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At the beginning of the sixties used E. C. COCKING (University of Nottingham) an enzyme preparation for the degradation of cell walls and yielded thus single protoplasts from tomato root tips, though it took until 1968 before I. TAKEBE et al. (Institute for Plant Virus Research, Aobacho, Chiba/Japan) developed a technique for the production of large amounts of active protoplasts from mesophyll cells of Nicotiana tabacum that became standard. It spread fast due to its simple use, and within short time succeeded a number of laboratories in the yield of protoplasts from tissues of different plant species. Protoplast preparation was at first done in two steps: |
- At first was the middle lamella dissolved by pectinases,
- then was the cell wall broken down by cellulase.
The enzymes required for this procedure are usually not pure, but crude extracts from certain bacteria and fungi. Nowadays is the method often shortened to just one operation in which a mixture of enzymes is applied. Due to the high osmotic pressure within the protoplast are isotonic media used. They contain usually mannitol and/ or sorbitol. Besides contains protoplast media a variety of inorganic salts and several vitamins. It facilitates the maintenance of protoplasts capable of dividing for several days. They can be cultivated on agar dishes where they grow into small visible calli.
Among the most striking properties of active protoplasts is the fast regeneration of a cell wall that does always precede protoplast division. It seems as if the development of a cell wall was a causal precondition of both cell division and development of polarity. Polarity and positional information are lost during protoplast generation. Both have to be developed anew, if a new plant is going to regenerate.
Protoplasts can be used for the production of 'vacuoplasts' (pure vacuoles) and subprotoplasts that consist of nucleus and plasma alone. The single fractions can be separated from each other and concentrated by density gradient centrifugation. What is the use of protoplasts?
- Protoplasts can regenerate into complete plants.
- Protoplasts of the same or different origin can fuse with each other. The fusion product may even regenerate into a plant. This is also called somatic hybridization.
- The protoplast can take up macromolecules (nucleic acids and proteins), viruses, cell components like chromosomes and chloroplasts by phagocytosis.
- Protoplasts are suitable for the study of the molecular architecture of plant cells
I. TAKEBE, G. LABIB and G. MELCHERS (Max-Planck-Institut für Biologie, Tübingen) regenerated complete plants from tobacco protoplasts in 1970.
Y. Y. GLEBA (Academy of Science of the Ukrainian SSR, Kiew) developed a technique for the cultivation of isolated protoplasts in micro-droplets. A number of thus cloned protoplasts regenerated into complete plants.
By now have protoplasts from a number of dicotyledonous angiosperms been regenerated into fertile plants. Solanaceae pose the smallest problems. In contrast regenerated protoplasts from leguminosae or monocots into complete plants only after the development of costly procedures that do still not always succeed. Regeneration requires an addition of phytohormones to the medium. It looks as if the mesophyll cells of monocots would loose the respective receptors. This may also be the reason why hormone-like herbicides impair dicots but not monocots (cereals). On the other hand is the lacking ability to regenerate into protoplasts of economically important cultivated plants a large handicap when trying to transfer the knowledge gained with this method to the agricultural practice.
The aggregation of two or more protoplasts is not enough to start a fusion. Protoplast surfaces bear strong negative charges. In contrast to animal cells is the surface charge not caused by sialic acid residues but by phosphate groups. Intact protoplasts in suspension do thus repel each other. They can be very impressively linked and fused by the addition of calcium ions or polyethylenglycol. Electric fields are an alternative.
Fusion of Protoplasts in an Electric Field (electrofusion; H.-U. KOOP, H.-G. SCHWEIGER, 1985)
Sexual hybridization occurs when haploid cells generated in a previous meiosis fuse. The fusion of somatic diploid cells should generate a tetraploid fusion product provided that the nuclei fuse, too. If this is the case, it is spoken of a synkaryon. A fusion product where the nuclei stay separate is called a heterokaryon.
For conditions comparable to sexual hybridization is haploid starting material needed. S. GUHA and S. C. MAHESHWARI (Botanical Institute of the University of Delhi, 1966) developed a medium for the cultivation of anthers from Datura innoxia. The anthers grew into seedling-like plantlets also called embryoids. These descendants of meiosis that did not develop further into pollen turned out to be 'plants out of gones'. They had not the diploid number of chromosomes normal for plant tissues but only a haploid set. Gones is the name for the (haploid) products of meiosis (independent of the sex). By now have haploid plants from a whole number of different plant species, both mono- and dicots, been cultivated successfully. They are used as starting material for the production of protoplast.
