Contents:IntroductionIsolation of Protoplast Fusion products - the hybrids and cybrids techniques of protoplast fusionProcedure for successful somatic hybridization Applications
Protoplast FusionPROTOPLAST FUSION:•A protoplast is a plant, bacterial or fungal cell that had its cell wall completely or partially removedusing either mechanical or enzymatic means. Protoplasts: Have their cell wall entirely removed Spheroplasts: Have their cell wall only partially removed•More generally protoplast refers to that unit of biology which is composed of a cells nucleus andthe surrounding protoplasmic materials.•Protoplast are naked spherical cells obtained from plants by removing of cell wall and it iscultivated in liquid as well as on solid media.•The protoplast an excellent tool for the synthesis of novel combination of genes and are essentialpart of the overall process required for the genetic manipulation of plants.
Protoplast FusionISOLATION OF PROTOPLAST:Protoplast can be isolated from almost all plant parts i.e.roots,leaves,fruits,tubers, root nodules,endosperm,pollen mother cell, callus andsuspension culture.Protoplasts are isolated from cells by two methods-MECHANICAL METHOD MECHANICAL METHOD:ENZYMATIC METHOD The cells are kept in a suitable plasmolyticum & cut with a fine knife, so that protoplast are released from cells cut through the cell wall, when the tissue is again deplasmolysed. This method is suitable for isolation of protoplasts from higher plant tissue such as leaf, bulb scale, fruit epidermis,raddish roots
Protoplast Fusion DISADVANTAGE- It yields a very small number of protoplasts after a rather tedious procedure. It is not suitable for isolating protoplasts from meristmatic & less vacuolated cells.A. Tissue is cut along the dotted line B. Release of Protoplasts from Damaged cells
Protoplast FusionEnzymes for the preparation of protoplasts•Cell walls are made of a variety of polysaccharides. Protoplasts can be made bydegrading cell walls with a mixture of the appropriate polysaccharide-degradingenzymes:•During and subsequent to digestion of the cell wall, the protoplast becomes verysensitive to osmotic stress. This means cell wall digestion and protoplast storage mustbe done in an isotonic solution to prevent rupture of the plasma membrane. Type of cell Enzyme Plant cells Cellulase, pectinase, xylanase Gram-positive bacteria Lysozyme (+EDTA) Fungal cells Chitinase
Method of isolation of protoplastFig. Method of isolation of protoplast
A. Shoot Culture B. Remove Leaves C. Cut LeavesG.Protoplasts F. Centrifuge 500g 1 minReady for E. Isolated Protoplasts D. Incubate inPurification Enzyme 8
Purification, culture and regeneration of protoplastsFig: Purification, culture and regeneration of protoplasts
PURIFICATION:Only two commonly used methods:-1.Sedimentation & washing2.Flotation1. In this method, the crude protoplasts suspension is centrifuged at low speed (50-100g for 5 min). The intact protoplasts form a pellet and supernatant containing cell debris can be pipetted off. The pellet is gently resuspended in fresh culture media plus mannitol and rewashed. This process is repeated two or three times to get relatively clean protoplast preparation.2. Protoplasts being lighter (low density) then other cell debris, gradients may be used, which will allow the protoplasts to float and the cell debris to sediment. A concentrated solution of mannitol, Sorbitol and sucrose (0.3-0.6M) can be used as a gradient and crude protoplasts suspension may be centrifuged in this gradient at an appropriate speed. Protoplasts can be pipetted off from the top of the tube after centrifugation.
FUSION PRODUCTS - THE HYBRIDS AND CYBRIDSFUSION PRODUCTS - THE HYBRIDS AND CYBRIDSFusion of cytoplasm of two protoplasts results in coalescence of cytoplasms. Thenuclei of two protoplasts may or may not fuse together even after fusion ofcytoplasms.The binucleate cells are known as heterokaryon or heterocyte .When nuclei are fused the cells are known as hybrid or synkaryocyte .Only cytoplasms fuse and genetic information from one of the two nuclei is lostis known as cybrid i.e. cytoplasmic hybrid or heteroplast .
TECHNIQUES OF PROTOPLAST FUSION1. Spontaneous fusion2. Mechanical fusion3. Induced fusion1. During isolation of protoplasts for culture, when enzymatic degradation of cellwalls is affected, some of the protoplasts, lying in close proximity, may undergofusion to produce homokaryons or homokaryocytes, each with 2-40 nuclei.The occurrence of multinucleate fusion bodies is more frequent, when protoplasts areprepared from actively dividing cells.
Somatic hybridization is generally used for fusion of protoplasts either from twodifferent species (interspecific fusion) or from two diverse sources belonging to thesame species.To achieve this objective, spontaneous fusion may be of no value, and induced fusionrequiring a suitable agent (fusogen) is necessary. In animals, inactivated Sendai virus isneeded to induce fusion.NaNO3 treatmentThis method was successfully utilized fro fusion of protoplasts from root tips of oat andmaize seedlings but is not preferred due to low frequency of fusion, particularly whenhighly vacuolated mesophyll protoplasts are used
Treatment with calcium ions (Ca++) at high pH.This method involves spinning (centrifugation the protoplasts in a fusion inducingsolution (0.05M CaCl2 2H2O in 0.4M mannitol at pH 10.5) for 30 minutes at 50g,after which the tubes are placed in a water bath (37 C) for 40-50 minutes.This leads to fusion of 20-50% of the protoplasts. The details of the protocol aredescribed by Bhojwani and Razdan (1983). The method was found to be superior toother methods in some cases, but high pH was toxic in other cases.
Polyethylene glycol (PEG) treatmentSince 1974, protoplast fusion has been successfully achieved in several crops, usingpolyethylene glycol (PEG) as a fusogen. The technique gives high frequency offusion with reproducible results and involves low cytotoxicity.The technique can be used for fusion of protoplasts from unrelated plant taxa (e.g.soybean – tobacco, soybean – maize, and soybean – barley), from unrelated animaltaxa and also between those from animal and plant cells.
Electrical fusionIf Protoplasts are placed into a small culture vessel containing electrodes and apotential difference is applied, then the protoplasts will line up between the electrodes.If now an extremely short, electric shock is applied, protoplasts can be induced tofuse.
Protoplast Fusion Induced by the Application of Electric Shock
Fig. Fusion of protoplasts of potato and tomato, and production of hybrid plant (pomato).
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 (NaNO3), maintenance of high pH 10.5 and temperature 37°C (as a resultof fusion of protoplasts viable heterokaryons are produced. PEG induces fusion of plantprotoplasts and animal cells and produces(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 hybridcolonies.(vii) Transfer of mature plants from the regenerated callus.
APPLICATION OF SOMATIC HYBRIDIZATION AND CYBRIDIZATION1.Somatic cell fusion appears to be the only means through which two differentparental genomes can be recombined among plants that cannot reproduce sexually(asexual or sterile).2. Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can be fusedto produce fertile diploids and polyploids.3. Somatic cell fusion overcomes sexual incompatibility barriers. In some casessomatic hybrids between two incompatible plants have also found application inindustry or agriculture.4. Somatic cell fusion is useful in the study of cytoplasmic genes and their activitiesand this information can be applied in plant-breeding experiments.