Cell wall remodelling involving galactomannan de-branching influence Agrobacterium tumefaciens-mediated transformation of Coffea canephora somatic embryos

Abstract

Direct somatic embryogenesis is favoured over indirect methods for the in vitro propagation of Coffea canephora, as the frequency of somaclonal variation is usually reduced. Ethylene action inhibitors improve the tissue culture response and thus silver nitrate (AgNO3) is used for direct somatic embryogenesis in coffee. It was observed that silver thiosulphate (STS) that is a more potent ethylene action inhibitor, induced a much robust response in C. canephora cotyledonary leaf explants with 7.49 ± 0.57 and 7.08 ± 0.12 embryos/explant at 60 and 80 µM AgNO3, respectively compared to 3.3 ± 0.18 embryos/explant at 40 µM AgNO3. Transient transformation indicated that STS improved the transformation potential of embryos by enhancing Agrobacterium tumefaciens adherence to surfaces. In vitro adherence assays demonstrated that the cell wall material from STS-derived embryos provide a better substratum for adherence of Agrobacterium. Furthermore, blocking this substratum with anti-mannan hybridoma supernatant negatively effects the adherence. The presence of galactose and mannose residues in the decomposed cellulose fraction of STS treated somatic embryos are indicative of de-branching and re-modelling of galactomannan in response to ethylene inhibition. Genes of mannan biosynthesis, degradation and de-branching enzyme were affected to different extents in embryos derived in AgNO3 and STS containing somatic embryogenesis medium. The results indicate that ethylene-mediated cell wall galactomannan remodelling is vital for improving the transgenic potential in coffee.