system. The conservation of germplasm can be done by two methods.
1. In-situ preservation: preservation of the germplasm in their natural environment by establishing biospheres, national parks etc.
2. Ex-situ preservation: in the form of seed or in vitro cultures.
Seeds from the most common material to conserve plant germplasm, their method have the following disadvantages:
- Some plants do not produce fertile seeds.
- Loss of seed viability
- Seed destruction by pests, etc.
- Poor germination rate.
- This is only useful for seed propagating plants.

In vitro preservation by tissue culture has several advantages over seed preservation.
- Small areas can store large amount of material.
- Protection from environmental methods.(4)

Disadvantages:

 It is a costly process. The purpose of cryopreservation is to retain the valuable cell line for the required purpose the routine techniques of culture maintenance are expensive and time consuming. Typically, a cell suspension culture would need to be transferred every 7-10 days and callus culture every 14-30 days. Furthermore, the maintenance for cultures in the fast growing state bears the risk of possible loss through contamination or equipment failure. Besides these technical aspects, it should be kept in mind that plant cells growing in the dedifferentiated stage are genetically unstable. The factors in influencing conservation by sub culturing is:

-Somaclonal variation
-Mass invasion by microbes
-Destroying the whole parent culture.
-Sub culturing leads to selection for good growth, neglecting productivity. .

Selection of plant material:
Tissues must be selected from healthy plants and in case of in vitro material culture parameters should be optimized before cryopreservation the morphological and physiological conditions of the plant material influence the ability of explants to survive freezing at -1960C. Different types of tissues can be used for cryopreservation such as plant organs (ovules, anther/pollen, embryos and endosperm), plant cells somatic embryos, and protoplast, etc. in general, small, young, richly cytoplasm meristematic cells survive better than the larger, highly vacuolated cells. A number of cell suspensions of medicinal plants have been successfully frozen(4):
  
 
PlantCulturecryoprotectantThawing ( 0C)

Anisodus
AcutangulusCell suspensionDMSO or lactal bumin hydrolysate -196
Atropa
BelladonnaCell suspensionDMSO (5%)  37
A. belladonnaprotoplastsDMSO +mannitol  35
Catharanthus
RoseusCell suspensionDMSO(5%)+IM sorbitol  40
Citrus sp.Ovule/nucellar embryoDMSO-7% +sucrose- 7%  35-40
Cinchona ledgerianaCallusMannitol + DMSORapid thawing

The uniform suspension composed of  small groups of cytoplasmic, meristematic callus are more suitable than highly vacuolated cells which have water content and they should be late lag phase or exponential phase actively growing suspension are able to withstand freezing much better than relatively old culture. A high density to cells per ampoule yield better results.
Callus derived from tropical plants is more resistant to freezing damage. A rapidly growing stage of callus shortly after 1 or 2 weeks of subculture is best for cryopreservation. The old cells at the top of callus and blackened area should be avoided. Cultured cells are not ideal for freezing. Instead, organized structures such as shoot apices, embryos or young plantlets are preferred.

Pregrowth:
Pre-growth involves cold hardening treatments or the application of additives known to enhance plant stress tolerance e.g., abscisic acid, proline, trehalose. Partial tissue dehydration can be achieved by the application of osmotically active compounds. The addition of low concentration of DMSO (1-5%) during pre-growth often improves shoot tip recovery, e.g.:
(1)Catharanthus. roseus cells were precultured in a medium containing 1M sorbitol before freezing.
(2)Digitalis cells were precultured on 6% mannitol medium for 3 days before freezing
(3)Nicotiana sylvestris with 6% sorbitol for 2-5 days before freezing

Cryoprotective Treatments:
There are two potential sources of cell damage during cryopreservation.
(1)Formation of large ice crystals, inside the cells, leading to rupture of organelle and the cell itself.
(2)Intracellular concentration of solutes increases to toxic levels before or during freezing as a result of dehydration(4).

