Protoplast Fusion – An Innovating Technique in Plant Genetics

 

Abstract

Protoplasts are the spherical membrane-covered plant cells whose cell wall is removed by mechanical procedures or digestive enzymes. They are isolated from a broad spectrum of plant varieties. Protoplasts fusion is a valuable tool to transmit genes with desired characteristics and produce hybrids containing valuable traits and enhanced quality. In this review, we have discussed the mechanism of protoplast fusion and different methods by which this phenomenon can be achieved. Moreover, by the end of this study, you will also learn about the importance of protoplast fusion in various biotechnological applications.

Introduction

Protoplast fusion or somatic fusion is an innovative technique of genetic engineering, which allows us to enhance the quality and quantity of plants and overcomes numerous sexual barriers in plant breeding.

What is a Protoplast?

Before you learn about protoplast fusion, you need to know what a protoplast is basically. Usually, a cell wall encloses the internal components of plant cells to shape, support, and protect the cell. However, if you remove that cell wall either mechanically or enzymatically, protoplasts will be released. 

Cellulose and pectinase enzymes are typically involved in the enzymatic digestion of the cell wall. Cellulase enzymes dissolve the cellulose in the cell wall while pectinase degrades the pectin that holds cells together.

After removing the cell wall, the resulting membrane-bound spheres are protoplasts that possess the remaining functional components of the cell except for the cell wall. 

What is Protoplast Fusion?

Protoplast fusion is a novel genetic modification tool that fuses two distinct genetically originated protoplasts from different somatic cells to produce a new hybrid plant with the characteristics of both parent plants. 

Researchers have produced hybrids among similar species, like between flowering and non-flowering potato plants, as well as between two distinct species are also produced, like wheat Triticum and rye Secale to generate Triticale. (Begna, 2020)

By this technique, you can transmit genes of your required quality and quantity into the hybrid protoplast. Hence by using this tool, you can produce favorable characteristics in the hybrid plant, for example, improved yield of bioproducts, rapid growth rate, enhanced quality of protein, nitrogen fixation, pests’ resistance, and resistance against plant diseases and severe environmental conditions. (Habte et al., 2018) It can also generate a combination of desirable nuclear and cytoplasmic traits that are not typically obtainable by traditional breeding. Moreover, this method also allows us to do genetic modification in vegetatively propagated crops and create hybrids among sexually incompatible strains. (Anthony et al., 1995) 

Mechanism of Protoplast Fusion

As the degrading enzymes remove the cell wall of the protoplast, a new cell wall regeneration process initiates almost immediately, and the cell wall gets regenerated within few days of removal. Hence, the experiment of protoplast fusion is started quickly after the isolation of protoplasts.

According to Verma et al., the mechanism by which two protoplasts get fused is not yet completely known. However, to explain the mechanism of this phenomenon, researchers have given several explanations, as described below.

Physical contact among protoplasts is essential in the initial level of protoplast fusion. However, there are negative net charges at the plasma membrane surface due to the presence of phospholipids that generates an effect of repulsion between both the cells, so their physical contact cannot occur naturally.

Fusogen agents are used to overcoming the problem of repulsion during chemical fusion. It involves a high molecular weight polymer polyethylene glycol which has similar polarity as the molecules of the phospholipid membrane. PEG binds to this phospholipid membrane, causing aggregation of cells resulting in the formation of a bridge connecting both the protoplasts. Moreover, the hydrophilicity of this reagent removes the water content from protoplasts. Thus, this decreases the turgidity of the system and forms tighter packing among protoplasts. The addition of high pH divalent ions like Ca++ neutralizes the typical negative charge on the membrane surface and reduces the electrostatic force to ease the close contact between agglutinated protoplasts. It also assists in the formation of pores so that fusion can occur.  With the elimination of PEG from protoplasts, the membranes rupture and transfer the genetic material; as a result, the fusion of protoplasts occurs quickly.

In electrical fusion, the polarity of a non-uniform electric field is temporarily adjusted via dielectrophoresis, generating a passage between the protoplasts. Then electrical impulses are applied to produce a rearrangement in the lipid bilayer and create water-soluble pores in the membrane. As a result, the cells enter the fusogenic state, and the fusion of protoplasts occurs. (Echeverri et al., 2019)

During the last stage of the fusion process, the molecular distance among the protoplasts becomes 10A or less. The cytoplasmic bridge expands; the fused protoplasts round off and produce spherical heterokaryon or homokaryon.

Methods of Protoplast Fusion

There are two main methods, by which protoplasts can be fused,

1. Spontaneous Fusion

During isolation of protoplasts, sometimes spontaneous fusion occurs in which two or more protoplasts spontaneously fuse. In this case, if protoplasts belong to similar parental plants, just a simple physical contact is enough to cause spontaneous fusion. In this phenomenon, a multinucleate protoplast will be produced by the fusion of two protoplasts whose plasmodesmata remained interconnected. This type of fusion is strictly intraspecific and hence only gives rise to homokaryons.

After the complete isolation of protoplasts, spontaneous fusions do not occur in any plant except the lily family. In this family, there is a possibility that spontaneous fusion might occur among protoplasts that are isolated from microsporocytes of few plants. This type of fusion causes intergeneric fusion. For example, the microsporocyte protoplast of Lolium longiflorum spontaneously fused with the protoplast of Trillium kamtschaticum. 

2. Induced Fusion

In this type of fusion, chemical agents are used for inducing fusion, mostly between protoplasts isolated from different sources. Due to negative charges on their plasma membrane, such protoplasts do not fuse easily, so external means are used to induce fusion. An example of such agents is PEG (polyethylene glycol). 

This technique is crucial in the case of sexually incompatible plants and is mostly employed to fuse distinct genotypes behind the boundaries of the sexual rule.

