A high-throughput seed germination assay for root parasitic plants

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.

Triazolide strigolactone mimics as potent selective germinators of parasitic plant Phelipanche ramosa

“…The assay was carried out using slightly modified published procedure . Fifty microliters of incubated seeds were transferred into each well of 96‐well plate.…”

Phenotypical and biochemical characterisation of resistance for parasitic weed (Orobanche foetidaPoir.) in radiation-mutagenised mutants of chickpea

“…Broomrape seed viability was assessed as 72.0 ± 1.1% by measuring germination rate in response to root exudates of the susceptible cv. Amdoun, as described by Pouvreau et al …”

New Strigolactone Analogs as Plant Hormones with Low Activities in the Rhizosphere

“…hermonthica.). After 4 d, the methylthiazolyldiphenyltetrazolium bromide assays, as high-throughput seed germination assay recently described (Pouvreau et al, 2013), were carried out according to Mosmann (1983) with minor modification of the solubilization buffer (Triton X-100 10%, HCl 0.04 mol L -1 in isopropanol). Each germination assay was repeated at least three times.…”