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Paper Roll Culture and Assessment of Maize Root Parameters
玉米根系的卷纸培样和参数评估   

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Abstract

Selection for genotypes with a vigorous root system could enhance the adaptation of maize under water and nutrient deficit soils. Although extensive genetic variation for root architecture has been reported (Kumar et al., 2012; Abdel-Ghani et al., 2014; Kumar et al., 2014; Pace et al., 2015), root traits have been seldom considered as selection criteria to improve yield in maize, mainly because characterization of root morphology in the field is laborious, inaccurate and time consuming (Tuberosa and Salvi, 2007). Characterization of root traits under hydroponic conditions in this case has the advantage of screening a high number of genotypes in a small space (in a growth chamber) within a short period of time (2-3 weeks). Thus, it saves the time and effort required for screening maize genotypes with vigorous root systems and might be helpful to monitor root development at different growth stages.

Materials and Reagents

  1. Regular weight (brown) germination paper 48.5” x 36.5”, custom-sized to 12” x 24” (Anchor Paper Company, catalog number: SD3836S )
  2. Small kitchen wire mesh strainer/sieve with handle (20 cm diameter)
  3. Small plastic cups, measuring boats (FisherbrandTM Hexagonal Polystyrene Weighing dishes) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 02-202-101 ), or double-faced filter paper for drying seed- should be the same number as the number of entries
  4. Waterproof pencil art grip aquarelle black (Faber-Castell, catalog number: 114299 ) and permanent marker (Sharpie®, Fine Point Permanent Marker, black)
    Note: Black marker works better as other colors fade faster.
  5. Plastic tags (5” x 5/8”) (International Greenhouse, catalog number: CN-1000 ) for labeling (optional if rolls are labeled)
  6. Rubber bands (OfficeMax Extra Long Rubber bands, or any other brand)
  7. Glassine bags (Seedburo S411 shoot bags, treated, 4” x 2-1/2” x 11”)
  8. Personal protective items: latex gloves, lab coat, closed shoes, mask goggles
  9. Maize seeds: Genotypes used in this protocol are pure lines obtained from the North Central Regional Plant Introduction Station in Ames, Iowa (Abdel-Ghani et al., 2013).
    Notes:
    1. Seeds should be multiplied under the same conditions to avoid differences due to the environment on the seed size.
    2. Seeds should display high germination percentages to keep a similar number of biological replications within experimental units.
  10. Chlorox® solution (6% sodium hypochlorite), household bleach (USA)
  11. Deionized sterile distilled water (ddH2O)
  12. Potassium nitrate (KNO3) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P-263-500 )
  13. Calcium nitrate [Ca(NO3)2] (Thermo Fisher Scientific, Fisher Scientific, catalog number: C109-3 )
  14. Monopotassium phosphate or potassium phosphate monobasic (KH2PO4) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP-362-500 )
  15. Magnesium sulfate (Thermo Fisher Scientific, Fisher Scientific, catalog number: M65-500 )
  16. Iron from iron chelate [Fe-EDTA, (Sigma-Aldrich, catalog number: E6760-100G ), Fe-DTPA, or Fe-EDDHA]
  17. Monocalcium phosphate or calcium phosphate monobasic [Ca(H2PO4)2] (MP Biomedicals, catalog number: 193803 )
  18. Calcium sulfate dihydrate (CaSO4·2H2O) (MP Biomedicals, catalog number: 191414 )
  19. Potassium sulfate (K2SO4) (Sigma-Aldrich, catalog number: P0772-1kg )
  20. Boric acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP168-1 )
  21. Manganese chloride-4 hydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: 221279-100g )
  22. Zinc sulfate-7 hydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251-100G )
  23. Copper sulfate-5 hydrate (Thermo Fisher Scientific, Fisher Science, catalog number: S25287A )
  24. Molybdic acid (H2MoO4) (Sigma-Aldrich, catalog number: 232084-100G )
  25. Hoagland’s nutrient solution
    1. High N (15 mM NO3-) Hoagland’s solution (see Recipes)
    2. Low N (1.5 mM NO3-) Hoagland’s solution (see Recipes)
    3. Micronutrient stock solution (1 L) (see Recipes)
  26. 30% ethanol (C2H6O) (commercial grade from any brand) (see Recipes)
  27. 2.5 g/L Fungicide solution Captan® (Bonide Products Inc.) (see Recipes)

Equipment

  1. 2 L capacity beakers (each beaker holds 8-10 paper rolls) (Coring, Pyrex® Griffin Beakers, catalog number: 10000-2L )
  2. 50 ml capacity beakers (Pyrex®, Griffin Beakers), for sterilizing and washing seeds (Sigma-Aldrich, catalog number: CLS100050 )
  3. Plant growth chamber (Conviron, model: PGC FLEX )
  4. Cold room (Rheem Puffer Hubbard environmental chamber) or refrigerator
  5. Autoclave (PRIMUS Sterilizer , model: PSS5 )
  6. Sensitive balance (Ohaus, model: AdventurerTM AR0640 )
    Note: This product has been discontinued.
  7. Flatbed scanner (Epson, model: Expression 10000 XL , or any other brand)
  8. Computer with flash drive and Windows operating system
  9. Fisher ScientificTM IsotempTM general purpose heating and drying oven (Thermo Fisher Scientific, Fisher Scientific, model: 15-103-0503) or any constant temperature oven/dryer
  10. Ruler or yardstick (Acme Westcott 15728 36” Aluminum Yard Stick)

Software

  1. WinRhizo (Regent Instruments, model: WinRhizo Pro 2009) or ARIA (Automatic Root Image Analysis) (Pace et al., 2014)

Procedure

This experiment was designed to test the performance of maize seedlings under contrasting level of nitrogen (N) levels (Abdel-Ghani et al., 2013). The seedlings should be exposed to Hoagland’s nutrient solution with high N (HN) and low N (LN) (Hershey, 1994). Nitrogen in Hoagland’s solution with HN contains 15 mM of NO3-, whereas the concentration of N in LN Hoagland’s solution is 1.5 mM (10% NO3-) (Abdel-Ghani et al., 2013; Abdel-Ghani et al., 2015). Other macro- and micro-elements should be constant in both nutrient treatments. All steps regarding paper roll preparation and culture are as follows (Also see Figure 1; steps A to D).


