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Plants use gravity as a guide for growth and development. Gravitropism, a gravity-directed growth process, directs upward shoot movement for efficient photosynthesis and gaseous exchange. In addition, it also directs downward growth of roots in soil, for assimilation of water and nutrients required for growth and development. Using time lapse video imaging this process can be efficiently studied in a real time scale. The analysis of the response under different conditions can help to unravel the mechanisms regulating gravitropism.

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Gravitropic Analysis of Tomato Seedlings using Time Lapse Video Imaging
延时视频成像法监测番茄幼苗的向地性

植物科学 > 植物生理学 > 植物生长
作者: Sulabha Sharma
Sulabha SharmaAffiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India
Bio-protocol author page: a2119
Kamal Tyagi
Kamal TyagiAffiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India
Bio-protocol author page: a2120
Yellamaraju Sreelakshmi
Yellamaraju SreelakshmiAffiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India
Bio-protocol author page: a2121
 and Rameshwar Sharma
Rameshwar SharmaAffiliation: Repository of Tomato Genomics Resources, Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, India
For correspondence: rameshwar.sharma@gmail.com
Bio-protocol author page: a2122
Vol 5, Iss 7, 4/5/2015, 2029 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1443

[Abstract] Plants use gravity as a guide for growth and development. Gravitropism, a gravity-directed growth process, directs upward shoot movement for efficient photosynthesis and gaseous exchange. In addition, it also directs downward growth of roots in soil, for assimilation of water and nutrients required for growth and development. Using time lapse video imaging this process can be efficiently studied in a real time scale. The analysis of the response under different conditions can help to unravel the mechanisms regulating gravitropism.
Keywords: Tomato(番茄), Auxin(生长素), Gravitropism(向地性), Timelapse photography(延时摄影), Plant movement(植物运动)

[Abstract]

Materials and Reagents

  1. Tomato seeds (Cultivar Arka Vikas)
  2. Sodium hypochlorite (Thermo Fisher Scientific, catalog number: 27096 )
  3. Agar (HIMEDIA, catalog number: PCT0901 )
  4. Distilled water
  5. 0.8% (w/v) agar (see Recipes)

Equipment

  1. Blue light (An alternate light source can also be used) for gravitropic analysis was obtained from blue LEDs (Light Emitting diodes, λmax 470 nm, Kwality Photonics). Photon fluence of light was measured with a light meter fitted with a Quantum sensor (Skye Instruments, UK).
  2. Spectrum of the blue light was measured using spectroradiometer purchased from International Light Technologies (model: RPS900W)
  3. Quickcam Pro 3000 or 4000 (Logitech) or any other suitable webcam for recording the response
  4. Germination paper (locally purchased) or blotting paper or Whatman paper
  5. Square germination boxes (dimension 10 x10 x 6 cm, Laxbro) / or square petriplates
  6. Square petriplate (dimension 12x12x2 cm, Genaxy, model: GEN-PTD-130SQ)
  7. Forceps from Pro’Kit® 1PK-125T (dimension 12 x1 x 4 cm)

Software

  1. SigmaPlot 10.0 (http://www.sigmaplot.com/products/sigmaplot/sigmaplot-details.php)
  2. ImageJ (http://imagej.nih.gov/ij/)
  3. Timelapse (http://tnlsoftsolutions.com/timelapsehome.php)

Procedure

  1. Plant growth conditions
    1. Tomato (Solanum lycopersicum L. cultivar Arka Vikas) seeds (Figure 1A) were surface sterilized in 20% (v/v) sodium hypochlorite for about 15 min followed by washing in running tap water to remove any trace of hypochlorite (this step can be performed preferably in a fume hood).
    2. The sterilized seeds were sown on two layers of wet filter paper in germination box (Figure 1B). (It is better to sterilize box and papers.)
    3. The seeds were germinated for 3 days in germination boxes (25 ± 2 °C).
    4. After emergence of the radical (Figure 2), equal sized seedlings were gently transferred on 0.8% (w/v) agar in petriplate and kept in darkness at 25 ± 2 °C for 2 days.
    5. Unless otherwise mentioned, for all experiments the seedling’s age was counted from the time of emergence of the radical. In all cases the average height of the etiolated seedlings used was 3.0 ± 0.3 cm (Figure 3).

