THE IMPORTANCE OF SALICYLIC ACID AND AN IMPROVED PLANT CONDITION IN DETERMINING SUCCESS IN AGROBACTERIUM-MEDIATED TRANSFORMATION

Abstract

A succesful Agrobacterium-mediated transformation requires a balanced interaction between the plant and Agrobacterium. We hypothesize that blocking salicylic acid (SA) biosynthesis could increase the plant's susceptibility and gene transfer events. In this study, the influence of the SA biosynthesis on transformation efficiency was investigated. We tested the effect of increasing concentrations of SA inhibitors such as 2-amidoindane-2-phosphonic acid (AIP) and paclobutrazol (PBZ) in Petunia hybrida and Nicotiana benthamiana. As a control, the effect of adding SA was examined. In addition, the effect of lipoic acid (LA) was studied. LA, an antioxidant, is considered to act as a general transformation enhancer by improving the plant's condition. Adding SA inhibitors should lead to improved conditions for Agrobacterium and adding LA should improve conditions for the plant. Explants from fresh leaves of P. hybrida and N. benthamiana were inoculated with A. tumefaciens strain AGL1(pBinGlyRed-Asc1) containing dsRed as a reporter gene. Transient dsRed expresion was determined at 14 days after co-cultivation by counting red fluorescing spots and transformation efficiency was determined from the number of explants with calli surviving on selection media at 30 days. In P. hybrida, 10 ?M AIP and PBZ resulted in the highest transient and stable transformation. However in N. benthamiana, only 50 ?M AIP showed a significant increase and only in transient expresion, and no evidence of any stimulatory effect of PBZ was observed. Adding SA always decreased the efficiencies in both plant species. Furthermore, 10 and 1 ?M LA significantly increased transformation efficiency in P. hybrida and N. benthamiana, respectively, with les browning and necrosis. Our preliminary results suggested that blocking SA biosynthesis promoted gene transfer but responses were variable and not equally succesful between the two plant species. However, repeating these experiments providing us with more data might clarify the picture further.