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A Method to Analyze Local and Systemic Effects of Environmental Stimuli on Root Development in Plants
环境刺激对植物根部发育的局部和系统效应的分析方法   

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Abstract

Root development in vascular plants is innately tied to the environment. However, relatively little attention has been paid toward understanding the spatial scales at which the root perceives and responds to external stimuli. While some environmental signals elicit global responses that affect root system architecture, others may have more localized effects. We have observed that various developmental processes can be induced or suppressed along the circumference of the main root depending on local contact with available water in a process termed hydropatterning (Bao et al., 2014). Our studies of hydropatterning indicate that the root can detect and respond to certain external stimuli at the resolution of the diameter of a single organ. In order to characterize developmental patterning at this spatial scale, we developed a procedure to vary environmental inputs across the circumferential axis of the root in vitro using agar media. Roots are grown between two blocks of agar media in a “sandwich”. Local environmental conditions can be varied depending on the composition of the media on either side. Stimuli that act locally can be distinguished from those that act systemically based on the developmental response of the root. Here we describe the overall method and provide an example of how it can be used to analyze lateral root patterning in Zea mays (maize) in response to an external water potential gradient. We also discuss how the method can be used more broadly for other plant species and environmental treatments.

Keywords: Plant-environment interactions(植物与环境的相互作用), Root development(根的发育), Lateral roots(侧根), Agar sandwich(琼脂三明治), Water stress(水分胁迫)

Materials and Reagents

  1. Germination paper (76 lb heavy weight seed germination paper) (Anchor Paper Company)
  2. 120 x 120 x 17 mm square plastic Petri plates (USA Scientific, catalog number: 5668-8102 )
  3. Aluminum foil
  4. Tri-fold paper towels (von Drehle Corporation, catalog number: 548-W )
  5. Silicone rubber (food-grade, high-temperature, 1/32” thickness, 50A medium hardness) (McMaster-Carr, catalog number: 86045K131 )
  6. Razor blades
  7. 50 ml conical tubes
  8. Filter paper, Grade 40 (Sigma-Aldrich, Whatman®, catalog number: Z241261 )
  9. Parafilm (VWR International, Bemis, catalog number: PM999 )
  10. Distilled water
  11. MS salts (Caisson Laboratories, catalog number: MSP01-50LT )
  12. MES hydrate (Sigma-Aldrich, catalog number: M2933 )
  13. Potassium hydroxide (1 M aqueous solution)
  14. Agar (BD, DifcoTM, catalog number: 214530 )
  15. Mannitol (PhytoTechnology Laboratories, catalog number: M562 )
  16. Maize kernels (B73 inbred line)
  17. Bleach (8.25% sodium hypochlorite solution)
  18. Tween 20 (Sigma-Aldrich, catalog number: P2287 )
  19. 70% (v/v) ethanol in water
  20. Superglue (Krazy Glue, catalog number: KG582 )
  21. Control agar media (see Recipes)
  22. Treatment agar media (see Recipes)
  23. Maize kernel sterilization solution (see Recipes)

Equipment

  1. Glass bottles (500 ml capacity or greater)
  2. pH meter
  3. Autoclave
  4. Laminar flow hood
  5. 4 °C cold room or refrigerator
  6. Glass beakers (500 ml capacity or greater)
  7. Hot water bath or hot plate
  8. Thermometer
  9. Timer
  10. Forceps
  11. Plant growth chamber (Percival, model number: CU41L4 )
  12. Permanent marker
  13. Microwave
  14. Ruler
  15. Computer equipped to run R or comparable statistical analysis software

Software

  1. R or comparable statistical analysis software (download link: https://cloud.r-project.org/)

Procedure

  1. Agar media preparation and sterilization of materials for seed plating and agar sandwich assembly
    Note: These steps should be performed no later than one day following seed sterilization (see below). These supplies can be stored for weeks; preparing them well ahead of time is recommended.
    1. Prepare control and treatment agar media
      Note: Four possible agar sandwich conditions can be constructed given two media types (block side/plate side: control/control, treatment/control, control/treatment, and treatment/treatment – see step C7 for further information). 1 L (15 plates) of each media type is sufficient to grow 12 plants per condition. This assumes two plants are used per plate, and includes extra plates for cutting agar blocks (see step C7). Amounts of media necessary will vary depending on desired sample size and number of conditions.
    2. Autoclave distilled water for 30 min at 121 °C on wet cycle. 500 ml autoclaved water is enough to sterilize ~100 seeds.
      Note: To save time, autoclave distilled water and agar media in the same cycle.
    3. Cut germination paper into 12 x 12 cm squares and wrap in aluminum foil. Two squares are sufficient to germinate 10-12 seeds.
    4. Fold paper towels along tri-fold creases and cut in half along the short axis. Wrap half-sheets in aluminum foil. Prepare one paper towel half-sheet for every 10-12 plants.
    5. Cut silicone rubber into 1.5 x 6 cm strips and wrap in aluminum foil. Cut two strips for each plant.
    6. Cut filter paper into ~4 x 4 cm squares and wrap in aluminum foil. Cut one square for each plant.
    7. Wrap 3-4 razor blades in aluminum foil.
    8. Autoclave germination paper squares, paper towel half-sheets, silicone rubber strips, filter paper squares, and razor blades at 121 °C for 20 min on dry cycle.
      Note: It is not recommended to autoclave these items along with agar media and distilled water, which should be autoclaved on wet cycle.

