Papaver rhoeas pollen SI bioassay with antisense oligonucleotide treatment in-vitro

As a transgenic approach with Papaver rhoeas is not possible, we used an alternative approach to knockdown PrPGAP1 by incubating P. rhoeas pollen with phosphorothioated antisense oligonucleotide designed against PrPGAP1^18,24. P. rhoeas pollen was hydrated for 0.5 h and incubated with poppy pollen growth medium (PrGM) containing 3 μM phosphorothioated oligonucleotides (Antisense-PrPGAP1 or Sense-PrPGAP1, designed using Sfold⁷⁶, Table S5) and 0.5% (v/v) cytofectin (T201007H, Genlantis) for 2 h at RT before SI induction. Pollen viability were assessed using FDA (2 μg.ml^-1) and PI (5 μg.ml^-1) 10 h after SI treatment. Pollen tube lengths were measured using Fiji (https://fiji.sc/)⁷⁷. Due to the limit of the imaging field size, pollen tubes which were longer than 600 μm (very rarely observed) were all recorded as 600 μm.