Hybrids have been produced either between different varieties of the same species e. During the enzyme treatment, protoplast from adjoining cells fuse through their plasmodesmata to form multinucleate protoplasts. Plant protoplasts materials round green lettuce, 1 leaf 21% sucrose solution, 0. This area is exciting because protoplasts from different origins can be fused together to form new genome combinations that cannot be obtained by conventional means. Simlarly, kuchko 1985 achieved somatic hybrid through interspecific cross of wild and cultivated variety of potatoes i. Protoplast fusion article about protoplast fusion by the. Cells cultured at a low temperature contained larger proportions of phospholipids of low phase transition point, the 1,2dilinoleoyltype, than those cultured at a normal temperature 25 c. Many inter and intra generic plants have been created by this tech nique2l. Plant protoplast fusion by electromanipulation sciencedirect.
This polymer binds to the lipid membrane of cells and thus induces fusion fusion takes place for 45 min in incubation at r. Protoplasts can be generated by stripping the cell wall from plant, bacterial, or fungal cells by mechanical, chemical or enzymatic means protoplasts differ from spheroplasts in that their cell wall has. Pdf plant regeneration from protoplast fusion in passiflora spp. While the production and vitality of wheat protoplasts were detected by the hemacytometer and the fda staining respectively. Isolation and fusion of protoplasts from mesophyll cells. Fusion was induced by adding a drop of 40% polyethylene glycol solution to the. Lf101 electro cell fusion generator electrodechamber. Factors influencing efficiency of protoplast isolation in. Somatic hybridization somatic hybridiztion refers to crossing of crop plants though fusion of somatic cell. Production of somatic hybrid plants through protoplast.
A mixture of carbohydrases can be used to degrade plant tissue, producing a protoplast suspension which can easily be seen under a. For protoplast fusion it is important that the cell wall of plant and. Protoplasts of both types are mixed in an equal proportion attaining a density of 5 x 10 4 to 2 x 10 5 protoplasts per ml. Protoplasts density is usually determined by haemocytometer. Protoplast technologyprotoplast, protoplast isolation. Plant protoplasts by teresa bengochea and john dodds 1986 chapman and hall. Protoplast isolation the source tissue is a common and important factor in successful poplar protoplast culture.
Production of somatic hybrid plants through protoplast fusion in. The effect of peg muting solution on fusion of protoplasts derived from cultured soybean cells and vicia haastana leaves eluting solution s percentage of heterokaryocytes to total surviving protoplasts and to soybean alone b,o. Plant protoplast fusion and somatic hybridization sciencedirect. Whole plants can be regenerated from protoplasts grown on solid or liquid media. Protoplast fusion is important in biochemical andgenetic engineering studies, butmanydetails regarding its mechanismare notknown. Peg solutions for protoplast fusion components peg solution pla p2 p3 peg 1540 0. Protoplast fusion one drop of dense protoplast suspension was pipetted onto the middle of the microscope slide and allowed the protoplasts to settle to the bottom for 5 min. Isolation of plant protoplasts by enzymatic method. Strain improvement of pleurotus species by protoplast fusion. After adhesion, membranes of protoplasts fuse in some localised areas and, eventually, the cytoplasm of the two protoplasts intermingle. Protoplast isolation and culture of dendrobium sonia bom 17. Impacts of banning protoplast fusion on the range of.
Among the many uses for these naked cells is transient or permanent genetic transformation by introduction of transgene dna, somatic hybridization by protoplast fusion of species or subspecies resistant to traditional crossbreeding, and isolation of sub. This chapter deals with the possible use of protoplast fusion for hybridization and genetic analysis of yeasts. Which plants have already been bred with the help of protoplast fusion. During fusion, two or more protoplasts come in contact and adhere with one another either spontaneously or in presence of fusion inducing chemicals. Electrofusion is a nontoxic technique that minimizes damage of the target protoplasts and results in significantly higher fusion frequencies than obtained by chemical fusion. Induced fusion mechanical fusion physical fusion of protoplasts under microscope by using micromanipulator and perfusion micropipette electrofusion fusion induced by electrical stimulation pearl chain of protoplasts is formed by low strength electric field 10kv m 1 fusion of protoplasts of pearl chain is induced by the application of high. Plant protoplasts can be fused and the fusion products cultured to produce somatic hybrid plants. Plant protoplast fusion and growth of intergeneric hybrid cells.