  2. Sterilization and germination of maize kernels
    1. Soak seeds for 4 h in distilled water in 50 ml conical tubes.
      Note: Use enough water to completely cover all of the kernels. For ease of handling, fill the tubes no higher than the 30 ml mark with kernels.
    2. Decant water. Remove tip caps from the kernels using a razor blade, ensuring that the embryo and endosperm of each kernel are not damaged (Figure 1A). Place kernels back in the 50 ml conical tube.
      Note: On occasion, the tip cap of the kernel may be too small or inconvenient to access without damaging other parts of the seed. In such cases, the excision step can be skipped.
    3. Incubate kernels in warm water (55-57 °C) for 5 min. Ensure that kernels are placed directly in contact with warm water by pouring some into the 50 ml conical tube containing the kernels before placing the tube in the water bath.
      Note: This can be done using a hot water bath, or by heating water in a glass beaker with a hot plate. Use of sterile water is not required at this point.
    4. Decant warm water. Rinse seeds once with room-temperature water.
    5. Incubate kernels in maize kernel sterilization solution for 20 min. Shake the tube occasionally (once every few minutes).
    6. Conduct all following steps in a laminar flow hood. Spray down all working surfaces and materials with 70% ethanol before placing into the hood.
    7. Decant maize kernel sterilization solution. Waste solution can be collected in a beaker.
    8. Rinse kernels 5-7 times with autoclaved distilled water.
    9. Plate seeds on autoclaved germination paper
      1. Place two germination paper squares into a plastic Petri plate. Place a paper towel half-sheet on top of the germination paper in the center of the plate (Figure 1A).
      2. Add 20 ml autoclaved distilled water to the plate to saturate the paper. Prepare enough plates for the number of kernels sterilized (10-12 kernels per plate).
      3. Fold back the paper towel half-sheet. Arrange 10-12 kernels in a row along the paper towel. Orient the kernels so that the cut ends point away from the paper towel (Figure 1B).
      4. Fold the paper towel half-sheet over the kernels. Smooth down the paper towel against the germination paper to limit movement of the kernels (Figure 1C).
      5. Seal the plates closed with Parafilm.
    10. Incubate plates vertically in a growth chamber for 2-3 days at 29 °C, 16 h light/8 h dark cycle. Orient the plates so that the cut ends of kernels are pointing toward gravity.


      Figure 1. Preparation of maize kernels for germination on sterile germination paper in square plates. A. A maize kernel with the embryo, endosperm, and tip cap outlined in black, cyan, and red, respectively. B. Square Petri plate containing germination paper and paper towel after soaking in water. C. Sterilized maize kernels lined up on germination paper. Note that the cut ends of the kernels point away from the paper towel that has been folded back. D. Germination paper plate after sealing with Parafilm. The paper towel has been folded over the kernels and smoothed down against the germination paper to keep the kernels in place. E. Germinated seedlings two days after plating.

  3. Agar sandwich assembly
    1. Conduct all steps in a laminar flow hood. Spray down all working surfaces and equipment with 70% ethanol before placing into the hood. Use forceps to manipulate materials such as agar, filter paper, silicone rubber, etc.
    2. Remove the Parafilm from the germination paper plates and fold back the paper towels. Check that primary roots have emerged and grown to a length of ~1 cm (Figure 1D).
    3. Take a plate of control agar media and cut a ~4 x 9 cm rectangle near one edge of the plate using an autoclaved razor blade. Remove and discard the agar in the rectangle. Ensure that the resulting hole is completely free of residual agar. This plate will be used for the “plate side” of the agar sandwich (Figure 2A).
    4. Place two squares of filter paper below the rectangular hole in the agar plate (Figure 2B). Smooth the paper down against the agar to remove any bubbles.
    5. Place silicone rubber strips to the left and right of the filter paper squares. Position the strips so that they just overlap the filter paper (Figure 2C).
    6. Glue the rubber to the agar using a small amount (1-2 drops) of superglue (Figure 2D).
      Note: Proceed carefully when positioning rubber strips, as superglue bonds very quickly and repositioning after the adhesive has set is challenging. Do not use too much superglue; although we rarely observe negative effects on the plant from superglue, root growth and development become strongly inhibited if the root comes into direct contact with either liquid or solid glue.
    7. Take another agar plate and cut it into 9 equally-sized squares using an autoclaved razor blade. These will be used for the “block side” of the agar sandwiches.
      Note: A printable template has been included in this protocol to assist with cutting agar blocks to the correct size (Figure 3).
    8. Remove two blocks from the plate in step C7 and place a filter paper square on each of them. Cut away any overhanging filter paper using an autoclaved razor blade (Figure 2E). Place the blocks paper-side-down into an empty plate lid to assist in cutting the filter paper.
    9. Position two seedlings in the agar plate from step C6 such that the kernels sit in the rectangular hole and the primary root tips are on top of the filter paper (Figure 2F). Avoid touching the primary root or coleoptile directly with the forceps.
    10. Glue down the agar blocks over the primary roots of the seedlings.
      1. Put a few drops of superglue on the exposed sides of the silicone rubber in the agar plate with the seedlings (Figure 2G).
      2. Lay the agar blocks across the silicone rubber rectangles with the paper side facing the seedlings (Figure 2H). Line up the blocks with the filter paper squares in the agar plate.
      3. Press the blocks against the silicone rubber rectangles so that the glue adheres them in place.
    11. Seal the agar sandwich plate with Parafilm.
    12. If a seedling comes out of the sandwich, perform either of the following:
      1. Reposition the seedling and slide the primary root further into the sandwich.
      2. Reposition the seedling so that the kernel is back in the rectangular hole in the plate. Cut a block of agar from an unused plate (either control or treatment media to match the media type on the plate-side of the seedling) and place it in a 50 ml conical tube. Heat the agar in a microwave until it melts (about 10-15 sec), and pipet the cooled molten agar solution around the kernel. After the agar solidifies, the seedling will be fixed in place.
    13. Label the plate with the agar sandwich condition (denoted as “block side/plate side”, e.g., “control/control”).
    14. Mark the positions of the primary root tips on the back of the plate using a permanent marker. Make sure the mark is visible from the other side of the plate (Figure 2I).


      Figure 2. Assembly of agar sandwiches. A. Plate-side agar media after removal of rectangular section of agar. B. Positioning of filter paper squares on plate-side agar media. C. Positioning of silicone rubber strips over filter paper squares. D. Applying superglue to one of the silicone rubber strips. Note that only a small volume of glue is required at each point of application. E. Agar blocks cut from a separate plate of media. Blocks are depicted sitting in an empty plate lid with filter paper squares applied. F. Maize seedlings in position in the plate containing the plate-side of the agar sandwich. G. Application of superglue to the silicone rubber strips before applying the agar block. H. Seedlings after both agar blocks have been applied and the plate has been sealed with Parafilm. I. Marks on the back of the plate indicating the positions of the primary root tips at the time of agar sandwich assembly.

