An efficient protocol for tissue sampling and DNA isolation from the stem bark of Leguminosae trees

Abstract

ABstRAct.Traditionally, molecular studies of plant species have used leaves as the source of DNA. However, sampling leaves from tall tree species can be quite difficult and expensive. We developed a sequence of procedures for using stem bark as a source of DNA from Leguminosae trees of the Atlantic Forest and the Cerrado. Leguminosae is an important species-rich family in these two highly diverse and endangered biomes. A modified CTAB protocol for DNA isolation is described, and details of the procedures for sampling and storage of the bark are given. The procedures were initially developed for three species, and then their applicability for 15 other species was evaluated. DNA of satisfactory quality was obtained from the bark of all species. The amounts of DNA obtained from leaves were slightly DNA from Leguminosae tree bark higher than from bark samples, while its purity was the same. Storing the bark frozen or by drying in silica gel yielded similar results. Polymerase chain reaction amplification worked for both plastid and nuclear genomes. This alternative for isolating DNA from bark samples of trees facilitates field work with these tree species.

Isolation of High-Quality DNA from a Desert Plant Reaumuria soongorica

“…Now there are many extraction methods of genomic DNA from biomaterials. The commonly employed DNA isolation methods involve the use of enzymes such as lysozyme and proteinase K (Lockhart et al, 1989), cetytrime-thylammonium bromice (CTAB) (Ghosh et al, 2009;Moyo et al, 2008;Khanuja et al, 1999;Novaes et al, 2009;Singh et al, 2000) or sodium dodecyl sulfate (SDS) (Kaufman et al, 1999;Dellaporta et al, 1983) treatment and extraction with organic solvents, detergent-induced lysis in conjunction with proteinase K and lysozyme (Perera et al, 1994) or lysis using guanidinium isothiocyanate (GITC)-containing solutions (Boom et al, 1990;Noordhoek et al, 1995;Chakravorty & Tyagi, 2001), among which, guanidinium thiocyanate has been shown to be a powerful agent in the purification of DNA because of its potential to lyse cells and its potential to inactivate nuclease (Boom et al, 1990;Chomczynski et al, 1987;Zeillinger et al, 1993). However, high amounts of gummy polysaccharides, polyphenols and other various secondary metabolites such as alkaloids, flavonoids, terpenes and tannins in the desert plants usually hamper the DNA isolation procedures and reactions such as DNA restriction, amplification and cloning (Moyo et al, 2008;Khanuja et al, 1999;Pang et al, 2011;Zhang K., 2011;Ji & Li, 2011).…”

Genomic DNA isolation of Acrocomia aculeata (Arecaceae) from leaf and stipe tissue samples for PCR analysis

“…The stipe is a good source of DNA for molecular biology and genetic studies due to its accessibility, especially in mature wild plants (Novaes et al, 2009). Moreover, lyophilization is a helpful tool when a large number of samples are collected.…”

Isolation of high quality and polysaccharide-free DNA from leaves of Dimorphandra mollis (Leguminosae), a tree from the Brazilian Cerrado

“…Thus, the DNA obtained from mature leaves of D. mollis was pure enough to be suitable for PCR amplifications. Novaes et al (2009) reported difficulty in amplifying DNA extracted from leaves of D. mollis. This problem was probably due to not using a suitable protocol for DNA extraction of the species.…”

“…However, reports about difficulties in isolating good-quality DNA are common in plants, especially in taxa from the Cerrado biome (Novaes et al, 2009;Silva, 2010;Moreira and Oliveira, 2011). Leaf tissues of different plant species have varying levels of polysaccharides, polyphenols, and other secondary metabolites that hinder the process of DNA purification and its use in molecular studies (Khanuja et al, 1999).…”