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This article throws light upon the three important applications of protoplasts hybridization.
The three important applications are: (A) Hybrids and Cybrids Production (B) Asymmetric Hybrids Production and (C) Direct DNA and Macromolecules Uptake.
A. Hybrids and Cybrids Production:
Protoplasts fusion technique during 1970s established that Para sexual hybrids can be produced by this method. Intra and inter-specific hybrids have been produced by this method in several Solanaceous plants like Datura, Petunia and Nicotiana species. By this method complete hybrid plants of intermediate characters were produced. Mostly amphidiploid plants were obtained.
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The partial transfer of cytoplasm (cytoplasmic characters), organelles uptake or loss of one set of chromosomes of one of the parent protoplasts leads to the formation of cybrid plants. This is not possible by conventional breeding programme. The donor-recipient protoplasts fusion system has been used to transfer chloroplasts and mitochondria in several crop plants.
It is established that mitochondria and chloroplasts have their own DNA and therefore, characters governed by this DNA are transferred and expressed. Under selective selection pressure, recombination between chloroplasts DNAs from different origin can occur with low frequency. In comparison to chloroplasts DNA, mitochondrial DNA undergoes more frequent and extensive recombination.
B. Asymmetric Hybrids Production:
Because of physicochemical nature of fusion, hybrid cells could be produced between distantly related taxa irrespective of their phylogenetic relationship. In majority of cases, these wide cell hybrids (hybrid of distantly related parents) showed instability in their chromosome constitution and to regenerate plants.
These plants do not contain complete set of chromosomes of both the parents and thus known as asymmetric hybrids, e.g.. In hybrid cells obtained in fusion between soybean x Nicotiana glauca, soybean x N. tabacum, carrot x tobacco, carrot x rice, Duboisia x tobacco and soybean x rice etc.
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In some intergeneric fusion combinations, hybrid plants with additive chromosome number were regenerated e.g., after fusion between potato and tomato, Arabidopsis and Brassica, Datura innoxia and Atropa balladonna, Lycopersicon esculentum and Solanum lycopersicoides, Eruca sativa and Brassica juncea and Lycopersicon esculentum and Solanum nigrum. In some of the cases, two parental diploid genome coexisted in the cybrid cells (amphidiploid). However, morphological abnormalities and sterility occurred in several of these intergeneric hybrids.
C. Direct DNA and Macromolecules Uptake:
Cell membranes are semi-solid structures as per the fluid mosaic model and fluidity of membrane is physiological characteristics. Transient aqueous pores in the bi-layer portion can make the membrane permeable for macromolecules. The formation of pores and repair of membrane pores (sealing) opens the new approach for introduction of nucleic acid molecules into cytoplasm and the nucleus.
The reversible, non-destructive permealization of membranes can be achieved by both the chemical treatments and action of short duration high electric pulses. Uptake of DNA molecules into a variety of plant protoplasts was successfully promoted by application of PEG treatment or electro-poration.
Plant protoplasts have the remarkable property to take up particles and macromolecules, like polystrene, latex particles, ferritin, proteins, DNA, viruses, bacteria or even whole chloroplasts and nuclei can be transplanted. Mayo and Cocking (1969) reported that the uptake is by pinocytosis, and demonstrated this by differential staining effects of phosphotungustic acid on the plasma lemma.
i. DNA Uptake:
The discovery that Diplococcus pneumoniae transforms itself by taking up exogenous DNA into its genomes has made genetic engineering a routine task in bacteria. New genes can be synthesized and DNA-RNA hybrids can be obtained. DNA can now be incorporated into the higher plant cells protoplasts. Doy et. al. (1972-73) transformed haploid tomato cell cultures by phage lambda (bacterial virus). Transformed cells can utilize galactose and lactose as carbon source for their growth. This is one of the approaches for obtaining transgenic plants (Table 30.2).
The transient expression of transferred gene in a few days permits the use of this technique for DNA uptake studies. It is evident from the data in the table that various marker genes have been used for transient expression. Production of transgenics requires the stable integration of the introduced DNA molecules into the genome of recipient species used as protoplasts source.
The use of various reporter genes as chloramphenicol acetyl transferase (CAT) and β-glucoronidase (GUS) also help in the identification of transformed cells (colonies). A reporter gene produces phenotypic expression (e.g. colour) to confirm the transfer of gene. This way, integration levels can be detected early in the transgenic cells (Fig. 30.5).
ii. Virus Uptake:
E.C. cocking reported that tomato fruit protoplasts take up TMV particles by pinocytosis. This system has been used to demonstrate mechanism of infection and nucleic protein replication.
iii. Bacterial Uptake and Nitrogen Fixing:
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Nitrogen fixing bacteria Rhizobium japonicum can be introduced into the pea leaf protoplasts during enzymatic digestion of the cell wall. This uptake occurs by invaginations of the plasma lemma during plasmolysis. Similarly, Davey and coworkers obtained protoplasts from root nodules cells packed with bacteria and try to fuse it with non-legume protoplasts. All these attempts to introduce nitrogen fixing genes/properties to non- legumes were not successful.
iv. Chloroplasts Transplantation:
Carlson incorporated the non-mutant tobacco chloroplasts into the cytoplasm of an albino protoplasts. Whole plant was regenerated later on. This may lead to improvement of plants for efficient photosynthesis.
v. Transplantation of Nuclei:
After smaller particles, nuclei were tried and they could be incorporated through plasma lemma without bursting it. Potrykus and colleagues transplanted nuclei from Petunia hybrida into protoplasts of Zea mays, Petunia hybrida and Nicotiana glauca. Further development of this technology led to the development of cloned sheep and monkey by nuclear transplantation.
