Striga seed germination bioassays
To evaluate the resistance of sorghum accessions, we screened 11lgs1 sorghum genotypes identified from genotypic screening of SAP
accessions for Striga germination induction. Shanqui Red,
PI533839, PI656054 were used as LGS1 controls while SRN 39 was used as
the lgs1 check.
Two Striga germination assays were used: (i) germination
frequency using extracted sorghum root exudate and (ii) agar gel assays
(Hess, Ejeta & Butler 1992). Root exudate germination assays were
performed according to Jamil et al. (2022). Striga seeds
were surface sterilized in 10% commercial bleach for 5 minutes followed
by rinsing thoroughly with sterile milliQ water and air drying in a
laminar flow for one hour. Striga seeds were then uniformly
distributed (approximately 50-100 seeds each) on 9mm-fibre filter paper
discs (Sartorius, Goettingen Germany) and put in 90mm-diameter
petri-plates lined with moistened filter paper (Whatman, Maidstone, UK).
Twelve (12) discs containing Striga seeds were transferred to
each plate, sealed with parafilm, and wrapped in aluminum foil. For
conditioning, Striga seeds were incubated at 30oC for 10 days. Afterwards, conditioned Strigaseeds were treated with root exudates collected from each sorghum (55 µL
per disc). Six discs were used per treatment. The petri plates were
sealed, wrapped in aluminum foil, and incubated for 24 h at 30oC to induce germination. Germinated and
non-germinated seeds were analyzed using the SeedQuant software (Braguyet al. 2021) by observing under a binocular microscope. The
germination frequency in percentage (GF %) was calculated for each
replicate using the formula:
\(\text{GF}=\left(\frac{\text{Ngs}}{\text{Nts}}\right)x100\) Where
Ngs is the number of germinated seeds per disc
and Nts is the total number of seeds per disc.
Agar gel assays were performed according to Hess et al . (1992).
Sorghum seeds were soaked in 70 % ethanol for 5 minutes and then in 1
% NaOCl solution for 20 mins. Seeds were rinsed thoroughly in sterile
double distilled water and transferred in petri dishes lined with moist
Whatman GFA filter paper (Meadow, UK). The plates were then wrapped in
aluminum foil and incubated at 28 °C for 48 hrs. Conditioned S.
hermonthica seeds (300 µl) were pipetted into petri dishes then water
agar (0.8 %) was dispensed over the seeds to achieve even distribution.
To the solidifying media, a pre-germinated sorghum seedling was placed
at the edge of each plate with the tip of the root pointing across the
plate. The gel-embedded host-parasite culture was incubated at 28 °C in
the dark for 72 hours and scored for induction of Striga seed
germination. Images were taken using a Leica MZ10F microscope and
analyzed for germination using imageJ Version 1.5i
(https://imagej.nih.gov/ij/).
The maximum germination distance (MGD), which is the distance between
the host root and the furthest germinated seeds, was used as a measure
of resistance.