Application of cellular manipulation protoplast isolation culture and fusion Introduction: The development of protoplast systems has increased

the versatility of plants for use in both biochemical and genetic research. They have become indispensable tools in genetic engineering and crop breeding Of all the possible starting points for plant genetic manipulation, only protoplasts offer the opportunity to take advantage of all the technologies now available. Since the first successful isolation of protoplasts by Cocking (1960), substantial progress has been made towards improving the technology. Principle of Protoplast culture The protoplast includes the plasmalemma and everything contained within ie.the entire cell without its inherent cellulosic cell wall. Fusion of protoplast is relatively a new versatile technique to induce or promote genetic recombination in a variety of prokaryotic and eukaryotic cells(Bhojwani S.S. et al 1977). Protoplast can be isolated from varieties of plant tissue. The basic principles of protoplast culture is the aseptic isolation of large number of intact living protoplast removing their cell wall and culture them on the suitable nutrient medium for their requisite- growth and development. In protoplast technology, from any two genotypically difference plant protoplasts are isolated from the somatic cell (diploid) are experimentally fuse to obtain parasexual hybrid protoplast. The hybrid protoplast contains hetroplasoic cytoplasm and fused parental nuclei. The fused protoplast is grown in vitro-with an aim to obtain hybrid plant. So in vitro fusion of plant protoplast derived either from somatic cell of plant is called somatic hybridization. The protoplast culture are produced experimentally by the removal of all wall by either enzymatically or mechanical means from the artificially plasmolysed plant cell. Experimentally produced protoplasts are known as isolated protoplasts. The success of a protoplast culture system primarily lies with consistent yields of a large population of uniform and highly viable protoplasts. Isolation of protoplast Protoplasts may be produced, under aseptic conditions, from a wide range of plant species either directly from the whole plant, or indirectly from in vitro cultured tissues. Isolated plant protoplast are cell from which the wall has been removed either mechanically or enzymatically . Protoplast can be prepared from a variety of tissue but among them mesophyll tissue from a wide range of plant has been proved to be the most ideal source of plant materical for protoplast isolation. Leaves of Nacotiam tabacum is highly standardized material for easy entry in to the art of protoplast isolation and culture.The essential step of the isolation of protoplast is the removal of the cell wall without damaging the cell or protoplast. There are two basic approaches for the enzymatic isolation of protoplasts: (a) the treatment of a plant tissue with a mixture of pectinase and cellulase so as simultaneously to macerate, or separate, cells and degrade their walls (Power & Cocking, 1970); and (b) the sequential (two-step) method involving the production of isolated cells which in turn are converted into protoplasts by a cellulase treatment (Nagata & Takebe, 1970). Since removal of the cell wall results in loss of wall pressure upon the cell, protoplasts are isolated and maintained in hypertonic plasmolytica provided by a balanced inorganic salt medium or monosaccharide sugar solution. Mannitol,

Isolation and Culture of Protoplasts

Enzymes used for breaking of cell walls: For protoplast fusion it is important that the cell wall of plant and microorganisms is degraded .So various enzymes used for this process. cellulase and pectinase or macerozyme acting on plant cell wall. Bacterial cell wall are degraded by the action of lysozyme. Fungal wall degraded by Novozyme -234 which includes glucanase and chitinase. Streptomyces cell wall degraded by action of lysozyme and achromopeptidase (Narayanswamy S 1994) ( Jogdand S.N 2001). Method of Protoplast Fusion Protoplast fusion is a physical phenomenon. During fusion , two or more protoplast come in contact and adhere with on another either spontaneously or in presence of fusion including chemicals. Protoplast fusion can be broadly classified into two categories: Spontaneous fusion: Protoplast during isolation often fuse spontaneously and this phenomenon is called spontaneous fiusion. During the enzyme treatment,protoplast from adjoining cells fuse through their plasmodesmata to form multinucleate protoplasts. Induced fusion: Fusion of freely isolated protoplasts from different sources with the help of fusion inducing chemicals agent s is known as induced fusion. Normally isolated protoplast do not fuse with each other because the surface of isolated protoplast carries negative charges (10mV to -30mV ) around the outside of the plasma membrane. And thus their is a strong tendency in the protoplast to repel each other due to their same charges .So this type of fusion needs a fusion inducing chemicals which actually reduce the electronegativity of the isolated

