Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in Encephalartos

Abstract

Premise Plants with stiff, leathery leaves pose a challenge for standard DNA extraction protocols. These tissues are recalcitrant to mechanical disruption via TissueLyser (or analogous devices) and are often high in secondary metabolites. These compounding factors result in low yields, which may be sufficient for PCR amplification but are generally inadequate for genomic applications that require large quantities of high‐quality DNA. Cycads in the genus Encephalartos exemplify these challenges, as this group of plants is fortified for life in harsh, dry habitats with notoriously thick and rigid leaves. Methods and Results Using a DNA extraction kit, we tested three methods of mechanical disruption and examined the differences between stored vs. freshly collected samples and mature vs. senescing leaflets. We found that the manual method of pulverizing tissue yields the highest concentrations of DNA, and that both senescing leaflets and leaflet tissue that has been stored for extended periods yield sufficient DNA for genomic analyses. Conclusions These findings shed light on the feasibility of using senescing leaves and/or tissue that has been stored on silica for long periods of time when attempting to extract large amounts of DNA. We provide here an optimized DNA extraction protocol that can be applied to cycads and other plant groups with tough or rigid leaves.

Emerging methods in botanical DNA/RNA extraction

“…To address plants that are recalcitrant due to sclerophylly, Jones et al ( 2023 ) investigate the effects of mechanical tissue disruption, storage time, and leaflet age on DNA extraction quality and concentration using the thick and rigid leaves of the cycad genus Encephalartos . In cycads, recalcitrant leaf tissue yielded greater concentrations of DNA when tissue was manually disrupted using a mortar and pestle vs. bead‐based disruption, although DNA purity was unaffected by the disruption method.…”

“…The time of storage in silica gel ranged from less than a year to a decade, with no significant effect on resulting DNA concentration. Finally, Jones et al ( 2023 ) reveal that both senescing and young leaflets could adequately yield sufficient, pure DNA for downstream applications.…”