  2. Analysis of gravitropism/gravistimulation
    1. The gravitropic response of tomato seedlings was examined under omnilateral blue light (white light can also be used). To eliminate any phototropic effect of light on seedlings the time lapse imaging can be done using Infra-red LEDs as a light source. Figure 4 shows the placement of camera and seedlings. Seedlings were placed at a horizontal distance of 20 cm from the camera lens. The light source was 20 cm above the seedlings.
    2. Gravity response in the hypocotyls was measured using 2-d-old etiolated seedlings, grown in square petriplates filled with 0.8% (w/v) agar (Figure 5A).
    3. At the time of gravistimulation, plates were turned at an angle of 90° to make the seedlings horizontal, and the plates were arranged in a rack (Figure 5B).
    4. Seedlings were photographed using a Quickcam Pro 3000 at every 10-min interval for 120 minutes for hypocotyl gravitropism. Figure 5C shows the hypocotyl bending after 120 min of blue light exposure.
    5. For each experiment, 10 seedlings were used and the experiment was repeated 3 to 4 times.
    6. For root gravitropism, seeds with just emerged radicles were sown on 0.8% (w/v) agar in petri plates and plates were kept vertically under darkness (as above). After 36 h, when roots elongated to ca. 2 to 2.5 cm, the plates were rotated by 90° to make seedlings horizontal. The images were recorded every 10 min for 6 h, or 12 h or 24 h (depending upon the experiment) using a Quickcam Pro 3000. Each petriplate contained 4-6 seedlings, and the experiment was repeated about 4 times (Figure 5D).

  3. Light sources
    In the current experiment we used omnilateral low fluence blue light (λmax 470 nm) obtained using blue LEDs to capture the movement of the seedlings with respect to gravity. The experiment can also be done in infrared light for eliminating any likely effect of plant photoreceptors i. e. under complete darkness. Unless otherwise mentioned, “low-fluence blue light” (3 µmol m-2 s-1) was used to provide minimum light to camera to capture the images.

  4. Time lapse video imaging and analysis
    1. Time lapse images were captured using Quickcam-Pro 4000 (see Whippo and Hangarter, 2003) attached to the computer.
    2. For creating a time lapse video image (Video 1), frames were extracted at specific time points using either MGI VideoWave4 PC video editing (USA) or Time lapse imaging software from the movie. Images were captured at 10 min interval after gravistimulation or after orienting plate by 90°. The time lapse frames were fused to create a video using the above software.
    3. The background illumination was provided by using low fluence blue light LEDs. It can be replaced by infrared LEDs (λmax 940 nm).
    4. The movie shows the negative gravitropic response of hypocotyls of tomato seedlings after the seedlings were horizontally oriented. After a lag period (~20 min), gravitropism resulted in differential growth of hypocotyls leading to bending of seedlings away from gravity vector. Within an hour, the hypocotyls reorient to vertical growth, and regain the same growth orientation that was before onset of gravistimulation.
    5. Angle measurements
      The curvature angles were calculated after subtracting the zero point images from the images obtained at defined time points. All measurements were performed using NIH ImageJ software and e-Ruler r. The graph was plotted using SigmaPlot 10.0. Figure 6 shows snapshot of angle measurement using the ImageJ software.
    6. Figure 7 shows the time course of gravitropic curvature of two day-old etiolated seedlings.

Representative data



Figure 1. A. Seeds were selected for the experiment. B. The seeds after treatment with 20% (v/v) sodium hypochlorite for 15 min. The treatment with sodium hypochlorite leads to thinning of seed coat and coiled embryo like structure is visible leading to uniform germination of the seeds.


Figure 2. 3-days-old etiolated germinated seeds of tomato. After emergence of radicle from seed, the seeds were transferred on to 0.8% (w/v) agar.


Figure 3. Two day-old etiolated seedlings of tomato. Seedlings of same height were used for the analysis.


Figure 4. Graphical illustration of time-lapse recording setup used for analyzing gravitropic response. The entire set-up was enclosed in wooden chamber placed in a dark room. The images were recorded at defined interval by a computer connected to the camera. Computer was placed outside the wooden chamber and the computer screen was either switched off or covered with a black cloth during the entire duration of recording.


Figure 5. A. The seeds after emergence of radicle were grown on 0.8% (w/v) agar in a petriplate. B. After two days, the petriplate was rotated at 90° for gravistimulation. C. Negative gravitropism of hypocotyl after 2 h. D. Positive gravitropism of root tip towards gravity. The white arrow in picture indicates the direction of gravitational field.


Figure 6. The yellow lines in right snapshot shows the measurement of hypocotyl angle by ImageJ software. The white arrow in picture indicates the direction of gravitational field.


Figure 7. Time course of hypocotyl gravitropic curvature in two-day old etiolated tomato seedlings. The curvature angles of individual hypocotyls were measured at defined time intervals for 120 min by time lapse photography. The data points represent the mean value ± S.E of 10 individual seedlings from three independent experiments.