Figure 1. Summary of steps for paper rolls preparation and culture. A. Kernels are surface sterilized with 6% sodium hypochlorite, washed with distilled water and dried out. Four sterilized maize kernels of similar size are placed on a double layer of brown filter papers pre-moistened with fungicide solution Captan®. B. Rolled germination papers are kept in 2 L glass beakers containing Hoagland’s nutrient solution with high nitrogen (HN) and low nitrogen (LN). C. Rolls should be kept for 14 days in a controlled growth chamber. D. Seedling after 14 days of incubation in the controlled growth chamber, under LN and HN treatments.

  1. Maize kernels sterilization
    1. Kernels are surface sterilized in 20 ml beakers with Chlorox® solution (6% sodium hypochlorite) for 15 min at room temperature. Chlorox should cover the kernels in the beaker and beakers should be manually shaken for 3-4 times.
    2. Chlorox should be drained out first, and then the seeds should be washed with ddH2O three times. Small sterilized sieve (20 cm in diameter) can be used to drain out water after each wash.
    3. After washing, kernels should be kept on a double-faced brown filter paper and left for 10 min until the seeds are dry. Small plastic cups or measuring boats can also be used to dry seeds.

  2. Growing seeds in paper rolls
    1. Brown germination paper should be cut down into 20 x 20 cm sheets and pre-moisturized with fungicide solution Captan® (2.5 g/L) to eliminate the possibility of any fungi development during seedling development. The brown paper should be moistened with fungicide solution by soaking the paper in the solution. Excess fungicide solution should be removed by pressing on the soaked papers by hand. The paper rolls should be labeled either with a permanent (water proof) pencil by writing directly on brown sheets and/or by attaching labels with each roll.
    2. Four sterilized maize kernels of similar size are placed 4 cm away from the top edge of a double layer of filter papers. Kernels are placed 4 cm apart and leaving 4 cm from left and right edges, covered with another filter paper, then wrapped into rolls, about 5 cm thick. The roll should be kept secure with a rubber band. Two-L capacity glass beakers containing autoclaved Hoagland’s nutrient solution with HN or LN should be filled to one half (about 400 ml), and consequently brown paper rolls should be placed vertically in the beakers, making sure that the seeds are on top and not submerged in the solution. About one half of the length of the rolls should be emerged in the solution. Eight to ten rolls could be placed per beaker. All steps were illustrated in Figure 1A-D.

  3. Growing conditions
    Rolls should be kept in a controlled growth chamber under the following conditions (Figure 1D): a photoperiod of 16/8 h (light/darkness) at 25/22 °C with photosynthetically active radiation of 200 µmol photons m-2 s-1. The relative humidity in the growth chamber should be maintained at 65%. Nutrient solution should be daily added to maintain the solution level in the beakers at 400 ml during the experiment. Seedlings should be kept 14 days in the growth chamber. Thereafter, maize root architecture related traits could be recorded either manually or using image analysis software.

  4. Recording maize root architecture
    After 14 days of incubation of maize kernels grown under HN and LN levels, the nutrient solution should be removed and replaced with about 400 ml of 30% ethanol and the samples should be stored in a cold room, only to be taken out for measuring, scanning, and drying. This is done to prevent further growth in order to preserve the roots and to record root data at the same time point. However, this step is not necessary if all roots can be scanned in one day. For measuring root traits using software, scan the roots and save the images using a flatbed scanner, as much as possible, make sure that roots do not overlap for ease in measurement. Out of the 4 seedlings per entry, 3 that look similar would be measured. Scanning should be done before drying the roots.
    1. Manually recorded traits: the root of individual seedlings should be separated into three parts by a blade, namely, primary root, seminal roots and crown roots (Figure 2). Maize root traits could be recorded by a ruler or gravimetrically. The lengths of primary root, seminal roots and crown roots can be recorded by a ruler. Seminal root number and crown root number can be recorded by counting up the rising roots. Fresh weight of shoots and roots could also be recorded using sensitive balance. Fresh roots are put in glassine paper bags (10 x 20 cm) and oven dried at 80 °C for at least 48 h. Dry weight measurements can be taken after drying using a sensitive balance.


      Figure 2. Maize root system: embryonic roots (primary roots and seminal roots) and postembryonic roots (shoot born crown roots and lateral roots). Crown roots are responsible for the major part of the water and nutrient uptake. All these roots are usually formed below the soil.

    2. Traits recorded by WinRhizo program: total number of root tips, forks, and crossings, total root length, root surface area, root volume and root average diameter could be measured using image analysis software. The software cannot distinguish primary, seminal, crown, or lateral roots by itself. As mentioned in the previous step, the roots have to be divided into primary, seminal, and crown roots; then, specific root measurements can be done. Steps of root imaging analysis using WinRhizo software are presented in Figures 3-17.
      1. Turn the scanner power on, open WinRhizo program and select the scanner that will be used (should be highlighted), then click “Select” (Figure 3).


        Figure 3. Scanner (image source) selection in WinRhizo

      2. The title window for WinRhizo will open. Click “Ok” (Figure 4).


        Figure 4. WinRhizo Startup page. Click “Ok” to continue.

