Improved maceration techniques to study the fruit vascular anatomy of grape

The visualization and evaluation of fruit vascular bundles in grape are indispensable for understanding the development and growth of the fruit. The vascular bundles in grape are as thin as human hair, and strongly adhere to flesh cells, making it difficult to isolate individual intact vessel elements. Currently there is little information about methods to characterize grape vascular tissue. In this study, we describe an easy and effective technique to visualize the xylem cell structure of the ‘Red Globe’ grape ( Vitis L.). The intact vascular bundles of the grape were isolated through enzymatic degradation of flesh cells by hydrolases which were effective in removing flesh cells from vascular bundles. This enabled the illustration of the fine surface structure of vessel elements and their arrangement in the vascular bundles of the grape. This modified method to separate the vascular bundles into individual vessel elements was more effective than former methods of manually shaking to isolate individual vessels. Clear images of xylem vessel arrangement and structural characteristics of individual vessel element were acquired by light microscopy (BX51, Olympus, Tokyo, Japan), transmission electron microscopy (Tecnai 12, Philips, Netherlands) and scanning electron microscopy (GeminiSEM 300, Carl Zeiss, Germany). In addition, the 3D structure of vessel elements was observed using confocal laser scanning microscopy (Zeiss: model: LSM-880, Oberkochen, Germany). The imaging techniques for visualizing and analyzing the structure of xylem vessels in grape described in this study are an improvement of past methods. An effective method to isolate grape vascular bundles from flesh cells was also developed, which enables the imaging of the fine surface structure of vessel elements and their arrangement in grape vascular bundles. By adjusting the process of maceration of vascular bundles, an easy and effective method was developed to gently disrupt the plant tissue and isolate individual vessel elements. These improved techniques are suitable to observe the intact morphology of vascular bundles in the grape, improve experimental efficiency and accelerate new discoveries on the anatomical structure of the grape.