Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava

摘要

Agrobacterium-mediated transformation of friable embryogenic calli (FECEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FECEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking ~6 months, that optimizes Agrobacterium-mediated transformation of FECEC from cassava model cultivar TMS60444. We cocultivate the FECEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FECEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FECEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.