      3. Scanning and saving root images
        1. Place up to 3 roots on the scanner. The root system can be entirely scanned or separate scans of dissected root parts could be performed in the case of a very dense and compacted root system. Make sure that the roots from one plant are not intertwined with those of other plants, as this affects the analysis.
        2. From the main tab, click “Image”, then click “Acquire Image” (Figure 5).


          Figure 5. Image acquisition in WinRhizo. After clicking on “Image>Acquire Image,” scanning of roots commences.

        3. The root images will show on the screen once scanning is done. Check the images (Figure 6).


          Figure 6. Scanned roots image preview from WinRhizo software

        4. Save the scanned image by clicking “Image,” then “Save Displayed Image”.
      4. Analyzing scanned root images
        1. Make a new file where the root parameter data will be stored. From the main tab, click “Data”, then click “New File.” If you have previously started the analysis, then select “Open file” (Figure 7).


          Figure 7. Creating/opening a file where the root parameter data will be saved. If analysis is done for the first time in a particular experiment, select “Image>New File…” If analysis for the same experiment has started. Select “Image>Open File…”.

        2. Select the location where the file will be saved. It is advisable to save the file in the folder where the images are. Type the desired file name, then click “Save.” The data file will be in text format (.txt) (Figure 8).


          Figure 8. Selecting the directory/folder where the data file will be saved

        3. To get the scanned images, click “Image”, the “Origin.” A new window (“Image Origin”) will appear. Select Disk (Figure 9).


          Figure 9. Selecting the source of the root images for analysis (Click on “Image>Origin”)

        4. To acquire the saved images, click on the floppy disk icon on the upper left side of the screen (Figure 10).


          Figure 10. Acquiring the previously scanned images for analysis. Click on the floppy disk icon.

        5. Click on the image to be analyzed, then click “Open” (Figure 11).


          Figure 11. Selecting the image to be analyzed by clicking on the file thumbnail

        6. Zoom the image out so that the whole image can be seen on the screen. In this example, the image was zoomed out to 1/8th of its original size (Figure 12).
          1)     Original size (Figure 12A)
          2)     Zoomed-out image (Figure 12B)


          Figure 12. Image of scanned roots for analysis. A. Original size; B. Image zoomed out to 1/8th of its size to show all roots.

        7. Select the root to be analyzed by first clicking either: 1). Rectangular selection or; 2). Free form selection for closely-spaced roots (Figure 13).


          Figure 13. Selecting individual roots for analysis. 1). Rectangular selection; 2). Free form selection.

        8. A window will appear. Label the root beside “Identification” and write your name as “Operator” Then click “Ok” (Figure 14).


          Figure 14. Labeling the individual roots

        9. Repeat steps D2 vii-viii until all the roots are analyzed (Figure 15).


          Figure 15. End of root image analysis, indicated by green outlines and labels at the upper left side for each root

        10. The output file looks like this: It is in text (*.txt) format; open the file using MS Excel and save as Excel spreadsheet to be able to organize (sort, filter) and make calculations (e.g., average) with the data. Root morphological data from individual plants can be found in the following columns (Figure 16):
          Length (cm) (Column 16/P) – Total root length (cm)
          ProjArea (cm2) (Column 18/R) – Total root projected area (cm2)
          SurfArea (cm2) (Column 20/T) – Total root surface area (cm2)
          AvgDiam (mm) (Column 22/V) – Average root diameter (mm)
          LenPerVol (cm/m3) (Column 24/X) – Total root length per cubic meter of soil (cm/m3)
          RootVolume (cm3) (Column 26/Z) – Total root volume
          Tips (Column 29/AC) – Number of tips
          Forks (Column 30/AD) – Number of forks
          Crossings (Column 31/AE) – Number of crossings


          Figure 16. Sample output file (in *.txt format) from root imaging analysis using WinRhizo software (step D2x)

    3. Traits recorded by ARIA program: steps of root imaging analysis using ARIA program are presented in Figure 17. The measurements are done by the software all at once. After loading the images and clicking on the primary root on the image (the same can be done for other roots), the software will start measuring all traits at the same time. The following traits and their corresponding description can be measured using ARIA (Pace et al., 2014).
      1. Total root length (TRL) – Cumulative length of all the roots in centimeters
      2. Primary root length (PRL) – Length of the primary root in centimeters
      3. Secondary root length (SEL) – Cumulative length of all secondary roots in centimeters
      4. Center of mass (COM) – Center of gravity of the root.
      5. Center of point (COP) – Absolute center of the root regardless of root length.
      6. Center of mass (Top) (CMT) – Center of gravity of the top 1/3 of the root (Top).
      7. Center of mass (Mid) (CMM) – Center of gravity of the middle 1/3 root (Middle).
      8. Center of mass (Bottom) (CMB) – Center of gravity of the bottom 1/3 root (Bottom).
      9. Center of point (Top) (CPT) – Absolute center of the root regardless of root length (Top).
      10. Center of point (Mid) (CPM) – Absolute center of the root regardless of root length (Middle).
      11. Center of point (Bottom) (CPB) – Absolute center of the root regardless of root length (Bottom).
      12. Maximum number of roots (MNR) – The 84th percentile value of the sum of every row.
      13. Perimeter (PER) – Total number of network pixels connected to a background pixel.
      14. Depth (DEP) – The maximum vertical distance reached by the root system.
      15. Width (WID) – The maximum horizontal width of the whole RSA.
      16. Width/Depth ratio (WDR) – The ratio of the maximum width to depth.
      17. Median (MED) – The median number of roots at all Y-location.
      18. Total number of roots (TNR) – Total number of roots.
      19. Convex area (CVA) – The area of the convex hull that encloses the entire root image
      20. Network area (NWA) – The number of pixels that are connected in the skeletonized image
      21. Solidity (SOL) – The fraction equal to the network area divided by the convex area
      22. Bushiness (BSH) – The ratio of the maximum to the median number of roots.
      23. Length distribution (LED) – The ratio of TRL in the upper one-third of the root to the TRL.
      24. Diameter (DIA) – Diameter of the primary root.
      25. Volume (VOL) – Volume of the primary root
      26. Surface area (SUA) – Surface area of the primary root.
      27. SRL – Total root length divided by root system volume


        Figure 17. Root image analysis using ARIA software (step D3) 

Representative data

Representative data showing seedling growth of genotypes PHZ51, B73 and Mo17 under LN and HN levels are shown in Figure 18. Maize genotypes responded to N deficiency by increasing the root:shoot (R:S) ratio. Lines presented in Figure 18 displayed a higher R:S ratio under LN as compared with HN treatment. To absorb sufficient amount of N under LN, maize plants adapt to N starvation by increasing the root volume and decreasing the aerial vegetative growth.


