Citation: Lukši´ c, K.; Zduni´ c, G.; Mucalo, A.; Marinov, L.; Rankovi´ c-Vasi´ c, Z.; Ivanovi´ c, J.; Nikoli´ c, D. Microstructure of Croatian Wild Grapevine (Vitis vinifera subsp. sylvestris Gmel Hegi) Pollen Grains Revealed by Scanning Electron Microscopy. Plants 2022, 11, 1479. https://doi.org/10.3390/ plants11111479 Academic Editor: Agnes Farkas Received: 2 May 2022 Accepted: 26 May 2022 Published: 31 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Microstructure of Croatian Wild Grapevine (Vitis vinifera subsp. sylvestris Gmel Hegi) Pollen Grains Revealed by Scanning Electron Microscopy Katarina Lukši´ c 1 , Goran Zduni´ c 1, *, Ana Mucalo 1 , Luka Marinov 1 , Zorica Rankovi´ c-Vasi´ c 2 , Jelena Ivanovi´ c 2 and Dragan Nikoli´ c 2 1 Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21 000 Split, Croatia; katarina.luksic@krs.hr (K.L.); ana.mucalo@krs.hr (A.M.); luka.marinov@krs.hr (L.M.) 2 Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Belgrade, Serbia; zoricarv@agrif.bg.ac.rs (Z.R.-V.); angelina.ivanovic.95@gmail.com (J.I.); nikolicd@agrif.bg.ac.rs (D.N.) * Correspondence: goran.zdunic@krs.hr; Tel.: +385-21-434-453 Abstract: Wild grapevine (Vitis vinifera subsp. sylvestris Gmel Hegi) is dioecious with male and female plants, whereas domesticated grapevine is mostly hermaphrodite with self-fertile hermaphrodite flowers. The pollen morphology of wild grapevine has been poorly studied. There is no detailed palynological study of V. sylvestris in Croatia and neighboring countries. Here, scanning electron microscopy (SEM) was used to analyze the pollen of V. sylvestris from male and female individuals growing at two natural sites in Croatia. The selective APT3 marker was used to confirm the flower phenotype with the genetic background. SEM analysis showed that the pollen grains of V. sylvestris were isopolar and radially symmetrical, with foveolate perforated ornamentation, regardless of the flower type of the individuals. All male flowers were 3-colporate and prolate in shape, whereas female individuals varied from subprolate to spheroidal and had inaperturate pollen grains. Pollen shape, dimensions and exine ornamentation proved very informative, and here we address the most polymorphic traits in the analyzed V. sylvestris individuals. Principal component analysis (PCA) and clustering based on pollen morphology variables clearly differentiated individuals by their flower type, and no grouping specific to population was observed, pointing to the conserved pollen structure of V. sylvestris. The results indicate the need to continue the palynological study of V. sylvestris and serve as a good phenotypic basis for functional genetic studies on genes involved in pollen morphology and function. Keywords: Vitis vinifera subsp. sylvestris; flower morphology; pollen structure; SEM 1. Introduction The Eurasian grapevine (Vitis vinifera L.) includes two subspecies: wild (Vitis vinifera L. subsp. sylvestris Hegi Gmelin, hereafter V. sylvestris) and domesticated grapevine (Vitis vinifera L. subsp. vinifera, hereafter V. vinifera). Both subspecies are diploid (2n = 38), sexually compatible, and prefer cross-pollination [1]. Despite sexual compatibility, in prac- tice, spatial dislocation between natural V. sylvestris habitats and vineyards and differences in flowering time between the two subspecies often result in low pollen exchange [2]. The wild grapevine is dioecious and presumed to be an ancestor of domesticated grapevines [3]. The crucial difference between the two subspecies is in flower morphol- ogy. Domesticated grapevine has mostly hermaphroditic and, in some cultivars, female- type flowers, whereas wild grapevine exhibits male or female flowers. Recent transcrip- tomic studies showed that all three types of flowers begin their development as perfect hermaphroditic flowers [4–6]. In later stages of flower specification, male flowers show a reduced pistil, style and stigma, whereas females have shorter, backward-reflexed or shriveled stamens with sterile pollen [6,7]. Plants 2022, 11, 1479. https://doi.org/10.3390/plants11111479 https://www.mdpi.com/journal/plants