Extraction of high-quality DNA from ethanol-preserved tropical plant tissues.
BACKGROUND
Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species.


RESULTS
Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA.


CONCLUSIONS
Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue.
Eduardo A
EA
Bressan
Mônica L
ML
Rossi
Lee T S
LT
Gerald
Núcleo de Pesquisa em Tecnologia e Inovação para Sustentabilidade da Agricultura, Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av, Centenário, 303, CP 96, 13400-970 Piracicaba, SP, Brazil. figueira@cena.usp.br.
Antonio
A
Figueira
D018744:DNA, Plant / Q000302:isolation & purification*; D000431:Ethanol / Q000737:chemistry*; D005615:Freezing; D018515:Plant Leaves / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D010944:Plants / Q000378:metabolism*; D011309:Preservation, Biological; D014329:Tropical Climate
D018744:DNA, Plant; D000431:Ethanol