A high-throughput seed germination assay for root parasitic plants

Jean-Bernard Pouvreau,Philippe Delavault,Mathieu Gauthier,Zachary Gaudin,Marc-Marie Lechat,Philippe Simier,Bathilde Auger

doi:10.1186/1746-4811-9-32

Abstract

BackgroundSome root-parasitic plants belonging to the Orobanche, Phelipanche or Striga genus represent one of the most destructive and intractable weed problems to agricultural production in both developed and developing countries. Compared with most of the other weeds, parasitic weeds are difficult to control by conventional methods because of their life style. The main difficulties that currently limit the development of successful control methods are the ability of the parasite to produce a tremendous number of tiny seeds that may remain viable in the soil for more than 15 years. Seed germination requires induction by stimulants present in root exudates of host plants. Researches performed on these minute seeds are until now tedious and time-consuming because germination rate is usually evaluated in Petri-dish by counting germinated seeds under a binocular microscope.ResultsWe developed an easy and fast method for germination rate determination based on a standardized 96-well plate test coupled with spectrophotometric reading of tetrazolium salt (MTT) reduction. We adapted the Mosmann’s protocol for cell cultures to germinating seeds and determined the conditions of seed stimulation and germination, MTT staining and formazan salt solubilization required to obtain a linear relationship between absorbance and germination rate. Dose–response analyses were presented as applications of interest for assessing half maximal effective or inhibitory concentrations of germination stimulants (strigolactones) or inhibitors (ABA), respectively, using four parameter logistic curves.ConclusionThe developed MTT system is simple and accurate. It yields reproducible results for germination bioassays of parasitic plant seeds. This method is adapted to high-throughput screenings of allelochemicals (stimulants, inhibitors) or biological extracts on parasitic plant seed germination, and strengthens the investigations of distinctive features of parasitic plant germination.

Highlights

Some root-parasitic plants belonging to the Orobanche, Phelipanche or Striga genus represent one of the most destructive and intractable weed problems to agricultural production in both developed and developing countries
To make easier and faster measurements for high-throughput studies, we developed a standardized 96-wells plate germination test coupled with spectrophotometric reading of methylthiazolyldiphenyl-tetrazolium bromide (MTT) reduction [20]
We demonstrate here that the MTT method can be adapted for high-throughput germination bioassays, giving an efficient tool for detailed investigations on seed germination, as presented for example in this paper as a proof of concept with data on the effects of Abscissic acid (ABA) level on P. ramosa seed germination

Summary

Introduction

Some root-parasitic plants belonging to the Orobanche, Phelipanche or Striga genus represent one of the most destructive and intractable weed problems to agricultural production in both developed and developing countries. Broomrape seed germination can be prevented in the rhizosphere by inhibitors including trigoxazonane present in root exudates of the allelopathic plant Trigonella [11], several trichotecenes produced by the potential biocontrol fungi agents, Myrothecium verrucaria and Fusarium compactum [12], 7-hydroxylated simple coumarins and both naringenin and gallic acid present in root exudates of resistant sunflower and pea, respectively [13,14,15]. In this context, many investigations are conducted on the identification and the characterization of the germination stimulants There is a great agronomical challenge to control these parasitic weeds, either by preventing seed germination through biocontrol agents or intercropping with allelopathic plants, or in contrast by promoting seed germination in the absence of host plants by cropping false hosts in order to reduce the seed bank of the soils [2]

Methods

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Discussion

Conclusion