protoplast and allow them to fuse with each others(Narayanswamy S 1994). The isolated plant protoplasts can be induced to fuse by three ways, (1) Chemical fusion (2) Chemo fusion (3) Electro fusion Mechanical fusion: In this process the isolated protoplast are brought into intimate physical contact mechanically under microscope using micromanipulator or perfusion micropipette. Chemo-fusion Several chemical have been used to induced protoplast fusion. Sodium nitrate(NaNO 3) polyethylene glycol (PEG), calcium iron(Ca2+), polyvinyl alcohol etc. The simplest and least expensive method to achieve protoplast fusion is by chemical method, of which the use of polymer polyethylene glycol (PEG) is the best known. The function of the PEG is the alter the membrane characteristic so that the protoplast become sticky and it the protoplasts are allowed to come into contact they will adhere together and the contents will fuse. Chemofusion is a non specific,inexpensive,can cause massive fusion product,can be cytotoxic and non selecetive and having less fusion frequency. Electro-fusion When protoplast that are in contact are subjected to suitable external electric field, which causes the formation of transient reversible pores in the plasma membranes they can fuse together. Protoplast have a negative surface change that helps repel neighbouring protoplasts. Protoplast can now be fused a large scale by electrical methods using charmer in which the cells are exposed to small electrical current. Recently, mild electrical stimulation is being used to fuse protoplasts. This technique is known as electro-fusion of protoplasts. Two classes capillary microelectrodes are placed in contact with the protoplast. An electrical field of low strength (10Kvm-1) gives rise to dielectrophoretic dipole generation within the protoplast suspension. This lead to pearl chain arrangement of protoplasts. The number of protoplast within the pearl chain depend upon the population density of the protoplast and the distance between the electrodes. When the protoplast are in contact, the plasma membranes break and fuse forming a continuous membrane a round the pearl chains. In compare, the electrical method of fusion is much more production then the chemical method as up to 50% of protoplast may be as heterokaryons. Mechanism of protoplast fusion: The mechanism of protoplast fusion is not fully known .Several explanations have been put forward to understand the mechanism of protoplast fusion. Some are explained here: When the protoplasts are brought into close proximity ,this is followed an induction phase thereby changes induced in electrostatic potential of the membrane results in fusion . After the fusion,The membranes stabilizes and the surface potential returns to their former state. Other literature showed when the protoplasts are closely adhered ,the external fusogens cause disturbance in the intramembranous proteins and glycoproteines.This increases membrane fluidity and creates a region where lipid molecule intermix,allowing coalescence of adjacent membranes.The negative charge carried by protoplast is mainly due to intramembranous phosphate groups .The addition of Ca ++ ions causes reduction in the zeta potential of plasma

membrane and under this situation protoplasts are fused(Peberdy J.F 1980).The highmolecular weight polymer (1000-6000) of PEG acts as a molecular bridges connecting the protoplasts. calcium ions linked the negatively charged PEG and membrane surface .On elution of the PEG ,the surface potential are disturbed ,leading to intramembrane contact and subsequent fusion, Besides this ,the strong affinity of PEG for water may cause local dehydration of the membrane and increase fluidity,thus inducing fusion. Somatic hybridization The results obtained so far from somatic hybridization represent that it is possible to recover fertile and stable amphidiploid somatic hybrids after protoplast fusion. Procedure for successful somatic hybridization is as below: (i) isolation of protoplasts from suitable plants, (ii) mixing of protoplasts in centrifuge tube containing fugigenic chemicals i.e. chemicals promoting protoplast fusion, such as polyethylene glycol (PEG) (20%, W/V), sodium nitrate (NaNO 3), maintenance of high pH 10.5 and temperature 37C (as a result of fusion of protoplasts viable heterokaryons are produced. PEG induces fusion of plant protoplasts and animal cells and produces heterokaryon (Davey et al, 1978), (iii) wall regeneration by heterokaryotic cells, (iv) fusion of nuclei of heterokaryon to produce hybrid cells, (v) plating and production of colonies of hybrid cells, (vi) selection of hybrid, subculture and induction of organogenesis in the hybrid colonies, and (vii) transfer of mature plants from the regenerated callus. Schieder (1982) discussed four major aspects of protoplast fusion as (i) Production of fertile amphidiploid somatic hybrids of sexually incompatible species is achieved. Induced fusion of protoplasts from two genetically different lines of species must results in a variety of homo-as well as heterokaryotic fusion products (heterokaryon or heterokaryocytes). Selection of a few true somatic hybrid colonies from the mixed population of regeneration protoplast is a key step in successful somatic hybridization technique; (ii) production of heterozygous lines within one plant species which normally will be propagated only vegetatively e.g. potato; (iii) he transfer of only a part of genetic information from one species to another using phenomenon of chromosome elimination, and (iv) the transfer of cytoplasmic genetic information from one to a second line or species. It has been possible to transfer useful genes (e:g nif genes, disease resistance genes, rapid growth genes) from one species to another, thereby, to widen the genetic base for plant breeding. Applications Gamborg and Nabors (1987) have described a number of variations in cell fusion as firstly, fusion of two protoplasts each from cells with normal parental ploidy, secondly, fusion of two protoplasts each obtained from haploid cell sources (the fusion of two diploid cells of Solarium sp. would yield a tetraploid which is the normal ploidy for potato, and thirdly, fusion of a protoplast with an enucleated protoplast. The result of this type of fusion would bring about cybrid with a single nucleus of one parent and the respective ploidy. Carlson (1972) succeeded in carrying out protoplast fusion of Nicotiana glauca x N.

