Somatic Hybridization

 

The conventional method to improve the characteristics of cultivated plants has been sexual hybridisation. The major limitation of sexual hybridization is that it can be performed with in plants species or very closely related species. This restricts the improvements that can be done in plants.

In this respect cell fusion offers a novel approach to distant hybridiziation or somatic hybridization. Within the cell wall are the living contents of the cell, called the protoplast. These contents are bounded by a single, two layered cell membrane. The protoplasts contains the cytoplastm, which in turn contains various membrane-bound organelles and vacoules, as well as the nucleus, which is the hereditary unit of the cell.

 In vitro fusion of isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plants is called as somatic hybridization.

Plant protoplasts are of immense utility in somatic plant cell genetic manipulations and improvements in crops. Thus, protoplast provide a novel opportunity to create cells with neo genetic constitution.

 The application of protoplast technology for the improvement of plants offers fascinating option to complement conventional breeding programs. The ability of isolated protoplasts to undergo fusion and take up macromolecules and cell organelles offers many possibilities in genetic engineering and crop improvement (Bhojwani et al . 1977). The experiments involving protoplasts consist of three stages –

i.  protoplast isolation

ii.  protoplast fusion

iii.  development of regenerated fertile plants from the fusion product (Hybrid).

History of Somatic Hybridization

 1) The term protoplasts was introduced in 1880 by Hanstein.

 2) The first isolation of protoplasts lasts was achieved by Klercker (1892) employing a mechanical method.

 3) In 1960 cocking used an enzymatic method for the removal of cell wall.

 4) Takabe et.al were successful to achieve the regeneration of whole, tobacco plant from protoplast.

5) In 1980 started protoplast fusion to improve plant genetic material and the development of transgenic plants.

Isolation of protoplasts:

Protoplasts are isolated by two techniques.

1. Mechanical Method.

2 Enzymatic Method.

Mechanical Method:

Klercker in 1892 pioneered the isolation of protoplasts by mechanical methods.

A small piece of epidermis from a plant is selected.

The cells are subjected to plasmolysis. This causes protoplasts to shrink away from the cell walls

The tissue is dissected with needle to release the protoplast. In this process some of the plasmolyzed cells were cut only through the cell wall, releasing intact protoplasts while some of the protoplasts may be damaged inside many cells. Protoplasts that were trapped in a cell and only the corner had been cut off could be encouraged to come out by reducing the osmolarity slightly to force the protoplasts swell to force their way out of the cut surface. The released protoplasts then have to be separated from damaged ones and cell debris.

The Protoplasts that are isolated through mechanical method having less number of viability cells.

It is restricted to certain tissues with vacuolated cells.

Disadvantages

• •  Lower protoplast yield.

•  Labour intensive method.

•  Protoplast obtained has low viability.

•  Method is applicable only to vacuolated cells.

 

 Enzymatic Method:

In 1960, E.C. Cocking demonstrated the possibility of enzymatic isolation of a large number of protoplasts from cells of higher plants. This method involves leaf sterilization followed by peeling of the lower epidermis to release cells which are plasmolyzed and added to enzyme mixture followed by harvest of protoplast as shown in. Either of the procedures for enzymatic isolation can be used: sequential enzymatic hydrolysis or mixed enzymatic hydrolysis.

The advantage of enzymatic method include good yield of viable cells and minimal (or) no damage to the protoplasts.

Protoplasts can be isolated from a wide variety of tissues and organs that include leaves, roots, shoot apices, fruits, embryos and microspores.

1.Two step (or) Sequential Method:

Firstly, cells are separated by the use of a maceration enzyme – a pectin hydrolyzing enzyme such as, macerozyme or Pectolyase. Once the cells are separated, due to degrading of middle lamella,  they are washed in CPW solution free of enzymes but containing plasmolyticum by gentle centrifugation (100g). The pellet is retained and resuspended in the second enzyme like, cellulases and hemicellulases, used to hydrolyse the remaining cell was component. These second enzymes removes the cell wall proper. Once the protoplasts are released they are washed with CPW to remove the debris.

