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Content archived on 2022-12-23

Intergeneric and interspecific protoplast fusion in red algae

Objective

- Red algae produce a variety of compounds of commercial interest. Most important among these products are polysaccharides used as gelling agents, such as agar-agar, carrageenan, etc. Yet, cultivation of red algae under controlled conditions is still a problem and restricted to very few species. Likewise, the breeding of superior culture varieties is not yet possible;
- The overall aim of the project is thus to make interesting species of red algae amenable to tissue culture techniques, and to combine traits of different genera by somatic hybridisation via protoplast fusion. Porphyridium spec. and Rhodella reticulata are unicellular red algae that can be easily cultivated, and Gracilaria tikvahiae is a major species for commercial polysaccharide production. The more specific long term goal of the project is to obtain unicellular agar-agar producing algae through intergeneric protoplast fusion of Porphyridium and Gracilaria. This will be achieved in a stepwise approach, starting from the isolation and regeneration of protoplasts in each species, and then proceeding from intraspecific to interspecific, and finally intergeneric fusions. This also involves analytical aspects, such as physiological, biochemical and genetic characterisation of parental strains and fusion progeny.
Results so far

- Production of mutant strains
Apart from a previously isolated herbicide (sulfometuron methyl, SMM) resistant strain (van Moppes et al. 1989), and the pigment mutants described in Sivan & Arad (1993), new mutants of Porphyridium were obtained which are resistant against the herbicides diuron and atrazin. These strains were characterised for photosynthetic properties, pigment and cell wall composition (Sivan & Arad, 1995).

- Protoplast isolation
As described in Sivan et al. (1992), protoplast isolation from Porphyridium was achieved with an enzyme preparation from a mixture of soil bacteria selected for this purpose. The same strategy was applied to isolate new bacterial strains for the digestion of Rhodella reticulata cell walls. The efficiency of these new enzymes in digesting the extracellular polysaccharide of Rhodella was optimised with respect to salinity, temperature and pH. So far, the activity is not as stable as desired, and the bacteria have not yet been identified.

- Protoplast fusion
The first intraspecific fusions of Porphyridium protoplasts are described in Sivan et al. (1995), and Sivan & Arad (1996). The fusion was achieved by a PEG/heat shock treatment. Hybrids were identified by the combination of parental traits, i.e. complementation of different phycoerythrin deficiency mutations, or double resistance against diuron and SMM. Cytological, biochemical, and molecular evidence (by randomly amplified polymorphic DNA - polymerase chain reaction, RAPD-PCR) clearly demonstrated genetic transfer, genetic complementation, and the completion of the parasexual cycle in the fusion progeny. For electrofusion, some basic parameters, such as fusion medium, cell alignment and (irreversible) breakdown voltage were established. So far, cell fusion has been hampered by incomplete cell wall removal, which is more critical for this method than for chemical fusion. A further improvement of protoplast isolation will be needed to successfully employ electrofusion techniques.

- Biochemical characterisation of cell walls
Detailed chemical analysis has been performed mainly on the soluble, extracellular polysaccharide of Porphyridium (Arad 1988). The complete removal of the cell bound glycoproteins by SDS-PAGE and subsequent staining for sugar moieties. Digoxigenin labelling was used for a general overview, and lectin affinity for subsets of specific residues. The comparison of patterns obtained with undigested and digested cells, as well as total cells, secreted compounds, and purified cell walls, aims at the identification of components that may be unique to the cell bound wall layer and thus resistant to digestion with the bacterial enzymes. A well-defined screening of additional enzymes capable to remove these residues is now possible.

Follow-up

- The isolation of protoplasts from Porphyridium (and other species) will be further optimised to allow somatic hybridisation by electrofusion as well as chemical fusion with PEG;
- The labelling techniques developed for the detection of glycoproteins after electrophoresis can be modified for cytochemical staining of sections for electron microscopy. This kind of ultrastructural analysis will improve our understanding of cell wall structure and biosynthesis in unicellular algae. This is an important aspect also with regard to the introduction of cell wall properties of higher red algae (Gracilaria) into these organisms;
- When protoplasts of Rhodella are readily available, interspecific fusions will be performed with Porphyridium protoplasts. This should verify the viability of interspecific hybrids, as well as the ability of the unicellular algae to recombine with and express "foreign" genes. After that the stage will be set for intergeneric fusion attempts.
- Culture of red algae, so far : Porphyridium and Rhodella;
- Production and characterisation of mutants, as well defined mutations provide genetic markers to distinguish parental strains from somatic hybrids after fusion;
- Isolation of protoplasts by selection of soil bacteria that produce enzymes capable of digesting the complex cell wall material of the unicellular algae;
- Biochemical analysis of cell wall composition by affinity labelling, denaturing polyacrylamide gel electrophoresis (SDS-PAGE), and enzymatic deglycosylation, in order to optimise the digestion strategy;
- Fusion of protoplasts by chemical (polyethylene glyco, PEG) and physical (electrofusion) methods;
- Selection, regeneration and characterisation of fusion products with respect to physiological, biochemical and genetic properties.

Topic(s)

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Call for proposal

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Coordinator

Eberhard-Karls-Universität Tübingen
EU contribution
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Address
Auf der Morgenstelle 1
72076 Tübingen
Germany

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Total cost
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