Versatile DNA extraction from diverse plant taxa using ionic liquids and magnetic ionic liquids: a methodological breakthrough for enhanced sample utility

Abstract

Background: There is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications.

Results: In a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant, Arabidopsis thaliana (L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastid rbcL and nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis.

Conclusions: The method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics.

Keywords: Ionic liquids; Magnetic ionic liquids; Matrix solid phase dispersion; Miniaturization; Plant DNA isolation; Sequencing; qPCR.

Fig. 1

Schematic diagram illustrating the (a) IL- based VA-MSPD approach and (b) direct IL- based extraction for the isolation of DNA from 1.5 mg of plant tissue using 15 µL of IL / MIL. (adapted from [23])

Fig. 2

Cq values as a measure of amplification success for (a) the ITS marker and (b)  rbcL marker derived from qPCR amplification of plant DNA extracted by the IL-VA-MSPD procedure employing 1.5 mg of treated plant tissue and 15 µL of [P_6,6,6,14⁺] [NTf₂^-] IL. Extractions were carried out in triplicate. (Cq > 30 is considered as delayed amplification) Note: *Complete inhibition of PCR was observed of Quercus macrocarpa therefore rbcL amplification was not carried out. ♦ A Cq value was not determined due to delayed amplification

Fig. 3

Effect of the plant matrix on the amplification of non-target 98 bp BRAF DNA template. A volume of 1 µL of 10.2 fg/µL non-target 98 bp DNA template (BRAF) was spiked into the qPCR assay and amplified in the presence of (a) 0.5 µL of the IL-DMSO-Water extract and (b) 0.5 µL of the MIL-DMSO extract containing plant DNA. All experiments were carried out in triplicate. Note: *Complete inhibition of PCR was observed

Fig. 4

Stability of extracted DNA over time from 1.5 mg of treated plant tissue using 15 µL of [P_6,6,6,14⁺] [NTf₂⁻] IL. The MSPD procedure was used in the extraction and DNA was stored in IL-DMSO-water mixture at room temperature. Stability evaluated in terms of Cq values as a measure of amplification success for the rbcL marker. All experiments were conducted in triplicate. Note: *A Cq value was not determined after 2 weeks due to diminished fluorescence in the amplification curves

Fig. 5

Stability of extracted DNA over time from 1.5 mg of treated plant tissue using 15 µL of [P_6,6,6,14⁺] [Ni(hfacac)₃⁻] MIL. The MSPD procedure was used in the extraction and DNA was stored in Ni MIL-DMSO mixture at room temperature. Stability evaluated in terms of Cq values as a measure of amplification success for the rbcL marker. All experiments were conducted in triplicate. Note: *A Cq value was not determined after 1 week due to diminished fluorescence in the amplification curves. (Stability tests were not performed for Lilium henryi and Pennisetum glaucum due to the delayed or no amplification in the initial experiments)