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, whic...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2023-05-01
|
| Series: | Applications in Plant Sciences |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/aps3.11525 |
| _version_ | 1797800187511439360 |
|---|---|
| author | Maia M. Jones Nathalie S. Nagalingum Vanessa M. Handley |
| author_facet | Maia M. Jones Nathalie S. Nagalingum Vanessa M. Handley |
| author_sort | Maia M. Jones |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-13T04:30:18Z |
| format | Article |
| id | doaj.art-c6d4739651f849ffa8480e73fc3a5dd5 |
| institution | Directory Open Access Journal |
| issn | 2168-0450 |
| language | English |
| last_indexed | 2024-03-13T04:30:18Z |
| publishDate | 2023-05-01 |
| publisher | Wiley |
| record_format | Article |
| series | Applications in Plant Sciences |
| spelling | doaj.art-c6d4739651f849ffa8480e73fc3a5dd52023-06-19T16:55:29ZengWileyApplications in Plant Sciences2168-04502023-05-01113n/an/a10.1002/aps3.11525Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in EncephalartosMaia M. Jones0Nathalie S. Nagalingum1Vanessa M. Handley2California Academy of Sciences 55 Music Concourse Drive San Francisco California 94118 USACalifornia Academy of Sciences 55 Music Concourse Drive San Francisco California 94118 USACalifornia Academy of Sciences 55 Music Concourse Drive San Francisco California 94118 USAAbstract 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.https://doi.org/10.1002/aps3.11525cycadsDNA extractionrecalcitrant tissuestored tissue |
| spellingShingle | Maia M. Jones Nathalie S. Nagalingum Vanessa M. Handley Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in Encephalartos Applications in Plant Sciences cycads DNA extraction recalcitrant tissue stored tissue |
| title | Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in Encephalartos |
| title_full | Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in Encephalartos |
| title_fullStr | Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in Encephalartos |
| title_full_unstemmed | Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in Encephalartos |
| title_short | Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in Encephalartos |
| title_sort | testing protocols to optimize dna extraction from tough leaf tissue a case study in encephalartos |
| topic | cycads DNA extraction recalcitrant tissue stored tissue |
| url | https://doi.org/10.1002/aps3.11525 |
| work_keys_str_mv | AT maiamjones testingprotocolstooptimizednaextractionfromtoughleaftissueacasestudyinencephalartos AT nathaliesnagalingum testingprotocolstooptimizednaextractionfromtoughleaftissueacasestudyinencephalartos AT vanessamhandley testingprotocolstooptimizednaextractionfromtoughleaftissueacasestudyinencephalartos |