langsdorffii. Simlarly, Kuchko (1985) achieved somatic hybrid through inter-specific cross of wild and cultivated variety of potatoes i.e. Solarium tuberosum x S. chacoense. Intergeneric hybrids which may be difficult to achieve by sexual crosses have been obtained through protoplast fusion e.g. tomato + potato, Datura + Atropa, barley+wheat, barley+ rice, wheat+ oat, and sugarcane + sorghum.With regard to inter familial cell hybrids, there have been very few successful experiments to obtain such hybrids, for example, hybrids of Glycine sp + Nicotiana species, (Kao, 1977; Chien et at, 1982). Based on detailed studies on inter familial hybrids, it has been concluded that inter familial hybrids of plant cells are genetically unstable and show species specific elimination/ reconstruction of chromosome belonging to one of the parents; this was usually the parent represented by the mesophyll cells in hybridization process. These hybrids are incapable of regeneration and morphogenesis into plants (Gleba, 1985). Similarly, intertribal hybrids were studied more widely due to their higher genetic stability; the first intertribal cell hybrid was obtained by Gleba and Hoffmann (1978) from callus cells of Arabidopsis thaliana crossed with mesophyll protoplasts of turnip (Brassica compestris). Fusion of protoplasts of potato and tomato, and production of hybrid plant (pomato).

Fusion products the hybrids and cybrids Fusion of cytoplasm of two protoplasts results in coalescence of cytoplasms. The nuclei of two protoplasts may or may not fuse together even after fusion of cytoplasms. The binucleate cells are known as heterokaryon or heterocyte .When nuclei are fused the cells are

known as hybrid or synkaryocyte (Constabel, 1978), and when only cytoplasms fuse and genetic information from one of the two nuclei is lost is known as cybrid i.e. cytoplasmic hybrid or heteroplast (Doods and Roberts, 1985). There are some genetic factors which are carried in cytoplasmic inheritance, instead of nuclear genes, for example, male sterility in some plants. Susceptibility and resistance to some of the pathotoxins and drug are controlled by cytoplasmic genes. Therefore, production of cybrids which contain the mixture of cytoplasms but only one nuclear genome can help in transfer of cyloplasmic genetic information from one plant to another. Thus, informations of cybrid can be applicable in plant breeding experiments. (Vasil, 1982). Hybrid/cybrid production through protoplast fusion.

In China cybrid technology in rice is a great success. Such plants are very useful in producing hybrid seeds without emasulation, However, cybrid technology has successfully been applied to carrot, Brassica sp, Citrus, tobacco and sugar beet (Balasubramanian et al. 1996). Protoplast fusion may be used to produce interspecific or even intergeneric hybrids. Protoplast fusion becomes an important tool of gene manipulation because it breakdown the barriers to genetic exchange imposed by conventional mating systems. Protoplast fusion technique has a great potential for genetic analysis and for strain improvement.It is particularly useful for industrially useful microorganisms(Murlidhar R.V and Panda T. 2000) Inter-specific and inter-generic fusion achievements Cross Oat Brassica sinensis Torrentia fourneri Crossed with Maize B. oleracea T. bailloni

Brassica oleracea B. campestris Datura innoxia Atropa belladonna Nicotiana tabacum N. glutinosa Datura innoxia D. candida Arabidopsis thaliana Brassica campestris Petunia hybrida Vicia faba Biotechnological applications of protoplast fusion Protoplasts contained all the intracellular organelles of cells and form a vital link in transfer of micromolecules between cyto organelles,currently most of the laboratories engaging in fungal genetics are using gene manipulation procedures based on protoplasts. Therefore to further improve the genetic properties of these strains using protoplast fusion are attempt to develop methods for preparation and regeneration of protoplasts. The process involves protoplast mutagenesis,transformation and protoplast fusion ( Evans D.A.1983). The direct bioconversion of cellulosic materials to ethanol by the intergeneric fusants between T.reesei and Saccharomyces cerevesie appears to be are of the best technique for an alternative approaches for ethanol production Also this process is helpful in the production of a complete set of cellulases by the protoplast fusion of T.reesei and A.niger (one produced more amount of endo and exoglucanase and other produced more - glucosidase(Ahmed M and Berkley EI 2006). Advandages By protoplast fusion it is possible to transfer some useful genes such as diesese resistance,nitrogen fixation ,rapid growth rate ,more product formation rate,protein quality,frost hardiness,drought resistance,herbicide resistance ,heat and cold resistance from one species to another. Limitations : Poor regeneration of hybrid plants Non-viability of fused products Not successful in all plants. Production of unfavorable hybrids Lack of an efficient method for selection of hybrids No confirmation of expression of particular trait in somatic hybrids