One step (or)simultaneous method:

This is the preferred method for protoplast isolation. Plant tissues are plasmolyzed in the presence of a mixture of pectinases and cellulase. Allow it for centrifugation. Thus, inducing simultaneous separation of cells and degradation of their walls to release the protoplasts directly in a single step.

Protoplasts are present in supernatent and pellet contain debris and dead cells.

It is essential to ensure that the isolated protoplasts are healthy and viable so that they are capable of undergoing sustained cell divisions and regeneration. By using certain dyes, we can examine the protoplasts. Flouresecence diacetate, Evan’s blue, phenol, safranin dyes are used.

In this tests FDA turn green, Evan's blue turn blue, safranin turns Red  colour in the case of non viable protoplasts. Viable protoplasts are unstained.

Protoplast purification

Enzyme treatment results in suspension of protoplast, undigested tissues and cellular debris. This suspension is passed through a metal sieve or a nylon mesh (50-100 µm) in order to remove undigested large debris. The filtered protoplast-enzyme solution is mixed with a suitable volume of osmoticum, solution is centrifuged to pellet the protoplasts, pellet of protoplast is resuspended in osmoticum of similar concentration as used in enzyme mixture. The protoplast band is sucked in Pasteur pipette and is put into other centrifuge and finally suspended in culture medium at particular density.

 

 Culture of Protoplasts:

The very first step in protoplast culture is the development of a cellwall around the membrane of protoplast. This is followed by cell division that give rise to a small colony with suitable manipulations of nutritional and physiological Conditions. The cell colonies may be grown continuously as culture or regenerated to whole plants.

Protoplasts are cultured either in semisolid agar or liquid medium.

Culture Medium:

The culture Medium with regard to nutritional components and osmoticum are briefly described.

Nutrient Medium:

1. Mostly MS, B5 Media with suitable modifications are used.

2. The Medium should be devoid of ammonium and the quantities of iron and zinc should be less.

3. The concentration of calcium should be 2-4 times higher than used for cell cultures. This is required for generation.

4. High auxins / Kinetin ratio is suitable to induce cell divisions while high kinetin/ auxin ratio is required for regeneration.

5. Glucose is preferred carbon source by protoplasts

Osmoticum and osmotic Pressure

Osmoticum broadly refers to the reagents chemicals that are added to increase the osmotic pressure of a liquid. The isolation and culture of protoplasts require osmotic protection until they develop a strong cell wall.

Addition of an osmoticum is essential for both isolation and culture media of protoplasts to prevent their rupture. The osmotica are of two type-non- ionic and ionic.

1. Non-ionic: The non-ionic substances most commonly used are soluble carbohydrates such as mannitol, sorbitol, glucose, fructose, galactose and sucrose. Mannitol being metabolically inert is most frequently used.

Importance of Protoplast Culture

The isolation and fusion of protoplast is a fascinating field in plant research. Protoplast isolation and their cultures provide millions of single cells for a variety of studies. Protoplasts have a wide range of applications.

1. The protoplast in culture can be regenerated into a whole plant.

2. Hybrids can be developed from protoplast fusion.

3. It is easy to perform single cell cloning with protoplast

4. Genetic transformations can be achieved through genetic engineering of protoplast DNA.

5. Protoplasts are excellent materials for ultrastructure studies.

6. Isolation of cell organelles and chromosomes is easy from protoplasts.

7. Protoplasts are useful for membrane studies.

8. Isolation of mutants from protoplast cultures is easy.

 Protoplast Fusion

The conventional method to improve the characteristics of cultivated plants has been sexual hybridisation. The major limitation of sexual hybridization is that it can ben perfomed with in plants species or very closely related species. This restricts the improvements that can be done in plants.

In vitro fusion of isolated protoplasts to form a hybrid cell and its subsequent development to form a hybrid plants is called as somatic hybridization.

