Introduction
- The process of fusion of protoplasts isolated from somatic cells of two different plant species/cultivars to regenerate hybrid plants is called as Somatic hybridization.
- Plant protoplasts offer exciting possibilities in the fields of somatic cell genetics and crop improvement.
- In somatic hybridization the nucleus and cytoplasm of both parents are fused in the hybrid cell. Sometimes, nuclear genome of only one parent but cytoplasmic genes (plastome) from both the parents are present in the fused hybrid, cybrid or cytoplasmic hybrid.
- Protoplast fusion technique can be used to overcome the barriers of incompatibility and acts as a method for the genetic manipulation of plant cells.
- It provides us with an opportunity to construct hybrids between taxonomically distant plant species beyond the limits of sexual crossability. It also creates cells with new genetic, nuclear as well as cytoplasmic constitutions that otherwise cannot be obtained.
- Somatic hybridization involves the fusion of protoplasts, selection of hybrid cells and identification of hybrid plants.
Overview of protoplasts
- Protoplast: Plant cells without the cell wall
- Mechanical and Enzymatic preparation of Protoplasts.
- Purification of the Protoplasts
- Protoplast viability and culture
- Factors that affect protoplast cultures
- Applications of protoplasts.
Protoplast: introduction, isolation, purification, culture and applications
check this post
Methods of Protoplast Fusion
- Spontaneous Fusion
- Induced Fusion
- Chemofusion
- Electrofusion
Spontaneous Fusion
- Plant cells are connected by plasmodesmata
- Protoplast by chance come close to each other and fuse together automatically, without any apparent external stimulus.
- Resulting product is called as homokaryons or homokaryocytes.
- May result in multinucleated structures.
- Frequency may be higher, when protoplast are prepared from actively dividing cultured cells.
Induced Fusion
Fusion of freely isolated protoplasts from different sources with the help of fusion inducing chemical agents is known as induced fusion. To achieve this, a suitable agent (fusogen) is added to fuse the plant protoplasts of different origins. The different fusogens employed are: NaNO3, artificial seawater, lysozyme, high pH/Ca++, polyethylene glycol, antibodies, concavalin A, polyvinyl alcohol, electrofusion dextran and dextran sulphate, fatty acids and esters
The isolated plant protoplasts can be induced to fuse by three ways:
- Mechanical Fusion
- Chemo-Fusion
- Electro Fusion
Mechanical Fusion
In this process, the isolated protoplasts are brought into intimate physical contact mechanically under microscope using micromanipulator and perfusion micropipette. This micropipette is partially blocked within 1 mm of the tip by a sealed glass rod. In this way the protoplasts are retained and compressed by the flow of liquid. By this technique occasional fusion of protoplast has been observed.
Chemo-Fusion
Chemical fusogens cause the isolated protoplasts to adhere to one another and leads to tight agglutination followed by fusion of protoplast.
- Fusion Induced by Sodium or Potassium Nitrate
- Fusion Induced by Calcium Ions at High pH
- Fusion Induced by PEG
Fusion Induced by Sodium or Potassium Nitrate
Fusion of isolated onion sub-protoplasts plasmolysed with sodium salts was achieved for the first time by Kiister (1909). Subsequently, Michel (1937) demonstrated fusion between protoplasts using potassium nitrate as plasmolyticum. Power et al (1970) reported sodium nitrate induced fusion of cereal root proto-plasts.
By this method, equal densities of protoplasts from two different sources are mixed and then centrifuged at 100 g for 5 minutes to get a dense pellet. This is followed by addition of 4 ml of 5.5% sodium nitrate in 10.2% sucrose solution to resuspend the protoplast pellet. The suspended protoplasts are kept in water-bath at 35° C for 5 minutes and again centrifuged at 200 g for 5 minutes. The pellet is once again kept in water-bath at 30°C for 30 minutes.
Fusion of protoplast takes place at the time of incubation. The, pellet is again suspended by 0.1% sodium nitrate for 5-10 minutes. The protoplasts are washed twice with liquid culture medium by repeated centrifugation. Finally, the protoplasts are plated in semisolid culture medium. The frequency of fusion is not very high in this method. Yet it is useful only for the protoplasts derived from meristematic cells
Fusion Induced by Calcium Ions at High pH
In 1973, Keller and Melcher (from Germany) developed a method to effectively induce, fusion of tobacco protoplast at high temperature (37° C) in media containing high concentration of Ca2+ ions, (i.e., calcium chloride) at a highly alkaline condition (pH 10.5).
