Fig. 3. Phenotypic analysis of Sli transgenic and CRISPR–Cas9 knock-out lines.

a Design of Sli pAGM:CRISPRΔSli construct. Four sgRNAs target the first exon of PGSC0003DMG400016861. Protospacer Adjacent Motifs are shown in red. b Fluorescence microscopy of self-pollinated styles from Sli transgenics, knock-outs, and untransformed controls. Wild-type B665 shows complete self-pollen tube growth through, but in knock-out line B665ΔSli-2 self-pollen tubes growth is arrested before the pollen tubes can reach the ovaries. Wild-type B666 and B667 show self-pollen tubes growth arrest, but transformation with pBINPLUS-Sli enables self-pollen tubes growth to the ovary. Each style in the image is composed of separate microscopy images of which the contrast and brightness levels were adjusted. The styles shown are representative of three independent experiments. c Pollen tube growth phenotypes of Sli transgenics, knock-outs and untransformed controls on a 0–4 scale: 0: no pollen tubes reach the ovary, 1: fewer than 25% of pollen tubes reach the ovary, 2: between 25 and 50% of pollen tubes reach the ovary, 3: between 50 and 75% of pollen tubes reach the ovary, and 4, more than 75% of pollen tubes reach the ovary. The boxes indicate the median and lower (25%) and upper (75%) quantiles, the whiskers indicate the smallest and largest observations. These data were generated in one greenhouse experiment with 3–5 clones of each genotype and are consistent in two independent experiments with the same genotypes. d PAGE analysis of PCR products from lines obtained after transformation of genotypes B663 and B665 with pAGM:CRISPRΔSli. Six regenerants show INDELs in the targeted area and are labeled B663ΔSli-1 and B665ΔSli-1 to B665ΔSli-5. The bands without labels are from regenerants in which pAGM:CRISPRΔSli did not induce INDELs. Additional lanes in the gel image were excised at the positions indicated by the vertical dashed lines. The horizontal dotted line represents a fragment size of 136 bp.