Cryoprotectants are categorized as:
- Penetrating: exert their protective colligative action
- Non-penetrating: effect through osmotic dehydration.

(a) Virtification: Avoidance of ice formation in biological tissues exposed to low and ultra low temperature reduces damage. This can be achieved through vitrification, a process in which ice formaton cannot take place because the aqueous solution is too concentrated to permit ice crystal nucleation. Instead, water solidifies into an amorphous “glassy” state.

(b) Cryoprotective dehydration: if cells are sufficiently dehydrated they may be able to withstand immersion in liquid nitrogen without further application of traditional cryoprotectant mixtures. Dehydration can be achieved by growth in the presence of high concentration of osmotically active compounds (sugars, polyols) and / or air desiccations in a sterile flow cabinet or over silica gel. Dehydration reduces the amount of water available for the ice formation.

(c) Encapsulation and dehydration: this involves the encapsulation of tissues in calcium alginate beads which are pregrown in liquid culture media containing high concentrations of sucrose. The beads are transferred to a sterile airflow in a laminar cabinet and desiccated further. After these treatments the tissues are able to withstand exposure to liquid nitrogen without application of chemical cryoprotectants.

Addition of cryoprotectants controls the appearance of ice crystals in cells and protects these cells from the toxic solution effect. A large number of heterogeneous groups of compounds have been shown to possess cryoprotective properties with different efficiencies.e.g. glycerol, DMSO, etc. cryoprotectants depress both the freezing point and the super cooling point of water, i.e., the temperature at which the homogeneous nucleation of ice occurs thus retarding the growth of ice crystal formation. DMSO has excellent cryoprotective properties and it is very widely employed for cryopreservation of cultured cells.(4)

Cryoprotectants used in cryopreservation:
 
Freezing and storage:
The type of crystal water within stored cells is very important survival of the tissue. Three different types of freezing procedures have been developed(21).

Rapid freezing:
The plant material is placed in vials and plunged into liquid nitrogen and decrease of -300 to -10000C/min or more occurs. The quicker the freezing is done, the smaller the intracellular ice crystals are. A somewhat slower temperature decrease is achieved when the vial containing plant material is put in the atmosphere over liquid nitrogen (-10 to -700C/min). Dry ice instead of nitrogen can also be used in a similar manner. It is also possible that ultra rapid cooling may prevent the growth of intracellular ice crystals by rapidly passing the cells through the temperature zone in which lethal ice crystal growth occurs. This method is technically simple and easy to handle. It has been successfully used for the cryopreservation of shoot tips of potato, strawberry, brassica spieces, and somatic embryos.

Slow freezing
The tissue is slowly frozen with a temperature decrease of 0.1-100C/min from 0oC to -1000C and then transferring to liquid nitrogen. Survival of cells frozen at slow freezing rates of -0.1to-100C/min may involve some beneficial effects of dehydration, which minimizes the amount of water that freezes intracellular. Slow cooling permits the flow of water from the cells to the outside, thereby promoting extra cellular ice formation instead of lethal intracellular freezing. It is generally agreed that upon extra cellular freezing the cytoplasm will be effectively concentrated and plant cells will survive better when adequately dehydrated. This method has been successfully employed for cryopreservation of meristems of peas, potato, cassava and strawberry, etc.

Stepwise freezing:
A slow freezing procedure down to -20 to -400C, a stop for a period of time (approximately 30 min) and then additional rapid freezing to -1960C is done by plunging in liquid nitrogen. A slow freezing procedure initially to -20 to -400C permits protective dehydration of the cells. An additional rapid freezing in liquid nitrogen prevents the growing of big ice crystals in the biochemical important structures(21).

Storage:
the frozen cells/tissues are immediately kept for storage at temperature ranging from -700C to -1960C. A liquid nitrogen refrigerator running at 1500C in the vapor phase or -1960C in the  liquid phase is ideal for this purpose. The temperature should be sufficiently low for long term storage of cells to stop all metabolic activities and prevent biochemical injury. Long term storage is done at -1960C. thus as long as regular supply of liquid nitrogen is ensured in liquid nitrogen refrigerator, it is possible to maintain the frozen material with little further care.

