SOMATIC CELL FUSION PROTOPLAST FUSION Group Members Muhammad

SOMATIC CELL FUSION (PROTOPLAST FUSION)

Group Members • • • Muhammad Ahmad Aghna Naveed Suniza Dar Sarmad Basit Kiran Ali Maria Gillani

What is protoplast? • A protoplast is a plant, bacterial or fungal cell that had its cell wall completely or partially removed using either mechanical or enzymatic means.

What is PROTOPLAST Fusion ? • Protoplast fusion is a type of fusion in which two protoplasts (I. e a cell without cell wall) are fused or combined together(under in vitro conditions) in such a way that the new hybrid cell is formed as a result of the fusion , which consist of characteristics of both the cells due to genetic modification.

Types of protoplast fusion : 1) Spontaneous fusion: Spontaneous fusion also known as mechanical fusion is the type of fusion in which the two protoplasts are fused together by bringing them closer and disrupting their cell membranes. 2) Induced fusion : induced fusion is the type of fusion in which either chemical agents (I. e chemofusion) or electric current (I. e electrofusion) is used to fuse the two protoplasts.

HISTORY • In 1847 Theodore Schwann observed that in certain cells the walls and cavities of the cells coalesce(fuse) together. It was this observation that provided the first hint that cells fuse. • The word protoplast was first introduced in 1880 by Hastein. • First isolation of the cell wall from plant cell to create protoplast was achieved by Klercker in in 1892 by mechanical method.

• The second isolation of cell wall from the plant cell was done by cocking in 1960 through enzymatic method. • The first plant regeneration was done by Nagata and Takebe in 1971. • The first induced fusion ( I. e chemo fusion) was introduced by Kao and Michayluk in 1974.

SOMATIC HYBRIDIZATION TECHNIQUE 1 2 3 4 • Isolation of protoplast • Fusion of the protoplasts of desired species/varieties • Identification and Selection of somatic hybrid cells • Regeneration of hybrid plants

Protoplast isolation Outer most covering in plant cell is cell wall which is made up of cellulose, hemicellulose, pectin, etc which is rigid and is not easily breakable. So we have two approaches to get a cell which only contains protoplast. 1) Mechanical: In this process a force is applied to the cell and the cell wall breaks. For example in almonds and peanuts. 2) Enzymatic: In this case, certain enzymes are used which dissolve the cell wall. The enzymes used are cellulose, pectinase, etc. And example is egg.

• Enzymatic approach is more used than the mechanical approach because in mechanical approach the force/pressure can sometimes damage the cell and the pressure does not remain constant for all the cell walls.

• Isolation of protoplasts is readily achieved by treating cells/tissues with a suitable mixture of cell wall degrading enzymes. • Usually, a mixture of pectinase or macerozyme (0. 11. 9%) and cellulose (1 -2%) is appropriate for most plant materials. Hemicellulase may be necessary for some tissues. • The p. H of enzyme solution is adjusted between 4. 7 and 8. 0 and the temperature is kept at 25 -30°C. The osmotic concentration of enzyme mixture and of subsequent media is elevated to stabilize the protoplasts and to prevent them from bursting. • Protoplasts have been isolated from virtually all plant parts, but leaf mesophyll is the most preferred tissue, at least in case of dicots, for this purpose

Fusion of the protoplasts of desired species/varieties • Polyethylene glycol (PEG) induced protoplast fusion is the most commonly used as it induces reproducible high frequency fusion accompanied with low toxicity to most cell types. The protoplast mixture is treated with 28 -50% PEG (MW 1, 500 -6, 000) for 15 -30 min, followed by gradual washing of the protoplasts to remove PEG; protoplast fusion occurs during the washing.

• Electrofusion: A more selective and less drastic approach is the electrofusion technique, which utilizes low voltage nonuniform alternating electric current pulses to bring the protoplasts in close contact. Fusion of protoplasts is brought about by a short pulse of high voltage.

