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

摘要

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