A transient transformation system for duckweed (Wolffia columbiana) using Agrobacterium-mediated gene transfer

Since duckweed (Lemnaceae family) is a valuable target plant for various ap plications including waste water treatment and food purposes, the expressio n of homologous or heterologous proteins may offer an extended range of app lication. Therefore, the feasability of transformation of Wolffia columbian a (Lemnaceae) by Agrobacterium tumefaciens-mediated gene transfer has been elucidated. Several methods were tested to increase the accessibility of th e plant cells for the infecting Agrobacterium tumefaciens strain LBA4404, h arboring p35SGUSINT : corundum- and gold particle-treatment, vacuum infiltr ation and disintegration of the fronds. The resulting overall transformatio n efficiency was higher than without any treatment, reaching an average of 3.9% of all fronds showing GUS staining. Induction of Agrobacterium's vir g enes by media conditions as well as the presence of 0.6 M mannitol during i nfection resulted in a clear increase of transformation efficiency. Max. ap prox. 30%, average 15-20% of fronds showing GUS staining were obtained both with corundum-treated as well as with vacuum infiltrated fronds, but trans formation pattern was different. Whereas in the former mainly epidermal and subepidermal cells were transformed, the latter showed, in addition, trans formed inner frond cells, including the meristematic region. Disintegration of the fronds, followed by vacuum infiltration, led to whole GUS-stained a reas of the frond fragments. The results as such and the observed transform ation patterns will serve as a basis for offering good conditions either in the in vivo - or the in-vitro-regeneration of transgenic duckweed fronds.