Equal densities of protoplasts are taken in a centrifuge tube and the protoplasts are spun at 100 g for 5 minutes. The pellet is suspended in 0.5 ml of medium. 4ml of 0.05M CaCl22H20 in 0.4M mannitol at pH 10.5 is mixed to the protoplast suspension. The centrifuge tube containing protoplasts at high pH/Ca2+ is placed in the water bath at 30° C for 10 minutes and is spun at 50 g for 3-4 minutes. This is followed by keeping the tubes in water bath (37°C) for 40-50 minutes. About 20- 30% protoplasts are involved in this fusion experiment.
Fusion Induced by PEG
In 1974, Kao and Michayluk from Canada discovered another fusion inducing chemical polyethylene glycol (PEG) which is the most effective agent discovered so far. Many fusion experiments are performed by a polyethylene glycol.
PEG induces protoplast aggregation and subsequent fusion. But the concentration and molecular weight of PEG are important with respect to fusion. A solution of 37.5% w/v PEG of molecular weight 1,540 or 6,000 aggre-gates mesophyll and cultured cell protoplasts during a 45 minutes incubation period at room temperature.
Fusion of protoplast takes place during slow elution of PEG with liquid culture medium. Carrot protoplast can be fused by 28% PEG 1540 and the fusion can be promoted by Ca2+ ion at the concentration of 3.5 mM. But higher concentration of Ca2+ ion (10 or 50 mM) has been considered beneficial.
In some studies, high pH/Ca2+ and PEG method have been combined. By this method, the agglutination of protoplasts can be brought about using sufficient quantities (0.1-5 ml) of protoplast in centrifuge tube or micro densities (150 µ l) of protoplast on a coverslip. The PEG method has been modified slightly to fuse higher plant protoplast
The modifications are given below:
- PEG is more effective when it is mixed with 10-15% dimethyl sulfoxide (DMSO).
- Addition of concanavalin A (Con A) to PEG increases protoplast fusion frequency.
- Sea water has been used alone or in combination with PEG to fuse protoplasts.
The PEG method has been widely accepted for protoplast fusion because it results in reproducible high-frequency heterokaryon formation, low cytotoxicity to most cell types and the formation of binucleate heterokaryons. PEG-induced fusion is non specific and is therefore useful for interspecific, intergeneric or interkingdom fusions.
Electro Fusion
Electro fusion is a modern technique of protoplast fusion which involves the use of mild electrical fields in protoplast suspension for inducing protoplast fusion. This technique is very easy, simple and fast. It is often more efficient than chemical induced fusion (chemo fusion).
Electrofusion is also applicable to those species whose protoplasts exhibit a severe toxic response to polyethylene glycol used for chemo fusion. The origin of electrofusion is based on biophysical studies of cell membrane.
The first report of electrofusion came from Senda et al. in 1979. They used microelectrodes to apply a 5-12 μAmp DC pulse to adhering pairs of Rauwolfia protoplasts to induce fusion. However, their fusion yields were low – restricted to just single protoplast pairs. This limited the utility and scalability of the approach. In 1981, Zimmermann and Scheurish significantly improved on the method to enable large scale electrofusion of plant protoplasts.
Electrofusion steps
- Electro fusion is a modern technique that uses mild electric fields to induce fusion between protoplasts in suspension. It is easier, simpler, and faster than chemical fusion methods.
- It involves two main steps:
- An alternating current (AC) field causes the protoplasts to align in chains between electrodes in a “pearl chain arrangement”. This brings the cell membranes into close contact.
- A brief high voltage direct current (DC) pulse induces membrane breakdown and fusion at the contact points.
- The AC field frequencies used are typically 0.5-1 MHz with field strengths of 100-200 V/cm. The DC fusion pulses are around 1000 V/cm applied for 10-15 μs.
- Cell alignment happens through “dielectrophoresis” – the AC field causes charge separation at the cell surface, resulting in attractions between adjacent cells.