Figure 18. Performance of three maize genotypes (PHZ51, B73 and Mo17) under low and high nitrogen levels

Recipes

  1. Hoagland’s nutrient solution
    Note: This is made essentially according to Hoagland and Arnon (1950). This solution is used to culture plants in hydroponic medium.
    1. High N (15 mM NO3-) Hoagland’s solution
      5 ml of 1 M (M = Molar) potassium nitrate
      5 ml of 1 M calcium nitrate
      1 ml of 1 M monopotassium phosphate
      2 ml of 1 M magnesium sulfate
      1 ml of micronutrient stock solution (see Recipe 1c below)
      5 ml of 1,000 mg/L iron from iron chelate (Fe-EDTA, Fe-DTPA, or Fe-EDDHA)
      Add ddH2O to complete the volume to 1 L
    2. Low N (1.5 mM NO3-) Hoagland’s solution
      10 ml of 0.05 M monocalcium phosphate
      200 ml of 0.01 M calcium sulfate dihydrate
      5 ml of 0.5 M potassium sulfate
      2 ml of 1 M magnesium sulfate
      1 ml of micronutrient stock solution (see Recipe 1c below)
      5 ml of iron chelate stock solution as for HN
      Add ddH2O to complete the volume to 1 L
    3. Micronutrient stock solution (1 L)
      2.86 g boric acid
      1.81 g manganese chloride-4 hydrate
      0.22 g zinc sulfate-7 hydrate
      0.08 g copper sulfate-5 hydrate
      0.02 g 85% molybdic acid
      Add autoclaved ddH2O to complete the volume to 1 L
  2. 30% ethanol (1 L)
    Add gradually 300 ml ethanol in 500 ml of ddH2O by stirring
    Add ddH2O until final volume is 1,000 ml
    Store at 4 °C
  3. 2.5 g/L Fungicide solution Captan®
    Dissovle 25 g of Captan® in 500 ml of ddH2O by stirring
    Add ddH2O until final volume is 1,000 ml
    Store at room temperature

Acknowledgments

This protocol was developed while Dr. Adel Abdel-Ghani was a visiting Fulbright Postdoctoral Fellow and during the sabbatical leave granted from Mu’tah University, Jordan during the academic year 2011-2012 at Iowa State University (ISU), Ames, USA. Authors would also like to thank USDA’s National Institute of Food and Agriculture (project number: IOW05180) and RF Baker Center for Plant Breeding for funding this work.

References

  1. Abdel-Ghani, A. H., Kumar, B., Pace, J., Jansen, C., Gonzalez-Portilla, P. J., Reyes-Matamoros, J., San Martin, J. P., Lee, M. and Lubberstedt, T. (2015). Association analysis of genes involved in maize (Zea mays L.) root development with seedling and agronomic traits under contrasting nitrogen levels. Plant Mol Biol 88(1-2): 133-147.
  2. Abdel-Ghani, A. H., Kumar, B., Reyes-Matamoros, J., Gonzalez-Portilla, P. J., Jansen, C., San Martin, J. P., Lee, M. and Lubberstedt, T. (2013). Genotypic variation and relationships between seedling and adult plant traits in maize (Zea mays L.) inbred lines grown under contrasting nitrogen levels. Euphytica 189(1): 123-133.
  3. Arana, M. V., Sanchez-Lamas, M., Strasser, B., Ibarra, S. E., Cerdan, P. D., Botto, J. F. and Sanchez, R. A. (2014). Functional diversity of phytochrome family in the control of light and gibberellin-mediated germination in Arabidopsis. Plant Cell Environ 37(9): 2014-2023.
  4. Hoagland, D. R. and Arnon, D. I. (1950). The water-culture method for growing plants without soil. Calif Agric Exp Stn Circ 347:1-32.
  5. Kumar, B., Abdel-Ghani, A. H., Pace, J., Reyes-Matamoros, J., Hochholdinger, F. and Lubberstedt, T. (2014). Association analysis of single nucleotide polymorphisms in candidate genes with root traits in maize (Zea mays L.) seedlings. Plant Sci 224: 9-19.
  6. Kumar, B., Abdel-Ghani, A. H., Reyes-Matamoros, J., Hochholdinger, F. and Lubberstedt, T. (2012). Genotypic variation for root architecture traits in seedlings of maize (Zea mays L.) inbred lines. Plant Breeding 131(4): 465-478.
  7. Pace, J., Gardner, C., Romay, C., Ganapathysubramanian, B. and Lubberstedt, T. (2015). Genome-wide association analysis of seedling root development in maize (Zea mays L.). BMC Genomics 16: 47.
  8. Pace, J., Lee, N., Naik, H. S., Ganapathysubramanian, B. and Lubberstedt, T. (2014). Analysis of maize (Zea mays L.) seedling roots with the high-throughput image analysis tool ARIA (Automatic Root Image Analysis). PLoS One 9(9): e108255.
  9. Tressler, W. L. and Williams, T. (1937). Hydroponics solution used for daphnia culture. Science 86(2229): 273-274.
  10. Tuberosa, R. and Salvi, S. (2007). From QTLs to genes controlling root traits in maize. Scale and Complexity in Plant Systems Research: Gene-Plant-Crop Relations 21: 15-24.

