Plant transformation using an agrobacterium. Tumefadens ti-plasmid vector system

Abstract

A plasmid, pDUB116 Was constructed, containing the Nos-NPT dominant selectable marker for expression in plants which was capable of being mobilised to Agrobacterium tumefaciens and forming cointegrates with pGV3850 and pGV3851. The frequencies of cointegrate formation of these plasmids were determined and the cointegrate structure established by Southern blotting. Inoculation of Kalanchoe diaigremontiana leaves and Nicotiana tabacum stems in vivo showed that pGV3851 is only weakly oncogenic. A fully oncogenic Ti plasmid (pTiGEl) was constructed which was capable of forming cointegrates with pDUB116 and suitable for use in in vivo plant transformation systems. A. tumefaciens GV3101 [pGV3850] was found to be highly resistant to cefotaxime and carbenicillin in plant tissue culture media, preventing its use in in vitro plant transformation proceedures. A. tumefaciens strains were therefore screened for sensitivity to a number of antibiotics, two of which, augmentin and timentin, were found to be inhibitory to the growth of A. tumefaciens but non-inhibitory to callusing, shooting or rooting of N. tabacum in tissue culture.The effect of the SV40 enhancer on the expression of the Nos promoter was investigated by constructing integrating plasmids (pDUBllG derivatives) with the SV40 enhancer 5' and 3'. and in both orientations with respect to the Nos-NPT gene. These plasmids were mobilised to A. tumefaciens and cointegrates with pTiGEl selected, characterised by Southern blotting, and inoculated in vivo onto leaves of K. diaigromentiana. Extracts from the resultant callus tissues were found to contain no detectable NPT activity in all cases. The same constructs were used to transform N. tabacum in vitro by a leaf disc transformation method and callus was selected on hormone free media and kanamycin. The callus induced by the constructs containing the SV40 enhancer showed no significant increase over the control construct, indicating that the SV40 enhancer does not function in these plants. Further improvements to the pBR322-homology mediated Ti vector system were made by constructing a new oncogenic Ti vector. pTiGE2. which has a smaller T-DNA containing a single copy of pBR322. giving a more defined T-DNA which is easier to analyse after cointegrate formation. New T-DNA integrating vectors containing the LacZ insertional inactivation region from pUC18 were constructed giving more unique restriction enzyme sites and making the selection of recombinants easier. A chimaeric CAMV-pea lectin gene was constructed and subcloned into a T-DNA integrating vector. Resultant cointegrates with pGV3850 were characterised and transgenic A'', tabacum plants regenerated. The T-DNA structure and copy number in the transgenic plants were investigated. Expression of the CAMV-pea lectin gene was characterised by northern blotting, western blotting, haemagglutination and ELISA and showed the gene to be expressed constitutively at high levels and the protein to be processed correctly. The subcellular site of lectin deposition in transgenic tobacco root was found to be the vacuole. Plants expressing lectin at high level were screened for resistance to a root-knot nematode and found not to be resistant to infection.