Selection of Hybrid Cells The protoplast suspension recovered after a treatment with a fusion inducing agent (fusogen) consists of the following cell types: (i) unfused protoplasts of the two species/strains, (ii) products of fusion between two or more protoplasts of the same species (homokaryons), (iii)‘hybrid’ protoplasts produced by fusion between one (or more) protoplast(s) of each of the two species (heterokaryons)

• In somatic hybridization experiments, only the heterokaryotic or ‘hybrid’ protoplasts, protoplasts particularly those resulting from fusion between one protoplast of each of the two fusion partners, are of interest. • However, they form only a small proportion of the population (usually 0. 5 -10%). Therefore, an effective strategy has to be employed for their identification and isolation. This step is called the selection of hybrid cells, is the most critical, and is cells still an active area of investigation. • Some visual markers, markers e. g. , pigmentation of the parental protoplasts may be used for the identification of hybrid cells under a microscope; these are then mechanically isolated and cultured.

• Several workers have attempted to devise systems, which specifically select for hybrid cells. In simple words, these systems exploit some properties (usually, deficiencies) of the parental species, which are not expressed in the hybrid cells due to complementation between their genetic systems. • Phytotoxins, antibiotics, specific amino acids can also be used to identify hybrids. • A more general and widely applicable strategy, strategy but demanding more work than the previous approach, is to culture the entire protoplast population without applying any selection for the hybrid cells. All the types of protoplasts form calli; the hybrid calli are later identified on the basis of callus morphology, chromosome constitution, protein and enzyme banding patterns, etc

Regeneration of Hybrid Plants • Once hybrid calli are obtained, plants are induced to regenerate from them since this is a prerequisite for their exploitation in crop improvement. Further, the hybrid plants must be at least partially fertile, fertile in addition to having some useful property, to be of any use in breeding schemes. The culture techniques have been refined to a state where plant regeneration has been obtained in a number of somatic hybrids

CHARACTERISTICS OF HYBRIDS AND CYBRIDS Somatic hybridization produces symmetric or asymmetric hybrids, or cybrids. 1) Symmetric Hybrids: • These contains the somatic chromosomes of both the parental species. These are very significant as they show all the properties exhibited by parent species. • Many symmetric hybrids have been produced between sexually incompatible species. Some of these possess and express useful genes and are fertile; these have been ordered into breeding programs. Symmetric hybrids provide the following opportunities.

i. Hybrids between Nonflowering and Male Sterile Strains: • Somatic hybrids can be produced between a nonflowering and a male sterile strain of a crop species. Such hybrids have been produced in potato: the fusion parents were a male sterile and a nonflowering dihaploid clone, while some of the somatic hybrids were male fertile. ii. Superior Somatic Hybrids: • Some somatic hybrids may possess such desirable features that may make them commercially useful. For example, Datura hybrids D. Their alkaloid content is 20 -25% higher than those in the parent species hence they are industrially useful for producing scopolamine. • It can also generate some novel materials that may be very useful in genetic, physiological, biochemical and other studies.

• Asymmetric Hybrids: • These are those hybrids which preserve the genetic material of one parent organism. The chromosome content of other parent species is lost. • For example, protoplasts of a nitrate reductase mutant of Nicotiana plumbaginifolia were fused with irradiated protoplasts of Atropa belladona-, the somatic hybrids were selected on nitrate medium. The hybrids contained 4 or 6 N. plumbaginifolia genomes plus 6 -26 A. belladona chromosomes (many of these were deleted chromosomes).

• Somatic Cybridization : • The process of protoplast fusion resulting in the development of cybrid is called as Cybridization. • CYBRID: Plants or cells which containing nucleus of one species but cytoplasm from both the parental species. For production of a cybrid cell uses two parental cells but should be one complete cell (cytoplasm with nucleus) and other should be without nucleus or in active nucleus.

Aims of Cybridization: To obtain biological information on intra- and intergeneric transfer of organelles. To obtain cytoplasm gene of one species to another species which have nucleus and cytoplasm This is also a way for those species which do not perform sexual reproduction, so this process provides opportunity to make a desire species with combination of both species.