简介

对象上下文(OIC)任务是广泛使用的对象识别(OR)任务的变体(Dix和Aggleton,1999)。 OIC任务利用啮齿动物具有探索新环境和物体的自然倾向的事实。海马似乎在OIC任务中发挥重要作用(比原始OR任务更重要),其中动物应该能够区分两个熟悉的对象,其中一个在与训练试验不同的背景下(Ennaceur和Aggleton,1997; Bermudez-Rattoni等人,2005; Albasser等人,2009; Roozendaal等人, 2010; Banks等人,2014; Bermudez-Rattoni,2014)。识别存储器包括多个附加组件,诸如项目与其上下文,地点等的关联。 (Bussey ,1999,2000)。在这里,我们基于早期的报告(Dix和Aggleton,1999; Eacott和Norman,2004; Balderas等人,2008; Barsegyan等人)描述了小鼠中OIC任务的一个版本。,2014; Kanatsou 等人,2015a; Kanatsou 等人,2015b)。...

材料和试剂

  1. 常规重量(棕色)发芽纸48.5"×36.5",定制尺寸为12"×24"(Anchor Paper Company,目录号:SD3836S)
  2. 小厨房丝网过滤器/带手柄的筛(直径20厘米)
  3. 小的塑料杯,测量船(Fisherbrand TM六角形聚苯乙烯称量皿)(Thermo Fisher Scient,Fisher Scientific,目录号:02-202-101)或用于干燥种子的双面滤纸 与条目数
    相同的数字
  4. 防水铅笔艺术握持水彩画黑色(Faber-Castell,目录号:114299)和永久标记(Sharpie ,Fine Point Permanent Marker,黑色)
    注意:黑标记效果更好,因为其他颜色褪色更快。
  5. 塑料标签(5"x 5/8")(国际温室,目录号:CN-1000)用于标签(如果卷标有标签,则可选)
  6. 橡胶带(OfficeMax Extra Long橡皮筋或任何其他品牌)
  7. 玻璃纸袋(Seedburo S411拍摄袋,处理,4"×2-1/2"×11")
  8. 个人防护用品:乳胶手套,实验服,闭合鞋,口罩护目镜
  9. 玉米种子:本方案中使用的基因型是从位于爱荷华州阿姆斯市的北部中央区域植物介绍站获得的纯品系(Abdel-Ghani等人,2013)。
    注意:
    1. 在相同条件下种子应该被繁殖以避免由于环境对种子大小的差异。
    2. 种子应该显示高的萌发百分比,以在实验单位内保持相似数量的生物复制。
  10. Chlorox ®溶液(6%次氯酸钠),家用漂白剂(美国)
  11. 去离子无菌蒸馏水(ddH 2 O)
  12. 硝酸钾(KNO 3)(Thermo Fisher Scientific,Fisher Scientific,目录号:P-263-500)
  13. 硝酸钙[Ca(NO 3)2](Thermo Fisher Scientific,Fisher Scientific,目录号:C109-3)
  14. 磷酸二氢钾或磷酸二氢钾(KH 2 PO 4)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP-362-500)
  15. 硫酸镁(Thermo Fisher Scientific,Fisher Scientific,目录号:M65-500)
  16. 来自铁螯合物的铁[Fe-EDTA(Sigma-Aldrich,目录号:E6760-100G),Fe-DTPA或Fe-EDDHA]
  17. 磷酸一钙或磷酸二氢钙[Ca(H 2 PO 4)2](MP Biomedicals,目录号:193803)
  18. 硫酸钙二水合物(CaSO 4·2H 2 O)(MP Biomedicals,目录号:191414)
  19. 硫酸钾(K 2 SO 4)(Sigma-Aldrich,目录号:P0772-1kg)
  20. 硼酸(Thermo Fisher Scientific,Fisher Scientific,目录号:BP168-1)
  21. 氯化锰-4水合物(MnCl 2·4H 2 O)(Sigma-Aldrich,目录号:221279-100g)
  22. 硫酸锌-7水合物(ZnSO 4·7H 2 O)(Sigma-Aldrich,目录号:Z0251-100G)
  23. 硫酸铜-5水合物(Thermo Fisher Scientific,Fisher Science,目录号:S25287A)
  24. 钼酸(H 2 MoO 4)(Sigma-Aldrich,目录号:232084-100G)
  25. Hoagland的营养液
    1. Hoagland的解决方案(见配方),高N(15mM NO 3 - )
    2. Hoogland解决方案(见配方)低N(1.5mM NO 3)
    3. 微量营养素储备溶液(1 L)(参见配方)
  26. 30%乙醇(C 2 H 6 O)(任何品牌的商品级)(参见配方)
  27. 2.5g/L杀菌剂溶液Captan (Bonide Products Inc.)(参见配方)

设备

  1. 2L容量烧杯(每个烧杯装有8-10个纸卷)(Coring,Pyrex Griffin Beakers,目录号:10000-2L)。
  2. 用于灭菌和洗涤种子(Sigma-Aldrich,目录号:CLS100050)的容量为50ml的烧杯(Pyrex?,Griffin Beakers)
  3. 植物生长室(Conviron,型号:PGC FLEX)
  4. 冷室(Rheem Puffer Hubbard环境室)或冰箱
  5. 高压釜(PRIMUS Strerilizer,型号:PSS5)
  6. 敏感平衡(Ohaus,型号:Adventurer TM AR0640)
    注意:此产品已停产。
  7. 平板扫描仪(Epson,型号:Expression 10000 XL或任何其他品牌)
  8. 带闪存驱动器和Windows操作系统的计算机
  9. 通用加热和干燥箱(Thermo Fisher Scientific,Fisher Scientific,型号:15-103-0503)或任何恒温烘箱/干燥器
    的Fisher Scientific TM />
  10. 尺子或尺子(Acme Westcott 15728 36"铝杖棒")

