Optimizing a DNA Extraction Protocol for Date Palm (Phoenix dactylifera) using Liquid Nitrogen

Abstract

Date palm (Phoenix dactylifera) is a dioecious fruit plant belongs to the family Arecaceae. These plants are grown in coastal areas in North-Eastern and South-Eastern regions of Sri Lanka. Date palm fruits have a high demand in globally as well as in local food industry.Date palm cultivators face a problem in identification of its sex because date palm has no distinguishable morphological features for sex determination until the sexual maturity is attained after 5-8 years of cultivation. In recent years, prior identification of gender specific DNA sequences have efficiently facilitated the sex identification of immature plants to avoid unnecessary management of male plants. Isolation of high-quality DNA is vital for these kinds of molecular biological applications. Therefore, this study was aimed at optimizing a widely used CTAB protocol (Doyle & Doyle) for date palm by introducing liquid nitrogen lysis step, increased volume of cell lysis buffer, increased concentration of Beta Mercaptaethanol, repeated choloroform-phenol extraction step and repeated ethanol washing steps at the end. The purity and concentration of isolated DNA were determined by using NanodropTM 2000 Spectrophotometer. Optical density at 230nm, 260nm and 280 nm were recorded and A260/280, A260/230 ratios were calculated. Further, DNA samples were checked in 0.8% agarose gels. 25 out of 33 samples gave acceptable values for A260/280 and A260/230, as ~1.8 and ~1.9, respectively which confirmed the absence of protein and other possible contaminants in the final elute. Clear bands of the gel electrogram confirmed the presence of genomic DNA with high purity.