In this way the protoplasts are retained and compressed by the flow of liquid. This micropipette is partially blocked within 1 mm of the tip by a sealed glass rod. In this process, the isolated protoplasts are brought into intimate physical contact mechanically under microscope using micromanipulator and perfusion micropipette. The isolated plant protoplasts can be induced to fuse by three ways: Mechanical Fusion: The fundamental objectives of somatic hybridization are mainly based on induced protoplast fusion. This technique has the possibility and ability to combine different genotype beyond the limits imposed by sexual process. So this type of fusion needs a fusion inducing chemical agent or system which actually reduces the electro-negativity of the isolated protoplasts and allows them to fuse with each other.Īctually, induced fusion is a highly important and a valuable technique because the protoplast from widely different and sexually incompatible plants can be used by this procedure. Normally, isolated protoplasts do not fuse with each other because the surface of the isolated protoplast carries negative charge (-10 mV-30 mV) around the outside of plasma membrane and thus there is a strong tendency for protoplasts to repel one another due to their same charges. Induced Fusion :įusion of freely isolated protoplasts from different sources with the help of fusion inducing chemical agents is known as induced fusion. Perhaps spontaneous fusion has some practical importance for chromosome doubling. Spontaneous fusion of two or more adjoining somatic protoplasts is of no practical use, but this may be important in studies of the nature and function of plasmodesmata, the physiology and control of mitosis in multinucleated cells and nuclear fusion. the spontaneous fusion of microsporocyte protoplast of Lolium longiflorum and Trillium kamtschaticum. This type of spontaneous fusion has been used to produce inter generic fusion, e.g. An exception is the protoplast from microsporocytes of some plants of lily family where the freely isolated protoplast fuses spontaneously. The protoplasts, once they are freely isolated, do not fuse spontaneously with each other. Spontaneous fusion is strictly intraspecific and gives rise to homokaryon. Finally a complete coalescence of adjacent cell takes place. These improved procedures for protoplast isolation and regeneration proved to be suitable for other streptomycete species.Electron microscopic studies have shown that as the cell wall are enzymatically degraded, the plasmodesmatal connection between the adjacent cells enlarge due to removal of its constriction and the enlargement of pit fields.Įventually, the greater enlargement of plasmodesmata allows the entry of organelles into neighbouring cells. clavuligerus protoplasts resulted from these experiments. An improved regeneration medium (R6) and protocol which supported higher and more consistent levels of regeneration of S. clavuligerus, and other potentially advantageous components and conditions, in an attempt to raise the regeneration frequency of the protoplasts. A systematic survey was made of the components of regeneration medium R5, previously used for S. The latter promoted improved protoplast viability. Protoplasts with the highest regeneration frequency were isolated from mycelium, grown in a two-stage culture system (without glycine), using lysozyme dissolved in a sucrose osmoticum containing 1% bovine serum albumin. An investigation of protoplast formation and cytology was made to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Reports of studies on the regeneration of protoplasts from Streptomyces clavuligerus are limited and for this reason the experiments described in this paper were carried out. The regeneration of streptomycete protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation.
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