软件

  1. WinRhizo(Regent Instruments,型号: WinRhizo Pro 2009 )或 ARIA(自动根图像分析)(Pace et al 。,2014)

程序

该实验设计用于在对比的氮(N)水平下测试玉米幼苗的性能(Abdel-Ghani等人,2013)。幼苗应暴露于具有高N(HN)和低N(LN)的Hoagland营养液(Hershey,1994)。在具有HN的Hoagland氏溶液中的氮含有15mM的NO 3+,而LN Hoagland's溶液中的N浓度为1.5mM(10%NO 3 - sub> - )(Abdel-Ghani等人,2013; Abdel-Ghani等人,2015)。其他宏观和微观元素在营养处理中应该是恒定的。关于纸卷准备和培养的所有步骤如下(还参见图1;步骤A至D)

图1.纸卷准备和培养步骤的总结 A.将玉米用6%次氯酸钠进行表面灭菌,用蒸馏水洗涤并干燥。将四个相似大小的灭菌的玉米籽粒放置在用杀真菌溶液Captan预湿润的双层棕色滤纸上。 B.将卷发芽纸保存在含有具有高氮(HN)和低氮(LN)的Hoagland营养液的2L玻璃烧杯中。 C.卷须在受控生长室中保存14天。 D.在受控生长室中在LN和HN处理下培养14天后的幼苗

  1. 玉米籽粒灭菌
    1. 在室温下,将核在20ml烧杯中用Chlorox溶液(6%次氯酸钠)表面灭菌15分钟。 Chlorox应该覆盖烧杯中的内核,烧杯应该手动摇动3-4次
    2. 首先应该排出氯氧化物,然后将种子用ddH 2 O 3洗涤三次。小的无菌筛(直径20cm)可以在每次洗涤后用于排出水。
    3. 洗涤后,应将玉米放在双面棕色滤纸上,放置10分钟,直至种子干燥。小塑料杯或测量船也可用于干燥种子
  2. 在纸卷中种植种子
    1. 将棕色发芽纸切成20×20cm的片,并用杀真菌溶液Captan(2.5g/L)预湿润以消除任何真菌发展的可能性 在幼苗发育期间。棕色纸应该用杀真菌溶液浸湿,将纸浸泡在溶液中。应通过用手压在浸泡的纸上除去过量的杀真菌剂溶液。纸卷应该用永久(防水)铅笔标记,直接在棕色纸上书写和/或在每卷上贴上标签。
    2. 将相似大小的四个灭菌的玉米籽粒置于离双层滤纸的顶部边缘4cm处。将核放置4cm,离开左边缘和右边缘4cm,用另一滤纸覆盖,然后包裹成约5cm厚的卷。卷筒应该用橡胶带固定。含有高压灭菌的Hoagland营养液(含有HN或LN)的两升容量玻璃烧杯应当填充至一半(约400ml),因此棕色纸卷应该垂直放置在烧杯中,确保种子在顶部,而不是浸没在溶液中。大约一半的辊长度应该在溶液中出现。每个烧杯可放置八至十个卷。所有步骤如图1A-D所示
  3. 生长条件
    辊应该保持在受控的生长室中在以下条件下(图1D):在25/22℃下16/8小时(光/暗)的光周期,具有200μmol光子m -2的光合有效辐射 s -1 。生长室中的相对湿度应保持在65%。应该每天添加营养溶液,以在实验期间将烧杯中的溶液水平维持在400ml。幼苗应在生长室中保持14天。此后,可以手动或使用图像分析软件记录玉米根系结构相关性状
  4. 记录玉米根构造
    在HN和LN水平下生长的玉米籽粒孵育14天后,应去除营养溶液并用约400ml的30%乙醇代替,并且样品应储存在冷室中,仅取出用于测量,扫描和干燥。这样做是为了防止进一步生长以保留根并在相同的时间点记录根数据。但是,如果可以在一天内扫描所有根,则不需要此步骤。为了使用软件测量根性状,使用平板扫描仪扫描根并尽可能地保存图像,确保根不重叠,以便于测量。在每个条目的4个幼苗中,测量看起来相似的3个。在干燥根之前应进行扫描。
    1. 手工记录的性状:个体幼苗的根应该用刀片分为三部分,即主根,根根和冠根(图2)。玉米根性状可以通过直尺或重量分析记录。主根,根根和冠根的长度可以用尺子记录。半数根数和冠根数可以通过计数上升根来记录。还可以使用敏感平衡记录嫩芽和根的新鲜重量。将新鲜根置于玻璃纸袋(10×20cm)中,并在80℃下烘干至少48小时。干燥后可以使用敏感的天平测量干重。


      图2.玉米根系:胚根(初级根和精根)和胚后根(茎生根冠根和侧根)。冠根负责水和营养物摄取的主要部分。所有这些根都通常在土壤下形成
    2. 由WinRhizo程序记录的性状:根尖,叉和杂交的总数,总根长度,根表面积,根体积和根平均直径可使用图像分析软件测量。软件不能自己区分主,根,冠或侧根。如上一步所述,根必须分为原,根和冠根;然后,可以进行特定根测量。使用WinRhizo软件进行根成像分析的步骤如图3-17所示。
      1. 打开扫描仪电源,打开WinRhizo程序并选择要使用的扫描仪(应突出显示),然后单击"选择"(图3)。


