Comparison of the efficiency of some of the most usual DNA extraction methods for woody plants in different tissues of <em>Vitis vinifera</em> L.

Abstract

&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Aim&lt;/strong&gt;: To compare different methods for extracting DNA from non-recalcitrant and recalcitrant tissues of &lt;em&gt;Vitis vinifera&lt;/em&gt; woody plants and propose a modification of a previously published method to reduce the time and cost of extraction.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Methods and results&lt;/strong&gt;: DNA was extracted from young and mature leaves as well as from stems and seeds using some of the most common methods of DNA isolation and two commercial kits. Another commercial kit, which does not require DNA extraction prior to PCR, was also used. Only two methods provided adequate results in all tissues. Other methods were only applicable to some tissues and some did not yield any functional DNA in any tissue. A modification of the method reported by Marsal &lt;em&gt;et al&lt;/em&gt;. (2011) is proposed to reduce handling time and cost.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Conclusion&lt;/strong&gt;: All of the methods studied here use a surfactant to improve the extractions. For DNA extraction from recalcitrant tissues to be optimal, it is best to use a combination of dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammonium bromide (CTAB). The changes made to the protocol reported by Marsal &lt;em&gt;et al&lt;/em&gt;. (2011) enable functional DNA to be obtained from leaves in only 90 minutes and at very low cost (17 €/8 samples). However, this method cannot adequately isolate DNA from recalcitrant tissues (stems and seeds) and so, for this type of sample, we would recommend using the original method.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Significance and impact of the study&lt;/strong&gt;: Nowadays, handling time and cost are key factors in selecting the most suitable DNA extraction method. This study compares not only the effectiveness of the various methods but also the handling time and cost. It also proposes a modification of the fastest and most economic DNA extraction method for leaves so that handling time and processing cost will be reduced even further.&lt;/p&gt;

Effect of plant tissues on DNA quantity and quality of barley (Hordeum vulgare) validating through PCR technique

“…This suggested that this DNA extraction protocol is effective to isolated DNA from matured and young leave. Similar result was also described by Marsal et al (2013) based on their studies on woody plants. Similarly, Marsal et al (2011) reported that young and matured leave provide good result than the recalcitrant tissues (seeds and stems).…”

“…However, the DNA contaminants have varied along with plant tissue type and even along with the different species (Lucas et al, 2019). Many studies are carried out for plant DNA extraction either to optimize the DNA extraction protocol or to select best plant tissue for quality DNA extraction (Hasan et al, 2012;Marsal et al, 2013). But optimizing DNA extraction protocol…”

“…Despite of this limitation, DNA isolation directly from seeds provides some advantages including less-time and effort, particularly for large experiments. Indeed better quality and quantity DNA extraction has been possible yet using Cetyl tri-methyl ammonium bromide (CTAB) protocol (Boiteux et al, 1999;Hasan et al, 2012;Marsal et al, 2013). Elsalam et al (2011) was able to extracte high molecular DNA (100 to 200 ng per 100 mg) using the standard CTAB protocol from germinated wheat seeds with minimal contamination of polysaccharides and polyphenol compounds.…”

“…Research done by Sahu et al (2012) indicates that obtaining good quality DNA is difficult from matured leave. However, Marsal et al (2013) and Marsal et al (2011) reported that matured leave were source of good quality and yield of DNA suitable for PCR amplification.…”

Molecular characterization of <em>Vitis vinifera</em> L. local cultivars from volcanic areas (Canary Islands and Madeira) using SSR markers

“…DNA was extracted using the method described by Marsal et al (2013) (based on Fort et al (2008) and Marsal et al (2011) protocols). The grapevine genepool was genotyped at 20 SSR markers, which were selected for their capacity for discrimination and polymorphism in agreement with previous studies: VVS2, VVS3, VVS29 (Thomas and Scott, 1993); VVMD5, VVMD6, VVMD7 (Bowers et al, 1996); VVMD27, VVMD28, VVMD36 (Bowers et al, 1999b); VrZAG21, VrZAG47, VrZAG62, VrZAG64, VrZAG79, VrZAG83 (Sefc et al, 1999); scu06vv (Scott et al, 2000); VvUCH11, VvUCH12, VvUCH19 (Lefort et al, 2002); VChr19a (Cipriani et al, 2010).…”