In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens

Abstract

CROWN GALL, the plant tumour of many dicotyledonous and some monocotyledonous species, is caused by Agrobacterium tumefaciens infection after wounding¹. Crown gall cells, unlike cells derived from healthy plants, have the ability to proliferate indefinitely in culture without the growth hormones auxin and cytokinin². The properties of the tumours, such as morphology (rough or smooth type). and the synthesis of the unusual aminoacid derivatives octopine or nopaline, are determined by the tumour-inducing (TI). plasmid present in the virulent bacterial strains^3,4. The presence of at least a part of the plasmid has been demonstrated in the DNA of the tumour cultures⁴. Transcription of this fragment was also demonstrated in the cells of the tumour tissues^5,6. These findings seem to confirm the involvement of the TI plasmid in crown gall disease. However, it is not known if introduction of the TI plasmid alone into the plant cells is sufficient to obtain transformation of healthy plant cells into tumour cells. A fundamental approach to this question involves the development of an in vitro transformation system which can use TI plasmids of A. tumefaciens as vectors of genetic material in experiments to modify the genetic information of plants. We report here the transformation of tobacco cells by virulent strains of A. tumefaciens, the isolation and characterisation of transformed calli and the regeneration of shoots with a distinct tumour marker.