Plant Sciences

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Browsing Plant Sciences by Advisor "Botha-Oberholster, A. M."

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    Assessing genetic diversity and identification of Microcystis aeruginosa strains through AFLP and peRRFLP analyses.
    (University of the Free State, 2003-12) Oberholster, Paul Johan; Grobbelaar, J. U.; Botha-Oberholster, A. M.
    English: There are 150 cyanobacterial genera and approximately 2 000 species known in the world. More than 40 of these have toxin producing strains. Cyanobacteria, commonly known as blue-green algae, are often present in small numbers together with a diverse assemblage of other photosynthetic algae that naturally occur in surface water worldwide. However, under conditions of warm temperatures, minimal water movement and elevated concentrations of phosphorus in a water body, cyanobacteria may frequently become dominant and form thick scums of floating algal cells. These dense aggregations of floating cells, termed 'blooms', presents a number of water quality problems; most often offensive odours and tastes, and sometimes biotoxins that can be divided into alkaloid neurotoxins and cyclic peptide hepatotoxins, commonly from the genus Microcystis and released in waterbodies. The neurotoxins act chiefly at neuromuscular junctions and cause rapid death because of respiratory paralysis. The hepatotoxins act on the hepatocyte cytoskeleton and cause intrahepatic haemorrhage and centrilobular necrosis. Clinically the hepatotoxin most often causes peracute or acute death, or subacute poisoning with signs such as icterus and hepatogenous photosensitivity. Currently cyanobacterial taxonomy does not provide an unequivocal system for the identification of toxigenic and bloom-forming genus Microcystis. The ambiguities that exist in the cyanobacterial taxonomy are due to the expressed variability, minor morphological and developmental characteristics that are used for identification. In this study geographically unrelated axenic strains of Microcystis aeruginosa were obtained from the Pasteur Institute, France (PCC); the National Institute for Environmental Studies, Japan (NIES); the Institute of Freshwater Ecology, UK (CCAP); the Pflanzen Physiologisches Institut, Universitat Gottingen, Germany (SAG) and the University of the Free State, South Africa (UV) culture collections. Nonaxenic strains were collected from Hartbeespoort, Rietvlei and Roodeplaat Dams in South Africa. After screening 20 primer combinations on a subset of strains eight IRDye700™-labeled EcoR1 primer pairs were selected for amplified fragment length polymorphism (AFLP) analysis to determine the genetic relationship of these geographically unrelated strains. A total of 909 bands were amplified from the eight primer combinations, of which 665 were informative, 207 non-informative and 37 monomorphic, with an average of 83.12 polymorphic bands per primer combination. The genetic relationship among all the Microcystis aeruginosa strains based on the combination of data obtained with the eight primer combinations was analysed employing the Unweighted Pair Group Method using Arithmetic Means (UPGMA) algorithm and presented as a dendrogram. In the dendrogram, the strains from Rietvlei (UP01) and Hartbeespoort Dams (UP04) grouped together and were thus genetically closer to each other, than to the strain from the Rhoodeplaat Dam (UP03). The Japanese strains (NIES88, NIES89, NIES90, NIES99, NIES299) also grouped separate from the other strains, with NIES90 and NIES299, genetically closest to each other. Interestingly, Microcystis aeruginosa strain PC7806 that originated from The Netherlands, also grouped within this group. Microcystis aeruginosa strains CCAP1450/1 (UK), UV027 (South Africa) and PC7813 grouped together, and are genetically closer to the UP-strains, than any of the other strains. In the present study, AFLP analysis proved useful for the identification of genetic diversity and analysis of population structure within Microcystis aeruginosa. In order to link the identification of strains with toxicity, the utility of the mcyB gene sequence for identification of strains was tested. Based on conserved motifs present in known sequences of mcyB four primer pairs were designed. Using the primer pairs Tax 3P/2M, Tax 1P/1M, Tax 7P/3M and Tax 10P/4M, the mcyB gene from PCC7813 and UV027 were sequenced, resulting in fragments of 2174 and 2170 base pairs in size, respectively. The obtained sequences were analyzed using nucleotide BLASTN annotation of the Basic Local Alignment Search Tool (BLAST). The sequence alignment indicated high homology to other published sequences in GenBank (AY034601 for pee7813 and AY034602 for UV027; e-value = 0.0). Upon further analysis of the sequences it was obvious that there are several base differences between the sequences of the two strains, which led us to investigate the potential of using differences in restriction sites, and thus insertions/deletions (indels) in nucleotide sequence to discriminate between the other M. aeruginosa strains, as well