- Fusion occurs through “electroporation” – the DC pulse transiently opens pores in the membrane, allowing reorganization into a fused membrane when pores open at cell contact sites.
- Low conductivity media is optimal to allow sufficient dielectrophoretic force. Some Ca2+ can help stabilize membranes.
- High cell densities or a small amount of PEG can also facilitate cell contacts.
- Electrofusion yields higher efficiencies than chemical fusion methods in many species and avoids toxicity issues and it’s faster.
Identification and selection of hybrid cells
Following fusion treatment, the protoplast population consists of parental types, homokaryotic fusion products of parental cells, heterokaryotic fusion products or hybrids and a variety of other nuclear cytoplasmic combinations.
Identification and recovery of protoplast fusion products have been based on observation of visual characters, or hybrid cells display genetic complementation for recessive mutations and physiological complementation for in vitro growth requirements. In complementation, fusion of two protoplasts each carrying a different recessive marker, will generate a fusion product which is functionally restored since each parent contributes a functional allele that corrects the respective deficiency of the other parent. Methods of identification includes the folllowing:
- Chlorophyll deficiency complementation
- Auxotroph complementation
- Complementation of resistance markers
- Use of metabolic inhibitors
- Use of visual characteristics
Verification and Characterization of Somatic Hybrids
Protoplasts from any two species can be fused together. However, there are a number of limitations to widespread utilization of somatic hybridization in higher plants, including aneuploidy, species barriers to hybridization, and the inability to regenerate plants from protoplasts. Hybrids must be verified as products of somatic fusion of two different protoplasts through various mathods:
- Morphology
- Isoenzyme analysis
- Chromosomal constitution
- Molecular techniques
- Genetic characterization
Cybrids
Somatic hybrids can be obtained where nucleus is derived from one parent and cytoplasm is derived from both, thus producing cytoplasmic hybrids, also called as cybrids. Early segregation of nuclei in a fused product can be stimulated and directed so that one protoplast contributes the cytoplasm while the other contributes the nucleus both nucleus and cytoplasm. There are different ways of inactivating, the nucleus of one protoplast can be inactivated. Thus, there will be fusion between protoplasts containing the full complement of nucleus, mitochondria and chloroplasts with functional cytoplasmic component of second parent.
- The method of combining the nuclear genome of one parent with the cytoplasmic genome (chloroplast / mitochondria) of any/both parent is called cybridization. Hybrid produced are called as Cybrids.
- Cybrids has cytoplasmic genome of one parent and nuclear genome of 2nd parent.
Application of cybrids would be the directed transfer of cytoplasmic male sterility or herbicide resistance from a donor to a recipient crop plant species. Transfer of cytoplasmic male sterility (CMS) from N. tabacum to N. sylvestris by protoplast fusion was first reported. Resistance of plants to herbicide atrazine has been transferred from Brassica campestris to B. napus via fractionated protoplast fusions.
Types of Hybrids
- Symmetric Hybrids
- Asymmetric hybrids
Symmetric Hybrids
- Hybrids would have the full genome of both the parents.
- Produced in several species like citrus, solanum, chrysanthemum etc.
- Usually results in production of polyploids, several allotetraploids in citrus species. Tetraploids*diploids will produce triploids (seedless).
- Gene conflict may arise and causes genetic imbalance, eg. fruits may present undesirable characteristics such as irregular, thick skin which, to some degree, limits their utilization.
- Significant amount of unwanted genetic material is involved.
- Results in
- Recalcitrant calli and Abnormal growth
- Low regeneration and low fertility.
- Difficulty in rooting.
Asymmetric Hybrids
- The nuclear genome of one parent may partially be eliminated.
- Chromosome elimination can be spontaneous or induced artificially.
- Asymmetric hybrids has higher regeneration capacities and better survivability. Regenerated plants has increased fertility.
Potential and applications of somatic hybridization
- Production of novel interspecific and intergeneric crosses between plants that are difficult or impossible to hybridize conventionally. It overcomes sexual incompatibility barriers. For example, fusion between protoplasts of (tomato) and (potato) created the pomato.