Thawing:
The temperature and rate at which tissues are thawed is dependent on the freezing method. Thawing is particularly critical in vitrified tissues as ice crystallization can occur during re-warming. In conventional freezing methods, thawing is usually achieved by plunging the cryovials into sterile water maintained at 40-450C. once all the ice has melted the samples are removed. Tissues which have been frozen by encapsulation and/or dehydration are frequently thawed at ambient temperatures. Vitrified and excessively dehydrated tissues may require a sequential lowering of media osmoticum during thawing.

Cryopreservation equipment:
Simple freezing chambers utilizing a cooled solvent system may be used. However, a typical, modern programmable freezer comprises a liquid nitrogen cooled sample chamber, to which nitrogen is pumped from an accessory Dewar or storage tank. Cooling rates, holding times, and terminal transfer temperatures are programmed, and chamber and sample temperatures are monitored by thermocouples. Models can be purchased from several companies.

The following equipment is required for cryopreservation.
1. A reliable source of liquid nitrogen 
2. Safety equipment (gloves, apron, face shield, pumps for dispensing liquid nitrogen from a large storage dewars, trolleys for the transport of dewars)
3. Small (1-2 litre) liquid nitrogen resistant dewar(s)
4. Dewar(s) for the routine storage of liquid nitrogen
5. Dewar(s) for the long-term storage of specimens-these may be equipped with an alarm system which is activated if the liquid nitrogen level falls below a critical level
6. Cryovials, straws, boxes, canes, racks
7. A refrigerator (-200 C)
8. A programmable freezer with dewar and pump
9.  A water-bath for thawing at 40-500 C

Cryopreservation of callus and cell suspention:
The cell suspension of Datura stramonium frozen at the rate of 1.C/min in the presence of 7% DMSO gave the best results. Periwinkle (catharanthus roseus) cell cultures subjected to cooling at the rate of 0.50C/min to -400C and then immersed in liquid nitrogen showed viability of about 60% of the controls (Chen et al., 1984). The cell cultures of Digitalis lanata survived upto 50%, and showed stability of biotransformation potential after cryostorage. The capacity to transform a-methyldigitoxin to beta-methyldigoxin remained unchanged. Likewise callus and cell suspension of Anisodus acutangulus retained their biosynthetic ability of hyoscyamine and scopolamine after freezing. The storage at -196.C was better than at -200C, callus cultures gave better results than cell suspensions. Lactalbumin hydrolysate used as cryoprotectant was similar to DMSO, but glycerin was ineffective.
Dioscorea deltoidea and Panax ginseng are two other species of medicinal value for the production of diosgenin and saponin respectively, and which have yielded excellent results. Preculture and hardening of Panax cells in the presence of 7%- 25%sucrose at 2-100C resulted in considerable increase in their survival. As a cryoprotectant, sucrose was best for Panax, whereas 7% DMSO gave optimal results in Dioscorea, and they retained biosynthetic potential for diosgenin, sitosterol, stigma sterol, and other metabolites.
Cinchona callus preculturedon 3 days in a medium containing 5% PVP, and cryoprotected with CB5 +  mannitol + DMSO, frozen slowly in liquid nitrogen vapors, transferred to liquid nitrogen and then rapidly thawed, gave rise to slowly growing calli on agar medium. However, the meristems did not survive after freezing(20).          
  
13.APPLICATION OF BIOTECHNOLOGY:

It is applied successfully in various fields:
1) Pharmaceutical industry
2) Agriculture
   a) Tissue culture and crop improvement
   b) Biological control of insects,pests and pathogens
   c) Biofertilizer and biogas development
3) Reproduction
4) Pollution control
5) Forestry development
7) Fishery development
Agriculture
-Decrease in fertilizer output
-Increase tolerance of plants to environment stress,
-Rapid reforestation and forest regeneration,
-To improve the nutrition quality of plants(1).

