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Basal medium formulation strongly affects transformation efficiency in maritime pine
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International audience. Methods for genetic transformation of maritime pine embryonal masses (EM) using Agrobacterium tumefaciens (At) have been independently established at 2 laboratories (FCBA and INRA) in France. The two procedures appeared roughly similar but technical differences were noticed during the decontamination and washing of co-cultivated EM, the subsequent reactivation culture step and the selection phase of transgenic lines. One major difference was the basal formulations of media used for At-mediated transformation development at FCBA (mDCR, modified Gupta and Durzan medium) and INRA (mLV, modified Litvay medium). Joined experiments were carried out in order to get insights on factors affecting the transformation rate based on a comparison of both procedures (one model genotype, C58pMP90 At strain, modified pCambia1301 binary vector harbouring the uidA-intron, hpt and bar genes). Our ultimate goal is to foster the transgenic approach as an efficient tool for establishing direct linkages between genes and phenotypes. We showed that the efficiency of the selected transformation procedures is tightly correlated with the basal medium formulation. Using mLV medium, currently recognized in both laboratories as a superior mineral base for maritime pine somatic embryogenesis, transformation yields were higher using the INRA procedure following either hygromycin or phosphinothricin selection. In contrast full advantage of the FCBA procedure could only be raised by using mDCR basal medium instead of mLV. In conjunction with strong phosphinothricin selection (1 mg l-1) the mDCR-based FCBA procedure was revealed as the best option to transform one model genotype of maritime pine (89.6 ± 18.4 phosphinothricin-resistant lines per g EM).
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