Early sex identification by leaflet distance in plantlets of Cycas revoluta

In dioecious plants, the process of sex determination usually occurs during the reproductive stage. However, it can be challenging to determine the sex of long-lived plants with long generation times. This is true for Cycas revoluta, which produces toxic seeds in female plants, leading to a preference for male plants in public green spaces. In this study we aim to identify a morphological trait that can be used to distinguish between the sexes in C. revoluta at a very early stage of plant growth. We sampled C. revoluta seedlings/plantlets at three different early growth stages and identified the sex of individuals by PCR amplification of a male-specific molecular marker. On the same plants, we measured morphological traits, including the distance between leaflets on the leaf rachis, perimeter, area, number of indents, and leaf complexity, and evaluated their correlation with the sex of the individuals. Among all measured traits, the medium distance between leaflets on the leaf rachis was found to be a sexually dimorphic morphological trait in Cycas plantlets in all three growth stages. In particular, the distance between leaflets on the leaf rachis in the upper part of the leaf in the first stage revealed no overlap between the sexes. Whenever available, morphological traits for sex differentiation in greenhouses and plant nurseries can represent a rapid and economic tool when coupled with automatized image recognition systems.