Figure 4.

Overview of the eight-stage procedure for Agrobacterium­mediated transformation. (a) Removal of seed coat, sterilization, and germination. (b) Preparation of Agrobacterium tumefaciens, culturing in LB liquid medium, transfer, and shaking to OD = 0.6–0.8. (c) Explant preparation, excision of embryos from the germinated seeds, cutting of cotyledons in half transversely, and selection of proximal parts with U-shaped ends as explants. (d) Infection, scratching of the proximal regions of explants with a micro-brush, and sonication. (e) Vacuum infiltration. Vacuum treatment was applied with a syringe. (f) Co-cultivation with A. tumefaciens in the dark. (g) Shoot regeneration and screening. The dark-cultured explants were washed with sterilized water, then transferred to shoot induction medium. Explants regenerated for about 4 weeks and were then screened for positive buds by fluorescence microscopy. (h) Root regeneration. The positive buds were removed and transferred to rooting medium.