Nopaline Strains Research Articles

Identification of an Agrobacterium tumefaciens pTiB6S3 vir region fragment that enhances the virulence of pTiC58

The Agrobacterium tumefaciens octopine strain B6S3 and the nopaline strain C58 were compared for their ability to induce opine synthesis on Kalanchoe daigremontiana stem fragments. Whereas B6S3 induced high levels of octopine synthesis, C58 induced only low levels of nopaline synthesis. However, C58-induced nopaline synthesis was greatly increased by mixed infection with B6S3. This effect (called “helper-effect”) was shown to be due to the activity of a 5 kb fragment from the virulence region of the B6S3 Ti plasmid, since incorporation of this fragment into the C58 plasmid enabled C58 to induce high levels of nopaline synthesis in the absence of a helper strain. The 5kb region contains the vir F locus, as defined earlier (Hooykaas et al. 1984b). A possible correlation between the helper function and vir F is discussed. Our results show that large differences in virulence on particular host plants exist between natural Agrobacterium strains and can be overcome by mixed infections.

Sexual transmission of T-DNA in abnormal tobacco regenerants transformed by octopine and nopaline strains of Agrobacterium tumefaciens

Transformed shoots regenerating from Crown gall tissues were produced by in vitro infection of regenerating tobacco protoplasts with Agrobacterium tumefaciens. As these shoots were abnormal in their growth and were unable to form roots, they were grafted onto normal tobacco, resulting in the formation of flowering shoots. Sexual crosses between different grafts and selfing produced two different types of seedlings. Some developed into normal, untransformed plants whereas others failed to develop a root system and remained very small. The abnormal seedlings were shown to consist of transformed cells since they expressed the opine marker. The suppression of root formation and the production of opines were 100 percent linked. The analysis of the progeny indicated that the shoots derived from these tumour lines were hemizygous for the T-DNA linked genes and expressed these genes as a dominant locus. The opine-positive shoots were shown to contain a deleted T-DNA and also some T-DNA specific transcripts: the transcript for octopine synthase or LpDH and two other transcripts, one of which is known to be involved in the suppression of root formation. The same transcripts could also be detected in the sexual progeny. These observations confirm that some active tumour-controlling genes can be sexually transmitted and do not drastically interfere with seed development, germination and early shoot formation. The formation of roots by these seedlings is however severely suppressed.

Growth requirements ofArabidopsis thaliana crown galls

Crown galls induced onArabidopsis thaliana plants by octopine or nopaline strains ofAgrobacterium tumefaciens were grownin vitro on different media. Dark growth of all tumor tissues was strictly hormone-dependent. In contrast, hormonal autonomy was observed in the light where crown gall calli readily differentiated into teratomas and (sometimes fertile) plants. Differentiating tissues always grew more vigorously than subtended calli. The growth of transformed calli was stimulated by vitamins and partly inhibited by growth regulators in concentrations used for the maintenance of untransformed calli. Crown gall calli, teratomas and sometimes regenerated plants were shown to express lysopine or nopaline dehydrogenase activities.

Four new opines from crown gall tumours —Their detection and properties

We report four new opines that have been discovered in sterile crown gall tumour tissue. These compounds have been partially characterised as phosphorylated sugar derivatives. Two were detected in tumours induced by nopaline strains of Agrobacterium tumefaciens and two in tumours induced by an agropine strain. These compounds are involved in interesting interactions with the nucleotide bacteriocin, agrocin 84 and we propose that they be called agocinopines.

Induction and in vitro culture of Arabidopsis Thaliana crown gall tumours

Tumours were induced on Arabidopsis thaliana by wounding the stem and infecting with Agrobacterium tumefaciens strains T37, C58 or Ach5. After about three weeks the tumours were excised and cultured in vitro on solidified medium without growth hormones. Both octopine (Ach5) and nopaline (T37 and C58) strains induce unorganised tumours on the plant. After excision and in vitro culture the T37 and C58 tumours immediately differentiate into teratomata and normal plants. The Ach5 tumours grow mainly as undifferentiated tissue, but also give rise spontaneously to teratomata and to a lesser extent to normal plants. The tumourous state of the callus tissue and teratomata was confirmed by the presence of octopine and nopaline.