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Environment

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Published: February 15th, 2021

DOI:

10.3791/62227

1Department of Plant and Soil Sciences, University of Delaware, 2National Synchrotron Radiation Lightsource-II, Brookhaven National Laboratory

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

Roots and their rhizospheres are dynamic, heterogeneous, and critically important for understanding how plants obtain mineral nutrients and contaminants1,2,3. Roots are the primary pathway by which nutrients (e.g., phosphorus) and contaminants (e.g., arsenic) move from soil to plants and thus understanding this process has implications for food quantity and quality, ecosystem functioning, and phytoremediation. However, roots are dynamic in space and time growing in response to nutrient acquisition needs and they often vary in function, diameter, and structure (e.g., lateral r....

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1. Preparation of slam-freezing equipment

  1. Place two copper blocks (~5 cm x 5 cm x 15 cm) horizontally inside of a clean cooler capable of holding liquid nitrogen and pour enough liquid nitrogen to submerge the blocks. Once the bubbling subsides, place two spacers on top of one copper block at each end.
    NOTE: The spacer height determines the height of the sample to be frozen; this example uses a 2 cm spacer to create cubes approximately 3 cm x 3 cm x 2 cm. The volume of the liquid nitrogen will depend o.......

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This method allows for preservation of roots and chemical species in the roots and rhizosphere of wetland plants and into the bulk soil. In this work, the method was used to evaluate As speciation and co-localization with Fe and Mn oxides and plant nutrients in the rhizosphere of rice (Oryza sativa L.). Rice was grown at the RICE Facility at the University of Delaware where 30 rice paddy mesocosms (2 m x 2 m, 49 plants each) are used to grow rice under various soil and water management conditions with the goal o.......

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This paper describes a protocol to obtain preserved bulk soil + rhizospheres of wetland plant roots using a slam-freezing technique that can be used for elemental imaging and/or chemical speciation mapping.

There are several benefits of this method over existing methods. First, this method allows the simultaneous investigation of roots and the surrounding rhizospheres. Methods currently exist to preserve and chemically image roots out of their soil environment by washing away the soil and pres.......

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The authors acknowledge a joint seed grant to Seyfferth and Tappero to support collaboration between the University of Delaware and Brookhaven National Laboratory. Parts of this research used the XFM (4-BM) Beamline of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704.

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Name Company Catalog Number Comments
Copper blocks McMaster Carr 89275K42
Diamond blade Buehler 15 LC, 102 mm x 0.3 mm operation speed: 225 rpm
Epoxy forms Struers 40300085 FixiForm
Epoxy Epotek 301-2FL
Superglue Loctite 404
Thin sectioning machine Buehler PetroThin
Wet saw Buehler IsoMet 1000

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