Applicability of the co-inoculation technique using Agrobacterium tumefaciens shooty-tumour strain 82.139 in silver birch

Abstract

In the co-inoculation technique, genetic transformation is performed using a mixture of Agrobacterium strains – shoot regeneration is induced by the wild-type strain 82.139, while the transferable genes are provided in a binary plasmid by another, non-oncogenic Agrobacterium strain. The aim of the present work was to study the applicability of co-inoculation under both in vitro and greenhouse conditions for in planta transformation in silver birch (Betula pendula Roth). In addition to the original method, several modifications of the technique including an genetically engineered 82.139 strain harbouring the binary pGUSINT were tested. The co-inoculations resulted in a gall formation frequency of 52–94% with greenhouse seedlings, and 4–63% with tissue-cultured plantlets, the shoot induction percentage varying by 0–13 in the greenhouse and 42–75 in vitro. PCR analysis verified that the majority of the regenerated material was non-transgenic, with a few individuals showing integration of the oncogenic T-DNA. According to the histochemical tests, however, some of the numerous differentiating buds and small shoots on gall tissues were transgenic, and contained the GUS reporter gene. The results show that it would have been necessary to apply selection pressure during differentiation in order to recover shoots transformed with the desired genes from the binary plasmid. The morphology and growth of all the regenerated plantlets was normal, suggesting that the oncogenic T-DNA was not expressed even though it was present. In conclusion, it was possible to obtain transgenic silver birch plantlets using the A. tumefaciens strain 82.139, but the co-inoculation method is not directly applicable as in planta transformation protocol.