Viedo 1. Time-lapse video imaging of negative gravitropism of tomato seedlings

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Notes

  1. The age of the seedlings is an important factor for the reproducibility of the experiment. The seedlings should be of uniform length to reduce the error. Tomato seedlings taller than 3.5 cm show poor hypocotyl tropism.
  2. The treatment with sodium hypochlorite should be carefully monitored as efficacy of sodium hypochlorite is variable and depends on batch and company used. The longer hypochlorite treatment or stronger hypochlorite solution treatment can kill the seeds.

Recipes

  1. 0.8% (w/v) agar
    Weigh 0.8 gm agar
    Add 100 ml double distilled water
    Boil for 2-3 minutes in a beaker
    Cool down and pour into the square petriplate

Acknowledgments

Financial support provided by the Council of Scientific and Industrial Research (CSIR), New Delhi, in the form of SRF to Kamal Tyagi is gratefully acknowledged. This work was supported by Department of Biotechnology, New Delhi grant to RS and YS.

References

  1. Whippo, C. W. and Hangarter, R. P. (2003). Second positive phototropism results from coordinated co-action of the phototropins and cryptochromes. Plant Physiol 132(3): 1499-1507.

材料和试剂

  1. 番茄种子(栽培品种Arka Vikas)
  2. 次氯酸钠(Thermo Fisher Scientific,目录号:27096)
  3. 琼脂(HIMEDIA,目录号:PCT0901)
  4. 蒸馏水
  5. 0.8%(w/v)琼脂(参见Recipes)

设备

  1. 还可以从蓝色LED(发光二极管,λmax 470nm,Kwality Photonics)获得用于重力分析的蓝光(也可以使用替代光源)。 用量子传感器(Skye Instruments,UK)测量光子的光子强度。
  2. 使用从International Light Technologies(型号:RPS900W)购买的分光辐射计测量蓝光的光谱
  3. Quickcam Pro 3000或4000(Logitech)或任何其他适用的录制回复
    的网络摄像头
  4. 发芽纸(本地购买)或吸墨纸或Whatman纸
  5. 方形发芽箱(尺寸为10×10×6cm,Laxbro)/或方形平皿
  6. 方形培养皿(尺寸12×12×2cm,Genaxy,型号:GEN-PTD-130SQ)
  7. 来自Pro'Kit 1PK-125T(尺寸为12 x1 x 4厘米)的钳子

软件

  1. SigmaPlot 10.0( http://www.sigmaplot.com/products/sigmaplot/sigmaplot-details.php
  2. ImageJ( http://imagej.nih.gov/ij/
  3. Timelapse( http://tnlsoftsolutions.com/timelapsehome.php

程序

  1. 植物生长条件
    1. 番茄(番茄栽培种Arka Vikas)种子(图1A) 在20%(v/v)次氯酸钠中表面灭菌约15 min,然后在流动的自来水中洗涤以除去任何痕量 次氯酸盐(该步骤可以优选在通风橱中进行)。
    2. 将灭菌的种子播种在两层湿滤纸上 发芽箱(图1B)。 (最好对盒子和纸张进行消毒。)
    3. 种子在发芽箱(25±2℃)中发芽3天。
    4. 在出现根部(图2)后,等尺寸的幼苗 轻轻转移到培养皿中的0.8%(w/v)琼脂上并保持 黑暗在25±2℃下2天。
    5. 除非另有说明, 对于所有实验,从时间开始计数幼苗的年龄 出现激进。 在所有情况下的平均高度 使用的植物幼苗为3.0±0.3cm(图3)。

  2. 重力分析/重力分析
    1. 检查番茄幼苗的引力反应 全方位蓝光(也可以使用白光)。 消除任何 光向光的影响时间推移成像可以 使用红外LED作为光源。 图4显示 安置照相机和幼木。 将幼苗放在水平   距离相机镜头20厘米。 光源为20cm 以上的苗。
    2. 下胚轴中的重力反应是 使用2-d-老化的幼苗测量,生长在方形平皿中 填充有0.8%(w/v)琼脂(图5A)
    3. 在那个时间 重力下,将板以90°的角度转动, 幼苗水平,并将板排列在架子上(图 5B)。
    4. 使用Quickcam Pro 3000 at在幼苗上拍照 每10分钟间隔120分钟用于下胚轴向度。 数字   图5C显示在蓝光暴露120分钟后的下胚轴弯曲。
    5. 对于每个实验,使用10个幼苗并且实验重复3至4次。
    6. 对于根性向性,播种具有刚出芽胚根的种子   在培养皿和板中的0.8%(w/v)琼脂在垂直下方保持 黑暗(如上)。 36 h后,根伸长至 2〜2.5 cm,将板旋转90°以使幼苗水平。 的 图像每10分钟记录6小时,或12小时或24小时(取决于 实验)使用Quickcam Pro 3000.每个培养皿 含4-6株幼苗,实验重复约4次 (图5D)。

