Chemi cal methods of plant fusion were developed in the early 1970s, first using sodium nitrate as a fusogen e. For school work, this procedure does not require an aseptic environment if the protoplasts are not to be. Plant protoplast fusion protoplast fusion has many uses in plant research. Upon completion of this set of labs you should be able to use a centrifuge and understand its principle of operation.
Protoplast is a plant, bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means. Establishment of method for protoplast fusion with peg. Protoplast definition of protoplast by medical dictionary. Plant protoplasts materials round green lettuce, 1 leaf 21% sucrose solution, 0. Hybrids have been produced either between different varieties of the same species e. While the production and vitality of wheat protoplasts were detected by the hemacytometer and the fda staining respectively. The text also describes the production of haploid protoplasts from developing pollen grains.
A mixture of carbohydrases can be used to degrade plant tissue, producing a protoplast suspension which can easily be seen under a microscope. With the ability of isolating protoplasts from plant cells using enzymes in the early 1960 by e. Present chapter describes the methods of protoplast isolation, purification, culture techniques as also protoplast fusion techniques and somatic hybrid selection procedures. The uptake of isolated metaphase chromosomes has proven successful in plant protoplast. In 10 of the 12 reports listed in table 1, section d, the protoplast source was leaves from shoot cultures.
Following, fusion and fragmentation progress is summarized. This polymer binds to the lipid membrane of cells and thus induces fusion fusion takes place for 45 min in incubation at r. Abstract introduction why fuse isolated plant protoplasts. The book discusses isolation, fusion, and culture of higher plant protoplasts, lower plant protoplasts, and bluegreen algal protoplast. Isolation and fusion of protoplasts from mesophyll cells. Not for use in diagnostic, preclinical, or clinical procedures. Therefore this technology can not directly be assigned to the field of genetic engineering where the new combination of genetic material is performed at dna level.
Whole plants can be regenerated from protoplasts grown on solid or liquid media. Plant protoplasts are often used as experimental material without paying attention to the tissue they are isolated from a protoplast is a protoplast, whereas in other cases, they are considered not sufficiently able to reproduce the in planta situation. Somatic hybridization by protoplast fusion is a promising. Protoplast isolation the source tissue is a common and important factor in successful poplar protoplast culture. This lecture explains about the protoplast fusion technique also known as somatic cell fusion which allows us to produce plant cell hybrids with increased drought tolerance and immunity. Protoplast isolation and culture plant tissue culture.
Protoplast fusion, culture and regeneration prior to fusion, protoplasts from s. Isolation and fusion of protoplasts from the phytopathogenic fungus. Top 15 applications of protoplast culture plant tissue. Lowe2 1plant sciences division, school of biosciences, university of nottingham, sutton bonington campus, loughborough le12 5rd, uk 2school of biology, university of. Cell fusion nuclear transfer embryo manipulation hybridoma production mammalian cell transfection plant protoplast fusion stem cell production. Ultrastructure of fusion product between protoplasts from c3 and. Optimization conditions of wheat mesophyll protoplast. The following points highlight the two methods of protoplast fusion. Get a printable copy pdf file of the complete article 1. Somatic hybridization somatic hybridiztion refers to crossing of crop plants though fusion of somatic cell. Efficient intergeneric fusion of pea pisum sativum l. Thus, protoplast fusion can be used to introduce novel germplasm into breeding programs.
Thepercentage ofprotoplasts in the chamberthat fuse depends onthe uniformityofthe lengths ofthe pearlchains. 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. Protoplast fusion becomes an important tool of gene manipulation because it breakdown the barriers to genetic exchange imposed by conventional mating systems. Lf101 electro cell fusion generator electrodechamber. A bacterial cell from which the rigid cell wall has. Impacts of banning protoplast fusion on the range of. Pegmediated protoplast transformation with naked dna. Nootjaree tudses1, siripong premjet,2,3 and duangporn premjet1,3. Successful application of the fusion technique demands a protocol for plant regeneration from protoplasts. Unlike c3 plants, the released protoplasts of c4 plant. Protoplast fusion for the generation of unique plants davey. Cocking, the interest in genetic modification of somatic cells in higher plants has developed.
Gels were analyzed and photographed under uv light. Pdf monoploid potato genotypes represent plant material that is free from. Introduction to the protoplast lab college of saint. The fusion of somatic cell takes place through protoplasts. The protoplasts can be isolated from a variety of tissues including leaves, roots, in vitro shoot cultures, callus, cell suspension and pollen. Count the number of protoplasts and adjust the protoplast density to approx 1 x lo6 cells1ml see note 1. Production of somatic hybrid plants through protoplast. Protoplasts during isolation often fuse spontaneously and this phenomenon is called spontaneous fusion. Plant protoplast fusion and growth of intergeneric hybrid. Protoplast fusion has been used as a method to create mushroom hybrids especially when conventional method cannot be achieved. The main objective of protoplast culture lies in the possibilities of fusion of one protoplast with another. This procedure provides a valuable method for genetic information transfer and gene analysis.