Plant protoplasts are of immense utility in somatic plant cell genetic manipulations and improvements in crops. Thus, protoplast provide a novel opportunity to create cells with neo genetic constitution. Protoplast fusion is a wonderful approach to overcome sexual incompatibility between different species of plants

Spontaneous Fusion

Protoplast fusion could be spontaneous during isolation of protoplast or it can be induced by mechanical, chemical and physical means. During spontaneous process, the adjacent protoplasts fuse together as a result of enzymatic degradation of cell walls forming homokaryons or homokaryocytes, each with two to several nuclei. Simply physical contact is sufficient to bring about the spontaneous fusion among the similar parental protoplasts. The occurrence of multinucleate fusion bodies is more frequent when the protoplasts are prepared from actively dividing callus cells or suspension cultures. Since the somatic hybridization or cybridization require fusion of protoplasts of different origin, the spontaneous fusion has no value. To achieve induced fusion, a suitable chemical agent (fusogen) like, NaNO₃, high Ca²⁺, polyethylene glycol (PEG), or electric stimulus is needed.

Chemical Fusion

i.    Fusion by means of Sodium Nitrate (NaNO₃): It was first demonstrated by Kuster in 1909 that the hypotonic solution of NaNO₃ induces fusion of isolated protoplast forming heterokaryon (hybrid

The isolated protoplast are exposed to a mixture of 5.5% of NaNO₃ in 10% sucrose solution. Incubation is carried out for 5 minutes at 35⁰C, followed by centrifugation. The protoplast pellet is kept in water bath at 30⁰C for about 30 minutes.

This method was fully described by Evans and Cocking (1975), however this method has a limitation of generating few no of hybrids, especially when highly vacuolated mesophyll protoplasts are involved.

 

ii. High pH and Ca⁺⁺ treatment: This technique led to the development of intra- and interspecific hybrids. It was demonstrated by Keller and Melcher in 1973. The isolated protoplasts from two plant species are incubated in 0.4 M mannitol solution containing high Ca⁺⁺(50 mM CaCl₂.2H₂O) with highly alkaline pH of 10.5 at 37°C for about 30 min. Aggregation of protoplasts takes place at once and fusion occurs within 10 min.

iii. Polyethylene glycol treatment: Polyethylene glycol (PEG) is the most popularly known fusogen due to ability of forming high frequency, binucleate heterokaryons with low cytotoxicity. With PEG the aggregation occurred mostly between two to three protoplasts unlike Ca⁺⁺ induced fusion which involves large clump formation. The freshly isolated protoplasts from two selected parents are mixed in appropriate proportions and treated with 15-45% PEG (1500-6000MW) solution for 15-30 min followed by gradual washing of the protoplasts to remove PEG. Protoplast fusion occurs during washing. The washing medium may be alkaline (pH 9-10) and contain a high Ca⁺⁺ ion concentration (50 mM). This combined approach of PEG and Ca⁺⁺ is much more efficient than the either of the treatment alone. PEG is negatively charged and may bind to cation like Ca⁺⁺, which in turn, may bind to the negatively charged molecules present in plasma lemma, they can also bind to cationic molecules of plasma membrane. 

During the washing process, PEG molecules may pull out the plasma lemma components bound to them. This would disturb plamalemma organization and may lead to the fusion of protoplasts located close to each other. The technique is nonselective thus, induce fusion between any two or more protoplasts.

Electrofusion: 

The chemical fusion of plant protoplast has many disadvantages – (1) The fusogen are toxic to some cell systems, (2) it produces random, multiple cell aggregates, and (3) must be removed before culture. Compare to this, electrofusion is rapid, simple, synchronous and more easily controlled. Moreover, the somatic hybrids produced by this method show much higher fertility than those produced by PEG-induced fusion.

Zimmermann and Scheurich (1981) demonstrated that batches of protoplasts could be fused by electric fields by devising a protocol which is now widely used. This protocol involves a two-step process. First, the protoplasts are introduced into a small fusion chamber containing parallel wires or plates which serve as electrodes. Second, a low-voltage and rapidly oscillating AC field is applied, which causes protoplasts to become aligned into chains of cells between electrodes. This creates complete cell-to-cell contact within a few minutes. Once alignment is complete, the fusion is induced by application of a brief spell of high-voltage DC pulses (o.125-1 kVcm^-1). A high voltage DC pulses induces a reversible breakdown of the plasma membrane at the site of cell contact, leading to fusion and consequent membrane reorganization. The entire process can be completed within 15 min.