Comparison of Different Leaf Preservation Methods To Obtain High Quality DNA From Enset (Ensete Ventricosum (Welw.) Cheesman), A Native And Orphan Food Security Crop In Ethiopia

Abstract

\n Background: Enset (Ensete ventricosum (Welw.) Cheesman) is a staple food for more than 20 million Ethiopians and only cultivated in the native indigenous farming systems of Ethiopia. In contrast to other cultivated species in the Musaceae family, enset has been relatively little studied at the molecular level. Application of advanced molecular genetic techniques requires rapid extraction of DNA of high quality and quantity. Fresh, lyophilized tissues, as well as tissues stored in liquid nitrogen are mainly preferred to avoid DNA degradation, thus most of the DNA extraction protocols recommend these types of tissues as starting material. However, such sample processing techniques are difficult to utilize in many developing countries and at collection sites of many endemic plant species, underutilized or orphan crop species like enset. These situations necessitate the development of alternative protocols for leaf preservation and optimized methods for isolating high-quality DNA from dried or preserved leaf samples. Results: In this study, three different leaf preservation and two DNA extraction methods were compared. Fresh young leaf tissue was preserved using the minor modified saturated NaCl-CTAB solution, silica gel or 96% ethanol at ambient temperature for more than 35 days. Subsequently, DNA was extracted using either the DNeasy Plant Mini Kit or the CTAB method. As compared to silica gel and 96% ethanol, the minor modified saturated NaCl-CTAB solution preserved the quality, quantity, and integrity of enset genomic DNA. This method consistently produced genomic DNA of high-quality and quantity at affordable cost. The DNeasy Plant Mini Kit method was found to be more efficient than the standard CTAB method, being faster and producing genomic DNA of higher quality. Conclusions: Using saturated NaCl-CTAB solution is an accessible, efficient, scalable, and inexpensive way to preserve enset leaves during collection and transportation. The preservation protocol was validated for leaf tissues of all cultivated and wild enset, and Entada landraces. Genomic DNA of high quality and quantity was obtained from preserved enset leaves, which can be used for further downstream applications including PCR and sequencing.