    15. Repeat the above steps with the remainder of the germinated seedlings. Ensure that all possible media combinations are used.
      Note: Possible combinations are (block side/plate side): control/control, treatment/control, control/treatment, and treatment/treatment.
    16. Incubate plates vertically in a growth chamber at 29 °C, 16 h light/8 h dark cycle for 4-5 days, or until lateral roots have emerged along the lengths of the primary roots within the sandwiches.
    17. If any kernels have not germinated, or their primary roots are too short to put in agar sandwiches, check them again after one day.
      1. Leave the kernels on the germination paper plates. Cover them back up with a paper towel, reseal the plates with Parafilm, and incubate them in a growth chamber.
      2. Assemble more agar sandwiches if possible on the following day.
        Note: Maize kernels sterilized with the above method that do not germinate after 3 days are typically non-viable. Germination rates after 3 days can range from 70-100% given the quality of the seeds used.


        Figure 3. Printable template for cutting agar blocks from Petri dishes. This figure can be printed on an 8.5 x 11-inch sheet of paper and placed underneath Petri dishes to serve as a guide while cutting agar blocks (see step C7).

  4. Quantification of lateral root patterning from seedlings grown in an agar sandwich
    1. Remove the Parafilm from the Petri plate and open (Figure 4A).
    2. Using a permanent marker, mark on the primary roots where they first grew past the agar blocks (Figure 4B, white arrow).
    3. Carefully remove the agar blocks and filter paper using forceps and a razor blade.
    4. Use a permanent marker to mark the primary roots where the root tips were at the time of sandwich assembly (denoted on the backs of the plates) (Figure 4B, black arrow). Measure and record the distance between this mark and the mark where the root grew past the agar block with a ruler.
    5. Count the number of lateral roots that emerged on the side of the primary root that was facing the agar block (Figure 4C and 4D). Only count lateral roots that emerged between the two marks made in steps D2 and D4 (Figure 4C, white and black arrows).
      Note: If visualization of lateral roots using the naked eye is difficult, a low-magnification microscope can be used to assist in quantification.
    6. Carefully lift up the primary root and/or rotate the seedling and count the number of lateral roots that emerged on the side exposed to the media in the plate.
      Note: Some varieties of maize will accumulate anthocyanin pigmentation in air-exposed regions of the root (Figure 4C and 4D). This can serve as a useful boundary between the side of the root exposed to the block and the side exposed to the media in the plate.


      Figure 4. Disassembly of agar sandwiches and plant phenotyping. A. Plants in agar sandwiches five days after sandwich assembly (plate lid removed). B. Primary roots after removal of the agar blocks and overlying filter paper squares. Marks have been placed on the primary roots corresponding to the position of the root tip at the time of sandwich assembly (black arrows) and the point at which the root grew past the agar block (white arrows). C. Side view of the primary root. Lateral root quantification is done between the two marks on the primary root (denoted with arrows as in B). Two lateral root-forming zones are separated by a stripe of red anthocyanin pigmentation that developed in response to air exposure in the gap between the two sides of the sandwich. D. Diagrammatic representation of the different zones of the primary root. Lateral roots appear on the faces of the root exposed to agar media (Block Side and Plate Side), but not in the air-exposed regions (Air Gaps) which are marked with anthocyanin.

    7. Count the number of lateral roots that emerged on the sides of the root exposed to the air gap that was between the agar block and the media in the plate. Only count lateral roots between the two ink marks as described in step D5.
      Note: This count is usually close to 0 for most varieties of maize due to lateral root hydropatterning (Bao et al., 2014).
    8. Divide the number of lateral roots on each side of the root by the distance between the two marks (recorded in step D4) to calculate the lateral root density in units of lateral roots/cm.
    9. Average the lateral root density on each side of the root within each sandwich condition.
    10. Perform statistical analysis on lateral root density values. The steps to accomplish this are described in brief below. An example script written in R (R Core Team, 2015) has been provided with this protocol (Supplementary files 1 and 2).
      1. Reformat the data table for use in a linear model (Figure 5)
        1. The original data table contains separate columns for lateral root density on each side of the root. Convert this to a format in which density and side of the root are in separate columns. This can be accomplished using the “gather()” function in the “tidyr” package (Wickham, 2015). Once this is done, each row of the data table corresponds to a particular lateral root density value.
        2. Divide the condition column into two columns for the treatment on either side of the sandwich.
        3. Label the conditions on either side of the sandwich as either “cis” (on the same side of the root as the density value for the current row) or “trans” (on side of the root opposite that of the density value for the current row).


        Figure 5. Pre-processing of lateral root quantification data for ANOVA. One plant from one condition is shown as an example. Air Gap Count is dropped from the table since all counts were 0 in each condition, and lateral root densities were calculated by dividing counts by the length of the quantified region. Separate columns for the side of the root and density on that side were then created. Lastly, the condition column was reformatted as cis- and trans-condition categories. Note that the condition notation for the “Block” row is now the inverse of the notation for the “Plate” row.

      2. Perform an ANOVA to assess the effects of side of the root (block vs plate), cis-condition (control vs treatment), trans-condition (control vs treatment), and all possible interaction terms on lateral root density.
        1. The full linear model is written as: lateral root density ~side + cis-condition + trans-condition + side:cis-condition + side:trans-condition + cis-condition:trans-condition + side:cis-condition:trans-condition + residual error.
        2. Since each plant in the data set is measured twice (once on each side of the sandwich), an error term to account for within-plant variation must be included. This can be done using the “nlme” package (Pinheiro et al., 2015).
        3. The ANOVA test will assign P-values for statistical significance for each term in the model. A P-value less than 0.05 can be accepted as statistically significant. The interpretation of significance of each term in the model is explained below.
      3. Analyze the output from the ANOVA test
        1. A significant effect of the side of the root (block vs plate), either alone or as an interaction with other variables, indicates that the two sides of the sandwich differ from one another. This may be an indicator of a technical artifact resulting from improper sandwich assembly.
        2. A significant effect of the cis-condition term indicates that the treatment has a local effect on lateral root development.
        3. A significant effect of the trans-condition term indicates that the treatment has a non-local effect on lateral root development. This is one indicator that treatment applied to the roots is having systemic effects on lateral root patterning.
        4. A significant cis-condition:trans-condition interaction term indicates that the response on one side of the root depends upon the conditions experienced on the other side. This is another indicator that the two sides are not responding autonomously to the local treatment.
      4. If any significant terms are observed from the ANOVA test, perform pairwise comparisons between conditions of interest. This can be done using the “multcomp” package (Hothorn et al., 2008).

Data analysis

See Figure 6A for an example graph of average lateral root densities in different agar sandwich conditions. Table 1 contains the output from an ANOVA conducted in R. Figure 6B summarizes results of post-hoc testing after observation of a significant cis-condition:trans-condition interaction term in the ANOVA.

