The below mentioned article provides an overview on Protoplast Fusion and Somatic Hybridization.
Plant breeders sometimes face difficulties due to sexual incompatibility. This difficulty can be removed by protoplast fusion. By this method wider crosses are possible. Even crosses between plants showing physical and chemical incompatibilities are possible by protoplast fusion.
In 1909 Kunster reported random fusion between mechanically isolated protoplasts. Method of fusion of naked protoplasts of somatic cells (Fig. 24) was developed in 1969. This is called ‘Para sexual hybridization’. In 1972 Bhojowari and Cocking used isolated protoplasts from pollen grains for this purpose.
Some of the terms used in somatic hybridization are defined here. When two or more protoplasts fuse then their cytoplasm always fuse, but the nuclei may or may not fuse. A cell having similar nuclei is called a homokaiyon and a cell having dissimilar nuclei is called a heterokaryon or heterokaryocyte.
When two nuclei of a heterokaryon fuse then a true hybrid protoplast or synkaryocyte is formed. If in a binucleate heterokaryon one of the nuclei disappears then it is called a cybrid or a cytoplasmic hybrid or a heteroplast, as the cytoplasms remain in a fused state.
In animal cells cell fusion was first reported by G. Barki, S. Sorieul and F. Cornefert (’60). They mixed cancerous cells of two different mouse and after few months hybrid cells were formed. In the hybrid cell, chromosomes of both parents were present. During division of such hybrid cells some chromosomes were omitted.
Later hybridization of cells of different species was successfully done. Hybrid cells of mouse and rat were observed. Hybridization was also attempted between cells of man and rat.
Such hybrid cells showed more similarities with rat cells, because during division of hybrid cells human chromosomes were generally omitted. Out of the 46 human chromosomes the hybrid cells contained only 2-15 human chromosomes.
Y. Okada of Osaka University reported that in presence of ‘Sendai’ strain of Para influenza virus cell fusion occurs at a higher frequency. Later H. Haris and J. F. Watkins (’65) of Oxford University used ‘sendai virus’ inactivated by UV ray for different types of cell fusion.
By using sendai virus, fusion of isolated protoplasts of animal cells was recorded. But this virus is ineffective for fusion of isolated protoplasts of plant cells. During digestion of cell walls by enzymes two adjacent protoplasts may fuse. Such fusion of protoplasts of the same tissue is always intraspecific. But this type of fusion is very rare as negative charge exists on the surface of the protoplasts.
Thus two protoplasts usually repel each other. Presence of fusigenic agents lower this charge. Molecular alteration in the bilayer structure of the plasma membrane is needed for protoplast fusion. Power and coworkers (’70) reported successful protoplast fusion of higher plants (root protoplasts of oat) in presence of sodium nitrate—a fusigenic agent.
Isolated protoplasts are placed on a medium containing sodium nitrate solution of proper osmotio concentration or a solution of sodium carbonate and proper osmoticum at 37°C for five minutes. The tubes are centrifuged at low speed and the protoplasts aggregate at the base of the tubes. The tubes are kept in darkness and left undisturbed at 37°G for 30 minutes.
By the action of sodium nitrate protoplasts adhere. In those regions where plasma membrane of the protoplasts are in close contact, there fusion of plasma membrane occurs and plasmodesmata like connections are established.
These cytoplasmic connections gradually expand and fuse with one another forming broader connections. Fused protoplasts become more or less spherical or oval. Two or more protoplasts may fuse together (Fig. 25).
By using sodium nitrate only few heterokaryons are formed. To overcome this difficulty from 1974 onwards polyethylene glycol (PEG) is widely used instead of sodium nitrate. By using PEG heterokaryons occur in greater frequency.
By its use fusion of protoplasts between two plant cells or two animal cells or between an animal cell and a plant cell was successful. Hybrid cells between soya bean-maize, soybean-pea and soya bean-barley were obtained by using PEG.
In this method at first concentrated suspensions of two types of protoplasts are mixed. Then to this 15% PEG solution is mixed in the ratio of 1: 3 (protoplast suspension: PEG). Isolated protoplasts of two different plants are centrifuged at 75-100 ×g for ten minutes in presence of PEG. The protoplasts fuse readily. This results in various fusion products, such as, homokaryons, heterokaryons, multiple fusion products, cybrids.
Protoplasts of microspores or meiocytes may fuse in absence of any fusigenic substance. Ito (’73) used grooved slides for fusion of protoplasts of meiocytes of some plants of Liliaceae.
Isolated protoplasts in enzymatic solution aggregate at the base of the groove. When the slide is gently tapped, the protoplasts fuse readily. Fused protoplasts are recognizable by their larger size. Fusion may occur between two or more protoplasts.
Fused or un-fused protoplasts are re-suspended, their viability and density are determined. The mixture is plated. This fusion product if forms callus then from this plantlets may be formed. High pH (about 10.5), high temperature (37°C) and high concentration of calcium ions (50 mM CaCl2, 2H2O) also help protoplast fusion.
Isolation of hybrids:
Protoplasts with distinct pigments on fusion produce hybrids which can be isolated by visual method. The hybrids can also be separated by a method of fluorescent cell sorting. Another method of hybrid isolation is growing in a medium which helps selective growth (biochemical method).
Hybrid protoplasts formed by the fusion of protoplasts of Nicotiana glauca and Nicotiana langsdorfii can be isolated by biochemical method (Fig. 26). The parental protoplasts fail to grow in an auxin free medium, but in such a medium hybrid cells can grow. Thus, in an auxin free medium only hybrid cells can survive. These cells form callus, which on sub-culturing form hybrid plants.
Hybrids can also be isolated by using biochemical mutants. Petunia hybrida cannot grow on a medium containing actinomycetes D. P. parodii can grow on such a medium, but its callus cells cannot produce entire plants.
When hybrids are formed by protoplast fusion between these two species, then such hybrids can grow on actinomycetes D containing medium and are also capable of regenerating whole plants.
Significance:
(1) Protoplast fusion has great significance in somatic hybridization, plant breeding and virus infection studies.
Takebe and coworkers (’71) obtained entire plants from hybrid protoplasts of tobacco. Later entire plantlets were obtained by this method in Citrus, Daucus, Solanum and Petunia.
Carlson and coworkers (’72) obtained hybrid plants of Nicotiana glauca and N. langsdorfii by protoplast fusion. Hybrid cells of Brassica campestris and Arabidopsis thaliana were successfully cultured by Gleba and Boffmann (’78, ’79). From such hybrid cells, they were able to produce hybrid plants.
(2) By protoplast fusion cytoplasmic male sterility can be transferred from one plant to another within a species or between two species. Medgyesy (’80) successfully transferred streptomycin resistance property from Nicotiana tabacum to N. sylvestris. This character is controlled by chloroplast DNA.