A rapid method of fruit cell isolation for cell size and shape measurements
A rapid method of fruit cell isolation for cell size and shape measurements
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DOI：
10.1186/1746-4811-5-5
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发表时间：
2009-04-29
期刊：
影响因子：
5.1
通讯作者：
Schaffer, Robert J.
中科院分区：
文献类型：
--
作者：
McAtee, Peter A.;Hallett, Ian C.;Schaffer, Robert J.
背景：细胞大小是肉质水果的一个结构成分，对果实大小和质地等重要性状有重要影响。目前有许多测量细胞大小的方法；大多数依赖于组织切片或用细胞壁降解酶或化学物质消化组织来释放单个细胞。这两种方法都不是分析大量水果的理想方法，因为这两种方法都需要相当长的时间来准备组织，而目前的细胞壁消化方法需要24到48小时。结果：为了开发一种更快速地测量果细胞大小的方法，我们开发了一种方案，将果胶溶解在植物细胞壁的中层，将单个细胞释放到缓冲溶液中。小水果样品在0.05M的Na2CO3溶液中，与0.3M的甘露醇渗透平衡，在不到30分钟的时间内产生良好的细胞分离，几乎没有细胞损伤。将化学处理与煮沸相结合的优点是细胞很快就会被杀死。这阻止了在分离过程中可能发生的细胞形状变化。用该方法对苹果品种‘太平洋玫瑰’和‘Jazz’(TM)的圆形和角形细胞进行了观察。利用该技术对5个不同苹果品种的细胞大小进行了深入分析。使用公共领域的ImageJ软件测量细胞大小。对于每个品种，至少测量了1000个细胞，发现每个品种表现出不同的细胞大小分布。品种间细胞大小相似，果肉硬度与细胞大小之间无相关性。这一方案在番茄、岩瓜和猕猴桃等其他肉质水果的组织上进行了测试。结果表明，所有类型水果的果肉组织都能很好地进行细胞分离，显示了该方法的广泛实用性。结论：我们开发了一种从肉质水果中分离单个细胞的方法，减少了果实细胞分离所需的时间。用该方法研究了5个不同苹果品种的细胞大小和形状的差异。虽然不同品种之间的坚实度与细胞大小无关，但角细胞较多的苹果似乎更坚固。
Background: Cell size is a structural component of fleshy fruit, contributing to important traits such as fruit size and texture. There are currently a number of methods for measuring cell size; most rely either on tissue sectioning or digestion of the tissue with cell wall degrading enzymes or chemicals to release single cells. Neither of these approaches is ideal for assaying large fruit numbers as both require a considerable time to prepare the tissue, with current methods of cell wall digestions taking 24 to 48 hours. Additionally, sectioning can lead to a measurement of a plane that does not represent the widest point of the cell.Results: To develop a more rapid way of measuring fruit cell size we have developed a protocol that solubilises pectin in the middle lamella of the plant cell wall releasing single cells into a buffered solution. Gently boiling small fruit samples in a 0.05 M Na2CO3 solution, osmotically balanced with 0.3 M mannitol, produced good cell separation with little cellular damage in less than 30 minutes. The advantage of combining a chemical treatment with boiling is that the cells are rapidly killed. This stopped cell shape changes that could potentially occur during separation. With this method both the rounded and angular cells of the apple cultivars SciRos 'Pacific Rose' and SciFresh 'Jazz'(TM) were observed in the separated cells. Using this technique, an in-depth analysis was performed measuring cell size from 5 different apple cultivars. Cell size was measured using the public domain ImageJ software. For each cultivar a minimum of 1000 cells were measured and it was found that each cultivar displayed a different distribution of cell size. Cell size within cultivars was similar and there was no correlation between flesh firmness and cell size. This protocol was tested on tissue from other fleshy fruit including tomato, rock melon and kiwifruit. It was found that good cell separation was achieved with flesh tissue from all these fruit types, showing a broad utility to this protocol.Conclusion: We have developed a method for isolating single cells from fleshy fruit that reduces the time needed for fruit cell separation. This method was used to demonstrate differences in cell size and shape for 5 different apple cultivars. While firmness between the different cultivars is independent of cell size, apples with more angular cells appear to be firmer.