Agrobacterium-mediated transformation of Pinus radiata organogenic tissue using vacuum-infiltration

An Agrobacterium tumefaciens-mediated transformation protocol was developed for detached cotyledons of Pinus radiata zygotic embryos resulting in up to 55% of cotyledons transiently expressing the reporter gene uidA. Transient expression of uidA was improved when detached cotyledons were pre-cultured on half strength medium containing cytokinin for 7 days, wounded by vortexing and then vacuum-infiltrated in a solution of A. tumefaciens. The transformation protocol was applied both to cotyledons and also to the apical meristematic dome which was the portion of the embryo remaining after cotyledons were detached, and from which the apical shoot and axillary shoots regenerate. Molecular analysis of putatively transformed shoots regenerated either adventitiously from cotyledons or via axillary shoots from apical domes, indicated the presence of uidA and nptII genes by PCR in some of these shoots. Biochemical analysis of putatively transformed shoots using nptII ELISA indicated that they contained the nptII enzyme. However, Southern hybridisation indicated stable integration of nptII only in one shoot which was regenerated from an apical dome. Shoots regenerated from cotyledons appeared to exhibit chimeric expression and were not stably transformed. Based on a comparison of time for regeneration, technical difficulty, molecular and biochemical analysis, apical domes may be more suitable as explants for transformation and subsequent regeneration of transclones than detached cotyledons.