The following points highlight the top fifteen applications of protoplast culture. They are discussed here below:
1. Study of Osmotic Behaviour
2. Study of IAA Action
3. Study of Plasma-lemma
4. Study of Cell Wall Formation
5. Organelle Isolation
6. Study of Morphogenesis
7. Virus Uptake and Replication
8. Study of Photosynthesis from Isolated Protoplast
9. Isolation of Bacteriods from Root Nodule Protoplast
10. Induction of Mutation and Genetic Variability
11. Microorganism Transplantation
12. Implantation of Chloroplast
13. Transplantation of Nuclei
14. Transplantation of Chromosome
15. Somatic Hybridization
Application # 1. Study of Osmotic Behaviour:
Influence of different environmental factors on the osmotic behaviour can be studied using plant protoplasts.
Application # 2. Study of IAA Action:
When growth promoters like IAA are applied to plants, they act directly on plasma membrane of the cell and increase the permeability of the membrane to water resulting in cell elongation. This can be established by the use of protoplast in vitro.
When IAA is applied to the plasmolyticum containing protoplasts they expand rapidly and finally burst due to too much vacuolation (Cocking and Hall, 1974). Further, it can be verified by using anti-auxins that suppress this bursting, indicating that the site of action of IAA is the plasma-lemma of the plant cell.
Application # 3. Study of Plasma-lemma:
When newly released protoplasts are placed in hypotonic solution or plain water, the protoplasts burst within a second or if the protoplasts are dropped from a certain height on a glass slide, the same result will happen. So by this process, plasma-membrane can be isolated very easily from protoplast and a number of study on plasma-membrane can be investigated.
Application # 4. Study of Cell Wall Formation:
The early deposition of cellulosic micro-fibril and their orientation at the protoplast surface can be followed using both light and electron microscope and has also provided much basic information concerning cell wall biology.
Application # 5. Organelle Isolation:
Protoplasts are very convenient material for the isolation of chloroplasts, mitochondria, nuclei and even chromosomes. It has been demonstrated that chloroplasts particularly isolated from cereal protoplast have higher capacity for CO2 fixation than those obtained by mechanical grinding.
Application # 6. Study of Morphogenesis:
Isolated protoplast provides an ideal single cell system. Under suitable condition, protoplast regenerates its own wall and become the walled cells. Cell division followed by plant regeneration may occur from such unique single cell system either through organogenesis or embryogenesis.
Plant regeneration is very important as well as significant for fusion experiment and for the experiment of genetic modification in protoplasts.
Application # 7. Virus Uptake and Replication:
The plant virus interrelationships in the past were not clearly known due to lack of suitable experimental systems that can easily infect the cells. But after the innovation of protoplast isolation and its culture, this problem is almost solved. Protoplast can directly be inoculated with pathogenic virus in the medium. The process of uptake of virus particle, their replication inside the protoplasts and their mode of action at the molecular and cellular level are made possible by the aid of protoplasts.
Application # 8. Study of Photosynthesis from Isolated Protoplast:
Elegant experiments to investigate various biophysical and biochemical aspects of photosynthesis in C3 and C4 plants have been carried out by a number of workers using protoplasts.
Application # 9. Isolation of Bacteriods from Root Nodule Protoplast:
Viable bacteriods from root nodules of legumes has been isolated by first preparing nodule protoplast and then rupturing them either mechanically or by lowering suddenly the concentration of the plasmolyticum in the surrounding medium. This method ensures the freedom of the preparation of bacteria from the infection thread.
Application # 10. Induction of Mutation and Genetic Variability:
It has been repeatedly observed that plant cell in culture show a wide range of genetic diversity. This phenomena can be exploited by plant breeders and geneticists for inducing variability in protoplast culture. The recessive characters can be detected in the regenerated plants derived from haploid protoplasts. Therefore, haploid protoplast would make an ideal system for studying the effect of irradiation and for the induction of mutation by plating them in media supplemented with various chemical mutagens.
From this method, mutant line can be selected.