Table 1. Output from analysis of variance in R. The cis-condition had a significant effect on lateral root density (P < 0.05), which supports the hypothesis that the treatment has a local effect on development. The trans-condition and the cis-condition:trans-condition interaction term were also significant, indicating that the treatment also had a systemic effect on development. All terms which contain “side” as a variable are not significant (P > 0.05), indicating that the effects of the treatment did not depend on the method of application in the agar sandwich (block or plate agar media). numDF and denDF denote numerator and denominator degrees of freedom, respectively.

 

Figure 6. Representative data and statistical analysis. A. Maize seedlings were treated with either control or mannitol agar media in 4 agar sandwich conditions. Average lateral root densities on each side of the root are shown. The Air Gap density was 0 in each condition. Error bars = standard error, n = 6 plants per condition. B. Summary of data from post-hoc testing. Since there was a significant effect of the cis-condition:trans-condition term on lateral root density and no significant effect of the side of the sandwich (block vs plate) (see Table 1), all pairwise comparisons between the different treatment categories were performed using the "multcomp" package in R. Similar groups are denoted with the same letter (P < 0.05 for significant difference). The response to control media was independent of the treatment on the other side of the root, indicating that the root responded locally to this condition. Contrastingly, the response to mannitol differs depending on whether the other side of the root is treated with mannitol or control media, indicating that mannitol can have systemic effects on development. Error bars = standard error, n = 12 observations per category.

Notes

  1. The agar sandwich procedure was adapted from a procedure used with rice seedlings (Karahara et al., 2012).
  2. We found that combining heat treatment along with incubation in bleach to be the most effective method of maize kernel sterilization. The heat treatment protocol was adapted from Daniels (1983). The sterilization and seed germination conditions described here have been optimized for maize kernels. Studies involving other plant species may require modifications to these steps to ensure adequate sterility and germination rates (e.g., omission of the heat treatment, addition of an incubation in ethanol, and/or use of different types of germination media to support growth of early seedlings).
  3. This method is broadly applicable to looking at gradients of essentially any chemical agent that can be applied in gel media. While we have primarily focused on differences in water availability by applying osmolytes on the treatment side (e.g., mannitol or polyethylene glycol), varying concentrations of nutrients such as nitrate and phosphate could also be investigated.
  4. Applying this technique to other plant species is mainly limited by the diameter of the primary root. The root diameter must be greater than or equal to the thickness of the silicone rubber used to separate the two agar media in the sandwich. If the root is thinner than gap between the two sides of the sandwich, then adequate contact with both media conditions will not occur. Narrower materials could be used as spacers between the two agar media. However, this increases the likelihood of a film of water forming between the two agar surfaces, which would gradually dissipate any chemical gradients across the sandwich.
  5. If the two media on either side of the sandwich come into direct contact with one another, any differences in concentrations of water-soluble chemicals will equilibrate over time. In order to control for this possibility when examining the effects of different water potentials, we measured the water potential of the media on either side of the sandwich using a vapor pressure osmometer to demonstrate that it was stable during the experiment (Bao et al., 2014). Studies of other chemical gradients may require alternative methods of quality control.
  6. In our hands, the side of the root exposed to the applied agar block occasionally has fewer lateral roots than the other side, even when the media composition is the same. We attribute this to inadequate contact of the block with the root, as evidenced by increased anthocyanin accumulation on the block side (an indicator of air exposure in maize primary roots). Significant effects of the “side” term in an ANOVA test can also help to identify when this occurs. This can be compensated by increasing the sample size in each sandwich condition, and by taking greater care during assembly of the agar sandwiches.
  7. The use of filter paper to separate the agar from the root is done to prevent the root tip from growing into either side of the sandwich. The filter paper can be omitted, but the likelihood of the root penetrating into the agar media increases. Additional caution should be exercised to ensure that the two types of agar on either side of the sandwich do not contact one another (see Note 5).

Recipes

  1. Control agar media
    1. ½x MS salts + 0.5 g/L MES hydrate
    2. Adjust pH to 5.7 using potassium hydroxide solution.
    3. Add 2% (w/v) agar and autoclave for 30 min at 121 °C on wet cycle.
    4. Pour media into plastic plates in laminar flow hood after cooling, 65 ml/plate. Do not discard the plastic sleeve used for shipping the plates. Volumes can be measured using a sterile 50 ml conical tube.
    5. After media solidifies, wrap plates in plastic sleeves provided with the plates and store inverted at 4 °C.
    Note: Media with agar concentrations lower than 2% w/v will be difficult to cut into blocks and manipulate when assembling agar sandwiches.
  2. Treatment agar media
    1. ½x MS salts + 0.5 g/L MES hydrate + 220 mM mannitol
    2. Adjust pH to 5.7 using potassium hydroxide solution.
    3. Add 2% (w/v) agar and autoclave for 30 min at 121 °C.
    4. Pour media into plastic plates and store as above for control agar media.
  3. Maize kernel sterilization solution
    20% (v/v) bleach + 0.01% (v/v) Tween 20 in distilled water
    Prepare fresh immediately before each sterilization. 15 ml is sufficient for up to 30 kernels.

Acknowledgments

This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1147470. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation.

References

  1. Bao, Y., Aggarwal, P., Robbins, N. E., Sturrock, C. J., Thompson, M. C., Tan, H. Q., Tham, C., Duan, L. N., Rodriguez, P. L., Vernoux, T., Mooney, S. J., Bennett, M. J. and Dinneny, J. R. (2014). Plant roots use a patterning mechanism to position lateral root branches toward available water. Proc Natl Acad Sci U S A 111(25): 9319-9324.
  2. Daniels, B. A. (1983). Elimination of Fusarium moniliforme from corn seed. Plant Disease 67(6): 609-611.
  3. Hothorn, T., Bretz, F. and Westfall, P. (2008). Simultaneous inference in general parametric models. Biom J 50(3): 346-363.
  4. Karahara, I., Umemura, K., Soga, Y., Akai, Y., Bando, T., Ito, Y., Tamaoki, D., Uesugi, K., Abe, J., Yamauchi, D. and Mineyuki, Y. (2012). Demonstration of osmotically dependent promotion of aerenchyma formation at different levels in the primary roots of rice using a 'sandwich' method and X-ray computed tomography. Ann Bot 110(2): 503-509.
  5. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., Heisterkamp, S. Willigen, B. V. and R-Core (2016). nlme: linear and nonlinear mixed effects models. R package version 3.1-128.
  6. R Development Core Team. (2015). R: A language and environment for statistical computing. R Foundation for Statistical Computing.
  7. Wickham, H. (2016). tidyr: easily tidy data with spread() and gather() functions. R package version 0.6.0.