- Somatic hybridization for gene transfer
- Disease resistance: The families Solanaceae and Brassiceae contain the most commonly used species for somatic hybridization. Both interspecific and intergeneric hybrids have been obtained. Many disease resistance genes viz. potato leaf roll virus, leaf blight etc. have been transferred to Solanum tuberosum from other species where normal crossings would not be possible due to taxonomic or other barriers.
- Abiotic stress resistance: Work related to somatic hybridization for abiotic stress has been mainly done on families Brassicaceae, Solaneae and relates to cold and frost resistance. Rokka developed somatic hybrids between cultivated potato (S. tuberosum) and wild relative (S. acaule) possessing several disease and early frost resistance characters.
- Quality characters: Somatic hybrids produced between B. napus and Eruca sativa were fertile and had low concentration of erucic acid content. This hybrid material has been introduced into breeding programme. Likewise, nicotine content character has been transferred to N. tabacum.
- Cytoplasmic male sterility: Several agriculturally useful traits are cytoplasmically encoded, including some types of male sterility and certain antibiotic and herbicide resistance factors. Earle (1997) produced cold tolerant cytoplasmically male sterile (cms) cabbage (Brassica oleracea ssp. capitata) by the fusion of cabbage protoplasts with cold tolerant ogura CMS broccoli lines.
- Production of auto tetraploids: Somatic hybridization can be used as an alternative to obtain tetraploids and, if this is unsuccessful, colchicine treatment can be used.
- Protoplasts of sexually sterile (haploid, triploid, aneuploid, etc.) plants can be fused to produce fertile diploids and polyploids.
- Hybridization becomes possible between plants that are still in the juvenile phase.
- Production of heterozygous lines within a single species that normally could only be propagated by vegetative means, e.g. potato and other tuber and root crops.
- Somatic cell fusion is useful in the study of cytoplasmic genes and their activities. This information can be applied in plant breeding experiments.
- The production of unique nuclear-cytoplasmic combinations. Evidence from a number of somatic hybrids suggests that although two types of cytoplasm are initially mixed during protoplast fusion, resulting in heteroplasmons, eventually one parent type cytoplasm predominates, resulting in cytoplasmic segregation. Mitochondrial and chloroplast recombination has also been reported to result in unique nuclear- cytoplasmic combinations. These unique combinations using protoplasts will aid the development of novel germplasm not obtainable by conventional methods
Problems and limitations of somatic hybridization
- Application of protoplast methodology requires efficient plant regeneration from protoplasts. Protoplasts from any two species can be fused. However, production of somatic hybrid plants has been limited to a few species.
- The lack of an efficient selection method for fused product is sometimes a major problem.
- The end products after somatic hybridization are often unbalanced (sterile, misformed, and unstable) and are therefore not viable, especially if the fusion partners are taxonomically far apart
- Somatic hybridization of two diploids leads to the formation of an amphidiploid which is generally unfavorable (except when tetraploids are formed intentionally). For this reason in most cases, the hybridization of two haploid protoplasts is normally recommended.
- Regeneration products after somatic hybridization are often variable due to somaclonal variation, chromosome elimination, translocation, organelle segregation etc.
- It is never certain that a particular characteristic will be expressed after somatic hybridization.
- The genetic stability during protoplast culture is poor.
- To achieve successful integration into a breeding programme, somatic hybrids must be capable of sexual reproduction. In all cases reported, somatic hybrids containing a mixture of genes from two species must be backcrossed to the cultivated crop to develop new varieties. All diverse intergeneric somatic hybrids reported are sterile and therefore have limited value for new variety development.
- To transfer useful genes from a wild species to a cultivated crop, it is necessary to achieve intergeneric recombination or chromosome substitution between parental genomes.
References
- Introduction to Plant Biotechnology,Third Edition, H.S. Chawla. ISBN 978-1-57808-636-8
- Woo, Hyun-A & Ku, Seong & Jie, Eun Yee & Kim, HyeRan & Kim, Hyun-Soon & Cho, Hye Sun & Jeong, Won-Joong & Park, Sang Un & Min, Sung Ran & Kim, Suk. (2021). Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus. Scientific Reports. 11. 15120. 10.1038/s41598-021-94597-4. ↩︎