Assessing Plant Root Viability: Method Comparison and Development

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Assessing Plant Root Viability: Method Comparison and Development

July 10, 2014

We use measurements of root viability to assess overall plant vigor and the extent of pathogen
or chemical damage. In the context of invasion and restoration – we can use root viability to
better understand the efficacy of weed management practices and restoration treatments.

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Assessing Plant Root Viability: Method Comparison and Development

In this experiment we compared two methods used to assess root viability; TTC reduction and FDA fluorescence microscopy. We adapted the latter to create a high-throughput, inexpensive, reliable method for use with the plate reader. We then tested the three methods with apple, fescue (bunch grass), and knapweed roots collected from MPG.

TTC – Colorless triphenyl tetrazolium chloride (TTC) is reduced by living roots to red formazan (TF). Following a method of Comas (et al. 2000), we incubated roots in TTC for 20 hours and then extracted TF from the roots using ethanol. We then measured TF concentration. FDA Microscopy – Fluorescein diacetate (FDA) is membrane-permeable. Once inside a living cell, intracellular esterases hydrolyze FDA producing the fluorescent compound fluorescein. We stained pea roots with FDA (Noland (et al. 1997), mounted them on slides, and used a fluorescence microscope to score % viability based on the proportion of intercepts that fluoresced (top and center right). FDA Plate Reader – We reviewed the literature and adapted the microscopy method above for use with our plate reader. FDA stained roots were loaded into multi-well plates (bottom left) and compared to known fluorescein concentrations (bottom right).

Root viability values correlated well between the microscopy and plate reader method, suggesting that measuring fluorescence with the plate reader accurately reflects root viability

We also compared the time and monetary investments per sample among the three methods. Our novel plate reader method provided accurate root viability measures in half the amount of time at a similar cost.

The plate reader and microscopy method were in agreement and showed that fescue roots were more viable than knapweed roots, whereas TTC showed the opposite. This is problematic given that both TTC and FDA methods are frequently used to assess root viability.

Our novel FDA Plate Reader method is cost effective, reliable, and saves time. In the near future, we plan to use this method to evaluate the direct effects of herbicide on roots as well as AM mediated uptake and transfer of herbicide to plant roots.