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Quantifying Fruit Dehiscence Using the Random Impact Test (RIT)
采用随机碰撞试验(RIT)定量测定果实开裂   

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Abstract

Fruit dehiscence is an important evolutionary and agronomic trait. For quantifying and comparing the exact fruit dehiscence capability between individual plants, the random impact test has been described (Morgan et al., 1998; Bruce et al., 2002; Arnaud et al., 2010). Here, we describe the random impact test optimized to measure dehiscence capability in the Brassicaceae plant Lepidium campestre (L. campestre). However, with slight alterations regarding agitation force, agitation time, and drying conditions, the test should be applicable to a wide range of plant species with dehiscent fruits.

Keywords: Random impact test(随机试验的影响), Fruit dehiscence(果开裂), Fruit opening(水果Opening)

Materials and Reagents

  1. L. campestre plants

Equipment

  1. An open container (e.g. Falcon tube)
  2. Desiccator or climate chamber allowing for the control of temperature and relative humidity
  3. A number of steel balls (in our case with a diameter of 5 mm)
  4. Mixer Mill (MM 400/Retsch) including two grinding jars (Figure 1)


    Figure 1. Mixer Mill (MM400/Retsch)

  5. A pair of tweezers

Software

  1. Microsoft Excel

Procedure

  1. Grow L. campestre plants until fruits are yellow and completely dry.
  2. Carefully harvest at least 60 fruits per plant without manually causing fruits to open.
  3. Place the fruits in an open container (e.g. Falcon tube) and keep them under constant environmental conditions (25 °C and 50% relative humidity) for at least 3 days to achieve consistent moisture content.
  4. Count 20 fruits and place them in a grinding jar of the mixer mill together with six of the 5-mm steel balls (Figure 2).


    Figure 2. 20 ripe fruits of L. campestre (left) placed in a grinding jar of the mixer mill together with six 5-mm steel balls (right)

  5. Carefully close the grinding jar, fasten it to the mixer mill and agitate for 5 sec at 9 hz.
  6. Remove the fruits from the grinding jar and determine the number of open fruits by counting the number of fully intact fruits (Figure 3).


    Figure 3. Fully intact fruits are counted and open fruits are removed

  7. Put the intact fruits back to the grinding jar and agitate at 9 hz for another 5 sec, then count again.
  8. Repeat these two steps (agitating and counting) another 4 times (or until no intact fruits are left) with agitation times of 10, 20, 40, and 80 sec (overall resulting in cumulative times of 5, 10, 20, 40, 80, and 160 sec).
  9. Plot the time (sec) against the number of open fruits to end up with a graph like shown in Figure 4.


    Figure 4. Cumulative agitation time is plotted against the number of open fruits

  10. Because during the following log transformation, you will run into trouble with the 0 data point, you add 1 (+1) to each time point (ending up with cumulative times of 6, 11, 21, 41, 81, 161).
  11. Then you apply the log10 to each time point to end up with a sigmoid graph like shown in Figure 5.


    Figure 5. Applying the log10 to each time point results in a sigmoid graph

  12. Sigmoid graphs can be linearized with the logit function. Thus you apply the logit to the number of open fruits. Because the logit is not defined for 0 and 100% (in our case 0 and 20) you end up with a near linear relationship of only 4 data points (Figure 6).


    Figure 6. Near linear relationship derived from a logit transformation of the number of open fruits

  13. A linear slope is fitted to the data (for example using the Excel ‘Trendline’-function) which gives you a linear equation (Figure 7).


    Figure 7. A linear slope is fitted to the data

  14. Now you want to calculate the halftime of dehiscence, which is the time point when half of the fruits (10 fruits) are open. The logit of 50% (in our case 10 fruits) equals 0. Thus, you have to calculate the x-intercept. In our example, the x-intercept is 2.4235/2.0408=1.1875.
  15. Finally, you just have to reverse the log transformation and the addition of 1 (see steps 10-11). Thus you calculate 101.1875 -1 = 14.3992, which is the dehiscence half-life in seconds.
  16. Repeat this measurement at least twice for each plant and calculate the mean half-life and standard deviation.

Acknowledgments

We thank Andreas Mühlhausen and Klaus Mummenhoff (Department of Botany, University of Osnabrück, Germany) for their kind cooperation in our project on fruit dehiscence and Thorsten Lenser for his help with data analysis. This protocol has been adapted based on previously published work (Morgan et al., 1998; Bruce et al., 2002; Arnaud et al., 2010). Our work was supported by a grant from the Deutsche Forschungsgemeinschaft to G.T. (TH 417/6-1).

References

  1. Arnaud, N., Girin, T., Sorefan, K., Fuentes, S., Wood, T. A., Lawrenson, T., Sablowski, R. and Ostergaard, L. (2010). Gibberellins control fruit patterning in Arabidopsis thaliana. Genes Dev 24(19): 2127-2132.
  2. Bruce, D., Farrent, J., Morgan, C. and Child, R. (2002). PA-precision agriculture: determining the oilseed rape pod strength needed to reduce seed loss due to pod shatter. Biosyst Eng 81(2): 179-184.
  3. Lenser, T. and Theißen, G. (2013). Conservation of fruit dehiscence pathways between Lepidium campestre and Arabidopsis thaliana sheds light on the regulation of INDEHISCENT. Plant J 76(4): 545-556.
  4. Morgan, C., Bruce, D., Child, R., Ladbrooke, Z. and Arthur, A. (1998). Genetic variation for pod shatter resistance among lines of oilseed rape developed from synthetic B. napus. Field Crops Research 58(2): 153-165.

