Mendelian transcmission of genes introduced into plants by the Ti plasmids of Agrobacterium tumefaciens

Abstract

Insertion of the bacterial transposon Tn7 was used to obtain mutants of an octopine Ti plasmid. Crown gall tumours induced on tobacco by an Agrobacterium tumefaciens strain carrying a particular mutant Ti plasmid (pGV2100) were found to give rise to shoots. These shoots were grown in vitro and one of them (rGV-1) was found to contain the T-DNA specific enzyme lysopine dehydrogenase (LpDH) and to form roots. After transfer to soil, rGV-1 developed into a morphologically and functionally normal tobacco plant. All cells of the regenerant and of vegetatively produced offspring were shown, by cloning of leaf protoplasts, to contain T-DNA and LpDH activity, rGV-1 and vegetatively produced offspring flowered normally. Plantlets obtained from haploid anther cultures were tested for LpDH activity. Forty-one percent of these plantlets were LpDH positive. Moreover, both self-pollination of rGV-1 and crosses between rGV-1 and normal tobacco plants showed that the LpDH character was transmitted both through the pollen and through the eggs of rGV-1 as a single dominant factor with Mendelian segregation ratios typical for monohybrid crosses. By repeated selfing, homozygous plants were obtained which bred true with respect to LpDH. The importance of these findings with respect to the use of Agrobacterium tumefaciens and Ti plasmids for genetic engineering in plants is discussed.

A DNA-binding protein factor recognizes two binding domains within the octopine synthase enhancer element.

“…The OCS gene is transferred and integrated with the T-DNA of Agrobacterium into the genome of plant cells during initiation of the crown gall tumor. The gene is not expressed in Agrobacterium but is expressed in the plant (Otten et al, 1981). Although the infectivity of Agrobacterium is limited generally to dicotyledonous plants, the transcriptional enhancer of the OCS promoter functions in both monocots and dicots and does not require any factors supplied by other genes of the bacterium (Hooykaas-van Slogteren et al, 1984;Ellis et al, 1987).…”

Expression of bacterial genes in plant cells.

“…Because of the high levels of phytohormones produced by crown gall tumor cells (18) they have generally proven recalcitrant to attempts to induce regeneration into whole plants (19,20). Exceptions to this are cases in which, as a result of aberrant integration or spontaneous deletion events, transformed cells have lost all or part of the Ti plasmid tumor genes and can now be regenerated (21,22). In addition, transformation of cells by weakly virulent, mutant Ti plasmids (23) and transformation by root-inducing (Ri) plasmids (24,25) have been shown to produce callus that can be regenerated into whole plants.…”

“…1010 Bq) for the unlabeled arginine in the assay buffer. The conditions for electrophoresis were as described (42) and the resulting electrophoretograms were exposed to x-ray film (Kodak, for [16][17][18][19][20][21][22][23][24] hr. The positions of octopine, nopaline, and arginine were established by their comigration with authentic standards.…”

Public Misunderstanding of Genetically Modified Organisms: How Science and Society are Interconnected

“…As we will point out later, this sledgehammer methods (in which mutations are created in a nonspecific region anywhere in the organism) are still favored, while the more modern, minimally invasive methods are heavily restricted. Modern plant cloning began with the detection of the soil bacterium Agrobacterium tumifaciens, which induces crown gall disease [10] , the detection of the Ti-plasmid [11] and cloning experiments with this plasmid [12] . The Ti plasmid randomly inserts DNA into the host plasmid, but in contrast to mutation breeding, only some genes in the host plasmid are modified.…”