This protocol improves considerably the contrast between the tissues of the parasitic plant and the host in samples that will be analyzed using microtomography. The technique described here speeds up the process of contrast perfusion through the sample, allowing the analysis of specific tissues and structures formed between the parasitic plant and the host. The vacuum approach is very straightforward and should offer no difficulties.
The perfusion apparatus, on the other hand, might be a bit challenging to set up. But practice beforehand could help. For small non-woody samples, the vacuum method can be used for application.
Whereas the profusion method should be used for samples that are larger and woody, including a segment of the host stem or root. If using the vacuum method, place the sample in the vial containing the contrasting solution. Then, place the vial in a vacuum chamber or desiccator connected to a vacuum pump.
Remove the lid from the vial and close the vacuum chamber or desiccator. Check that there are no cracks on the vacuum chamber or desiccator. Open the exhaustion valve of the chamber or desiccator to force the air out.
Turn on the pump and wait until the pressure reaches approximately 20 inches of mercury, or 10 psi. Close the chamber or desiccator exhaust valve to prevent air from reentering. Then quickly turn off the pump.
Leave the sample under vacuum for at least two hours. If using the profusion method, select the supply tank according to the sample size. For small samples, a 50 milliliter syringe without a needle can be used as a tank.
Connect one end of a transparent plastic tubing to the supply tank. Then connect the other end to a two-way or three-way valve. Connect a second tubing to a different outlet in the valve.
Secure the supply tank at an elevated position without disassembling the apparatus set up in the previous step. Close the three-way or two-way valve to prevent liquid from exiting the tubing system and pour the contrasting solution into the supply tank. Ensure there are no large air bubbles in the tubing system and close the valve again, leaving the apparatus in place.
To prepare the sample for profusion of the contrast solution, keep it submerged in liquid and cut off the tip of the proximal end of the host stem or root. Carefully open the valve to allow the contrasting solution to flow slowly and fill the plastic tubing connected to the tank while holding the open end of the system at a slightly elevated position to prevent the contrasting solution from spilling. Connect the proximal end of the host stem or root in the sample to the open end of the tubing system.
Let the solution perfuse the sample for at least two hours or until the solution accumulates inside the container. Close the valve and carefully disconnect the sample from the apparatus. Wash the sample by submerging it in water for two minutes.
Place the sample in a paper towel at room temperature to allow excess water to evaporate for two to five minutes without allowing the sample to dry out completely. Wrap the sample in a paraffin film and avoid folding the film on top of the sample. Micro-CT scanning can be leveraged to better understand the complex structures of parasitic plants and their interaction with hosts in a non-destructive, three-dimensional way.
The protocols described here work for different micro-CT systems. However, the settings and parameters depend on the system and the samples. 3D X-ray microscope images were as effective as anatomical sections observed under a light microscope to analyze tissue organization and topology at the parasite host interface.
Based on the bright white and dark gray color difference due to differential absorption of the contrast solution, it is possible to observe the abundance of parenchyma surrounding the vascular core of the haustorium. The vascular core is easily observed in cross-sections as two vascular strands are separated by parenchyma. The endoparasite Viscum minimum, growing inside a succulent host plant, showed parenchyma cells that store carbohydrates in the form of starch.
The difference in iodine absorption allowed the detection of the intricate web of cortical strands formed by the endoparasite within the host body. Results obtained for Cuscuta Americana and Struthantus marcianus help illustrate the convenience of the microtomography approach for small and larger samples respectively. The virtual serial sectioning Sibelium fungiform showed that the vessels in the host root bifurcate into the parasite tuber, and that the xylem continuity between the two plants is formed by vessel-to-vessel connection via perforation plates.
The procedure described here can be combined with other techniques, such as virtual segmentation, to provide new insights into the three dimensional structure of the connection between parasitic plants and their hosts. This technique has paved the way for analyzing different aspects of the biology of parasitic plants, including the development of endoparasites and the functionality of hyperparasitic connections.