简介

果实开裂是一种重要的进化和农艺性状。 为了定量和比较单个植物之间的确切的果实开裂能力,已经描述了随机影响测试(Morgan等人,1998; Bruce等人,2002; Arnaud 等,2010)。 在这里,我们描述了优化用于测量十字花科植物(Lepidium campestre)(L. campestre)中的开裂能力的随机冲击测试。 然而,对于搅拌力,搅拌时间和干燥条件的轻微改变,试验应该适用于具有开裂果实的宽范围的植物物种。

关键字:随机试验的影响, 果开裂, 水果Opening

材料和试剂

  1. L。 campestre 植物

设备

  1. 一个开放式容器(如 Falcon管)
  2. 干燥器或气候室,允许控制温度和相对湿度
  3. 许多钢球(在我们的例子中直径为5毫米)
  4. 混合机(MM 400/Retsch)包括两个研磨罐(图1)


    图1.搅拌机(MM400/Retsch)

  5. 一对镊子

软件

  1. Microsoft Excel

程序

  1. 成长L。 campestre 植物,直到水果变黄并完全干燥
  2. 仔细收获至少60个水果,每个植物,而不手动造成水果开放
  3. 将果实放置在开放容器(例如 Falcon管)中,并保持它们在恒定的环境条件(25℃和50%相对湿度)下至少3天以达到一致的水分含量。 >
  4. 计数20个水果,并将它们与六个5毫米钢球(图2)一起放入搅拌机的研磨罐中。


    图2. L的20个成熟果实。 campestre (左)与六个5-mm钢球(右)放在搅拌磨机的研磨罐中

  5. 小心地关闭研磨罐,将其固定在搅拌磨中,并以9hz搅拌5秒
  6. 从研磨罐中取出水果,并通过计数完全完整的水果的数量确定开放水果的数量(图3)。


    图3.完全完整的水果被计数,开放水果被除去

  7. 将完好的水果放回研磨罐中,以9hz再搅拌5秒钟,然后再次计数
  8. 重复这两个步骤(搅拌和计数)另外4次(或直到没有完整的水果留下),搅拌时间为10,20,40和80秒(总体导致累积时间为5,10,20,40,80 ,和160秒)。
  9. 绘制时间(秒)对开放水果的数量结束,如图4所示的图。


    图4.累积搅拌时间对开放水果数量进行绘制
  10. 因为在下面的日志转换过程中,您将遇到0数据点的问题,您可以为每个时间点添加1(+1)(以累积时间6,11,21,41,81,161结束)。< br />
  11. 然后,将日志 10 应用到每个时间点,最终得到如图5所示的sigmoid图。


    图5.对每个时间点应用日志 10 会生成Sigmoid图

  12. Sigmoid图可以用logit函数线性化。因此,您将logit应用于开放水果的数量。因为logit没有为0和100%(在我们的例子中是0和20)定义,所以最终只有4个数据点的近似线性关系(图6)。


    图6.从开放果实数的logit转换得到的近线性关系

  13. 使用线性斜率拟合数据(例如使用Excel'Trendline'函数),得到一个线性方程(图7)。


    图7.线性斜率适合数据

  14. 现在你想计算开裂的半衰期,这是一半的果实(10个水果)开放的时间点。 50%的logit(在我们的例子中是10个果实)等于0.因此,你必须计算x截距。在我们的例子中,x截距为2.4235/2.0408 = 1.1875
  15. 最后,你只需要反转日志转换和添加1(参见步骤10-11)。 因此,你计算10 1.1875 -1 = 14.3992,这是开裂半衰期,以秒为单位。
  16. 对每个植物重复该测量至少两次,并计算平均半衰期和标准偏差

致谢

我们感谢AndreasMühlhausen和Klaus Mummenhoff(德国Osnabrück大学植物系)在我们的水果开裂项目和Thorsten Lenser的合作,他对数据分析的帮助。 该协议已经基于先前公开的工作(Morgan等人,1998; Bruce等人,2002; Arnaud等人, > 2010)。 我们的工作得到了德意志研究所对G.T.的资助。 (TH 417/6-1)。

参考文献

  1. Arnaud,N.,Girin,T.,Sorefan,K.,Fuentes,S.,Wood,T.A.,Lawrenson,T.,Sablowski,R.and Ostergaard,L。 Gibberellins控制拟南芥中的水果图案 > Genes Dev 24(19):2127-2132。
  2. Bruce,D.,Farrent,J.,Morgan,C。和Child,R。(2002)。 PA精确农业:确定降低荚果破碎所致种子损失所需的油籽油荚强度。 Biosyst Eng 81(2):179-184。
  3. Lenser,T.和Theißen,G。(2013)。 保护水果开裂路径 植物J 76( 4):545-556。
  4. Morgan,C.,Bruce,D.,Child,R.,Ladbrooke,Z.and Arthur,A。(1998)。 从合成B开发的油籽油菜品系之间的荚果粉碎抗性的遗传变异。 58(2):153-165。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lenser, T. and Theißen, G. (2014). Quantifying Fruit Dehiscence Using the Random Impact Test (RIT). Bio-protocol 4(15): e1200. DOI: 10.21769/BioProtoc.1200.
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