Citrus transformation using mature tissue explants.

Mature tissue protocol for production of transgenic Citrus plants via Agrobacterium-mediated transformation uses explants derived from branches of mature, fruit-bearing trees. Through the multiple cleaning steps consisting of grafting of apical tip meristems on rootstock plants grown under sanitary conditions, "mother" plants are produced that will serve as a source of budding material. These buds are grafted onto rootstock plants grown under the same, highly sanitary conditions. Newly obtained, one meter tall, young grafted plants serve as a source of explants for co-incubation experiments with Agrobacterium. Following successful transformation with Agrobacterium, selected transgenic shoots are micrografted onto rootstock plants in vitro where they are allowed to grow for a couple of months. Grafted transgenic plantlet together with the associated rootstock plant is taken out of culture tubes, severed from the root, and regrafted in terra on a 1-year-old rootstock plant. With the application of proper horticultural techniques, such a plant will yield first fruit about 12-15 months later.