Agrobacterium-mediated Gene Transfer via Hypocotyls

1. Grow cotton seedlings aseptically in culture tubes.
2. Using strictly aseptic practices, transfer hypocotyls to aluminun foil cutting pads and cut into approximately 2 inch sections.
3. Transfer hypocotyls to Petri dishes containing Agrobacterium spp. plus Murashigee-Skoog Non-hormone (MSNH) solution. Further section tissue into approximately 5 mm explant sections. Tissue is wounded for generating Agrobacterium spp. infection sites.
4. Remove excess Agrobacterium spp. plus MSNH by blotting tissue on sterile filter paper.
5. Transfer explant sections to Petri dishes containing T2 medium. 20 explants per dish works well (Four rows of five).
6. Incubate Petri dishes for 3 days at 28 C for co-cultivation.
7. Remove explants, rinse in MSNH, blot on sterile filter paper, and transfer to MS2NK plus antibiotics. Incubate Petri dishes at 30 C in growth room for 3 weeks for callus initiation.