A modified staining technique for arbuscular mycorrhiza compatible with molecular probes
The effects of the different steps of the root staining on the arbuscular mycorrhizal (AM) fungal rDNA extraction and amplification have been assessed. The results obtained using molecular techniques are compared with those obtained from fresh, non-stained leek roots. A modified staining procedure that eliminates heating, the use of hydrochloric acid and trypan blue, has been proved to be the most adequate to observe the AM colonisation in different plant species with/without lignified roots allowing at the same time the subsequent rDNA extraction and amplification from the stained roots. The staining technique decreased the sensitivity of the process and a higher number of roots had to be used to obtain enough material for a positive amplification. The extraction and amplification process was reliable up to 3 days after staining. A week after staining, the amplification was not dependable and after 2 weeks there was no amplification from stained material.
KeywordsArbuscular mycorrhizal fungi Fungal detection DNA extraction Nested PCR Staining techniques
This research was supported by grants CGL2006-05648, RTA04-027-C2 and RTA2007-00039 from the Spanish Ministry of Science and Education (MEC).
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