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Redalyc.Overcoming DNA Extraction Problems from Carnivorous Plants Anales del Jardín Botánico de Madrid ISSN: 0211-1322 [email protected] Consejo Superior de Investigaciones Científicas España Fleischmann, Andreas; Heubl, Günther Overcoming DNA extraction problems from carnivorous plants Anales del Jardín Botánico de Madrid, vol. 66, núm. 2, julio-diciembre, 2009, pp. 209-215 Consejo Superior de Investigaciones Científicas Madrid, España Available in: http://www.redalyc.org/articulo.oa?id=55612913003 How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative Anales del Jardín Botánico de Madrid Vol. 66(2): 209-215 julio-diciembre 2009 ISSN: 0211-1322 doi: 10.3989/ajbm.2198 Overcoming DNA extraction problems from carnivorous plants by Andreas Fleischmann & Günther Heubl LMU Munich, Systematic Botany and Mycology, Menzinger Strasse 67, D-80638 Munich, Germany [email protected] Abstract Resumen Fleischmann, A. & Heubl, G. 2009. Overcoming DNA extraction Fleischmann, A. & Heubl, G. 2009. Superando problemas de ex- problems from carnivorous plants. Anales Jard. Bot. Madrid tracción de ADN de plantas carnívoras. Anales Jard. Bot. Madrid 66(2): 209-215. 66(2): 209-215 (en inglés). We tested previously published protocols for DNA isolation from Probamos algunos protocolos publicados previamente para el plants with high contents of polyphenols and polysaccharides aislamiento del ADN de plantas con alto contenido de polifeno- for several taxa of carnivorous plants. However, we did not get les y polisacáridos para varios táxones de plantas carnívoras. Sin satisfying results with fresh or silica dried leaf tissue obtained embargo, no conseguimos muy buenos resultados ni con tejidos from field collected or greenhouse grown plants, nor from de hojas frescas, ni con tejidos de hojas secadas en gel de sílice herbarium specimens. Therefore, we have developed a simple obtenidas de plantas colectadas en el campo o cultivadas en los modified protocol of the commercially available Macherey- invernaderos, ni de especímenes de herbario. Por lo tanto, he- Nagel NucleoSpin® Plant kit for rapid, effective and repro- mos desarrollado un protocolo sencillo, modificado del Mache- ducible isolation of high quality genomic DNA suitable for PCR rey-Nagel NucleoSpin® Plant kit disponible en el mercado para reactions. DNA extraction can be conducted from both fresh el aislamiento rápido, eficaz y reproducible de ADN genómico and dried leaf tissue of various carnivorous plant taxa, irrespec- de alta calidad conveniente para la reacción en cadena de la po- tive of high contents of polysaccharides, phenolic compounds limerasa. La extracción del ADN se puede realizar en tejidos de and other secondary plant metabolites that interfere with DNA hojas frescas o secas de varios táxones de plantas carnívoras, sin isolation and amplification. importar el grado de contenido de polisacáridos, compuestos fenólicos u otros metabolitos secundarios que interfieren con el aislamiento y la amplificación del ADN. Keywords: carnivorous plants, DNA extraction, polyphenols, Palabras clave: plantas carnívoras, extracción del ADN, polife- polysaccharides, secondary metabolites, viscous mucilage. noles, polisacáridos, metabolitos secundarios, mucílago viscoso. Introduction & Griesbach, 2001) and Roridula (Wollenweber, 2007). The carnivorous plant members of the order Many carnivorous plants show bright coloured Nepenthales (Aldrovanda, Dionaea, Drosera, Dro- leaves, which serve as visual attractants for insect prey sophyllum, Nepenthes and Triphyophyllum) are addi- (Juniper & al., 1989). Most pitcher plants (Cephalotus, tionally characterised by quinones like plumbagin Darlingtonia, Heliamphora, Nepenthes and Sarracenia) (Schlauer & al., 2005). The phenolic metabolite group often have dark red spots, markings, veins or bright of phenylethanoid glycosides is mainly restricted to the coloured leaves which serve as insect guides and prob- Lamiales, an observation supported by the presence of ably mimic flowers (Juniper & al., 1989). Quite often acetoside, which was found in both carnivorous fami- these colour marks result from polyphenolic secondary lies of this order, Byblidaceae and Lentibulariaceae metabolites (flavonoids or tannins). Polyphenolic com- (Schlauer & al., 2004; Schlauer & al., 2005). pounds are known from several carnivorous plants, for In those carnivorous plants that trap insects by example flavonoids occur in Sarraceniaceae (Sheridan sticky flypaper traps, such as Drosera, Drosophyllum, 210 A. Fleischmann & G. Heubl Triphyophyllum, Byblis and Pinguicula, the leaves are and problems with DNA extraction spurred the deve- usually densely covered with specialized glands