We really would suggest that you read the previous post on protoplast isolation and regeneration, before reading this post.
As mentioned in the last post, protoplasts are cells that have had their cell walls removed, but possess plasma membrane and all other remaining cellular components. This removal of the cell wall barrier allows the fusion of protoplast with another protoplast.
Protoplast fusion leads to the mix of genetic information. The transfer of nuclear and cytoplasmic genetic information between plants belonging to different species or genera are not possible by traditional ways of sexual crosses.
For eg, the genes responsible for certain features can be transferred from a wild growing plant into important agricultural crops using the protoplast fusion.
Following the protoplast fusion, the hybrid protoplast contains the mix of cytoplasm and two parental nuclei. The fused protoplast is grown in vitro with an aim to obtain a hybrid plant. This in vitro fusion of plant protoplasts obtained from somatic cells of the two desired plants is called somatic hybridization.
Let us learn how the protoplast fusion is carried out:
• Protoplast Fusion procedure:
As we know, from the previous post, the protoplasts can be obtained either by mechanical or enzymatic methods. The intact protoplasts initiate the process of regeneration of new cell wall immediately after the cell wall degrading enzymes are removed and within a few days the cell wall is regenerated. Hence the protoplast fusion experiments must be carried out immediately after the isolation of protoplasts.
• Mechanism of fusion
Protoplast fusion consists of 3 steps:
1. Aggluttination
Protoplasts have a net negative charge on the surface that causes it to to repel the surrounding protoplasts. Hence, the cell should be brought in close proximity of each other.
2. Adhesion
Adhesion involves lipid-lipid interaction between the two cell membranes.
3. Fusion
The final step involves rearrangement of the membranes to form pores which eventually expands and results into cytoplasmic mixing and hence the protoplast fusion.
• Types of fusion
1. Spontaneous protoplast fusion
Spontaneous protoplast fusion is characterized by fusion of two or more protoplasts. It occurs spontaneously in the nature. This occurs in the protoplasts which remained interconnected through plasmodesmas.
(Just for info: Read to know about plasmodesmata.)
This is common phenomenon in the protoplasts isolated from pollen mother cells, which can fuse in the absence of an inducer. The membrane structure of these cells may be differ from other somatic cells considering pollen mother cell gives rise to the germinating pollen grains, which are involved in the fertilization process and hence membrane fusion.
2. Induced protoplast fusion
The protoplast fusion can be induced either by some chemicals or by electric field. Firstly, the two plasma membranes are brought in contact and the protoplast are induced to create destabilized regions on the membrane. When two such regions of adjacent protoplasts coincide, the membrane fusion occurs and produces cytoplasmic bridges. This results in the mixing of the two cytoplasms. Slightly deplasmolysing conditions are favourable for the fusion to take place.
– Types of induced fusion:
1. Chemical fusion
This type of induced fusion occurs in the presence of a relatively high concentration of chemical fusogen (NaNO, CaNO, polyvinyl alcohol, polyethylene glycol) usually combined with high pH (9.0–10.5) and Ca ions. These factors disrupt the integrity of the cytoplasmic membrane, e.g. they change its surface charge. Different chemical fusogen and their mode of action are given below:
a. Sodium nitrate solution
A slightly hypotonic aqueous sodium nitrate solution reduces the negative charge on the outer membrane thus allowing adhesion of protoplasts. The presence of the Na+ ion induces localized realignment of the lipo-protein in the membrane which in turn allows fusion to occur when two such regions come into contact. Slightly deplasmolysing conditions facilitate cytoplasmic coalescence. However, sodium nitrate did not appear to be effective in the large-scale production of viable fusion products.