简介

维管植物中的根发育与环境固有关系。然而,相对较少的关注已经被用于理解根部感知和响应外部刺激的空间尺度。虽然一些环境信号引起影响根系统结构的全局响应,但其他环境信号可能具有更局部化的效应。我们已经观察到,根据在称为水图案化的过程中与可用水的局部接触,可以沿着主根的圆周诱导或抑制各种发育过程(Bao等人,2014)。我们对水印的研究表明根部可以检测和响应某些外部刺激在单个器官的直径的分辨率。为了表征在该空间尺度的发育图案化,我们开发了使用琼脂培养基在体外改变穿过根的周向轴的环境输入的程序。根在"三明治"中的两块琼脂培养基之间生长。局部环境条件可以根据任一侧的介质的组成而变化。局部作用的刺激可以与基于根的发育反应系统作用的刺激相区分。在这里我们描述了整体的方法,并提供了一个例子,它如何可以用于分析外部水势梯度响应中玉米Zea mays(玉米)的侧根图案。我们还讨论了该方法如何可以更广泛地用于其他植物物种和环境处理。

关键字:植物与环境的相互作用, 根的发育, 侧根, 琼脂三明治, 水分胁迫

材料和试剂

  1. 发芽纸(76磅重重的种子发芽纸)(Anchor Paper Company)
  2. 120×120×17mm正方形塑料培养皿(USA Scientific,目录号:5668-8102)
  3. 铝箔
  4. 三折纸巾(von Drehle公司,目录号:548-W)
  5. 硅橡胶(食品级,高温,1/32"厚度,50A中等硬度)(McMaster-Carr,目录号:86045K131)
  6. 剃刀刀片
  7. 50ml锥形管
  8. 滤纸,级别40(Sigma-Aldrich,Whatman ,目录号:Z241261)
  9. Parafilm(VWR International,Bemis,目录号:PM999)
  10. 蒸馏水
  11. MS盐(Caisson Laboratories,目录号:MSP01-50LT)
  12. MES水合物(Sigma-Aldrich,目录号:M2933)
  13. 氢氧化钾(1M水溶液)
  14. 琼脂(BD,Difco TM ,目录号:214530)
  15. 甘露醇(PhytoTechnology Laboratories,目录号:M562)
  16. 玉米粒(B73自交系)
  17. 漂白剂(8.25%次氯酸钠溶液)
  18. 吐温20(Sigma-Aldrich,目录号:P2287)
  19. 70%(v/v)乙醇/水
  20. Superglue(Krazy Glue,目录号:KG582)
  21. 控制琼脂培养基(参见配方)
  22. 处理琼脂培养基(见配方)
  23. 玉米核消毒液(见配方)

设备

  1. 玻璃瓶(500 ml以上容量)
  2. pH计
  3. 高压灭菌器
  4. 层流罩
  5. 4℃冷藏室或冰箱
  6. 玻璃烧杯(500 ml以上容量)
  7. 热水浴或热水板
  8. 温度计
  9. 计时器
  10. 镊子
  11. 植物生长室(Percival,型号:CU41L4)
  12. 永久标记
  13. 微波
  14. 标尺
  15. 配备运行R或类似统计分析软件的计算机

软件

  1. R或类似的统计分析软件(下载链接: https://cloud.r-project .org/

程序

  1. 琼脂培养基制备和种子电镀和琼脂夹心组件的材料灭菌
    注意:这些步骤应在种子灭菌后不超过一天执行(见下文)。这些用品可以储存数周;建议提前准备好他们。
    1. 准备控制和处理琼脂培养基
      注意:给定两种培养基类型(块侧/板侧:对照/对照,处理/对照,对照/处理和处理/处理 - 参见步骤C7以获得更多信息),可以构建四种可能的琼脂夹心条件。每种培养基类型的1L(15个平板)足以在每种条件下生长12株植物。这假设每个板使用两个植物,并且包括用于切割琼脂块的额外板(参见步骤C7)。所需的介质量将根据所需的样品大小和条件数量而变化。
    2. 在121℃下,在湿循环中高压灭菌蒸馏水30分钟。 500毫升高压灭菌水足以对〜100粒种子灭菌 注意:为了节省时间,在同一循环中使用高压灭菌蒸馏水和琼脂培养基。
    3. 将发芽纸切成12×12厘米的正方形,并包裹在铝箔中。两个方块足以发芽10-12粒种子
    4. 沿着三折褶皱折叠纸巾,沿短轴切成两半。 用铝箔包装半张。 每10-12株植物准备一张纸巾半张。
    5. 将硅橡胶切成1.5 x 6厘米的条带,然后用铝箔包裹。 每棵植物切两条。
    6. 将滤纸切成〜4×4厘米正方形,并包裹在铝箔中。 为每个植物切割一个正方形。
    7. 包裹3-4片剃刀刀片在铝箔。
    8. 高压灭菌纸张方格,纸巾半张,硅橡胶条,滤纸方格和剃刀刀片在121℃干燥循环20分钟。
      注意:不建议将这些物品与琼脂培养基和蒸馏水一起高压灭菌,应在湿循环中高压灭菌。

