Observation of Flowering Process of Grape(Vitis vinifera L.)—Insight into the Starting of Pollination of Flower Hiding in Calyptras(Cap)

It is generally agreed that many Vitis vinifera L.cultivars are self-fertile,where self-pollination often occurs before capfall in a process called cleistogamy.Therefore,it is difficult to identify the right time to remove stamens before self-pollination during the cross-breeding of grape.For this paper,we observed the process of grape flowering and measured the pollen viability and stigma receptivity of grape flowers of‘Shine Muscat’in order to identify the starting time of self-pollination before capfall and to provide useful information for improving the efficiency of cross-breeding.The results demonstrate that the anther is not cracked during the visible clusters and separated clusters stages.Meanwhile,in the separated floral buds,flowering begins,and full bloom stages,the pollen viability is 60.7%,73.2%and 80.3%,respectively;however,at the berry set stage,pollen viability drops to zero.The top of the mature stigma is composed of a layer of nearly cylindrical papillary cells,and the stigma receptivity for pollen changes with the development of flowers:in particular,no reaction was observed in the visible clusters stage;weak positive reaction at the separated clusters stage;strong positive reaction at the separated floral buds,flowering begins,and full bloom stages;and no reaction at the berry set stage.In the separated floral buds stage,pollen tubes were seen germinating in the style.In the flowering begins stage,more pollen tubes were observed at the entry of the ovary.During the full bloom stage,most pollen tubes elongated into the ovary base and some entered the pearl hole.At the berry set stage,newborn endosperm nucleus could be seen in the ovule.From the above,we can conclude that the initiation time of closed fertilization for‘Shine Muscat’grape can be judged as the separated floral buds stage,and it is best to discard the stamen before the separated floral buds stage when conducting cross-breeding.

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.

A Simple Clear Technique in Observing Vascular Development of Grape Ovary

The vascular system of the grapevine(Vitis vinifera L.)flower is a channel for transporting water and nutrients to the ovary.It plays an important role in the development of the ovary and fertilization through pollination.However,the vascular bundles in the flower are so tiny that they are difficult to sample and observe by traditional slicing techniques.In this study,‘Summer Black’grape flowers were selected as the test materials,and the tissue samples were treated by the optical clearing technique.After simple compaction,the structure and development of the vasculature were observed by common microscopy,fluorescence microscopy and laser confocal microscopy.The results showed that the transparency effects of 3%NaOH and a saturated trichloroacetaldehyde composite agreed well with the observations of the vascular structure and the developmental process of the flower in different periods.Moreover,the samples after optical clearing could be reconstructed in 3D,which helped us know more about its development and function.According to these observations,the vasculature of the‘Summer Black’flower can be divided into ovule vascular bundles,peripheral vascular bundles and central vascular bundles.The peripheral vascular bundles were composed of the first-order vascular bundles and the inferior vascular bundles which branched from the superior vascular bundles.These bundles branched in different directions with no discernible pattern.The two different branching methods were as follows.First,the inferior vascular bundle was directly connected to a superior vascular bundle.Secondly,some of the superior vascular bundles bent in different ways,forming the inferior vascular bundle connecting the superior vascular bundles by a metamorphosed vessel with a triangular shape.In a comparison of the developmental changes in various periods,the growth of vascular bundles at each period was directly proportional to the growth of the flower.Laser confocal scanning was used to explore the three-dimensional morphology of the peripheral vascular bundle and showed that the peripheral vascular bundle of grapes was not completely parallel to the flower’s epidermal cells.As a result,the optical clearing technique was convenient and authentic compared with the traditional slicing operation for tiny flower organs.With these advantages according to the observations,this study provides a feasible technique and useful information for the study of vascular bundle development in grape flower organs.