Can phagocytose plant cells

At the beginning of the sixties, E. C. COCKING (University of Nottingham) used an enzyme preparation to break down the cell wall and thus obtained individual protoplasts from the root tips of the tomato. But it wasn't until 1968 that I. TAKEBE et al. (Institute for Plant Virus Research, Aobacho, Chiba / Japan) a method that has become a standard method for obtaining large amounts of active protoplasts from mesophyll cells from Nicotiana tabacum developed. Because of its ease of use, it quickly found widespread use, and within a short period of time a number of laboratories managed to obtain protoplasts from a wide variety of tissues from various plant species. For the preparation of protoplasts, a two-stage process was initially developed in which

  1. the middle lamella is dissolved by pectinases, and then
  2. the cell wall is digested by cellulase.

The enzymes required for this are usually not pure preparations, but raw extracts from certain bacteria and fungi. Instead of the two-step process, the process is often shortened to one operation by administering an enzyme mixture. Because of the high osmotic pressure inside the protoplast, isotonic media must be used. The sugar alcohols mannitol and / or sorbitol are usually added to them. Otherwise, a protoplast medium contains a variety of inorganic salts and some vitamins. Protoplasts can be kept alive for days and retain their ability to divide. They can be cultivated on agar plates, where they grow into small visible colonies (calli), which brings us back to "microbiology" with its many possibilities.

One of the distinctive properties of active protoplasts is the rapid regeneration of a cell wall, which always precedes protoplast division. It looks as if wall formation is a causal prerequisite for cell division and the formation of a polarity. During the production of the protoplast, this was lost, as was the position information. Both must be re-acquired when regenerating to a plant.

Protoplasts are suitable for the production of "vacuoplasts" (i.e. pure vacuoles) and subprotoplasts, which only consist of a cell nucleus and a plasma rim. The individual fractions can be separated from one another and enriched by density gradient centrifugation. What are protoplasts used for?

  1. Protoplasts can be regenerated into whole plants.
  2. Protoplasts of the same or different origins can be fused with one another. If necessary, a plant can be regenerated from the fusion product (somatic hybridization).
  3. Macromolecules (nucleic acids and proteins), viruses, cell components (chromosomes, chloroplasts, etc.) can be absorbed by the protoplasts by phagocytosis.
  4. Protoplasts are useful for studying the molecular architecture of plant cells.

In 1970 I. TAKEBE, G. LABIB and G. MELCHERS (Max Planck Institute for Biology, Tübingen) regenerated a complete plant from tobacco protoplasts.

Y.Y. GLEBA (Academy of Sciences of the Ukrainian SSR, Kiev) developed a method for the culture of isolated protoplasts in microdroplets. A number of protoplasts cloned in this way could be made to develop into complete plants.

There are now a number of dicotyledonous angiosperms from whose protoplasts complete fertile plants have been regenerated. The Solanaceae are the least troublesome. Protoplasts from legumes and monocotyledons, on the other hand, could only be brought to the regeneration of whole plants after elaborate procedures and not always. An addition of phytohormones to the medium is necessary for regeneration. It looks like the mesophyll cells of the monocotyledons are losing the necessary receptors. This may also be the reason why hormone-like herbicides damage dicotyledons, but do not impair monocotyledon growth (cereals). On the other hand, the inadequate regenerative capacity of the protoplasts of economically important crops is a major handicap when it comes to implementing the experience gained with this method in agricultural practice.

An aggregation of two or more protoplasts is not enough to initiate a fusion. Protoplast surfaces are strongly negatively charged. In contrast to animal cells, the surface charge is not based on sialic acid residues, but is caused by phosphate groups. Intact protoplasts therefore repel each other in suspension. Mono- and polycations are suitable for neutralizing the surface charge. Calcium ions or polyethylene glycol are very effective, which means that the protoplasts can be crosslinked and fused. An alternative solution is to apply an electric field.

Fusion of protoplasts in an electric field (electrofusion) (H.-U. KOOP, H.-G. SCHWEIGER, 1985

In a sexual hybridization, haploid cells, which have arisen from a previous meiosis, fuse. In the fusion of somatic diploid cells one would have to expect a tetraploid fusion product, provided that the cell nuclei also fuse. If that is the case, it is called a synkaryon; the nuclei remain separate, by a heterokaryon.

