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定量分析拟南芥根系生长
生长的植物根系很容易测量，尤其在拟南芥中，因为增加的器官大小基本上限于一定范围。精确测量根尖生长，可用于在突变体生长过程中、转基因背景、或是实验处理响应方面，来确定生长活性（在某一特定时间的增长速度）。根系生长衡量方法有多种方式，最简单的就是将种子种在培养皿中，实时记录根尖生长，用直尺测量根系长度，然后将数据后输入Microsoft Excel分析，但这种方法在处理大量的幼苗时是繁琐，不正确的。另一种做法，就是本文所描述的，利用该植物生长的”快照”，就是用凝胶成像设备（例如，用一台摄像机从EB（ethidium-bromide）染色电泳凝胶捕获图像）。图像分析软体很随意，只要能让用户简单剪切和粘贴NIH-Image数据到Microsoft Excel就可。
材料
含0.8％琼脂（w/v）或植物凝胶的MS培养基（0.5x），种子（表面消毒）、培养皿（方形最好，没有也无所谓）、封口膜、直尺、统计分析软件（如微软的Excel）
设备 电脑（废话）、凝胶成像系统（这个操作具体视设备而定，询厂家）
恒温箱设至22°C-23°C时，记号笔（又是菲菲）
NIH-Image
是公共软件，可下载（还有一个指导手册）http://rsb.info.nih.gov/nih-image
方法
1、将表面消毒的种子摆在含0.8％琼脂（w/v）或植物凝胶的MS培养基（0.5x）。
2、加上1-2毫升的水，盖盖子，封口膜封口。光照培养48小时，4ºC。
3、恒温箱再培养，检查平板，以确保它们不干（有的凝结应该可以看见）。生长最好在22°C-23°C，一天。
4。此时根系已长到约1.5cm，转移最好的幼苗到新平板。选择健康的种苗与等根长度的5-7毫米相间种植。
5。监察生长，在12小时或24小时的间隔时间标记根尖位置。用直尺测量并记录数据。
6。置培养皿于摄像机下捕获图像，然后逐渐放大直到整个屏幕都是植物生长。记下放大率，拍照并保存为TIFF格式。（如果图象不能被保存在一个TIFF格式，打开该文件在AdobePhotoshop转换到TIFF格式）。注意：在整个实验中放大率必须保持恒定。
7、保存到NIH-Image，调整对比度以分清根系。进行一个周期的” sharpen”以提高根系的能见度的。
8、测量根尖生长。如果每时间点照一张相，打开所有图片并使用”pencil”工具，标志根尖在每次实验时间点上的位置，从而获得一张梯度图。
9、从工具板，可以设置光标为” straight line”，衡量尺度（例如，10毫米），下拉分析菜单至”set scale.”
10、返回到工具板，选择”freehand line”，并标记两个时间点的根系位置。测量键入 typing #1, 显示数据键入typing #2。
11、输出数据进行统计分析。退出NIH-Image前保存数据文件，并在Microsoft Excel重新打开定量分析根长度和增长率。
比较根系生长率时，要记得拟南芥在出芽后的前10d根系生长发育快速(Beemster and Baskin 1998)。
METHOD
1. Sow surface-sterilized seeds on 0.5X MS plates containing 0.8% (w/v) agar or Phytagar. 2. Add 1-2 mL of H2O to the bottom of each plate, replace the lids, and wrap the plates tightly with Parafilm. Hold each plate by its sidewalls and avoid touching the future bottoms of the plates. Keep the plates at 4ºC for 48 h (with illumination) in a vertical orientation. 3. Stand the plates in an incubator, still in a vertical orientation. Examine the plates regularly to ensure that they do not dry out (some condensation should be visible).
Growth is best at 22°C-23°C in a 16-h day/8-h night cycle. 4. After roots have grown to a length of ~1.5 cm, transfer the best seedlings to a new assay plate. Choose healthy seedlings with approximately equal root lengths. Space seedlings 5-7 mm apart. 5. Monitor growth (displacement of the position of the root tip) at 12-h or 24-h intervals by marking the position of the advancing root tip. Measure the displacement with a ruler and record the data for each time point. If large numbers of seedlings are being analyzed, use one of the following two methods: i. Use a fine-tipped marker pen to indicate the position of the root tip at the chosen time points. Capture the final image of the marked plate for analysis.
- Unless the Petri dish used is marked with a grid, make sure that an accurate indication of scale is marked on the base of the dish, e.g., a 5-mm bar. ii. Alternatively, and less conveniently, capture a video image of the seedlings at each specified time point.
- Again, make sure that an accurate indication of the scale is marked on the base of the dish. 6. Capture images by placing the Petri dish under the video camera and zoom in until the area where plants are growing covers the entire screen. Make a note of this magnification, capture the image and save it, preferably in a TIFF format. (If the image cannot be saved in a TIFF format directly, open the file in Adobe Photoshop and then convert it to a TIFF format).
The magnification must remain constant throughout the experiment. 7. Import the file into NIH-Image. Adjust the contrast of the image to distinguish the roots clearly. To enhance the visibility of the roots, undertake one cycle of “sharpen” using the Process pull-down menu. 8. Measure the advancement of the root tip. If taking one image per time point, open all images of a series and use the “pencil” tool from the Tools menu to mark the positions of the root tip at each experimental time point on the root captured on the last image. 9. From the Tools palette, set the cursor to the “straight line” and measure the scale (e.g., 10 mm) that was marked on the plate in Step 5 prior to image capture. Pull down the Analyze menu to “set scale.” 10. Return to the Tools palette to select “freehand line” and trace the outline of the root between two time point markings. Measure by typing 1, and display the data by typing 2. 11. Export the data for statistical analysis. Save the data file before exiting NIH-Image and reopen it in Microsoft Excel for quantitative analysis of root lengths and growth rates.
When comparing root growth rates, remember that Arabidopsis root growth accelerates developmentally during the first ~10 d following germination
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