Protoplasm fusion

Protoplasm fusion develp a hybrid plant

1. Protoplasm fusion Mrs. Praveen Garg VITS College, Satna

2. The protoplasm of a living plant or bacterial cell whose cell wall has been removed. Protoplasts  are the isolated cells whose cell wall is removed and are bounded by plasmalemma .  Protoplasts  can be the cells of plants, fungi or bacteria. These are also called as 'naked' cells.  Protoplasm  is the complex, semifluid , translucent substance that constitutes the living matter of plant and animal cells. Protoplasts are cells which have had their  cell wall  removed, usually by digestion with  enzymes . Cellulase   enzymes  digest the cellulose in plant  cell  walls while pectinase   enzymes  break down the pectin holding cells together. Once the  cell wall  has been removed the resulting protoplast is spherical in  shape . Introduction

3. The protoplasm consists of two major divisions in eukaryotes: the cytoplasm, and the nucleoplasm (cell nucleus). The cytoplasm is the eukaryotic cell's jelly-like material. Except for the nucleus it consists of the cytosol , the vesicles, the cytoskeleton, the inclusions and the organelles. Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both, a somatic hybrid. Protoplast fusion is a physical phenomenon. During fusion, two or more protoplasts come in contact and adhere with one another either spontaneously or in presence of fusion induc­ing chemicals.

4. Principle of protoplasm fusion The basic principle of protoplasm culture is the aseptic isolation of large number of intact living protoplast removing their cell wall and culture them on a suitable nutrient medium for their growth and development. Protoplast can be isolated from varieties of plant tissue such as leaf mesophyll and cell from liquid suspension culture.

5. Isolation of protoplast Young leaves of the plants are collected and wash with distilled water. Surface sterilization of leaves with sodium hypochlorite solution for 30 min. With the help of long sterilized forcep , one leaf is transferred on a sterile plate. Leaf pieces in enzyme solution are incubated in the dark at 24 C for 16-18 hrs for digestion. Digested leaf piece are gently agitated to facilitate the release of the protoplast. The protoplast suspension is then allowed to pass through a nylon mash to remove the larger pieces of undigested tissue. The filtrate is transferred to centrifuged tube and spin for 5 min, the protoplast form the pellet. Pellet is re-suspended in protoplast culture medium.

6. Methods of protoplasm fusion Protoplast fusion was reported by Kuster and Michel. 1) Spontaneous fusion: Protoplasts, during isolation, often fuse spontaneously. Simple physical contact is sufficient to bring about the spontaneous fusion among the similar parental protoplasts.  Protoplasts from adjoining cells fuse through their plasmodesmata to form a multinucleate protoplast. Spontaneous fusion is strictly intraspecific and gives rise to homokaryon .

7. 2) Induced fusion : It is the method of fusion of freely isolated protoplasts from different sources with the help of fusion inducing chemical agents. Normally, isolated protoplasts do not fuse with each other because the surface of the isolated protoplast carries negative charge. Thus, there is a strong tendency for protoplasts to repel one another due to their same charges. So this type of fusion needs a fusion inducing chemical agent, which actually reduces the electronegativity of the isolated protoplasts and allow them to fuse with each other. Induced fusion is a highly important and a valuable technique because the protoplast from widely different and sexually incompatible plants can be fused by this procedure.  This technique has ability to combine different genotype and the main objectives of somatic hybridization are mainly based on induced protoplast fusion.

8. Types of Induced fusion Induced fusion may be divided into three types: Mechanical fusion Chemo fusion Electrofusion

9. Mechanical fusion: In this process, the isolated protoplasts are brought into physical contact mechanically under microscope using micromanipulator and micropipette. Chemo fusion: In this process, the isolated protoplast is fused by the use of some chemical agent such as sodium nitrate, polyvinyl alcohol, polyethylene glycol, calcium ion etc. Chemical fusogens cause the isolated protoplasts to adhere to one another and leads to tight agglutination by fusion of protoplast. The adhesion of isolated protoplast takes place either due to reduction of negative charges of protoplast or due to attraction of protoplast by electrostatic forces caused by chemical fusogens .