Application # 11. Microorganism Transplantation:
Incorporation of microorganisms like bacteria, blue-green algae, yeast etc. into protoplasts has been attempted with the immediate objective of establishing endosymbiotic association with higher plant cells which may eventually yield a plant having some beneficial activity. Bacterial cell uptake by plant protoplasts has been investigated with species of Rhizobium and Spirillum.
There are reports based on ultra- structural examinations that bacteria enter the cells by endocytosis and may become embedded in vesicles in the cytoplasm of protoplasts. Similar uptake studies were performed with yeast and blue green algae cells.
Introduction of Anabaena variables and nitrogen fixing blue green algae Gleocapsa sp. into protoplasts has also been 15ported. However, nothing is known about the fate of the introduced microorganism, because there has been no reported evidence of survival or development of any organisms within the protoplasts.
Application # 12. Implantation of Chloroplast:
Plant protoplasts have ability to uptake the isolated chloroplasts by the process of endocytosis. Several reports have described uptake of chloroplasts. Chloroplasts isolated from Vaucheria dichotoma were implanted into carrot cell culture protoplasts. The chloroplasts may enter the cytoplasm enclosed in membrane-bound vesicles, although the enclosing membrane in some cases is absent.
Biological evidence of chloroplast gene expression was presented but the experiments have not been confirmed. The inability and ability of chloroplasts to survive and multiply in recipient protoplasts have not been unequivocally demonstrated, although limited replication has been reported.
Potentially the chloroplast uptake procedure offers an excellent approach to study chloroplast/ cytoplasm and nuclear interrelationships, genetics and physiological autonomy and specificity of functions of the organelles.
Application # 13. Transplantation of Nuclei:
Isolated nuclei can be introduced into the protoplasts. Both intra and inter-specific nuclear transplantation have been observed in Petunia hybrida, Nicotiana tabacum and Zea mays. Retention, normal function or degradation of the incorporated nuclei is not known. But it is really opening up new avenues for the study of nuclear- cytoplasmic interaction if fertile plants with foreign nuclei could be regenerated from such protoplasts.
Application # 14. Transplantation of Chromosome:
The uptake of isolated metaphase chromosomes has proven successful in plant protoplast. This procedure provides a valuable method for genetic information transfer and gene analysis.
Application # 15. Somatic Hybridization:
The main objective of protoplast culture lies in the possibilities of fusion of one protoplast with another. Normally isolated protoplasts do not fuse with each other, but in presence of fusion inducing agent like PEG, a protoplast of one species can be fused with one of a different species, thus a hybrid protoplast will be produced.
Now this hybrid protoplast regenerates a cell wall and its two nuclei fused together, then a somatic hybrid cell will be produced. Sometimes nuclei do not fuse and one nucleus of any one parent may be eliminated in the subsequent development stages. Thus hybrid cell is produced with the nuclear genome of any one partner and the cytoplasm of both parent.
A somatic hybrid plant can be regenerated from these cells. Thus the production of new hybrid without recourse to sexual reproduction will constitute one of the greatest potentials in plant genetics for crop improvement. A number of crosses including both inter and interspecific ones are not possible by normal pollination and so the hybrids cannot be produced.
This obviously limits the scope of the plant breeder. Somatic hybridization or protoplast fusion will provide an alternative way to overcome the limitations of conventional breeding. It is also possible to manipulate hybrid production such that desirable characters e.g. disease resistance, protein quality, nitrogen fixation and cold tolerance which are genetically controlled, could be transferred from one species to another. On the other hand, hybrid plant is also useful where some desirable characters are controlled by cytoplasmic genome e.g. male sterility.
A brief list of somatic hybrid plants raised through protoplast fusion is given below:
Sexually Compatible Combination:
Daucus carota + D. capillifohus, Nicotiana glauca + N. langsdorffi, N. tabacum + N. alata, Petunia parodit + P. hybrida, Solanum tuberosum + S. chacoense.
Sexually Incompatible Combination:
Datura innoxia + D. Candida Nicotiana, sylvestns + N. khightiana, N. tabacum + N. nesophila, Petunia parodit + P- parviflora.
Arabidopsis thaliana + Brassica campestris, Daucus carota + Aegopodium podagana, Solanum tuberosum + Lycopersicon esculentum.