Optimized flow cytometry protocol for assessing DNA content in Vanilla (Orchidaceae) species: Insights into crop wild relatives and a commercial hybrid

Abstract

Vanilla (Orchidaceae) is a highly valued spice used in a wide range of products. Although wild crop relatives of Vanilla planifolia, the primary cultivated species, may possess traits for crop improvement, they have received limited attention. This study presents an optimized and reproducible flow cytometry protocol adapted to Vanilla leaves, that effectively prevents nuclei clustering and raphide interference. Assessing nuclear C-DNA content and estimating DNA ploidy in succulent plant tissues pose significant challenges. The abundance of polysaccharides and raphides in Vanilla tissues often leads to inaccuracies in cytometric measurements and difficulties in instrument maintenance. To address this issue, we used a nuclei isolation buffer containing 2.0 % (v/v) detergent and utilized the clear upper phase of the filtrate after raphide sedimentation to obtain accurate nuclear DNA content values. We applied this protocol to estimate the C-DNA content of 12 Vanilla species native to Costa Rica and a widely cultivated commercial hybrid. The values ranged from 5.211 ± 0.008 pg in V. planifolia to 9.0 ± 1.3 pg in V. pompona, with no clear distinction between species of the subgenera Vanilla and Xanata. For the first time, we report the 2C-DNA content of V. costaricensis, V. dressleri, V. hartii, V. helleri, V. inodora, V. karen-christianae and the 'Vaitsy' hybrid. Our results confirm the occurrence of partial endoreplication in all samples analyzed. This study contributes to the understanding of less studied wild crop relatives of V. planifolia and provides an improved method for flow cytometric analysis that can be applied to tissues containing mucilaginous compounds and raphides of other plant species.