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

Abstract

Aim: To compare different methods for extracting DNA from non-recalcitrant and recalcitrant tissues of Vitis vinifera woody plants and propose a modification of a previously published method to reduce the time and cost of extraction.

Methods and results: 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 et al. (2011) is proposed to reduce handling time and cost.

Conclusion: 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 et al. (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.

Significance and impact of the study: 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.