In order for haploid plants to propagate by seeds do they first have to become diploid. This is achieved rather easily since colchicine was found out to be very suitable for this task in1937. Diploid plants thus generated have the advantage of being completely homozygous.
Haploid species of Nicotiana (N. tabacum, N. sylvestris) have first been used for intraspecific, later on also for interspecific protoplast fusion. The plants yielded by regeneration of such fusion products do not differ from sexually generated ones. Fusion products of parental varieties that complement their chlorophyll defects are especially suitable. They can be recognized by their green colour (G. MELCHERS and G. LABIB, Tübingen 1974).
Interspecific fusions can be generated rather easily. It is even possible to fuse plant protoplasts and animal cells (fibroblasts) and to keep the fusion product alive for several hours. Such experiments are not performed for regeneration purposes but in order to understand the co-operation of membranes or the translation of single plant genes within the animal cell's plasma. With few exceptions regenerate interspecific fusion products only, if sexual hybridization is successful, too.
Interspecific heterokaryons, especially those of not closely related species, do not form synkaryons. Nuclear division is asynchronous and as a consequence are chromosomes lost: the whole system looses its balance. Despite this have some somatic interspecific hybridizations been successful. G. MELCHERS succeeded in 1978 in the fusion of tomato and potato protoplasts. The fusion products (two German terms: meaning Tomate x Kartoffel or Kartoffel x Tomate = "Tomoffel" or "Karmate") regenerated into whole plants that flowered but did not develop fertile seeds.
The fusion product of Datura innoxia and Datura stramonium developed by O. SCHIEDER (Max-Planck-Institut für Züchtungsforschung, Köln) did even produce fertile seeds. It can therefore be regarded as a new species: Datura straubii. Datura - species are used for the production of medically important alkaloids hyoscyamine and scopalamine. Datura straubii has a larger growth capacity and a 20 - 25 percent higher alkaloid content than its two parental species.
Protoplasts can absorb foreign molecules and organelles by phagocytosis. This furnished proof that one cell can be infected by more than just one virus particle and that different strains of viruses can multiply within one cell. Furthermore were protoplasts transformed with Ti-DNA from Agrobacterium tumefaciens which made the system interesting for genetic engineering. The absorption of plastids permits an analysis of the co-operation between the nuclear and the plastidic genomes. Absorbed foreign genes can, too, make up for defects within the protoplast genome. The fusion with inactivated protoplasts of Physalis and Datura transferred a nitrate reductase activity to a mutant of Nicotiana tabacum with a deficient nitrate reductase. Transformation proved to be stable (R. P. GUPTA, M. GUPTA, O. SCHIEDER, 1982).
The use of modern analytic techniques like gel electrophoresis, evidence of enzyme activities within a gel or isoenzymes verifies the success of fusion experiments. Ribulose - 1,5 - bisphosphate carboxylase has an important marker function since it consists of nuclear and plastidic encoded subunits. At the same time offers the analysis of this protein information about the activities of the plastids and nucleus of the parental species within the fusion product.
After protoplast techniques had been established became the production and subsequent isolation of mutants important. Auxotrophic strains, i.e. strains that are dependent on the supply of certain nutrients were of special interest. Only few successes were achieved, one reason of which may be that a large part of angiosperm genomes are allopolyploid and that genetic information exists in several copies even in haploids. This may easily compensate for a defect gene. In addition are plants able to produce one and the same product by different metabolic pathways. A possible way out of the dilemma could be the use of 'monohaploid' stock plants (1 x instead of 1 n). In the case of cultivated plants would that mean that their original varieties have to be found first.
S. M. WICK et al. (Australian National University, Canberra, 1981) produced protoplasts from pre-fixed cells since the original cell shape is lost during protoplast generation from native cells. The thus generated protoplasts are dead, but they have kept their shape, and indirect immunofluorescence allows the depiction of the cell's structural elements' distribution. The use of lectins showed that the protoplast surfaces of different species carry different carbohydrate patterns and that the lectin-binding properties of the plasmalemma differ fundamentally from those of the intracellular membranes and the tonoplast.
© Peter v. Sengbusch - b-online@botanik.uni-hamburg.de