A New Way to Image Root Structure and Development

A cool new way to discern the effects of different growing conditions and/or genetic variations on a plant’s root structure has been discussed and tested in a recent article in Plant Methods.

This methods paper assesses the use of RhizoTubes, half metre high transparent tubes with a diameter of 18cm. Inside the tubes, pressed close to the outer surface, is a membrane consisting of an 18µm mesh which allows water, nutrients and microorganisms to pass through but stops soil or other growing media and plant roots from penetrating from alternative sides of the mesh. The result is an inner area holding the growing media for which the water content, nutrient content and microorganism content can be controlled and an outer area pressed against the outer tube in which the plant roots grow.

rhizotube-diagram

Figure from article. RhizoTube diagram showing inner and outer tubes and the membrane separating the root propagation area from the growing substrate.

By growing plants in the tubes the roots are forced to grow in a two dimensional matrix which is visible from the outside of the tube.

Plants can be grown in the RhizoTubes in greenhouse environments, allowing experimentation with temperature, humidity and the like and how these environments, interacting with differing genotypes or soil stress scenarios, affect root development and structure. Such insights may play a significant role in agriculture and our ability to predict what species or genetic modifications will be able to thrive in more difficult growing environments.

The RhizoTubes are mounted on roller conveyors and can be easily moved around. Most importantly, the tubes can be moved into an imaging area (named the RhizoCab) where a the root system is illuminated with red, green and blue wavelengths of light. The RhizoTube is rotated through 360 degrees and as it does so a high definition camera photographs the tube in one pixel wide increments. The result is amazing images like these:

rhizotubes

Figure from article. Examples of images (600) taken by RhizoCab of plant cultivated for 51 days (a), Pisum sativum plant (Cameor genotype) cultivated for 18 days with 10 meq soil mineral nitrogen (b) or without soil mineral nitrogen (c), Pisum sativum plant (Kayanne genotype) cultivated for 18 days without soil mineral nitrogen (d). Details of zone where either mycorhize can be seen or nodules (e) easily detected are indicated, with a resolution of 3600 (i.e. a pixel equals 7 µm)

As can be seen, the definition of the images of the root system are high enough for fine root structure and nodulation to be captured.

The aim of the device is to allow non-invasive assessment of root structures, avoiding the need to disturb the soil and roots of a plant under investigation as is required when such experiments are carried out in pots. The question the researchers were testing in this paper was whether the RhizoTubes distort normal growing patterns compared to growing the plants in a pot. Too much distortion and that results of experiments using the RhiztoTubes are unreliable.

Comparing RhizoTube growth with pot growth

Six experiments in total were performed with each experiment replicated in both RhizoTubes and pots. Set indicators were used in each experiment to compare the root growth between the two. The six experiments were:

  1. To test whether grapevine cuttings could survive and flourish when grown in RhizoTubes;
  2. To characterise root systems of pea roots and their nodulation between RhizoTubes and pots;
  3. To assess the effects of varying nitrogen availabilities on roots of different pea plants;
  4. To assess the differing responses to nitrogen availability on the crop Brassica napus and the weed Vulpia myuros;
  5. To assess what effect water deficit would have on pea roots and Medicago truncatula grown in the two containers; and
  6. To assess whether the presence of the membrane has any effect on the persistence of Psuedomonas fluorescences C7R12 on pea and wheat plants grown in RhizoTubes and whether the bacteria could be completely removed from the surface of the tubes after the experiment.

The results

In short, the differences between plants grown in the RhizoTubes to those grown in the pots were quite limited, but a couple of adjustments need to be made to ensure a like-for-like comparison in future experiments.

  1. The grapevine cuttings were able to develop in the RhizoTubes with comparable biomass ratios between the RhizoTube grown plantlets and those grown in pots.
  2. Pea root nodulation didn’t occur in either pots or the RhizoTubes when high levels of nitrogen were supplied and there was no difference in dry matter and root matter between the two containers. There was a difference in root length between the two containers, with the main roots from plants grown in the RhizoTube being 47% longer  and the number and length of first order roots emanating from the main root started decreasing further down the main root compared to that of the plants grown in pots. The second order root characteristics were comparable between the two containers.
  3. Adjusting nitrogen levels led to the nodulation of pea roots on both the main root and the lateral roots in both containers with Kayanne plants nodulating about 6cm higher in the RhizoTubes compared to the pots.
  4. Both the Brassica and Vuplia species tested developed a mess of thin, entangled roots which stopped the researchers from manually performing the root system architecture measurements. But it didn’t stop the RhizoCab taking some detailed pictures of the root structure. Although there were expected differences between the responses to nitrogen between the two plants, there was no significant difference between the growth of either plant in the differing containers.
  5. Performing a water stress experiment required a bit of fine tuning in the RhizoTube. Peas grown in RhizoTubes had lower (although only slightly lower) shoot and root growth although the same issue wasn’t found for Medicago. The authors hypothesise that although the RhizoTubes are suited to water stress testing of plants, fine tuning may be needed in how much and when water is provided in the tubes compared to the pots to ensure that a similar water level is attained. The same can also be said of nutrient provision in the tubes.
  6. The P. fluorescens was able to stably remain in the root system in the RhizoTubes and were the tubes were able to be disinfected of the bacteria at the end of the experiment.

The future

The RhizoTubesin combination with the RhizoCab imaging system appear to provide researchers the ability to monitor root differences between test subjects. As agricultural innovation continues to look for new ways to assist nutrient acquisition efficiency and adaptability to harsher growing environments, the ability to identify the phenotypic variation between genetically differing crops, and doing so with higher throughput than what exists at present, will certainly increase the rate of our knowledge gathering and application.

The main inhibitory issue remains the ability to phenotype roots of field plants, as opposed to pot plants. Perhaps the next iteration of this technology will be the capturing of 3D images in a similar device to that developed here.

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