A wound-induced promoter driving npt-II expression limited to dedifferentiated cells at wound sites is sufficient to allow selection of transgenic shoots

Abstract

There is much data to indicate that only a small number of cells in plant explants are competent for stable transformation by Agrobacterium. Circumstantial evidence suggests that certain cells reentering cell division at wound sites are competent for transformation by Agrobacterium. We have discovered a member of the intracellular PR gene family from asparagus (AoPR1) which is strongly expressed upon wounding and during the reactivation of the cell cycle in cultured asparagus cells, but which shows very little expression in intact plant tissues. The promoter from the AoPR1 gene was fused to an intron-containing GUS reporter gene and shown to be more strongly expressed than the commonly used CaMV 35S constitutive promoter in target cells for plant transformation. A transcriptional fusion of the AoPR1 promoter with an NPT-II gene was found to be a very efficient marker for the selection of transgenic tobacco callus. Expression of the AoPR1-NPT-II gene allowed efficient shoot formation on transgenic callus and efficient adventitious root formation on transgenic shoots. These latter observations provided firm evidence that transformation selection marker gene expression is most crucial at the early stages of the transformation process, during the establishment of transformed micro-calli.

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

“…The levels of Cry1Ac protein accumulated over a period of 72 h were sufficient to cause 100% within 48 h of the bioassay. Although low levels of Cry1Ac were present in the unwounded leaves at the beginning of the feeding bioassay, these levels were not high enough to cause larval mortality within 48 h. Taken together with previous results from AoPR1-GUS and AoPR1-NPT-II marker gene expression in plants suggests that the Bt endotoxin gene under the control of AoPR1 promoter in transgenic plants behaves in a manner similar to that of endogenous defence genes which are generally silent or expressed at very low levels in the absence of biotic or abiotic stress [4,9,16,17]. Wounding by insect bites and chewing triggered the expression of the cry1Ac gene in levels sufficient to kill the target insect pests.…”

“…To subclone the AoPR1 promoter (900 bp) into the plasmid pRD400 [18], the plasmid AoPR1 GUS-INT [16,17] carrying AoPR1 promoter was cut with BamHI and KpnI enzymes. AoPR1 promoter was subcloned into the plasmid pRD400 pre-digested with BamHI and KpnI.…”

“…Considering that AoPR1-directed GUS gene expression was mainly confined to wound sites, it was obvious that the AoPR1 promoter was transcriptionally more active, on a per cell basis, following wounding than CaMV35S promoter [11]. Although the AoPR1 promoter showed strong expression at wound sites and in developing callus tissue, it was virtually silent in non-wounded leaves, roots and tubers (the latter in the case of potato) tissue from the transgenic plants [16,17]. We report here the use of the AoPR1 promoter to limit the expression of a Bt gene to wound sites caused by insect invasion.…”

Plant Transformation by Coinoculation with a Disarmed Agrobacterium tumefaciens Strain and an Escherichia coli Strain Carrying Mobilizable Transgenes

“…However, the efficiencies reported here are sufficient for routine creation of transgenic plants in all but the most recalcitrant species. Further increases in transfer efficiency might be attainable by preconjugating the donor strains prior to infection or by using approaches that are useful for standard Agrobacterium-mediated plant transformation, such as overexpression of VirG and VirE in the disarmed Agrobacterium strain (42,48), adding acetosyringone to artificially stimulate expression of the vir regulon (49), or placing genes of interest under stronger or early-expressed promoters (14).…”

Visualisation of stromules in transgenic wheat expressing a plastid-targeted yellow fluorescent protein

“…The SnaBI site was recreated only when the GBSSI TP-EYFP fragment was inserted in the correct orientation. pJOD14-SCV is a binary super clean vector (Firek et al 1993) containing the rice Act1 promoter/ 5 0 intron and the nos terminator for constitutive expression, and the NPTII (neomycin phosphotransferase II) gene under the control of the subterranean clover stunt virus Sc4 promoter (Schünmann et al 2003) and arabidopsis FAD2 intron (Okuley et al 1994) for selection. The resulting plasmid pGBSSTP-EYFP was introduced into Agrobacterium tumefaciens supervirulent strain EHA105 (Hood et al 1993) by electroporation (Shen and Forde 1989).…”