The study of reactive oxygen species effects and dna decondensation, along with thorough phytohormone monitoring, are in our opinion the most promising research topics in the further strive for rationalization of protoplast regeneration. The relation between the composition of the phospholipid molecular species in a cell membrane and the velocity of protoplast fusion was studied using cells cultured at a low temperature 10 c. Plant protoplast fusion protoplast fusion has many uses in plant research. Fusion of proplast incudes three stages 1 agglutination 2 membrane fusion 3rounding off of the fused protoplast carlson et al 1972 first time fused the leaf protoplasts of nicotina gluca and n. Protoplasts are plant cells which have lost their cell wall. Protoplasts were considered essential for gene trans fer because the cell wall is usually impermeable to dna.
Read this article to learn about the isolation of plant protoplasts by enzymatic method, plant protoplast culture, somatic hybridization and fusion techniques used in plant protoplasts. Protoplast fusion definition of protoplast fusion by. Protoplast fusion would continue to be an existing area of research in modern biotechnology 11. Here protoplast preparation after fusion is suspended in kmc solution equal volume of 0. Several protoplast based genetic engineering techniques emerged, including protoplast fusion, dna microinjection, electroporation, and polyethylene glycol pegmediated. Carlson 1972 succeeded in carrying out protoplast fusion of nicotiana glauca x n. Protoplast fusion technology for somatic hybridisation in. Protoplast isolation science and plants for schools. The text also describes the production of haploid protoplasts from developing pollen grains. Protoplast fusion between haploid gametic and diploid somatic cells has been achieved which clearly demonstrate that fusion is independent of cell type. Electrofusion is preferred over chemical fusion, as fusion conditions are much easier to control duquenne et al. This chapter discusses plant protoplast fusion by electromanipulation.
In a method known as somatic cell hybridization or protoplast fusion, cell walls of different. Protoplast fusion can be broadly classified into two categories. Simply physical contact is sufficient to bring about the spontaneous fusion among the similar parental protoplasts. Protoplast surfaces bear strong negative charges, and intact protoplasts in suspension repel each other, hence fusion is accomplished by the addition of calcium ions or polyethylene glycol peg, or by using electric fields. Their attempts to obtain callus from protoplasts of c. Procedure for successful somatic hybridization is as below. Agricultural applications of plant protoplast fusion nature. Density gradient fractionation of protoplast suspension after fusion. Protoplast is a plant, bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means.
Protoplast fusion technology and its biotechnological. Pdf summary protoplasts were isolated from leaf explants ofpassiflora edulis var. Thepercentage ofprotoplasts in the chamberthat fuse depends onthe uniformityofthe lengths ofthe pearlchains. The application of this technique to citrus would be of great value for the im provement of this kind of fruit tree. Isolation and fusion of protoplasts from mesophyll cells of. Protoplasts are cells of plants, fungi, or bacteria in which the cell wall has been removed, but the plasma membrane is intact. Polyethylene glycol peg is the most widely used one. Intergeneric hybrids which may be difficult to achieve by sexual crosses have been obtained through protoplast fusion e. Isolation and callus production from cotyledon protoplasts. Optimization conditions of wheat mesophyll protoplast. Protoplast fusion protoplast fusion was performed according to a modified method of 1, 2.
Pdf protoplasts are the cells of which cell walls are removed and. Protoplast suspension was purified via repeated three times centrifugation in w5 medium 5 minutes at 100 g, supernatant removal, rewashing and subsequent centrifugation on sucrose gradient pelletier et al. This technique is used to create hybrids between plant species that cannot be crossed sexually. Fusion of protoplasts of potato and tomato, and production of hybrid plant pomato.
Protoplast fusion technology was employed to reconstruct fusants from this. The chemicals which promote fusion of protoplasts are called fusogenic agents. In a second study, tang and punja 1989 reported protoplast fusion of. Protoplast fusion technology vary protoplast from speciesgenera, fusion to create new genotypes, plant regeneration organogenesissomatic embryogenesis, isolated in large quantities, protoplasts must be totipotent. Protoplasts are isolated by two methods, mechanical and enzymatic. Some of the advantages of protoplast fusion are large scale reproducible isolation of protoplasts from various tissues and more or less universal application.