Below I have explained some most used types of induced fusion,

2.1.Chemo Fusion

In this fusion, the cells are submerged in an alkaline solution of 9.0 to 10.5 pH, along with the fusing agent, an osmotic regulator (to prevent any rupture of the protoplasts), as well as divalent cations (to enhance the fusion of membranes). 

Polyethylene glycol (PEG) is a fusogen that is generally used in most biotechnological applications. It is considered a universal fusing agent due to its superb stability in addition to its excellent binding action. Calcium ion is most frequently used as a divalent with PEG.

After the formation of hybrid, its phenotypic and genotypic ability is the main focus of study. (Echeverri et al., 2019)

 

2.3.Mechanically Induced Fusion

In this fusion, membranes of two isolated protoplasts are joined under the microscope by mechanical means. Mostly micromanipulators or perfusion micropipettes are used in this process. (Verma et al., 2008)

A sealed glass rod is utilized to shape the tip of the micropipette, which retains and compresses the protoplasts by the flow of liquid. Occasional protoplasts fusion is observed by this method. 

3.3. Electro-Fusion

Have you heard about the reversible electrical rupture of cell membrane? In this process, the membrane is polarized in an electric field at a voltage of 0.5-1.5 V, and within a short period, the membrane gets ruptured.  As the protoplasts get exposed to an electric field, their permeability reversibly increases, which results in a disturbance of electric charges on their surface, and fusion occurs between neighboring protoplasts.

This phenomenon does not distort the natural structure of the membrane. It gets restored within few seconds to minutes depending on the conditions of the experiment and the composition of the membrane.

Electrofusion is more advantageous as compared to chemo fusion because of its easier fusion conditions and faster procedure. Moreover, there is also no requirement of chemical agents, which may affect the viability of the cells. (Echeverri et al., 2019)

Application of Protoplast Fusion

 

Application of Protoplast Fusion

1.      A Tool For Genetic Engineering

Gokhale et al., in their article, hoped that protoplast fusion would open the door of novel scopes in the genetics of microorganisms fortifying the conventional techniques used in biotechnology. 

This technique allowed chromosome mapping of several organisms including, Streptomyces species, Staphylococcus aureus, B. stearothermophilus, and B.subtilis. Preliminary positions for 11 markers were identified on the genetic map of Streptomyces clavuligerus using protoplast fusion. (Gokhale et al., 1993)

Moreover, this technique also plays a critical role in the development of genetic recombination as well as hybrid strains in filamentous fungi. It is an important tool for creating desired properties in strains by combining genes from distinct species. (Verma et al., 2008).

2.      Disease Resistance

Protoplast fusion is a leading technique used to transmit the genes of disease resistance between various plants. It transfers disease-resistant genes between closely related species as well as distantly related ones. This tool also prevents any challenges faced during the introduction of genes. (Harms et al., 2018)

Biotic and abiotic constraint-resistant rootstocks were created in citrus plants by cybridization and somatic hybridization. That not only increased the yield but also improved the quality of the fruit. (Dambier et al., 2011).

1.      Enhancement in Nutritional Quality

The aquatic food chain is the primary natural source of nutrition. An example of such a nutritional source is Chlorella vulgaris species which are widely used as dietary supplements. Bioactive chemical components like vitamins, proteins, polysaccharides, lipids, and minerals are also produced by microalgal species. (Ishaku et al., 2020)

Lee and Tan used somatic hybridization and fusion to produce genetically enhanced traits in the algal strains. To improve the nutritional content of algae algae-algae protoplast fusion proves to be more efficient than conventional methods like selection and mutation. Its strong efficiency increases the production of nutritionally valuable metabolites. (Lee and Tan, 1988)

Conclusion

Protoplast fusion is an important biotechnological tool used for genetic modification and plant breeding. Various mechanical and enzymatic procedures remove the cell wall, and then the protoplasts are fused to transmit valuable traits and produce hybrids with desired characteristics. Researchers are using many methods employing different fusogen agents to carry out the phenomenon of protoplast fusion. Hybrid plants with favorable attributes like enhanced nutritional quality, improved yield, and disease and pest resistance have been produced. Moreover, protoplast fusion continues to be a critical investigation tool in the research sector of biotechnology. It is expected, in the future, this technique will be one of the most commonly applied research mechanisms for tissue culturists, molecular biologists, biochemical engineers, and biotechnologists.

 

References

• Begna, T. (2020). Review on Somatic Hybridization and Its Role in Crop Improvement.
• Habte-Tsion, H. M., Kumar, V., & Rossi, W. (2018). Perspectives of nonstarch polysaccharide enzymes in nutrition. In Enzymes in Human and Animal Nutrition (pp. 239-254). Academic Press.
• Anthony, P., Marchant, R., Blackhall, N. W., Power, J. B., & Davey, M. R. (1995). Chemical fusion of protoplasts. In Plant Tissue Culture Manual (pp. 1-15). Springer, Dordrecht.
• Chiang, M. S., Chong, C., Landry, B. S., & Crete, R. (1993). Cabbage: Brassica oleracea subsp. capitata L. In Genetic improvement of vegetable crops (pp. 113-155). Pergamon.
• Peng, Z., Tong, H., Liang, G., Shi, Y., & Yuan, L. (2018). Protoplast isolation and fusion induced by PEG with leaves and roots of tea plant (Camellia sinensis LO Kuntze). Acta Agronomica Sinica, 44(3), 463-470.
• Verma, Nitin, M. C. Bansal, and Vivek Kumar. "Protoplast fusion technology and its biotechnological applications." Chem Eng Trans 14 (2008): 113-120.

 

 By: Hadia Islam