In order to create conditions that are comparable to those of a sexual crossbreed, haploid starting material is required. For this purpose, S. GUHA and S. C. MAHESHWARI (Botanical Institute of the University of Delhi, 1966) developed a medium for the culture of the anthers ofDatura innoxia. Seedlings similar to small plants (embryoids) grew out of the anthers. Because they did not have the diploid chromosome number of normal plant tissue, but only the haploid number, these descendants of a meiosis, which did not develop into pollen grains, turned out to be "plants from gons". Gonen is the name for the (haploid) products of a meiosis (regardless of gender). It has meanwhile succeeded in producing haploid plants from a number of different plant species (di- and monocotyledons) and using them as starting material for the production of protoplasts.

In order to be able to propagate haploid plants through seeds, they must be made diploid. However, since colchicine was discovered for this task in 1937, this has not been difficult. One might ask what the advantage of this process is, and the answer is: The diploid plants produced in this way are homozygous in all their genes.

Haploids Nicotiana-Species (N. tabacum, N. sylvestris i.a.) were initially used for intra- and later also for interspecific fusions of their protoplasts. Plants produced by regeneration of fusion products are no different from those produced sexually. Parent strains with mutually complementary chlorophyll defects are suitable for the selection of fusion products; Fusion products can then be recognized by their green color (G. MELCHERS and G. LABIB, Tübingen 1974).

Interspecific fusions are relatively easy to produce. It is even possible to fuse plant protoplasts and animal cells (fibroblasts) and keep the fusion products alive for a few hours. Such experiments are of course not about attempts at regeneration, but about questions of the cooperation of the membranes with one another or the expressibility of individual genes of a plant cell in the animal cell plasma or vice versa. With a few exceptions, interspecific fusion products can only be regenerated if crosses on the sexual path are successful.

Interspecific heterocarias, especially in less closely related species, do not form syncaria, the nuclear divisions are asynchronous, chromosomes are lost, and the system is out of balance. However, there are already some successes in interspecific somatic hybridization. G. MELCHERS succeeded in 1978 in fusing tomato and potato protoplasts with one another and bringing the fusion product (Tomoffel. Karmate) to the regeneration of whole plants. The plants bloomed, but fertile seeds were not produced.

O. SCHIEDER (Max Planck Institute for Breeding Research, Cologne) was between Datura innoxia and Datura stramonium also successful in this point. The plants resulting from the fusion are fully fertile and therefore to be regarded as a new species: Datura straubii.DaturaSpecies are used to obtain the medically important alkaloids hyoscyamine and scopalamine. Datura straubii is more vigorous than the two original species, its alkaloids content is 20-25 percent higher than that of the parents.

Protoplasts can take up foreign molecules and organelles through phagocytosis. Among other things, this proved that a cell can be infected by more than one virus particle and that different virus strains can multiply in one cell. Transformation of protoplasts with Ti-DNA was also successful Agrobacteriumtumefacienswhich opened up a way to use the system for genetic engineering purposes and investigations. The inclusion of plastids allows the analysis of the cooperation between the nuclear and plastid genome. The inclusion of individual foreign genes can be used to repair a genetic defect in the protoplast genome. Thus, in a nitrate reductase-deficient mutant of Nicotiana tabacum, after fusion with inactivated protoplasts Physalis and Daturato transfer a nitrate reductase activity. The transformation proved to be stable (R. P. GUPTA, M. GUPTA, O. SCHIEDER, 1982).

The success of fusion experiments can be proven by using modern analytical methods (gel electrophoresis, detection of enzyme activities in the gel, isoenzymes, etc.). Ribulose-1,5-bisphosphate carboxylase plays an important marker role because it consists of nucleus-coded and plastid-coded subunits and because the analysis of this protein also provides information about the activities of plastids and the nucleus of the parent species in the fusion product.

After protoplast techniques were established, there was an interest in inducing mutations and isolating the mutants; Particular interest was given to the auxotrophic strains, i.e. those that are dependent on the supply of certain substances. There have been a few successes in this direction, but the number of failures is greater. One reason for this is presumably the fact that a large part of the angiospermegenome is allopolyploid and that the genetic information is present several times even in haploids, so that the defect in one sentence is compensated for by an intact gene in another. In addition, plants often get to the same product via different metabolic pathways. A possible way out of this would be the use of "monohaploid" starting forms (1x instead of 1n). In the case of cultivated plants, this means that one must first search for their original forms.

Examples of this have already been cited several times. Since the original cell shape is lost during protoplast formation from native cells, S. M. WICK et al. (Australian National University, Canberra, 1981) Protoplasts made from prefixed cells. Although they are no longer alive, they have retained their shape, and by using indirect immunofluorescence a picture of the distribution of structural elements in the cell can be obtained. By using lectins it could be shown that the protoplast surfaces of different species have different carbohydrate patterns and that the lectin binding properties of the plasma lemma differ fundamentally from the intracellular membranes and the tonoplast.