  2. 玉米粒的灭菌和发芽
    1. 在50ml锥形管中的蒸馏水中浸泡种子4小时 注意:使用足够的水,以完全覆盖所有的内核。为了易于处理,填充管不高于30毫升标记用内核。
    2. 淡水。使用剃刀刀片,从确保胚胎和胚乳胚胎和胚乳没有损坏(图1A)删除尖端帽的内核。将内核放回50 ml锥形管中。
      注意:有时,内核的尖端帽可能太小或不便于访问,而不会损坏种子的其他部分。在这种情况下,可以跳过切除步骤。
    3. 在温水(55-57℃)孵育谷粒5分钟。在将管放入水浴之前,将一些内容倒入含有内核的50ml锥形管中,以确保内核直接接触温水。
      注意:这可以使用热水浴,或通过用热板在玻璃烧杯中加热水来完成。此时不需要使用无菌水。
    4. 滗析温水。 用室温水冲洗种子一次。
    5. 在玉米核消毒溶液中孵育谷粒20分钟。 偶尔摇动管(每隔几分钟一次)。
    6. 在层流罩中进行所有以下步骤。 在放入发动机罩之前,用70%乙醇喷洒所有工作表面和材料。
    7. 滗析玉米仁消毒液。 废溶液可以在烧杯中收集
    8. 用高压灭菌蒸馏水冲洗核5-7次。
    9. 在高压灭菌的发芽纸上种子种子
      1. 将两个发芽纸方块放入塑料培养皿中。在纸板中心的发芽纸上放置一张纸巾半张(图1A)。
      2. 加入20毫升高压灭菌蒸馏水到板中以饱和纸。准备足够的数量的核仁灭菌(每板10-12粒)
      3. 折回纸巾半张。沿着纸巾连续排列10-12个内核。定向内核,使切割端指向远离纸巾(图1B)。
      4. 将纸巾半张放在内核上。将纸巾平稳地放在发芽纸上,以限制内核的移动(图1C)。
      5. 密封用石蜡膜封闭的平板。
    10. 在生长室中在29℃,16小时光照/8小时黑暗循环下垂直培养平板2-3天。定位板材,使切割的颗粒末端指向重力

      图1.制备用于在方形板上的无菌发芽纸上萌发的玉米籽粒A.具有分别以黑色,青色和红色描绘的胚,胚乳和尖端帽的玉米籽粒。 B.在水中浸泡后含有发芽纸和纸巾的方形培养皿。 C.灭菌的玉米粒排列在发芽纸上。注意,内核的切割端远离已经折回的纸巾。 D.用封口膜密封后的发芽纸板。纸巾已经被折叠在谷粒上并且平滑在发芽纸上以保持谷粒在适当的位置。 E.电镀两天后发芽的幼苗
  3. 琼脂夹心组件
    1. 在层流罩中进行所有步骤。在放入通风橱之前,用70%乙醇喷洒所有工作表面和设备。使用镊子操作材料,如琼脂,滤纸,硅橡胶,等。
    2. 从发芽纸板中取出石蜡膜并折回纸巾。检查初生根已经出现并长到〜1厘米的长度(图1D)。
    3. 取一块对照琼脂培养基,并使用高压灭菌剃刀刀片在板的一个边缘附近切割〜4×9厘米的矩形。删除并丢弃矩形中的琼脂。确保产生的孔是 完全不含残留琼脂。该板将用于琼脂夹心的"板侧"(图2A)。
    4. 将两个方格的滤纸放在琼脂板的矩形孔下方(图2B)。将纸张顺着琼脂平滑,以除去任何气泡。
    5. 将硅橡胶条放在滤纸方格的左侧和右侧。将条带定位,使其刚好与滤纸重叠(图2C)。
    6. 使用少量(1-2滴)超强力胶将橡胶胶粘到琼脂上(图2D) 注意:在定位橡胶条时要小心,因为超胶粘键非常快,并且在粘合剂固化之后重新定位是有挑战性的。不要使用太多的superglue;虽然我们很少观察到对超高浓度植物的负面影响,如果根直接接触液体或固体胶,根部生长和发育会被强烈抑制。
    7. 取另一块琼脂板,并使用高压灭菌剃刀刀片将其切成9个相等大小的正方形。这些将用于琼脂三明治的"块侧" 注意:此协议中包括可打印的模板,以帮助将琼脂块切割成正确的尺寸(图3)。
    8. 在步骤C7中从板上取下两个块,并在每个块上放置一个滤纸方格。使用高压灭菌剃刀刀片切掉任何悬垂的滤纸(图2E)。放置块纸 - 下降到空的板盖以帮助切割滤纸
    9. 将两个秧苗放置在来自步骤C6的琼脂板中,使得颗粒位于矩形孔中,并且主根尖位于滤纸的顶部(图2F)。 避免直接用钳子接触主根或胚芽鞘。
    10. 将琼脂块置于幼苗的主根上。
      1. 将几滴超强力胶放在琼脂板上的硅橡胶的暴露面上(图2G)。
      2. 将琼脂块放置在硅橡胶矩形上,纸面朝向幼苗(图2H)。 将块与琼脂平板上的滤纸方格对齐。
      3. 将这些块压在硅胶橡胶矩形上,使胶水粘附在其上。
    11. 用石蜡膜密封琼脂夹心板。
    12. 如果幼苗从三明治中出来,请执行以下任一操作:
      1. 重新放置苗,将主根进一步滑入三明治
      2. 重新放置幼苗,使内核回到板上的矩形孔中。 从未使用的板(控制或处理媒体,以匹配在培养皿的板侧的培养基类型)切成一块琼脂,并将其放置在50ml锥形管中。 在微波炉中加热琼脂,直到它融化(约10-15秒),并吸取冷却的熔融琼脂溶液在核心周围。 琼脂固化后,幼苗将固定在适当位置
    13. 用琼脂夹心条件(表示为"块侧/板侧",例如"控制/对照")标记板。
    14. 使用永久性标记标记板背面主要根尖的位置。确保标记从板的另一侧可见(图2I)。


      图2.琼脂三明治的装配 A.去除琼脂的矩形部分后的平板侧琼脂培养基。 B.在板侧琼脂培养基上定位滤纸方格。 C.将硅橡胶条定位在滤纸方格上。 D.对一个硅橡胶条施加超强力胶。注意,在每个应用点只需要少量的胶水。 E.从单独的培养基板上切下的琼脂块。块被描述坐在有被应用的滤纸纸正方形的一个空的板盖子。 F.玉米幼苗在含有琼脂三明治的板侧的板中的位置。 G.在施加琼脂块之前,将超纯胶应用于硅橡胶条。 H.已经施用两个琼脂块之后的幼苗,并且用Parafilm密封板。 I.在板的背面的标记指示在琼脂夹层组件时主要根尖的位置
    15. 对剩余的发芽幼苗重复上述步骤。确保使用所有可能的介质组合。
      注意:可能的组合(块侧/板侧):控制/控制,治疗/控制,控制/治疗和治疗/治疗。
    16. 在生长室中在29℃,16小时光照/8小时黑暗循环下垂直培养板4-5天,或者直到沿着三明治内的主根的长度出现侧根。
    17. 如果任何谷粒没有发芽,或者它们的主根太短,不能放入琼脂三明治,一天后再检查一下。
      1. 离开发芽纸板上的内核。用纸巾覆盖它们,用Parafilm重新密封板,并在生长室中孵育它们。
      2. 如果可能,在第二天装配更多的琼脂三明治。
        注意:用上述方法灭菌的玉米籽粒在3天后不发芽通常是不存活的。考虑到所用种子的质量,3天后的发芽率可以为70-100%