Usually a peg solution of about 2850% is used for protoplast fusion. Fusion products can be selected for on media containing antibiotics or herbicides. Protoplast isolation and culture plant tissue culture. Cells cultured at a low temperature contained larger proportions of phospholipids of low phase transition point, the 1,2dilinoleoyltype, than those. Protoplast fusion purified protoplasts were resuspended in 0,1. Fusion was induced by adding a drop of 40% polyethylene glycol solution to the settled protoplasts.
The preliminary identification of fusion product is done under microscope. Protoplast fusion has been used as a method to create mushroom hybrids especially when conventional method cannot be achieved. Protoplast fusion has been used successfully to transfer the cytoplasmic traits in various plants such as perennial ryegrass, rice, brassica. Establishment of method for protoplast fusion with pegmediated between jatropha curcas l. Plant regeneration from protoplast fusion inpassiflora spp. Peg polyethylene glycol fusion this chemical has been identified as a possiblepotent fusogen. Establishment of method for protoplast fusion with peg. Protoplasts during isolation often fuse spontaneously and this phenomenon is called spontaneous fusion. In 10 of the 12 reports listed in table 1, section d, the protoplast source was leaves from shoot cultures. Thus, protoplast fusion can be used to introduce novel germplasm into breeding programs. Protoplast fusion involves mixing of protoplasts from two different genomes and their fusion is carried out by spontaneous, mechanical or induced fusion techniques. Protoplast during isolation often fuse spontaneously and this phenomenon is called spontaneous fiusion.
Protoplast fusion was performed according to a modified method of 1, 2. As a result, genetic manipulation by protoplast fusion in vitro is now a realistic proposition. Often the presence of polyethylene glycol or electric fields will enhance the frequency of fusion. The following points highlight the two methods of protoplast fusion. After fusion, the acfield strength was reduced because this procedure was found to shorten the time required for the fusion product to round upas a single protoplast fig. These can then be induced to form shoots and roots and hybrid plants can be tested for desirable characteristics. The fusion of somatic cell takes place through protoplasts. Protoplast fusion regarding the low density of protoplasts obtained for ni637 as pv accession table 1, electrofusion could not be easily realized. Nov 16, 2012 protoplast fusion induced by the application of electric shock 18. Electrofusion, that is, cell fusion in an electric field, has become of great interest as a powerful technique for the fusion of plant, animal, and microbial cells as it is simpler, more rapid, and less harmful to the cells than the conventional polyethylene glycol peg method and is available for mass production of fusion. Protoplast fusion and somatic hybridization in vitro. Protoplast fusion provides an alternative way of producing hybrids for species which can not be crossbred. In plant breeding programs, cell fusion is used in many traditional breeding and hybridization programs as well as in general propagation using tissue culture. Wepreviously proposed that membranefluidity is determined bythe phase transition ofthe phospholipid molecularspecies 3, 17.
The research object of this study is ml71 wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. In one study, a fusion rate of 5 to 12% was reported between protoplasts of c. In the fusion unit the dna from the component cells may undergo recombination. Nootjaree tudses1, siripong premjet,2,3 and duangporn premjet1,3. Cuy501g4 ms gold block electrode 4mm gap, 160l cuy4984 ms stand model chamber electrode. Protoplast fusion and growth of hybrid cells 219 table 6. Further, most of the shoot cultures were from nonseedling trees, which allowed researchers. Plant protoplast fusion and growth of intergeneric hybrid. Choose from 15 different sets of protoplast flashcards on quizlet.
Pdf protoplast fusion technology and its biotechnological. Production of somatic hybrid plants through protoplast fusion. Therefore, chemical fusion was conducted to perform protoplast fusion between ni637 with all pp and pc accessions. The book discusses isolation, fusion, and culture of higher plant protoplasts, lower plant protoplasts, and bluegreen algal protoplast. It may allow the exchange of dna between unrelated. Somatic hybridization involves four discrete, yet interrelated, stages. Following, fusion and fragmentation progress is summarized. Mechanical isolation method of protoplasts is no more practically used. 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. Membranefluidity may affect the rate ofprotoplast fusion. Three main effectorsthe concentration of mannitol, enzymolysis time and centrifugal force, affect the production and vitality of protoplast.