J.-P. Hernalsteens

Genetic identification of functions of TL-DNA transcripts in octopine crown galls

The TL‐DNA in octopine crown galls encodes seven transcripts most, if not all, expressed from individual promoters. Site‐specific deletions and substitutions in the T‐region of the octopine plasmid pTiB6S3 indicate some of the functions of the TL‐DNA transcripts. Two of the seven genes are sufficient to allow tumorous growth. T‐DNA transfer and oncogenicity are controlled by different and independently acting functions. None of the transcripts of TL‐DNA appear to be essential for T‐DNA transfer. Four, possibly five, of the TL‐DNA transcripts act by suppressing organ development. Shoot and root formation are suppressed by the action of different transcripts.

An Agrobacterium-transformed cell culture from the monocot Asparagus officinalis.

Cultured stem fragments from the monocotyledonous plant Asparagus officinalis infected by the oncogenic bacterium Agrobacterium tumefaciens developed tumorous proliferations. This tissue was propagated in vitro on hormone‐free culture medium. The T‐DNA‐encoded markers nopaline and agrocinopine were unambiguously detected in these tissues. The data demonstrate that stable T‐DNA transfer as well as expression of T‐DNA genes is possible in at least some monocotyledonous plants. This opens new possibilities for plant genetic engineering using the Ti plasmid as a gene vector.

Vectors for cloning in plant cells

Publisher Summary This chapter discusses vectors for cloning in plant cells. The chapter presents recent octopine Ti plasmid-derived vectors and their use to transfer and express foreign genes in plants. Plant cells transformed with this avirulent tDNA retain their intrinsic capacity to redifferentiate into complete plants. Moreover, the tDNA vectors contain a dominant selectable marker gene, mainly a chimeric neomycin phosphotransferase gene that provides the transformed plant cell with a selectable kanamycin resistance trait.. pGV2260 contains the intact oir region, which encodes Ti plasmid functions necessary for tDNA transfer and/or integration. These functions can act in trans to the tDNA. The chapter discusses cointegration vectors and binary vectors. The chapter includes the steps to introduce a foreign gene into a Ti plasmid vector. The chapter also discusses the expression of chimeric genes in plant cells, plant transformation, the transformation of tobacco leaf fragments, the analysis of gene expression in transformed plants, neomycin phosphotransferase assay, and tDNA organization in transformed plants.

Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline crown gall tumours☆☆☆

Abstract Eight lines of nopaline crown gall tumours were analysed by Southern (1975) blot hybridization to determine the size, internal organization, boundaries, possible plant DNA integration and accuracy of transfer of the Ti-plasmid DNA segment (T-DNA) transferred from Agrobacterium tumefaciens to crown gall plant cells. The conservation of this T-DNA in tumour tissues and tissues derived from plants regenerated from crown gall teratomas was also studied. A defined plasmid segment (the T-region) of about 15 × 10 6 M r is accurately transferred and integrated into nuclear plant DNA without any major internal rearrangements. Furthermore, common composite fragments covalently linking the left and the right boundary of the T-region were observed, thus indicating either tandem duplications of integrated T-DNA segments or polymeric circles of T-DNA segments. The length of the transferred segment is not determined by size, since insertions in the T-region were found to be co-transferred with the T-DNA. The results indicate that sequences at the boundaries of the region may play a role in the transfer mechanism, although the right boundary could be replaced by a Tn1 insertion. Cells from plants regenerated from crown gall teratomas were shown to contain T-DNA without internal rearrangements but with minor modifications of the boundary fragments. In plants obtained from meiotic products of teratomaderived regenerated plants no T-DNA was observed.

Mendelian Transmission of Genes Introduced into Plants by the Ti Plasmids of Agrobacterium tumefaciens

SummaryInsertion 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.

Transfer of Ti plasmids between Agrobacterium strains by mobilisation with the conjugative plasmid RP4

SummaryThe P type conjugative plasmid RP4 has been shown to be able to promote the transfer of the Agrobacterium Ti-plasmid. The results provide additional evidence that agrocin 84 sensitivity, exclusion of phage AP1, ability to catabolize the guanidine derivatives octopine and nopaline and tumor inducing ability, are Ti-plasmid determined properties. Furthermore, the results strongly support the notion that at least part of the Ti-plasmid is transferred from the bacterium to the target plant cells, since it was demonstrated that Ti-plasmid linked genes specify the synthesis of octopine or nopaline by crown-gall tumor cells.

Agrocin 84 sensitivity: a plasmid determined property in Agrobacterium tumefaciens

SummaryIt was shown for some oncogenic Agrobacterium tumefaciens strains that agrocin 84 sensitivity is determined by the presence of a large closed circular DNA plasmid, called the Ti-plasmid. Whereas wild-type strain C58 is agrocin 84 sensitive, all Ti-plasmid cured derivatives were found to be fully resistant. Moreover all independently isolated agrocin 84 resistant colonies were stably non-oncogenic and plasmid negative. In a growth experiment carried out at 37° C it was shown that the kinetics of appearance of non-oncogenic cells on the one hand and of agrocin 84 resistant cells on the other were identical. The fact that not all oncogenic, plasmid harbouring, Agrobacterium tumefaciens strains are sensitive to agrocin 84, points to the possibility that the genes determining agrocin 84 sensitivity are not essential for tumour-inducing ability.

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.

Mutagenesis by insertion of the drug resistance transposon Tn7 applied to the Ti plasmid of Agrobacterium tumefaciens

Insertions of the linked trimethoprim and streptomycin resistance transposon Tn7 were isolated in a cointegrated plasmid formed between the Ti-plasmid of Agrobacterium tumefaciens strain B6S3 and the broad host range resistance plasmid RP4. By using the spontaneous dissociation of this cointegrate, we could demonstrate that these insertions had occurred into the Ti as well as into the RP4 part of the cointegrated plasmid. Among the insertions in the Ti part, mutants affected in different Ti plasmid-determined phenotypes, including non-oncogenic mutants, were detected. These mutants are useful for the physical mapping of these plasmid markers.

In vivo transfer of the Ti-plasmid of Agrobacterium tumefaciens to Escherichia coli

SummaryThe Ti-plasmids are naturally self-transmissible from their normal host Agrobacterium to E. coli. They are however unable to stably establish themselves as a replicon in E. coli. It is nevertheless possible to study the Ti-plasmids in E. coli with the help of Ti::RP4 cointegrate plasmids that transfer and maintain themselves very efficiently in E. coli. An E. coli harbouring such a Ti::RP4 plasmid is unable to catabolize octopine and unable to induce crowngall tumours on plants.