High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus.

In preliminary experiments on Agrobacterium-mediated transformation of citrus, we found transformation events occurring in callus formed from the cambium. Factors affecting Agrobacterium-sour orange (Citrus aurantium L.) interactions, such as culture medium, explant source and culture conditions, were studied to assess competence for transformation in such callus and to improve transformation frequency. Cell divisions and redifferentiation from the transgenic cells leading to transformation events were stimulated more by a combination of benzylaminopurine (BAP) + naphthalene-acetic acid (NAA) in the regeneration-selection medium than by BAP alone. Both age and source of the sour orange plant material affected transformation frequency. Explants from 4-month-old seedlings grown in the greenhouse showed higher transformation frequency than younger and older plant materials, indicating that they had a more suitable balance between dedifferentiated cells competent for transformation and Agrobacterium virulence. Enhancement of transformation frequency enabled us to incorporate the coat protein gene of citrus tristeza virus (CTV) in a sufficient number of sour orange plants to be able to evaluate this strategy for producing CTV-resistant plants.