as using the mcyB gene to discern between M. aeruginosa and M. wesenbergii in raw water samples. A vast number of restriction sites were identified with differences followed by restriction digest of the specific polymerase chain reaction (PCR) mcyB gene fragment. This work demonstrates that PCR assays provide a useful indicator of toxicity as well as the identification of taxonomical characteristics between laboratory cultures and environmental isolates. A number of questions arise from the present study and future research therefore needs to address the following issues: • Are there more than one Microeystis aeruginosa strain / "population" present at a given time in a specific water reservoir? Do these populations change through the season? What role does the individual populations play in a cyanobacterial bloom? Thus, the dynamics and structure of populations need to be clarified. • Which mcy gene in the cluster is mostly responsible for toxin production? Does the expression of the genes correlate with gene structure/sequence? What role does the environment play in determining the level of expression, and thus toxin production?
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    Micropropagation of Pinus species
    (University of the Free State, 1999) Jacoby, Angeline; Van der Westhuizen, A. J.; Botha-Oberholster, A. M.
    English: The aim of this study was to develop an effective protocol for the micropropagation of Pinus patuIa and P. radiata. Micropropagation procedures by means of somatic embryogenesis on solidified medium and in cell suspension cultures as well as organogenesis were investigated. In addition the possible relationship between phenolic, auxin and cytokinin content within cuttings and the tendency of these cuttings to root, were to be investigated. The cones of P. patuIa and P. radjata were collected for the somatic embryogenesis study, on a two weekly basis during the summer months of 1995 to 1997. Somatic embryonic cultures were initiated from the immature female gametophytes containing zygotic embryos. The embryonal suspensor mass (ESM) formed, was used as starting material for cell suspension cultures. Organogenesis included axillary and adventitious budding on hypocotyls and cotyledons respectively of young germlings deriving from seeds of open pollinated cones. Various techniques to sterilize the seeds were evaluated and it was found that 30% H202 (10 min.) proved most effective for P. patuIa, and 10% H202 (5 min.) was most effective for P. radiata. The initiation of somatic embryonic cultures was attempted on solidified modified Murashige and Skoog medium (MSG), Schenk and Hildebrandt (SH), Gresshof and Day (GO), Quoirin and Lepoivre (LP) and variations of the Douglas-fir Cotyledon Revised (OCR) media, each differing with regard to nitrogen sources and growth regulator composition. It was concluded that the most effective initiation media for P. patuIa were DCR1 and DCR5, and that DCR2 was most effective for P. radiata. Maintainance of the embryonic cultures was most successfully achieved for both species on ½ LP medium containing 3% maltose and no growth regulators. Maturation of P. radiata somatic embryos was achieved on solidified OCR2 medium supplemented with 1.3 rnql-¹ ABA, 30 gl-¹ glucose and 1% (mIv) activated charcoal. Attempts to mature P. patuIa embryos were unsuccessful. Embryonic cell suspension cultures were established in liquid GO, OCR, SH and ½ LP media. The best culture growth was achieved on ½ LP medium supplemented with 0,5 mql-¹ 2,4-0 and maltose as carbon source. Re-establishment of these cultures onto solidified ½ LP medium, supplemented with ABA, for further development and maturation was successful. Adventitious buds were induced on young (14 day old) cotyledons on nutrient (OCR) medium containing cytokinins (2 rnql-¹ BAP and 0.5 rnql-¹ Kin). In addition axillary buds were initiated on hypocotyls. A better success rate was obtained by axillary budding on hypocotyls than adventitious budding on cotyledons. Best elongation of the axillary buds was recorded on OCR medium containing no growth regulators. Rooting of these elongated shoots was subsequently successfully conducted on a GO medium supplemented with 0.5 rnql-¹ NMand 2 mgI-¹l IBA. An investigation on possible chemical markers of the rooting potential of cuttings was conducted on softwood cuttings of P. elliottii hybrids. The rooting percentage correlated inversely with the total phenolic content of the cuttings. According to TLC chromatograms for the separation of phenolic acids no special phenolic acid could be related to high or low rooting potential. Immunoassays were used to determine the endogenous auxin and cytokinin levels of cuttings. The rooting percentage correlated positively with the auxin concentration and negatively with the cytokinin concentration as expected. Results obtained in this study showed that the micropropagation of P. patuIa and P. radiata is feasible. These results contribute to a better understanding of micropropagation of Pinus species which has great potential for mass propagation demanded by forestry.