        图3. WinRhizo中的扫描仪(图像源)选择

      2. WinRhizo的标题窗口将打开。单击"确定"(图4)。


        图4. WinRhizo启动页。单击"确定"继续。

      3. 扫描和保存根图像
        1. 在扫描仪上放置最多3根。根系统可以被完全扫描,或者在非常密集和紧凑的根系统的情况下可以执行解剖根部部分的单独扫描。确保来自一个植物的根与其他植物的根不相互缠绕,因为这影响分析。
        2. 从主选项卡,单击"图像",然后单击"获取图像"(图5)

          图5. WinRhizo中的图像采集。点击"图像>采集图像"后,扫描根目录。

        3. 扫描完成后,根图像将显示在屏幕上。检查图像(图6)。


          图6.从WinRhizo软件扫描的根图像预览

        4. 通过单击"图像",然后单击"保存显示的图像"保存扫描的图像。
      4. 分析扫描的根图像
        1. 创建一个新文件,存储根参数数据。 从主选项卡,单击"数据",然后单击"新文件"。如果您以前开始分析,则选择"打开文件"(图7)。

          图7.创建/打开保存根参数数据的文件如果特定实验中是第一次进行分析,请选择"图像>新文件..." 实验已经开始。 选择"图像>打开文件..."。

        2. 选择保存文件的位置。建议将文件保存在图像所在的文件夹中。键入所需的文件名,然后单击"保存"。数据文件将是文本格式(.txt)(图8)。


          图8.选择保存数据文件的目录/文件夹

        3. 要获取扫描图像,请单击"图像","原点"。将出现一个新窗口("图像原点")。选择磁盘(图9)。


          图9.选择要分析的根图片的来源(点击"图片>原产地")

        4. 要获取保存的图像,请单击屏幕左上方的软盘图标(图10)。


          图10.获取先前扫描的图像进行分析。单击软盘图标。

        5. 单击要分析的图像,然后单击"打开"(图11)。


          图11.通过点击文件缩略图
          选择要分析的图像
        6. 缩放图像,以便可以在屏幕上看到整个图像。在此示例中,图像缩小到原始大小的1/8 th (图12)。
          1)     原始大小(图12A)
          2)     缩小图像(图12B)


          图12.用于分析的扫描根的图像 A.原始大小; B.图像缩小到其大小的1/8 th 以显示所有根
        7. 选择要分析的根,首先单击:1)。矩形选择或; 2)。自由形式选择紧密间隔的根(图13)。


          图13.选择单个根进行分析 1)。矩形选择; 2)。自由形式选择。

        8. 将出现一个窗口。在"识别"旁边标记根,并将您的名称写为"操作员",然后单击"确定"(图14)

          图14.标记个别根

        9. 重复步骤D2 vii-viii,直到分析所有根(图15)。


          图15.根图像分析结束,由每个根的左上侧的绿色轮廓和标签指示

        10. 输出文件如下所示:它是文本(* .txt)格式;使用MS Excel打开文件,并保存为Excel电子表格,以便能够组织(排序,过滤)和进行计算(例如。,平均)。来自单个植物的根形态数据可以在以下列中找到(图16):
          长度(cm)(柱16/P) - 根长度总和(cm)
          ProjArea(cm 2 )(栏18/R) - 总根投影面积(cm 2 )
          SurfArea(cm 2 )(栏20/T) - 总根表面积(cm 2 )
          AvgDiam(mm)(栏22/V) - 平均根直径(mm)
          LenPerVol(cm/m )(栏24/X) - 每立方米土壤的根部总长度(cm/m <3) 根体积(cm <3> )(第26/Z栏) - 总根体积
          提示(第29栏/AC) - 提示数量
          叉(列30/AD) - 货叉数量
          交叉(列31/AE) - 交叉数


          图16.使用WinRhizo软件进行根成像分析的示例输出文件(* .txt格式)(步骤D2x)

    3. 由ARIA程序记录的性状:使用ARIA程序的根成像分析的步骤在图17中示出。通过软件一次进行测量。 加载图像并单击图像上的主根(对于其他根也可以这样做),软件将开始同时测量所有性状。 以下性状及其相应描述可以使用ARIA来测量(Pace等人,2014)。
      1. 总根长(TRL) - 所有根的累积长度,单位为厘米
      2. 主根长度(PRL) - 主根长度,单位为厘米
      3. 次根长度(SEL) - 所有次根的累积长度,单位为厘米
      4. 质心(COM) - 根部的重心。
      5. 中心点(COP) - 根的绝对中心,与根长度无关。
      6. 质心(顶部)(CMT) - 根部顶部1/3(顶部)的重心。
      7. 质心(中)(CMM) - 中间1/3根(中间)的重心。
      8. 质心(底部)(CMB) - 底部1/3根部(底部)的重心。
      9. 中心点(顶部)(CPT) - 根的绝对中心,无论根长(顶部)。
      10. 中心点(中间)(CPM) - 根的绝对中心,与根长度无关(中间)。
      11. 中心点(底部)(CPB) - 根的绝对中心,而不管根长度(底部)。
      12. 最大根数(MNR) - 每行总和的第84百分位数值。
      13. 周长(PER) - 连接到背景像素的网络像素总数。
      14. 深度(DEP) - 根系统达到的最大垂直距离。
      15. 宽度(WID) - 整个RSA的最大水平宽度
      16. 宽度/深度比(WDR) - 最大宽度与深度的比率。
      17. 中值(MED) - 在所有Y位置的根的中值数
      18. 根总数(TNR) - 根总数。
      19. 凸面积(CVA) - 包围整个根图像的凸包区域
      20. 网络区域(NWA) - 在骨架化图像中连接的像素数
      21. Solidity(SOL) - 等于网络面积除以凸区域的分数
      22. Bushiness(BSH) - 最大值与根的中位数的比值。
      23. 长度分布(LED) - 根的上三分之一处的TRL与TRL的比率
      24. 直径(DIA) - 主根的直径。
      25. 体积(VOL) - 主根体积
      26. 表面积(SUA) - 主根的表面积。
      27. SRL - 根长度总和除以根系统量