b. Polyethylene glycol (PEG)
PEG was first used in 1974 by Kao and Michayluk to increase the frequency of the fused protoplasts of lucerne also known as alfalfa (Medicago sativa). It is available as polymeric compound in the range of molecular weights (200-20,000) (4000 and 6000 commonly used). PEG solutions induce tight adhesion of protoplasts owing to electrostatic forces. The presence of cations, such as Ca, in the PEG solution, enhances adhesion, as they form molecular bridge between the negatively polarized PEG molecule and the protein of the membrane. Water is also withdrawn from the protoplast by PEG, thus reducing the turgidity of the system and allowing closer packing of protoplasts. This dramatic disruption, and possible reversal, of the charge properties of the membrane after removal of PEG, leads to membrane fusion. PEG is more suitable for mesophyll protoplasts which are not damaged so much as during electrofusion (they can burst). Dehydration leads to asymmetry in the lipid packing pressure in the two leaflets of the membrane bilayer leading to formation of a single bilayer septum at a point of close apposition of two cell membranes. The single bilayer septum then decays during formation of the initial fusion pore. Agents that enhance or alleviate the dehydration-induced asymmetric packing stress will favor or inhibit fusion.
(Just for info: Read this paper on Polyethylene glycol mediated fusion of protoplasts from different species of Canna genus)
c. Solutions buffered at high pH and containing calcium ions also induces fusion.
This may resemble the action of PEG in reversing membrane charge properties in conjunction with the establishment of molecular bridges prior to membrane fusion.
2. Electrofusion
Electrofusion of the protoplasts occur in an equipment called the pulse generator, which is a source of short direct current, a switching unit for the application of alternating or direct pulses and a closet equipped with electrodes for the fusion. In electrofusion, the chains of protoplasts between the electrodes are characteristic. Protoplasts are in contact and exposed to a suitable electric pulse field. The electric pulses induce the development of temporary holes in the plasma membrane. The membrane functions as an insulator and has a high electric resistance. As difference of the potential is increased through the membrane, the voltage of the membrane collapses and a hole (pore) is formed. The critical value of voltage is between 0.5–1.5 V, depending on the composition of the cytoplasmic membrane or the type of cell. The intensity and length of the pulse needed for the fusion depends on the size of the protoplast. Viability and efficiency are increased if shorter pulses of higher voltage are used rather than longer pulses of lower voltage.
The advantages of electrofusion are Speed (By setting the electrodes efficiently, even great numbers of protoplasts may be exposed to the pulse field at the same time.), Simplicity, Synchrony, Ease of control, Exclusion of chemical fusogens.
• Products of fusion
By using any method or fusogens, three different products are formed after the fusion procedure are heterokaryon (binucleate) synkaryon/ hybrids or cybrids, homokaryon and the unfused donors
– Heterokaryon cells
Cell in which there are protoplasm, organelles and genetic material from both protoplast donors is called heterokaryon or hetero-karyocyte in the early fused state. It contains a mixture of cytoplasmic components, but the nuclei of both parents have not fused yet. Heterokaryon will then undergo cell division, during which it may rapidly lose one of the nuclei or nuclear fusion may occur and nuclear gene may be lost due to chromosomal elimination. When nuclei are fused the cells are known as hybrid or synkaryocyte
A type of heterokaryon cells are called Cybrids. In this one protoplast contributes to cytoplasm only and other donor will donate either only the nuclei or both the nuclei and cytoplasm (mitochondria and chloroplast genes). It can be done using lethal dose of X rays or gamma radiation (the nucleus of one species is inactivated before the fusion) followed by centrifugation. The economically important features controlled by cytoplasm can be transferred e.g. male sterility, some types of resistance to herbicides and resistance to diseases, the creation of nectar and resistance to fungal toxins
Several factors affect the survival of hybrids produced by the process of the protoplast fusion. First and foremost, the physiological and genetic differences of partner cells, i.e. the compatibility of the two donors. The quality of the donor protoplasts is also important. The protoplasts should be viable and not much damaged.