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    ItemOpen Access
    Molecular characterisation of toxin-producing and non toxin-producing strains of Microcystis aeruginosa
    (University of the Free State, 2001-05) Botes, Elsabé; Grobelaar, J. U.; Botha-Oberholster, A. M.
    English: The main aim of this study was to attempt to develop a molecular screening tool for naturally occurring blooms of M. aeruginosa based on the presence or absence of the gene mcyB. This peptide synthetase has previously been implicated in toxin production in M. aeruginosa (Dittmann et al., 1997). Geographically unrelated strains of M. aeruginosa were obtained from the Pasteur Institute, France; the National Institute for Environmental Studies, Japan; the Institute of Freshwater Ecology, UK; and the University of the Free State culture collections. Based on conserved regions present in known sequences of mcyB four primer pairs were designed. The strains were maintained under standard PCR reactions were performed and the fragments generated with the various primer pairs were compared with expected fragment sizes. PCR products of the expected size were amplified in both toxin-produêing strains with all four primer pairs, signifying that these toxin-producing strains possess a copy of mcyB. It was also possible to generate PCR fragments with three primer pairs from the non toxin-producing strain CCAP 1450/1. These results indicated that this strain contained at least partial elements of mcyB. Fragments amplified by PCR from toxin-producing strains were cloned into pGemT®-Easy (Promega) and sequenced. Basepair and translated amino acid alignment of the assembled fragments showed a high degree of homology with previously deposited sequences of mcyB in the Genbank database. A fragment amplified by PCR from strain PCC 7813 with primer pair Tax 7P/3M was randomly labelled and used as a probe to screen unrelated strains of M. aeruginosa for the presence of mcyB. This probe hybridised to a fragment of the expected size in all toxin-producing strains as well as the non toxin-producing strain confirming PCR results that all strains contain this particular portion of mcyB. A second probe generated from strain PCC 7813 with primer pair Tox 1P/1M representing the fragment of mcyB not amplified by PCR in strain CCAP 1450/1 was synthesised. This probe hybridised to a fragment of the expected size in all toxin-producing strains and the non toxin-producing strain. Hybridisation of this probe to PvuII digested DNA from CCAP 1450/1 indicated that there was enough target DNA in the CCAP 1450/1 genome for the Tox 1P/1M/PCC 7813 probe to hybridise to, hinting at the possibility that this strain also possess a complete copy of the gene. Crude cell extracts were made from all strains investigated and analysed by HPLC for the presence of microcystin-LR. Microcystin-LR was detected in all toxin-producing strains as well as the 'non toxin-producing' strain CCAP 1450/1. The Institute of Freshwater Ecology where this strain was obtained from was contacted and enquiries made. From replies received it became known that firstly, the Institute has never tested the strain for microcystin-LR production and that secondly, the strains are not monocul tures. The most probable explanation for the anomalous results gathered from strain CCAP 1450/1 is that a toxin-producing M. aeruginosa type dominated in the culture for the duration of this study.