that lopment of a more reliable method of obtaining high secrete a water-based viscous mucilage that contains quality DNA from leaves of carnivorous plants, irres - predominantly acidic polysaccharides (Juniper & al., pective of the plant tissue used for extraction. There- 1989; Rost & Schauer, 1977). In the two species of the fore, a standard protocol for quick and easy genomic pre-carnivorous genus Roridula from South Africa, DNA extraction from a NucleoSpin Plant kit the glue of the sticky traps is resin-based (Lloyd, (Macherey-Nagel, Germany) was modified to fulfil the 1934) and rich in phenolic compounds, such as specific needs when dealing with DNA isolation from flavonoids (Wollenweber, 2007). carnivorous plant material rich in secondary metabo- Both phenolic secondary metabolites and leaf se- lites, polyphenols and polysaccharides. cretions in the mentioned carnivorous plants impede the extraction of DNA from leaf tissue. During ho- Material and methods mogenization of the plant samples, phenolic compo- nents can become oxidized (Loomis, 1974). Oxi- Plant material dized polyphenols are known to bind covalently and Plant samples from herbarium specimens and living therefore irreversibly to DNA molecules and thus in- plants from tissue culture were obtained from various terfere with subsequent reactions such as DNA am- sources (see Table 1 for details). plification, restriction digest and cloning (Katterman & Shattuck, 1983; Stange & al., 1998; Porebski & al., Reagents and solutions 1997). Polysaccharides inhibit the enzymatic activity • Buffer C0 solution (cat. no. 740570.250, of several enzymes such as polymerases, ligases and Macherey-Nagel (M-N)). restriction endonucleases (Shioda & Marakami- • 5% sodium N-lauroyl sarcosine (w/v) (Sigma). Muofushi, 1987; Richards, 1988), including Taq poly- • 10% PVP (polyvinyl-pyrrolidone) (w/v) (Sigma). merase (Fang & al., 1992) and thus interfere with • 5 M NaCl. PCR (polymerase chain reaction). The result is either • RNase A solution (cat. no. 740505, M-N). a very low DNA yield or none at all, or if DNA can be • Buffer C4 solution (cat. no. 740935, M-N). extracted, the remaining secondary metabolites may • Phenol:chloroform (1:1 v/v). inhibit further steps in DNA amplification. For • Chloroform:isoamyl alcohol (24:1 v/v). exam ple, total genomic DNA isolated from bright • Ethanol (100%). coloured leaves of several species of Drosera, He- • Buffer CW (cat. no. 740932, M-N). liamphora and Sarracenia could not be amplified by • Buffer C5 solution (cat. no. 740931, M-N). PCR in our studies. Therefore, genomic DNA extrac- • Elution buffer CE (cat. no. 740570.250, M-N). tion in carnivorous plants was acquired from flower material in some cases (A. Fleischmann, unpu- PVP, sodium N-lauroyl sarcosine and NaCl solu- blished; Müller & al., 2004), which usually contains tions are sterilized by autoclaving. lower amounts of polyphenols and polysaccharides than the carnivorous leaves. Unfortunately, flower DNA extraction material is not always available in abundance and This method for DNA extraction is a modified ver- many crucial species only flower rarely in cultivation, sion of the NucleoSpin® Plant kit standard user ma- or removal of flowers from herbarium specimens is nual “Genomic DNA from Plant” (Macherey-Nagel, not allowed. 2007). Five different protocols for DNA isolation from plants with high concentrations of polyphenols and The plant samples are homogenized, for fresh leaf polysaccharides have been tested and compared when tissue preferably by grinding in a mortar under liquid conducting this study (Tel-Zur & al., 1999; Stange & nitrogen. Dried herbarium specimens or silica dried al., 1998; Porebski & al., 1997; Lodhi & al., 1994), in- material is best homogenized using an automatic cluding one protocol specifically designed for DNA retch. To achieve lysis of the plant cells, the (frozen) extraction from in vitro material of the carnivorous powder is transferred into a capped microfuge tube. plant Drosera rotundifolia (Bekesiova & al., 1999). Following the protocol of the manufacturer of the M- None of these methods, however, enabled us to obtain N NucleoSpin® Plant kit (Macherey-Nagel, 2007) total genomic DNA suitable for PCR from the carni- 400 µl buffer C0 (preheated to 45°C) are added. In vorous plant taxa used in this study. Consequently our addition to the original recipe, we add 94µl 5M NaCl, broad and long-term research on carnivorous plants 120µl of 5% sodium N-lauroyl sarcosine and 60µl Anales del Jardín Botánico de Madrid 66(2): 209-215, julio-diciembre 2009. ISSN: 0211-1322. doi: 10.3989/ajbm. 2198 DNA extraction from carnivorous plants 211 10% PVP to the suspension.
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