Enhancing T-DNA Transfer Efficiency in Barley (Hordeum vulgare L.) Cells Using Extracellular Cellulose and Lectin.

Abstract

A major limitation of transforming barley tissues by Agrobacterium tumefaciens is the low frequency of T-DNA transfer due to recalcitrance of barley as a host. The effect of extracellular cellulose and lectin on Agrobacterium transformation efficiency was investigated in this study. Barley callus cultures were transformed with the AGL1 strain containing the vector pBI121 in the presence of 10mgmL(-1) cellulose or 0.001, 0.05 and 0.1mgmL(-1) lectin. Addition of cellulose significantly (P ≤ 0.05) increased the number of GUS spots by 50% compared to standard conditions in the presence of only 200μM acetosyringone (AS). Frequency of G418-resistant aggregates on the surfaces of callus cultures was 29 and 71.5%, following AS and AS + cellulose treatments, respectively, after 4weeks of selection. Presence of 0.05 or 0.1mgmL(-1) lectin also increased the number of GUS spots and frequency of G418-resistant cells in the selection period, but the increase in blue spots was not significant. We examined the effect of lectin and cellulose on bacterial attachment to callus tissues. Both cellulose and lectin were found to have a significant positive effect on the numbers of bacteria attached to barley callus. Epifluorescence microscopy revealed that Agrobacterium cells had accumulated in the scaffolds of irregular fibrous cellulose with a mean particle size of 200μm. Expression of nptII in transformed callus lines confirmed the stable transformation of the gene. Our study showed for the first time the binding of Agrobacterium cells to fibrous cellulose and also demonstrated how polysaccharides and glycoproteins can be used to improve T-DNA transfer in monocotyledon transformation procedures.