        图17.使用ARIA软件(步骤D3)的根图像分析

代表数据

在LN和HN水平下显示基因型PHZ51,B73和Mo17的幼苗生长的代表性数据显示于图18中。玉米基因型通过增加根:苗(R:S)比率对N缺乏有反应。 与HN处理相比,图18中显示的线显示LN下的更高的R:S比。 为了在LN下吸收足够量的N,玉米植物通过增加根体积和减少空中营养生长来适应N饥饿。


图18.在低和高氮水平下的三种玉米基因型(PHZ51,B73和Mo17)的性能

食谱

  1. Hoagland的营养液
    注意:这基本上根据Hoagland和Arnon(1950)。 该溶液用于在水培培养基中培养植物。
    1. 高N(15mM NO <3> - )Hoagland解决方案
      5ml 1M(M =摩尔)硝酸钾 5ml的1M硝酸钙
      1ml 1M磷酸二氢钠
      加入2ml 1M硫酸镁 1毫升微量营养素储备溶液(见下面的配方1c) 5毫升来自铁螯合物(Fe-EDTA,Fe-DTPA或Fe-EDDHA)的1,000毫克/升铁
      添加ddH 2 O即可完成体积为1 L
    2. 低N(1.5mM NO <3> - )Hoagland's溶液
      10ml 0.05M磷酸二氢钙
      200ml 0.01M硫酸钙二水合物 5ml 0.5M硫酸钾 加入2ml 1M硫酸镁 1毫升微量营养素储备溶液(见下面的配方1c) 5ml HN螯合物储备溶液 添加ddH 2 O即可完成体积为1 L
    3. 微量营养素储备溶液(1 L)
      2.86g硼酸 1.81克氯化锰-4水合物 0.22克硫酸锌-7水合物 0.08克硫酸铜-5水合物 0.02g 85%钼酸 加入高压灭菌的ddH 2 O,使体积达到1 L
  2. 30%乙醇(1L) 通过搅拌加入在500ml ddH 2 O中逐渐加入300ml乙醇 添加ddH 2 O直到最终体积为1000ml
    存储在4°C
  3. 2.5 g/L杀菌剂溶液Captan ®
    通过搅拌,在500ml ddH 2 O中溶解25g Captan 添加ddH 2 O直到最终体积为1000ml
    在室温下贮存

致谢

该协议开发时,Adel Abdel-Ghani博士是访问富布莱特博士后研究员,并在约翰Mu'tah大学在2011-2012学年期间在爱荷华州立大学(ISU),美国阿姆斯,美国休假期间休假。 作者还要感谢美国农业部国家粮食和农业研究所(项目编号:IOW05180)和RF Baker植物育种中心为这项工作提供资金。

参考文献

  1. Abdel-Ghani,AH,Kumar,B.,Pace,J.,Jansen,C.,Gonzalez-Portilla,PJ,Reyes-Matamoros,J.,San Martin,JP,Lee,M.and Lubberstedt, )。  涉及玉米的基因的关联分析( Zea mays L.)根发育与在相反氮水平下的幼苗和农艺性状。植物分子生物学88(1-2):133-147。 >
  2. Abdel-Ghani,AH,Kumar,B.,Reyes-Matamoros,J.,Gonzalez-Portilla,PJ,Jansen,C.,San Martin,JP,Lee,M.and Lubberstedt,T。(2013) a class ="ke-insertfile"href ="http://link.springer.com/article/10.1007/s10681-012-0759-0"target ="_ blank">幼苗和成年植物性状之间的基因型差异和关系玉米( Zea mays L.)在对比氮水平下生长的近交系。 Euphytica 189(1):123-133。
  3. Aeroa,MV,Sanchez-Lamas,M.,Strasser,B.,Ibarra,SE,Cerdan,PD,Botto,JF and Sanchez,RA(2014)。  植物色素家族在拟南芥中控制光和赤霉素介导的萌发的功能多样性 a> Plant Cell Environ 37(9):2014-2023。
  4. Hoagland,DR和Arnon,DI(1950)。  "文化 生长植物无土壤的方法。 Calif Agric Exp Stn Circ 347:1-32。
  5. Kumar,B.,Abdel-Ghani,AH,Pace,J.,Reyes-Matamoros,J.,Hochholdinger,F。和Lubberstedt,T(2014)。  在玉米中具有根性状的候选基因中的单核苷酸多态性的关联分析( Zea mays )幼苗。 植物科学 224:9-19
  6. Kumar,B.,Abdel-Ghani,AH,Reyes-Matamoros,J.,Hochholdinger,F。和Lubberstedt,T。(2012)。  L.)近交系。 植物育种 131(4):465-478。
  7. Pace,J.,Gardner,C.,Romay,C.,Ganapathysubramanian,B.和Lubberstedt,T。(2015)。  玉米中幼苗根发育的全基因组关联分析( Zea mays L.)。 BMC Genomics 16:47.
  8. Pace,J.,Lee,N.,Naik,HS,Ganapathysubramanian,B。和Lubberstedt,T。(2014)。  用于水蚤培养的水培溶液。 Science 86(2229):273-274。
  9. Tuberosa,R。和Salvi,S。(2007)。  从QTL到 在植物系统研究中的规模和复杂性:基因 - 植物 - 作物关系21:15-24。
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引用:Abdel-Ghani, A. H., Sanchez, D. L., Kumar, B. and Lubberstedt, T. (2016). Paper Roll Culture and Assessment of Maize Root Parameters. Bio-protocol 6(18): e1926. DOI: 10.21769/BioProtoc.1926.
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