        图3.用于从培养皿切割琼脂块的可印刷模板。该图可印刷在8.5×11英寸的纸上,并放置在培养皿下方作为导向,同时切割琼脂块见步骤C7)。

  4. 从琼脂三明治中生长的幼苗侧根图案的量化
    1. 从培养皿中取出石蜡膜并打开(图4A)
    2. 使用永久标记,在主根上标记它们首先生长通过琼脂块(图4B,白色箭头)。
    3. 使用镊子和刀片小心地取出琼脂块和滤纸。
    4. 使用永久性标记标记根尖在夹层组装时(在板的背面上指示)的根部(图4B,黑色箭头)。测量并记录此标记与根部通过标尺生长经过琼脂块的标记之间的距离。
    5. 计数出现在面向琼脂块的主根侧的侧根的数目(图4C和4D)。只计算在步骤D2和D4中形成的两个标记之间出现的侧根(图4C,白色和黑色箭头)。
      注意:如果用肉眼观察侧根很困难,可以使用低倍率显微镜辅助定量。
    6. 小心提起主根和/或旋转幼苗,并计数在暴露于培养基中培养基的一侧出现的侧根数。
      注意:一些品种的玉米将在根部的暴露于空气的区域中积累花青素色素沉着(图4C和4D)。这可以用作暴露于块的根部的侧部和暴露于板中的介质的侧部之间的有用边界。


      图4.琼脂三明治和植物表型的拆解 A.夹心装配后五天(琼脂板去除)的琼脂三明治中的植物。 B.去除琼脂块和覆盖的滤纸方块之后的主根。标记已经放置在主根上,对应于夹层组装时根尖的位置(黑色箭头)和根生长经过琼脂块的点(白色箭头)。 C.主根的侧视图。横向根量化在主根上的两个标记之间进行(用B中的箭头表示)。两个侧根形成区由红色花青素色素化的条纹分开,所述红色花青素色素化响应于夹层物两侧之间的间隙中的空气暴露而形成。 D.主根不同区域的图示。横向根出现在暴露于琼脂培养基(块侧和板侧)的根的表面上,但不在标记有花青素的空气暴露区域(空气间隙)中。

    7. 计数出现在根暴露于琼脂块和培养基之间的空气间隙的根侧的侧根的数目。只计算两个墨水标记之间的侧根,如步骤D5中所述。
      注意:对于大多数玉米品种,由于侧根水成熟,这个计数通常接近于0(Bao等人,2014)。
    8. 将根部每侧的侧根数除以两个标记之间的距离(在步骤D4中记录),以计算以侧根/cm为单位的侧根密度。
    9. 平均每个夹心条件下根部每侧的侧根密度
    10. 对侧根密度值进行统计分析。下面简要描述实现这一点的步骤。此协议提供了一个使用R(R Core Team,2015)编写的示例脚本(补充文件 1 2 )。
      1. 重新格式化数据表以用于线性模型(图5)
        1. 原始数据表包含在根的每侧上的侧根密度的单独列。将其转换为密度和根的边在其中的格式 单独的列。这可以使用"tidyr"包中的"gather()"函数来实现(Wickham,2015)。一旦完成,数据表的每一行对应于特定的横向根密度值。
        2. 将条件列分成两列,用于夹心的任一侧上的处理。
        3. 将三明治任一侧上的条件标记为"顺式"(在根的相同侧与当前行的密度值)或"反式"(在根侧的与电流的密度值相反的一侧行)。


        图5. ANOVA的侧根定量数据的预处理。来自一个条件的一株植物作为实例显示。空气间隙计数从表中下降,因为在每个条件下所有计数为0,并且通过将计数除以定量区域的长度计算侧根密度。然后创建根侧和该侧的密度的单独的列。最后,条件列重新格式化为顺式和反式条件类别。请注意,"块"行的条件符号现在是"Plate"行的符号的逆。

      2. 进行方差分析以评估根侧(块与板),顺式条件(对照与治疗),反式条件(对照与治疗)和所有可能的相关项对侧根密度的影响。
        1. 全线性模型写为:侧根密度〜侧面+顺式条件+反式条件+侧面:顺式条件+侧面:反式条件+顺式条件:反式条件+侧:顺式条件:条件+残差。
        2. 由于数据集中的每个植物被测量两次(在夹心的每一侧上一次),因此必须包括考虑植物内变异的误差项。这可以使用"nlme"包(Pinheiro等人。,2015)。
        3. ANOVA检验将为模型中每个项的统计显着性分配 P 值。 P 值小于0.05可被接受为统计显着性。下面解释模型中每个项的显着性的解释。
      3. 分析ANOVA测试的输出
        1. 根部(块对板)的一侧的显着效应,单独地或作为与其它变量的相互作用,表明夹心的两侧彼此不同。这可能是因不当的夹心装配而产生的技术性痕迹的指示
        2. 顺式条件术语的显着效果表明治疗对侧根发育具有局部效应
        3. 反式条件术语的显着效果表明治疗对侧根发育具有非局部效应。这是一个指标,表明对根部的处理对侧根形态有系统的影响
        4. 显着的顺式条件:反条件相互作用项表示根的一侧上的反应取决于另一侧经历的条件。这是另一个指标,表明双方没有自主地对当地治疗作出反应。
      4. 如果从ANOVA检验观察到任何重要术语,则在感兴趣的条件之间进行成对比较。这可以使用"multcomp"包(Hothorn等人,2008)。

数据分析

关于不同琼脂夹心条件下的平均侧根密度的实例图,参见图6A。表1包含在R中进行的ANOVA的输出。图6B总结了在观察到显着顺式条件:ANOVA中的反条件相互作用项之后的事后检验的结果。

