Abstract
Rosemary plant is in high demand due to its application in traditional health care, food flavoring, fragrance and pharmaceutical industries. It contains high level of secondary metabolites which are responsible for its beneficial activities. Application of molecular techniques would facilitate the production of these substances and screening of accessions. The isolation of polymerase chain reaction (PCR) amplifiable genomic DNA is a pre-requisite for taking advantage of these technologies. Even though several DNA isolation protocols for plants with high level of secondary metabolites were developed, they may not permit optimal DNA extraction due to chemotypic variation within species. Extracting DNA from different rosemary accessions is a challenging task due to its high level of secondary metabolites. Therefore, this research is conducted with the aim of optimizing a reliable and rapid method suitable for extracting DNA from rosemary plants. The optimized protocol avoids the use of repeated toxic phenols, liquid nitrogen and large polypropylene tube. It is appropriate for both fresh and dry leaf samples. The quality of the obtained DNA was excellent as evident by A260/A280 ratio ranging from 1.7 to 1.89 and the concentration ranged from 195.8 to 2184 ng/µl. The success of this protocol indicated its applicability for other plants with high secondary metabolite contents. Key words: DNA isolation, secondary metabolites, rosemary, gel electrophoresis, polymerase chain reaction (PCR) amplification.
Highlights
Rosemary (Rosemarinus officinalis L.) is an aromatic, medicinal and spice herb that belongs to the Lamiaceae family (Elhassan and Osman, 2014)
DNA extraction method developed by diversity array technology (DArT, 2019), cetyl trimethylammonium bromide (CTAB) extraction method by Doyle and Doyle (1990) and a methods developed by Khanuja et al (1999) were employed for extracting DNA from rosemary accessions
Standardized DNA extraction procedure (i) 10 mg of -80°C frozen leaves were ground to fine powder by using pre chilled mortar and pestle. (ii) The powdered materials were transferred into 2 ml sterile Eppendorf tube and 1000 μl of freshly prepared extraction buffer which contains 100 mMTris-HCl, 25 mM EDTA, 1.5 M NaCl, 2.5% CTAB (w/v), 0.2% β-mercaptoethanol (v/v) and 1% PVP (w/v) was added and mixed by inverting the tubes slowly. (iii) After properly mixed, the samples were incubated at 65°C for 90 min in water bath (2 h incubation was employed for dry samples)
Summary
Rosemary (Rosemarinus officinalis L.) is an aromatic, medicinal and spice herb that belongs to the Lamiaceae family (Elhassan and Osman, 2014). The genus Rosemarinus includes Rosmarinus eriocalyx, Rosmarinus tomentosus, Rosmarinus lavandulaceus and Rosmarinus laxiflorus (Zaouali et al, 2010; Rosselló et al, 2006; Upson, 2006; Angioni et al, 2004; Elamrani et al, 2000; Arnold et al, 1997). Among all Rosmarinus species, only R. officinalis had gained medicinal, pharmaceutical and industrial importance. It is the most exploited species due to its valuable essential oil and phenolic contents (Zaouali et al, 2010).
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