• Selection of Somatic Hybrid:
Once the protoplast fusion procedure is done, the somatic hybrids immediately after the procedure should be selected appropriately. The different techniques for the selection are
1. Manual selection
In the manual selection, the visual identification of the fusants are done and they are then mechanically isolated with the help of micromanipulator (“fishing”). This technique is precise, direct and reliable but time-consuming. This method requires the fused protoplasts to be morphologically different or stained fluorescently.
Eg: Fusion of green mesophyll protoplasts containing chloroplasts with colourless protoplasts of cell cultures containing vacuoles or starch grains, or with protoplasts from etiolated tissues. The chloroplasts are visible in one part of the cell and the vacuoles or starch grains in the other part. Additional fusion of chloroplasts in the whole cell appears shortly after the fusion.
Double fluorescent staining for heterokaryons, e.g. one protoplasts stained with fluorescein diacetate other with rhodamine isothiocyanate
2. Flow cytometry
in flow cytometer the protoplasts flow fluently between the light source and fluorescence detectors, the flow is dispersed into droplets and the computer deflects electrostatically the droplets containing heterokaryons into different test tubes. Double fluorescent staining is used and selecting protoplasts during the flow through the capillary of the flow cytometer. Advantages of using flow cytometry is that a high number of fused protoplasts may be separated. This process is fully automatic and quick
• Identification of somatic hybrids after establishment
1. Morphological
The somatic hybrid is morphologically similar to both parents, such features are included in vegetative or floral morphology. E.g. in interspecies hybrids of tobacco Nicotiana tabacum + N. glauca. Some features present only in one parent are present in all hybrids and behave as dominant.
2. Cytological
The viability of pollen and the number of chromosomes. The somatic hybrids between more distant species have lower viability of pollen than the parent species. The number of chromosomes may be equal to the sum of chromosomes of both parents or completely different (asymmetric hybridization, complete chromosome set, individual chromosomes, fragmentation, polyploidy).
3. Biochemical identification
This includes analyses of isoenzymes, partial proteins, secondary metabolites, resistance of plants against viral infection and antibiotics or sensitivity to herbicides and fungal toxins.
4. Genetic analysis
Genetic analysis can be undertaken only if the hybrid plants are fertile. Many hybrid plants of distant related species are sterile. RFLP and RAPD can be used for the comparison of genotypes. Flow cytometry can be used to analyze the nuclear DNA to establish ploidy.
• Variability among somatic hybrids
The variability is higher in protoplast fusion hybrids, than in a comparable population of plants after sexual crossing. The four potential sources of variations are nuclear incompatibility, mitotic recombination, somaclonal variation and segregation of organelles.
• Utilization of somatic hybrids
Somatic hybrids can be further utilized for different purposes. However, the somatic hybrids must be capable of sexual reproduction to allow continuous supply of the somatic hybrids. The somatic hybrids must contain a mixture of genes from both parent donors, which is the main purpose of the whole fusion procedure. They must be capable of retrospective crossing into cultivated crops for the development of a new variety. Some hybrids have been confirmed to have acquired desirable characteristics from the donor cell, for e.g. the resistance to diseases as observed in the transfer of the resistance to TMV in somatic hybrids obtained by fusing protoplasts of Nicotiana tabacum and Nicotiana nesophyla.
• The Uses of Somatic Hybridisation
– The somatic hybridisation using protoplast fusion allows the bypassing of the sexual incompatibility and sexual sterility.
-It allows the study of cytoplasmic inheritance.
-The protoplasts can be utilized as material for mutagenic treatment due to absence of the cell wall.
-Also the protoplasts can take up the foreign genetic material.
The examples of the genes or traits transferred by the help of protoplast fusion are genes of resistance to different virus and fungal diseases from wildly growing species, cytoplasmic male sterility (CMS), resistance to stress including tolerance to salinity, cold, drought, resistance to insect parasites (synthesis of phytoalexins), and genes for synthesis of reserve proteins, vitamins, secondary metabolites of pharmaceutical importance.
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