Fig. 3.

Transformation through overexpression and CRISPR/Cas9 editing. (a) Gene editing of Sh1 from green millet through CRISPR/Cas9 with two gRNA targets. Three editing events (T₁, Csh1-1, Csh1-2, and Csh1-3) contained a 1-bp insertion, a 16-bp deletion, and a 19-bp deletion in the CDS of Sh1, respectively, which resulted in gene-frame shifts and early stoppage of translation. (b) The seeds remained firmly on the panicles of the plants from the three editing events (T₁), while all seeds were shed freely from the head of the control green millet plants. (c) The breaking tensile strength of seed detachment from the pedicel remained low in the control green millet plant but was greatly enhanced (P < 0.001) in the plants descended from the three editing events. The breaking tensile strengths were recorded in gravitational units of force (gf). (d) Three overexpression transgenic events were obtained resulting in plants (T₁, Osh1-1, Osh1-2, and Osh1-3) with much higher expressions in comparison to the control foxtail millet plant. (e) Plants resulting from the three overexpression events exhibited easy shattering, while the control foxtail millet was nonshattering. (f) Breaking tensile strengths of seed detachment from the pedicel were greatly decreased (P < 0.001) in the transgenic overexpression plants as compared to the nontransgenic foxtail millet plants. (g–n) Scanning electron microscopy images of the surfaces of the pedicels from the edited and control green millet plants (g, i, k, and m) and close-up views of the surfaces (h, j, l, and n). (o–v) Scanning electron microscopy images of the surfaces of the pedicels from the overexpression transgenic and control foxtail millet plants (o, q, s, and u) and close-up views of the surfaces (p, r, t, and v).