  3. 光源
    在当前实验中,我们使用使用蓝色LED获得的全方位低通量蓝光(λ 470nm)来捕获幼苗相对于重力的运动。 该实验也可以在红外光下进行,以消除植物光感受器的任何可能的影响。 在完全黑暗下。 除非另有说明,使用"低通量蓝光"(3μmolm -2 s -1 -1 )向照相机提供最小光以捕获图像。 />
  4. 时间流逝视频成像和分析
    1. 使用连接到计算机的Quickcam-Pro 4000(参见Whippo和Hangarter,2003)捕获时间推移图像。
    2. 为了创建时间流逝视频图像(视频1),帧是 在特定时间点使用MGI VideoWave4 PC视频提取 编辑(美国)或时间流逝成像软件从电影。 图片是   在重力刺激后或定向后以10分钟间隔捕获 板90°。 时间流逝帧被融合以创建视频使用 上面的软件。
    3. 背景照明由 使用低通量蓝光LED。 它可以由红外LED替代 (λmax 940nm)。
    4. 电影显示负向引力反应   的番茄幼苗下胚轴水平后下胚轴   面向。 在滞后期(〜20分钟)后,引力向导 下胚轴的差异生长导致幼苗弯曲离开 从重力矢量。 在一个小时内,下胚轴重新定向为垂直   生长,并恢复与发病前相同的生长方向   重力刺激。
    5. 角度测量
      曲率角 在从图像中减去零点图像之后计算 在定义的时间点获得。 所有测量使用 NIH ImageJ软件和e-Ruler r。 使用SigmaPlot绘制图   10.0。 图6显示了使用ImageJ的角度测量的快照 软件
    6. 图7显示了两个日龄匍匐幼苗的向地弯曲的时间过程。

代表数据



图1.选择种子用于实验。 B.用20%(v/v)次氯酸钠处理15分钟后的种子用次氯酸钠处理导致种皮变薄,可以看到盘绕的胚状结构,导致种子均匀发芽。


图2.番茄的3天龄的发芽的种子。从种子出现胚根后,将种子转移到0.8%(w/v)琼脂上。


图3.番茄的两日龄绿化幼苗。使用相同高度的幼苗进行分析。


图4.用于分析向性反应的延时记录设置的图形说明。整个设置被封闭在放置在暗室中的木室中。通过连接到照相机的计算机以限定的间隔记录图像。计算机放在木室外,在整个记录期间,计算机屏幕被关闭或用黑布覆盖。


图5。将胚根出芽后的种子在培养皿中的0.8%(w/v)琼脂上生长。 B.两天后,将培养皿旋转90°以进行重力刺激。 C.2小时后下胚轴的阴性引力。 D.根尖向重力的正向引力。图中的白色箭头表示重力场的方向

F 图6.右侧快照中的黄线表示ImageJ软件对下胚轴角度的测量。图片中的白色箭头表示重力场的方向。


图7.两日龄老化的番茄幼苗中下胚轴向弯曲的时间过程。 以定义的时间间隔通过时间流逝摄影测量单个下胚轴的曲率角120分钟。数据点表示来自三个独立实验的10个个体幼苗的平均值±S.E。

Viedo 1.番茄幼苗的负向引力的延时视频成像
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笔记

  1. 幼苗的年龄是实验重现性的重要因素。 幼苗应具有均匀的长度以减少误差。 高于3.5cm的番茄幼苗表现出较差的下胚轴向性
  2. 应仔细监测次氯酸钠的处理,因为次氯酸钠的功效是可变的,取决于所用的批次和公司。 较长的次氯酸盐处理或较强的次氯酸盐溶液处理可以杀死种子

食谱

  1. 0.8%(w/v)琼脂 称重0.8gm琼脂
    加入100 ml双蒸水
    在烧杯中煮沸2-3分钟
    冷却,倒入方形塔板

致谢

非常感谢科学技术委员会和工业研究委员会(CSIR),新德里以SRF形式向Kamal Tyagi提供的资金支持。 这项工作得到新德里生物技术部的支持,授予RS和YS。

参考文献

  1. Whippo,C.W。和Hangarter,R.P。(2003)。 第二个正向光趋向性来自光催化素和密码色素的协同作用。 Plant Physiol 132(3):1499-1507。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sharma, S., Tyagi, K., Sreelakshmi, Y. and Sharma, R. (2015). Gravitropic Analysis of Tomato Seedlings using Time Lapse Video Imaging. Bio-protocol 5(7): e1443. DOI: 10.21769/BioProtoc.1443; Full Text
  2. Whippo, C. W. and Hangarter, R. P. (2003). Secondpositive phototropism results from coordinated co-action of the phototropinsand cryptochromes. Plant Physiol 132(3): 1499-1507.




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