A novel autofluorescence-based selection of calli amenable to Agrobacterium-mediated transformation in onion (Allium cepa L.)

Abstract

Suppression of lachrymatory factor synthase (LFS) activity in onion (Allium cepa L.) is expected not only to reduce lachrymatory factor released from the disrupted tissues, but also to increase thiosulfinate and their derivatives associated with the characteristic flavor and various health effects. To test this hypothesis, we used transgenic methods to obtain non-lachrymatory onions in which the LFS gene was silenced by RNA interference (RNAi). In transformation experiments, we found that onion calli generated after preconditioning of the suspension-cultured calli on a solid medium were amenable to Agrobacterium-mediated transformation. These calli were observed without or with low levels of yellowish autofluorescence under the fluorescence microscope, and were easily distinguished from the recalcitrant calli having a characteristic yellowish autofluorescence. The frequency of transient green fluorescent protein (GFP) expression significantly increased when these amenable calli were infected with Agrobacterium harboring the plasmid carrying a GFP expression cassette. Southern blot analysis of adaptor ligation PCR products revealed that 4 independent transgenic lines were obtained. The successful transfer of the RNAi construct was confirmed by using PCR. Analysis of the 4 transgenic lines confirmed that the levels of LFS gene transcript, LFS protein, and LFS enzyme activity were reduced to 3.2–11.0%, 0.09–20.7%, and 0.3–10.1%, respectively, of the wild-type control. The thiosulfinate assay suggested that a significant increase in thiosulfinate formation could be expected in the onion bulb extracts in which LFS enzyme activity was suppressed to approximately 1% or less than that of the wild-type control.