Development of a high-throughput method to screen plant mutant libraries using synchrotron-based X-ray fluorescence microscopy (µ-XRF)

Peng Wanga , S. Chena, E. Lombib, F.-J. Zhaoa and P. M. Kopittkec

a College of Resources and Environmental Sciences, Nanjing Agricultural University, China

b Future Industries Institute, University of South Australia, Australia

c School of Agriculture and Food Sciences, The University of Queensland, Australia

p.wang3@njau.edu.cn

Numerous genes in plants control the uptake, translocation, and storage of elements, including the essential macro- and micro-nutrients and the various non-essential elements. However, few genes have been identified due to a lack of efficient method to screen libraries of mutated plants for individuals which have unusual characteristics in metal uptake and distribution. Screening requires the analysis of thousands of randomly mutated plants, but most techniques for quantifying metals are poorly suited for high-throughput analysis of large populations. Inductively coupled plasma mass spectrometry (ICP-MS) allows for quantitative and simultaneous measurement of a range of elements and is useful for determining the metal concentration in whole plants or organs, but it lacks spatial resolution. Identification of the spatial distribution of metals in specific tissues and organs would be more useful for the identification of unusual individuals. In addition, ICP-MS analyses are destructive and requires a comparatively large amount of work to prepare samples (e.g. grinding, acid digestion), especially when used for thousands of mutated plants. With the development of a new generation of fast fluorescence detector systems, synchrotron-based X-ray fluorescence microscopy (µ-XRF) provides a unique capability of obtaining the elemental distribution in vivo with minimal sample preparation, and importantly it is a rapid, sensitive and non-destructive method. In this study, we develop µ-XRF as a high-throughput method to screen mutant libraries of rice and Arabidopsis thaliana seed with the phenotype of altered metal accumulation / distribution in whole intact seeds. This is particularly important in plant biology given that the identification of genes involved in metal metabolism requires the ‘screening’ of thousands of genetic variants. Once identified, the casual gene(s) responsible for the phenotype will be further investigated using genetic and molecular biology methods.

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