表1. R方差分析的输出顺式条件对侧根密度有显着影响(

0.05),表明治疗的效果不依赖于在琼脂夹心(块或板琼脂培养基)。 numDF和denDF分别表示分子和分母自由度
 

图6.代表性数据和统计分析 A.在4个琼脂夹心条件下用对照或甘露醇琼脂培养基处理玉米幼苗。显示了根部每侧的平均侧根密度。在每种条件下,气隙密度为0。误差棒=标准误差,每种条件n = 6株。 B.事后测试的数据摘要。由于顺式条件:反式条件项对横向根密度有显着影响,并且夹心(块对板)侧没有显着影响(参见表1),不同处理类别之间的所有成对比较使用R中的"multcomp"包执行。类似的组用相同的字母表示(对于显着差异, P <0.05)。对于对照培养基的反应与在根的另一侧上的处理无关,表明根部局部地响应于该条件。相反,对甘露醇的反应根据是否用甘露醇或对照培养基处理根的另一侧而不同,表明甘露醇可对发育具有系统效应。误差线=标准误差,n =每个类别12个观察值。

笔记

  1. 琼脂夹心程序改造自用于水稻幼苗的程序(Karahara等人,2012)。
  2. 我们发现,结合热处理和漂白剂孵化是玉米核消毒的最有效的方法。热处理方案改编自Daniels(1983)。本文所述的灭菌和种子发芽条件已经针对玉米籽粒进行了优化。涉及其它植物物种的研究可能需要对这些步骤进行修改以确保足够的不育性和发芽率(例如,遗漏 热处理,在乙醇中添加孵育,和/或使用不同类型的发芽培养基以支持早期幼苗的生长)。
  3. 该方法广泛适用于查看可以施加在凝胶介质中的基本上任何化学试剂的梯度。虽然我们主要集中在通过在处理侧应用渗透剂(例如,甘露醇或聚乙二醇)的水可用性的差异,但也可以研究不同浓度的营养物,例如硝酸盐和磷酸盐。
  4. 将这种技术应用于其他植物物种主要受到主根直径的限制。根直径必须大于或等于用于分离夹层中的两种琼脂介质的硅橡胶的厚度。如果根部比夹层结构的两侧之间的间隙薄,则不会发生与两种介质条件的充分接触。较窄的材料可以用作两种琼脂培养基之间的间隔物。然而,这增加了在两个琼脂表面之间形成水膜的可能性,这将逐渐消耗穿过夹心的任何化学梯度。
  5. 如果夹心两侧的两种介质彼此直接接触,水溶性化学品浓度的任何差异将随时间平衡。为了在检查不同水势的影响时控制这种可能性,我们使用蒸汽压渗透计测量夹心的任一侧上的介质的水势,以证明其在实验期间是稳定的(Bao等人al 。,2014)。对其他化学梯度的研究可能需要替代的质量控制方法
  6. 在我们的手中,暴露于施加的琼脂块的根的一侧偶尔具有比另一侧更少的侧根,即使当培养基组成相同时。我们将这归因于块与根的不充分接触,如块侧的增加的花青素积累(玉米主根的空气暴露的指标)所证明的。 "方差分析"方法在方差分析测试中的显着影响也可以帮助确定何时 这发生。 这可以通过在每个夹心条件下增加样品大小以及在装配琼脂三明治时更加小心来补偿。
  7. 使用滤纸将琼脂与根分离,以防止根尖生长到夹层的任一侧。 滤纸可以省略,但是根部渗入琼脂培养基的可能性增加。 应当额外注意,以确保三明治两侧的两种类型的琼脂不会相互接触(见注5)。

食谱

  1. 控制琼脂培养基
    1. 1×MS盐+ 0.5g/L MES水合物
    2. 使用氢氧化钾溶液将pH调节至5.7
    3. 在湿循环中在121℃下加入2%(w/v)琼脂和高压灭菌器30分钟
    4. 冷却后,在层流罩中将培养基倒入塑料板中,65ml /板。 不要丢弃用于运输板的塑料套管。 体积可以使用无菌的50ml锥形管测量
    5. 培养基凝固后,将平板放在装有平板的塑料套中,并在4℃下倒置贮存。
    注意:琼脂浓度低于2%w/v的培养基将很难切成块,并在装配琼脂三明治时进行操作。
  2. 治疗琼脂培养基
    1. 1/2xMS盐+ 0.5g/L MES水合物+ 220mM甘露醇
    2. 使用氢氧化钾溶液将pH调节至5.7
    3. 在121℃下加入2%(w/v)琼脂和高压灭菌器30分钟
    4. 将培养基倒入塑料板中,如上所述存放于对照琼脂培养基上
  3. 玉米核消毒液
    20%(v/v)漂白剂+ 0.01%(v/v)吐温20的蒸馏水中 每次灭菌前立即准备新鲜。 15 ml足以满足30颗内核。

致谢

本材料基于国家科学基金会研究生研究奖学金资助号DGE-1147470支持的工作。在本材料中表达的任何意见,发现,结论或建议是作者的,不一定反映国家科学基金会的意见。

参考文献

  1. Bao et al。,2008; Wang et al。,2008; Wang et al。,2008; Wang,et al。 MJ和Dinneny,JR(2014)。  植物根使用图案化机制将侧根分支定位于可用水。美国国家科学院院刊111(25):9319-9324。
  2. Daniels,BA(1983)。  从玉米种子中消除 Fusarium moniliforme 。 植物病 67(6):609-611。
  3. Hothorn,T.,Bretz,F.和Westfall,P.(2008)。  一般参数模型中的同时推理。 Biom J 50(3):346-363。
  4. Karahara,I.,Umemura,K.,Soga,Y.,Akai,Y.,Bando,T.,Ito,Y.,Tamaoki,D.,Uesugi,K.,Abe,J.,Yamauchi, Mineyuki,Y。(2012)。  渗透依赖性使用"三明治"方法和X射线计算机断层摄影术在水稻的主根中促进不同水平的气胀形成。 Ann Bot 110(2):503-509。
  5. Pinheiro,J.,Bates,D.,DebRoy,S.,Sarkar,D.,Heisterkamp,S. Willigen,BV和R-Core(2016)。  nlme:线性和非线性混合效应模型 R包版本 3.1-128 。
  6. R开发核心团队。 (2015)。  R:用于统计计算的语言和环境。 的统计计算基础。
  7. Wickham,H.(2016)。  tidyr:使用spread()和gather()函数轻松整理数据。 R包 版本 0.6.0 。

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引用:Robbins II, N. E. and Dinneny, J. (2016). A Method to Analyze Local and Systemic Effects of Environmental Stimuli on Root Development in Plants. Bio-protocol 6(17): e1923. DOI: 10.21769/BioProtoc.1923.
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