Abstract
Molecular marker techniques have been widely used for cultivar identification of inbred date palms (Phoenix dactylifera L.; Arecaceae) and biodiversity conservation. Isolation of highly pure DNA is the prerequisite for PCR amplification and subsequent use such as DNA fingerprinting and sequencing of genes that have recently been developed for barcoding. To avoid problems related to the preservation and use of liquid nitrogen, we examined sterile sand for grinding the date palm leaves. Individual and combined effects of sodium chloride (NaCl), polyvinylpyrrolidone (PVP) and lithium chloride (LiCl) with the cetyltrimethylammonium bromide (CTAB) method for a DNA yield of sufficient purity and PCR amplification were evaluated in this study. Presence of LiCl and PVP alone or together in the lysis buffer did not significantly improve the DNA yield and purity compared with the addition of NaCl. Our study suggested that grinding of date palm leaf with sterile sand and inclusion of NaCl (1.4 M) in the lysis buffer without the costly use of liquid nitrogen, PVP and LiCl, provides a DNA yield of sufficient purity, suitable for PCR amplification.
Highlights
Most of the plant DNA isolation methods including commercial kits require grinding of the plant material in liquid nitrogen
The highly versatile cetyl trimethylammonium bromide (CTAB) method has been used for the extraction of DNA from various plant materials [4]
DNA extracted with the buffers B, C and D produced a higher yield compared with buffers A and E (Figure 1, upper panel)
Summary
Most of the plant DNA isolation methods including commercial kits require grinding of the plant material in liquid nitrogen. Date palm leaves are hard, fibrous and the extraction of genomic DNA from the leaves is difficult. To avoid the problems related with the preservation and use of liquid nitrogen, acid-washed sand or glass powder were used for grinding the leaves of date palm [2]. A combination of NaCl, PVP and LiCl has been used with the CTAB method for the isolation of genomic DNA from coniferous tissues (Taxus baccata) [8]. The individual effects of NaCl, PVP and LiCl as well as their typical combinations have not been tested for optimal isolation of genomic DNA from plant tissues. Our main objective was to optimize a simple, inexpensive and rapid procedure for DNA isolation from tough leaves (date palm) without compromising the yield and purity of DNA
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