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Molecular detection and diagnosis of fungal contaminants of recalcitrant seeds: Quercus robur L. acorns as a model system

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Recovery in vitro after cryostorage of excised embryonic axes from recalcitrant seeds is frequently compromised by the proliferation of seed-borne mycoflora. Current culture-based methods to ascertain the contamination status of seeds/axes prior to cryostorage are time-consuming and sometimes inaccurate. This study assessed the potential of the PCR as a faster, more sensitive, reliable alternative to detect fungal contaminants. Published universal primer DNA sequences were used to amplify the Internal Transcribed Spacer region (ITS) of the 28S rDNA gene from genomic DNA of the principal fungal contaminants, viz. Fusarium sp., Penicillium sp. and Aspergillus sp., of acorns of South African provenance. The sequences of the amplicons obtained by universal amplification were then used as a basis for the synthesis of genus-specific DNA primers. Subsequent assessment revealed that the primers were specific to the genus for which they had been designed and that fidelity was retained in a complex template consisting of a mixture of seed and fungal genomic DNA. The developed method was shown to be sensitive, the lowest detection limit being 0.1 ng of fungal DNA. The specificity and sensitivity of the PCR was confirmed by the successful detection and discrimination amongst fungi in the embryonic axes of experimentally inoculated seeds.
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Document Type: Research Article

Publication date: July 1, 2006

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  • Seed Science and Technology (SST) is one of the leading international journals featuring original papers and review articles on seed quality and physiology as related to seed production, harvest, processing, sampling, storage, distribution and testing. This widely recognised journal is designed to meet the needs of researchers, advisers and all those involved in the improvement and technical control of seed quality.
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