Observation of segmental membrane development and evaluation of mastication properties in two citrus tetraploids in comparison with their diploid parents

【Objective】Citrus is a widely cultivated fruit crop globally and is highly favored by consumers, with its fruit quality consistently being a focal point of research. As a key indicator for assessing citrus fruit quality, fruit mastication significantly influences consumers’taste experience. The purpose of this study was to conduct a comprehensive evaluation of mastication properties in fruits from two tetraploid (doubled diploid) citrus lines in comparison with their corresponding diploid parents, to investigate the effects of genome doubling on citrus fruit mastication, and to identify the key factors influencing these mastication properties. The findings aim to provide a solid theoretical foundation and practical guidance for improving mastication quality in polyploid citrus fruits and contribute to enhancing overall citrus fruit quality.【Methods】In this study, fruits from diploid varieties of Ponkan mandarin and Nadorcott tangor, along with their corresponding tetraploids counterparts, were selected as experimental materials. Fruit samples were collected over a period spanning from 60 to 240 days after flowering, with sampling intervals set at 30 and 60 days, respectively. A total of 24 healthy fruits with uniform size were collected from all directions around the canopy at each time point. Of these, 12 fruits were used for cytological observation and texture analysis, while the remaining 12 were used for segmental membrane isolation. When the segmental membranes were collected, they were immediately frozen in liquid nitrogen and stored for subsequent analysis of cell wall components. During the fruit ripening stage, sensory evaluation was conducted to assess the fruit mastication of the tetraploids in comparation with their diploid parents. Concurrently, physical properties such as segmental membrane hardness and shearing force were precisely measured using a texture analyzer. Furthermore, the contents of cell wall components, including pectin, lignin, cellulose and hemicellulose, were measured and analyzed. Finally, scanning electron microscopy was employed to examine developmental dynamics of the segmental membrane.【Results】Fruit sensory evaluation at maturity revealed that the mastication of the diploid Ponkan mandarin and Nadorcott tangor was superior to that of their tetraploid counterparts, suggesting that the segmental membrane thickness in tetraploids was greater than that of the diploid parents. The texture analyzer measurements revealed that both the hardness and shearing force of the segmental membrane in the tetraploid of Nadorcott tangor were significantly higher than that in its diploid parent at 150, 210, and 240 d after anthesis. For the tetraploid of Ponkan mandarin, no significance was observed between tetraploid and diploid in most developmental stages. The trends for both hardness and shearing force were consistent, showing a gradual decrease over time. To investigate the developmental dynamics of the segmental membrane in two tetraploids and their diploid parents, scanning electron microscopy was employed to examine the ultrastructure of the segmental membrane at 60, 90, 150, 210, and 240 d after flowering. The results revealed that the segmental membrane thickness in both tetraploids was consistently greater than that in their respective diploid parents across most of the developmental stages. Furthermore, in both tetraploids and their diploid parents, the segmental membrane displayed a trend of gradually thinning during fruit development. To elucidate the relationship between fruit mastication in tetraploids and their diploid progenitors, the cell wall composition (specifically cellulose, hemicellulose, lignin, and pectin) and the content of each component were analyzed at 90, 150, 210, and 240 days after flowering. The results indicated that the cellulose content in the segmental membranes of both tetraploids and their diploid parents varied across developmental stages, exhibiting an initial increase followed by a decrease. And the hemicellulose content in the segmental membrane initially decreased and subsequently increased. However, at each corresponding developmental stage, there were no consistent changing patterns in the cellulose and hemicellulose content between the tetraploids and their diploid parents. The trends in cellulose and hemicellulose content contrasted with the observed trend in segmental membrane thickness, indicating that these components were not the primary factors influencing fruit mastication in tetraploids. The lignin content in the tetraploid Nadorcott tangor was consistently higher than that in its diploid parent throughout the developmental period, peaking at 240 d after flowering. In terms of pectin content, protopectin levels in the segmental membrane of both tetraploids and their diploid parents were consistently higher than those of water-soluble pectin across all developmental stages. For protopectin, its content in the segmental membrane of tetraploid Nadorcott tangor was significantly higher than that of its diploid parent throughout each stage of fruit development. Nevertheless, there was minimal difference in protopectin content between the tetraploid of Ponkan mandarin and its diploid parent, which was in line with the results measured by the texture analyzer. The content of water-soluble pectin exhibited a continuous increase throughout development, with tetraploids showing higher levels than their diploid counterparts at almost stages. This difference became most pronounced at 240 d after anthesis. The total pectin content also exhibited an overall increasing trend, with tetraploids showing significantly higher levels than their corresponding diploid parents across nearly all developmental stages.【Conclusion】The thickness of the segmental membrane and protopectin content may be key factors contributing to mastication in tetraploid citrus fruits. This finding not only provides a clear direction for improving mastication quality in polyploid citrus but also establishes a crucial foundation for indepth investigation into the effects of genome doubling on fruit texture.