Protoplast Fusion and Somatic Hybridization | Biotechnology

In this article we will discuss about:- 1. Method of Protoplast Fusion 2. Mechanism of Protoplast Fusion 3. Identification and Selection 4. Verification and Characterisation.

Isolated protoplasts are devoid of cell walls which make them easy tools for under­going fusions in vitro. An important factor is that generally there is incompatibility barrier between two protoplasts of different species or genera. The process of fusion may be spontaneous or it may be induced (Fig. 20.5).

Method of Protoplast Fusion:

(a) Spontaneous Fusion Method:

During enzymatic degradation of cell walls some of the adjacent protoplasts may fuse together to form homokaryons, sometimes more than two protoplasts fuse together and form multinucleate cells by a phenomenon where expansion and subsequent coalescence of the plasmodesmata led to such cases. However the spontaneous fusion products do not regenerate into whole plants except for undergoing a few divisions.

(b) Induced Fusion Method:

Freshly isolated protoplasts can be induced to undergo fusion, irrespective of their origin with the help of different kinds of fusogen (fusion inducing agents) e.g., NaNO₃, lysozyme, High pH/Ca⁺⁺, polyethylene glycol (PEG), concavalin, polyvinyl alcohol, electro-fusion, dextran sulphate, etc.

Some of these treatments are discussed here:

(i) NaNO₃ Treatment:

Isolated protoplasts are suspended in mixture of 5.5% NaNCO₃ and 10% sucrose soln. and incubated in a water bath at 35°C for 5 min. and centrifuged for 5 min. at 200 x g, following centrifugation the supernatant is decanted and the pellet is incubated at 30°C for 30 min. During this period most of the protoplasts undergo cell fusion. After sometimes these are washed with osmoticum and then plated properly in culture medium.

(ii) Calcium Ions at High pH:

Here the isolated protoplasts are incubated in a solu­tion of osmoticum containing 0.05 M CaCl₂ at pH 10.5 (using 0.05 µ glycine— NaOH buffer) and at temperature 37°C. Aggregation of protoplasts generally takes place at once and fusion occurs within 10 mins. After this treatment, 20-50% of the protoplasts have been found to be involved in fusion.

(iii) PEG Treatment:

PEG has been found to be used as a fusogen in most of the suc­cessful cases of protoplast fusion. 1 ml of protoplast suspension is equally mixed with 1 ml of 28-56% PEG (1500-6000 MW) solution. The tube is then allowed to settle for 10 min to 40 min at room temperature.

Both the mol. wt. and the conc. of PEG used is critical in inducing successful fusions. After PEG treatment the elution of fusogen is done by using high pH/Ca⁺⁺ containing solution which is most effective in enhancing the fusion frequency and survivability of protoplas­ts (Fig. 20.6A).

(iv) Electro-fusion:

Protoplasts are placed into a small cell culture containing electrodes and a potential difference is applied due to which protoplasts line up between the electrodes. In this fusion method, two step procedures is followed: a low voltage and rapidly oscillating AC field is applied, which causes the protoplasts to become aligned into chains by cell to cell contact.

Second step is brief application of a high voltage DC pulse which induces reversible breakdown of the plasma membrane at the site of cell contact, leading to fusion and consequent membrane reorganisation. Heterokaryons produced by this electro-fusion divide normally in culture medium and have the capability of regenerating the plantlet (Fig. 20.6B).

Mechanism of Protoplast Fusion:

Protoplast Fusion Consists of Three main Phases:

(i) Agglutination or Adhesion:

Two or more protoplasts are brought into close proximity by using a variety of treatment like PEG, high pH, high conc. of Ca⁺⁺ ions, etc.

(ii) Fusion of Plasma Membrane at Localised Sites:

Membranes of protoplasts agglutinated by fusogen get fused at the point of adhesion, which results in the formation of cytoplasmic bridges between the protoplasts and fusion requires less than 10A distance between two membranes. The high pH and high Ca⁺⁺ ions have shown to neutralize the normal surface charge so that the agglutinated proto­plasts can fuse due to intermingling of lipid molecules in membranes.

(iii) Formation of Heterokaryon:

Rounding off of the fused protoplasts occur due to the expansion of cytoplasmic bridges forming spherical heterokaryon or homokaryon.

Identification and Selection of Hybrid Cells:

Following fusion treatment the protoplast population consists of parental type proto­plasts, homokaryotic fused products and also heterokaryotic fusion products. The propor­tion of viable heterokaryotic fusion generally is lower, identification and recovery of proto­plast fusion products have been based on observation of visual characters or hybrid cells may show genetic complementation for some growth requirements, etc.

(a) Visual Selection:

In most of the fusion programme generally the selection pro­cedure involves the fusion between a non-green protoplasts of one parent and the green protoplast of another parent, as this facilitate the visual identification of heterokaryons under light microscope. The non-green protoplasts may be avai­lable from callus tissue and the green protoplasts from leaf tissue (Fig. 20.7A-B).

(b) Selection by Complementation:

If the parental protoplasts from the two parents can be identified by biochemical marker then the heterokaryons can be selected easily by using the proper growth requirement in media.

(i) Complementation of Resistance Markers:

Dominant characters such as traits conferring resistance to antibiotics, amino acid analogues or toxic com pounds have been selected as potent markers. When the protoplasts from two lines are being fused together then the fused product can be selected in presence of both the metabolites because of double resistance as compared to single parent.

(ii) Use of Metabolic Inhibitors:

The parental protoplasts are treated with irre­versible biochemical inhibitor such as iodoacetate or di-ethyl-pyro-carbamate and following treatment only hybrid cells will be capable of division.

(iii) Auxotroph Complementation:

Where two parental protoplasts are both mutated for the same enzyme but both of them are of two different muta­tional types. So the fused and hybrid protoplasts could be grown easily by complementing each other capable of producing active enzyme.

(iv) Chlorophyll Deficiency Complementation:

This is the frequently used method for selecting the somatic hybrid where the normal plant protoplasts are allowed to fuse with an albino or mutated or chlorophyll deficient type of protoplast. The fused products or somatic hybrids must be able to produce the green colonies by complementation.

Verification and Characterisation of Somatic Hybrid:

Somatic hybrids after regeneration should be verified through clear demonstration of genetic contribution of both the parents.

(i) Morphological Characters:

In most of the cases the somatic hybrids bear the characters from the parents or sometimes they have the somatic or sexual cha­racters intermediate of both the parents. Such traits may be leaf size, leaf surface, pigment, flower shape, pollen character, etc.

(ii) Isoenzyme Analysis:

Electrophoretic banding patterns of isoenzymes have been extensively used to verify hybridity. Somatic hybrids may display isozyme band­ing of certain enzymes specific to either the parents or sometimes newer type of banding. The enzymes like esterase, isoperoxidase, phosphatase, alcohol dehy­drogenase, etc. are studied.

(iii) Chromosomal Constitution:

Counting chromosomes in presumed somatic hybrid is a reliable and easy method of detection. Cytologically the chromosome num­ber should be the sum of chromosome number of two parents. Besides the num­ber, the size and structure of the chromosomes of both the parents are accounted for verification of somatic hybrids.

(iv) Molecular Technique:

With the availability of numerous molecular markers such as RFLP, AFLP, RAPD, microsatellites, etc. the somatic hybrid identifica­tion has become more easier. PCR technology are being utilised for hybrid identification. Specific restriction patterns of chloroplast and mitochondrial DNA have been used with great advantage to characterise the somatic hybrids.