Tissue Culture And Crop Improvement:
          Biotechnical international has already constructed a strain of Rhizobium that is improved with respect to its ability to fix nitrogen. Certain genetically engineered organism of the type have been extensively tried out in the fields and cleared by the environmental protection agency in the USAThe phenomenon of induced resistance in plants has an intriguing similarity to immunization by vaccination in mammals. Several approaches may be used to genetically engineer crop plants with greater insect resistance.
          The prospect of utilization of bio-techniques broadly includes manipulation at tissue, cellular and DNA level of plant genetic resources for crop improvement.
Jojoba (simmondsia chinensis), a plant with appreciable non-edible oil, waxes and lubricant contents guayule (parthenium arogentatum) with useful biomass products (i.e. latex) have been propagated successfully through suitable in vitro technique.
Virus infection is a major problem in vegetative propagated plants. 
         This can be overcome by using plants through culture of meristem or shoot apex, which is free from pathogen. This technique has been successfully adopted for commercial production of virus free plants of citrus, sugarcane, cassava etc.
         Regeneration of plants form callus or plant protoplasts can result in recovery of somoclonal variants for useful agronomic traits, such as disease resistance for sugarcane and potato crops.
         Haploids are very important in genetic improvement of crops. Haploid plants obtained form anther cultures have led to development of improved cultivars of rice, wheat and tobacco in Japan and china.
         New species could be developed by somatic cell hybridization. There is already a new fruit in the market in the U.S. called nectarine, which is a hybrid between a plum and a peach(1).

Biological control of insects, pests and pathogens:
Success has been recorded in fighting frost with genetically altered bacteria belonging to the Pseudomonas species. The common pseudomonas contains a protein on their surface that acts as seeds for the formation of ice crystals when the temperature falls. These bacteria love to coat plants and the ice crystals thus formed damage the plant. The gene what has the blueprint of this protein has been snipped out of this bacterium through genetic engineering techniques. Therefore if enough of the bacteria depleted of this gene were made available in the soil, they would compete with the normal but damaging pseudomonil and reduce frost damage.
The pesticides of tomorrow will thus be biology or immunology based for such pesticides would be extremely specific and would destroy only the desired species. Several new microbial insecticides and acaricides have been developed prominent among them are Nosema locustae, a protozoan for grass hopper management, Hirsutella thomsonii, a fungus for mite control and a baculovirus for control of the European pine fly (new diprion sertifer) in Europe, two biotypes of the fungus Viticillium lecanii have been developed for management of white flls and aphids on glass-house crop cultures.
In Canada, the nuclear polyhedrosis virus of red headed pine saw fly is used against forest pest. Most of work now being done is directed on improving the defense mechanism in crops. for instance tri-plasmid of Agrobacterium and biotechnology toxin genes have been transferred to tobacco and tomato plants. Soma clones of crops resistant to Phytopthera alternaria have been isolated.

Use of medicinal plant cell cultures in biotechnology:
Plants have been the traditional source of biopharmaceuticals and use of cell and tissue culture is an alternate approach to obtain these compounds on a commercial scale without destroying the natural resources.
Culture of plant cells in vitro coupled with molecular biology techniques is an alternate source for the production of biopharmaceuticals.eg.Shikonine and Sanguinarine are produced on large scale with the help of biotechnology.
The global turnover of medicinal plants and their extractives is around USD 4 billion.
Some economically important plant based drugs obtained commercially.

CompoundPlant sourceTherapeutic use
Steroidal hormones (95% from Diosgenin)Dioscorea sp.Oral contraceptives, anabolics
Digitalis GlycosidesD.purpurea, D.lanataCardio tonic glycosides
CocaineErythroxylon cocoaLocal anaesthetic
ArtimisinineArtimisia annuaAnti-malarial
PodophyllotoxinPodophyllum paltatumAnti-cancer
  
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