10. Electro fusion: Electro fusion is a modern technique of pro­toplast fusion which involves the use of mild electrical fields in protoplast suspension for inducing protoplast fusion. This technique is very easy, simple and fast. It is often more efficient than chemical induced fusion (chemo fusion). In this protocol, fusion is a two-step process. First the protoplasts are put into a small fusion chamber containing parallel wires or plates which serve as electrodes . 

11. Fusion Induced by Sodium nitrate By this method, equal densities of proto­plasts from two different sources are mixed and then centrifuged at 100 g for 5 minutes to get a dense pellet. This is followed by addition of 4 ml of 5.5% sodium nitrate in 10.2% sucrose solution to re-suspend the protoplast pellet. The suspended protoplasts are kept in water-bath at 35° C for 5 minutes and again centrifuged at 200 g for 5 minutes. The pellet is once again kept in water-bath at 30°C for 30 minutes. Fusion of protoplast takes place at the time of incubation. The, pellet is again suspended by 0.1% sodium nitrate for 5-10 minutes. The protoplasts are washed twice with liquid culture medium by repeated centrifugation. Finally, the protoplasts are plated in semisolid culture medium. By this method, intra-and inter­specific fusions have been achieved 

12. Fusion Induced by Calcium Ions at High pH In 1973, Keller and Melcher developed a method to effectively induce, fusion of tobacco protoplast at high temperature (37° C) in media containing high concentration of Ca 2+  ions, (i.e., calcium chloride) at a highly alkaline condition (pH 10.5). Equal densities of protoplasts are taken in a centrifuge tube and the protoplasts are spun at 100 g for 5 minutes. The pellet is suspended in 0.5 ml of medium. 4ml of 0.05M CaCl 2 2H 2 0 in 0.4M mannitol at pH 10.5 is mixed to the protoplast suspension. The centrifuge tube containing protoplasts at high pH/Ca 2+  is placed in the water bath at 30° C for 10 minutes and is spun at 50 g for 3-4 minutes. Keep the tubes in water bath (37°C) for 40-50 minutes. About 20-30% protoplasts are involved in this fusion experiment.

13. Fusion Induced by PEG Many fusion experiments are performed by a polyethylene glycol. PEG induces protoplast aggregation and subsequent fusion. But the concentration and molecular weight of PEG are important with respect to fusion. A solution of 37.5% w/v PEG of molecular weight 1,540 or 6,000 aggregates mesophyll and cultured cell protoplasts during a 45 minutes incubation period at room temperature. In some studies, high pH/Ca 2+  and PEG method have been combined. By this method, the agglutination of protoplasts can be brought about using sufficient quantities (0.1-5 ml) of protoplast in centrifuge tube or micro densities of protoplast on a coverslip . The PEG method has been modified slightly to fuse higher plant protoplast.

14. The modifications are given below: PEG is more effective when it is mixed with 10-15% dimethyl sulfoxide (DMSO). Addition of concanavalin A (Con A) to PEG increases protoplast fusion frequency. Sea water has been used alone or in com­bination with PEG to fuse protoplasts.

15. 15% solution of Polyvinyl alcohol (PVP) in combination with 0.05 CaCl 2  and 0.3 M mannitol are used to fuse plant proto­plasts. Lectins are also known to agglutinate pro­toplasts. Various proteins are also used for aggluti­nation of protoplast. Other Chemicals

16. Protoplast fusion in plants are based on the following basic points. Protoplast isolation Fusion of protoplast, obtain from different species to produce heterokaryons . Cell wall regeneration by the fused product. Fusion of nuclei. Division of hybrid cells and their growth. Identification, selection or isolation of hybrid cell. Induction of organogenesis in the callus tissue derived from hybrid cells. Raising mature plants from regenerated shoot.

17. Importance of protoplast culture Protoplast fusion provides a method of combining the different genomes of different genera and species, with the potential of overcoming sexual incompatibility barrier between plants . By protoplast fusion, it is possible to transfer some useful genes such as disease resistance, nitrogen fixation, rapid growth rate, protein quality, frost hardiness, drought resistance etc. from one species to another and thereby widen the genetic base for plant breeding . Cybrids are generally produced due to elimination of total genome of one parent after the fusion of two protoplasts. If one parental nucleus completely disappears, the cytoplasm of the two parental protoplasts is still hybridized and the fusion product is known as cybrid or cytoplasmic hybrid or heteroplast

18. Thank you