Protoplast fusion in Animals • Protoplast fusion or Hybrid cells can be produced by fusing different types of somatic cells from two different tissues or species in a cell culture media. Chromosome or gene mapping via somatic cell hybridization technique is based on fusion of human somatic cell and mouse somatic cell. Usually human somatic cells like fibrocyte or leucocyte are fused with continuous cell lines of mouse. When somatic cells of human and mouse origin or cells of any two species of mammals or two cells of same species are mixed, spontaneously cell fusion occurs at very low rate. Therefore by adding ultraviolet inactivated Sendai (parainfluenza) virus or chemical compound known as polyethylene glycol (PEG) increases the somatic cell fusion by 100 to 1000 times.

Procedure: 1. Required type of human and mouse somatic cells are selected and then mixed together along with inactivated Sendai virus or PEG (polyethylene glycol); hence they enhance the cell fusion process. 2. After some time, mixture of all three types of cell such as human cell, mouse cell and hybrid cells are plated on a selective media. Commonly used selective media is known as HAT medium. This selective media only supports the multiplication or cell proliferation of hybrid cells. 3. Some clones are extracted from the selective media and are tested for both cytogenetic and appropriate biochemical analysis for the detection of investigating biological molecule, such as enzyme or protein or any traits.

4. Now the result is correlated with the presence or absence of a trait with the presence or absence of human chromosome or gene in the somatic hybrid cells. 5. Depending upon the correlation between the presence and absence of a human chromosome or gene with that of a trait in somatic hybrid cells, can be used in locating the gene present in the concerned chromosome or genetic material.

APPLICATIONS IN ANIMAL CELLS 1. Hybrid cells are used to study the gene expression. 2. Hybrid cells are used to study the basics of cell division 3. Hybrid cells can be used to study the transformation of normal cells into malignant cells. 4. Hybrid cells are used to obtain viral replication 5. Hybrid cells are used for chromosome or gene mapping 6. Hybrid cells are also used in the production of monoclonal antibodies

ADVANTAGES 1. Symmetric hybrids can be produced between species, which cannot be hybridized sexually. These hybrids can be readily used in breeding programmes for transfer of useful genes to crops or may be useful as new crop species. 2. Hybrids can be produced even between such strains, which are completely sterile, e. g. , monoploids 3. Cytoplasm transfers can be affected in one year, while backcrossing may take 5 -6 years. Even where backcrossing is not applicable, cytoplasm transfers can be made using this approach. 4. Mitochondria of one species can be combined with chloroplasts of another species. This may be very important in some cases, and is not achievable by sexual means even between easily crossable species. 5. Recombinant organellar genomes, especially of mitochondria, are generated in somatic hybrids and hybrids. Some of these recombinant genomes may possess useful features.

LIMITATIONS 1. Techniques for protoplast isolation, culture and fusion are not available for many important crop species like many cereals and pulses. 2. In many cases, chromosome elimination occurs from somatic hybrids leading to asymmetric hybrids. Such hybrids may be useful, but there is no control on chromosome elimination. 3. Many somatic hybrids show genetic instability, which may be an inherent feature of some species combinations.

4. Many somatic hybrids either do not regenerate or give rise to sterile regenerants. Such hybrids are useless for crop improvement. All interfamily somatic hybrids are genetically unstable and/or morphologically abnormal, while intergeneric and intertribal hybrids are genetically stable but produce abnormal and/or sterile plants or only teratomata. 5. No confirmation of expression of particular trait in somatic hybrids.

ANY QUESTIONS ?

Quiz • Q 1. Who observed that in certain cells the walls and cavities of the cells coalesce(fuse) together. a. Theodore schwann b. Paul gilbert c. Marie Curie d. Michael Roberts

Q 2. Name two steps of protoplast fusion. Q 3. Why is enzymatic approach more preffered than the mechanical approach? Q 4. Give two advantages of somatic hybridization Q 5. What are the limitations of somatic fusion? Any two