This chapter discusses plant protoplast fusion by electromanipulation. Large numbers of viable protoplasts of pea pisum sativum and grass pea. Protoplast fusion technology and its biotechnological. Peg polyethylene glycol fusion this chemical has been identified as a possiblepotent fusogen. Procedures for protoplast fusion chemical fusion of protoplasts electrical fusion of. As a result of improvements in regeneration of whole plants from isolated protoplasts and improvements in protoplast fusion technology, more new somatic. When colonies had developed, alginate beads were despolimeryzed using 20 mm sodium citrate solution in sorbitol 0,3 m, ph 7,4 and gently shaking. Protoplast isolation science and plants for schools. Methods of somatic hybridization procedure for successful somatic hybridization is as below.
To date, little research on protoplast culture has been reported in some orchid genera such as renantanda,12 dendrobium,3 aranda,46 phalaenopsis,7 and cymbidium. The research object of this study is ml71 wheat leaf, which is used to isolate protoplast with enzyme hydrolysis method. 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. Archaic term meaning the first individual of a type or race. 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. Protoplast fusion and growth of hybrid cells 219 table 6. Plant protoplasts by teresa bengochea and john dodds 1986 chapman and hall. Protoplasts are isolated by two methods, mechanical and enzymatic. Agglutination and fusion of plant protoplasts by polyethylene glycol.
Place the protoplast suspension on ice for 35 min see note 2. 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. Final concentration was protoplasts per millilitre. Three main effectorsthe concentration of mannitol, enzymolysis time and centrifugal force, affect the production and vitality of protoplast. Protoplast fusion and somatic hybridization in vitro. Protoplast fusion technique has a great potential for genetic analysis and for strain. As a result, genetic manipulation by protoplast fusion in vitro is now a realistic proposition.
Isolation of plant protoplasts by enzymatic method. An improved protocol for plant regeneration from leaf and. Protoplast production and isolation from etlingera elatior scielo. 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. 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. Impacts of banning protoplast fusion, 2008 8 it must be noted that protoplast fusion is carried out on the basis of vegetative plant cells.
However, the first step is to develop efficient protoplast isolation, culture and plant regeneration systems from protoplasts of the genotypes of interest 18. Fusion characteristics of plant protoplasts in electric. Protoplasts may also be used for plant breeding, using a technique called protoplast fusion. Simply physical contact is sufficient to bring about the spontaneous fusion among the similar parental protoplasts.
Protoplast preparation and fusion for protoplasting, single 4 mm plugs of trichoderma koningii mycelium from 48 hold pda cultures or t. Plant tissue culture pdf search engine pdf library site. Usually a peg solution of about 2850% is used for protoplast fusion. Genomic, transcriptomic and proteomic studies have led to better insights in fundamental processes such as cell wall. The electrical parameters important in the fusion of plant protoplasts aligned dielectrophoretically in highfrequency alternating electric fields have been established. This technique is used to create hybrids between plant species that cannot be crossed sexually. This system does not require proprietary fusion medium, but can be used with the cytofusion medium c for convenience. Protoplast culture for orchids is generally known to be difficult. Purchase recommend to your library advertising and corporate services. Isolation of protoplast protoplast can be isolated by following methods. Plant breeding programs are, therefore, attempting to increase e. Electrofusion is a nontoxic technique that minimizes damage of the target protoplasts and results in significantly higher fusion frequencies than obtained by chemical fusion.
The effect of high ph and calcium on tobacco leaf protoplast fusion. After adhesion, membranes of protoplasts fuse in some localised areas and, eventually, the cytoplasm of the two protoplasts intermingle. Plant tissue culture, third edition builds on the classroom tested, audience proven manual that has guided users through successful plant culturing a. Plant protoplast fusion by electromanipulation sciencedirect. Protoplasts are cells of plants, fungi, or bacteria in which the cell wall has been removed, but the plasma membrane is intact. Fusion is carried out by application of an electric current or by treatment with chemicals such as polyethylene glycol peg.
These can then be induced to form shoots and roots and hybrid plants can be tested for desirable characteristics. Protoplast fusion may be used to produce interspecific or even intergeneric hybrids. Pdf protoplast fusion for the production of intermonoploid somatic. Further, most of the shoot cultures were from nonseedling trees, which allowed researchers. Protoplast fusion would continue to be an existing area of research in modern biotechnology 11. 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. Protoplasts were aligned in an alternating electric field between two relatively distant 1 mm electrodes, by dielectrophoresis induced by field inhomogeneities caused by the.
1224 275 516 1012 69 180 966 858 60 378 1287 365 1492 948 1426 14 713 1021 884 1159 595 1387 723 1181 938 1408 1242 486 705 432