We crossed the Agrobacterium tumefaciens chrysanthemum strain ANTI, which harbors four plasmids, with the plasmid-free recipient C58.00RS, Transconjugants degrading the Amadori-opines chrysopine and deoxy-fructosyl-oxo-proline (dfop) harbored the Ti plasmid of ANTI, termed pAtANT4b, Upon transfer to the recipient strain C58.00RS, pAtANT4b (pTiANT4) and pANT4a (the largest of the four plasmids of ANTI) could cointegrate, The cointegration of the two plasmids occurs at various places of the pTiANT4, a feature that may affect several functions of the Ti plasmid (e.g., opine degradation). Transconjugants utilizing the opine deoxy-fructosyl-glutamine (dfg) always harbored the large pAtANT4a, Other Agrobacterium strains, including nonpathogenic strains such as C58C1, naturally degraded dfg, Remarkably, strain C58C1 carries a large cryptic plasmid termed pAtC58 that also encodes dfg degradation. A screening of physiological traits additionally revealed that this plasmid allows utilization of octopine as sole nitrogen source after mutation. All these results demonstrate that the larger plasmid of A, tumefaciens is a catabolic plasmid and that both the "cryptic" plasmid and Ti plasmid cooperate for opine degradation.

In the Sidi M’djahed nursery (Algeria), over 60,000 eucalyptus (Eucalyptus occidentalis) plantlets exhibited tumour-like growths localized at the crown of the plants that resembled crown galls caused by Agrobacterium tumefaciens. Bacteria colonizing the galls were isolated and purified. Most (22 out of 24) of the isolates had cultural and biochemical characteristics similar to those of strains of the biovar 1 of A. tumefaciens. Twenty out of 22 Agrobacterium isolates induced tumour formation on various test plants. In PCR experiments, DNA extracted from these virulent strains yielded an amplification signal corresponding to a 247-bp fragment located within the virulence region of nopaline type Ti plasmid. Consistent with this, the opine nopaline was detected in the tumours induced on test plants – but not on eucalyptus plants. Nopaline was degraded by the 20 pathogenic isolates that were also sensitive to agrocin 84, indicating the presence of a nopaline-type pTi in these strains. The chromosomal region encoding the 16S rRNA was analyzed in a sub-population of the pathogenic agrobacterial isolates. The analyzed strains were found to belong to the ribogroup of the reference strain B6. Interestingly, Eucalyptus camaldulensis and Eucalyptus cladocalyx grown in the same nursery and in the same soil substrate developed no galls.

Agrobacterium tumefaciens strain A281 carrying pTiBo542 was described as able to induce large and early-appearing tumours on a wider range of plants than other Agrobacterium strains. Here we show a strict correlation between the supervirulence of A281 and super-transformation ability of its disarmed derivative EHA105/p35SGUSINT. This strain produced much higher transformation than the disarmed derivative of the common strain C58, called C58(pMP90)/p35SGUSINT. Transformation frequency increased from six to 22-fold, depending on the citrus genotype. This increase permitted to regenerate for the first time, whole transgenic plants from lemon, alemow and Cleopatra mandarin. Since the chromosomal background from C58 as well as the T-DNA from p35SGUSINT are identical for both bacterial strains, the super-transformation ability of EHA105/p35SGUSINT in citrus may be attributed to the vir region of pTiBo542. In addition, introduction of extra copies of virG from pTiBo542 into either EHA105/p35SGUSINT or C58(pMP90)/p35SGUSINT further increased transformation, demonstrating the importance of vir induction, and mainly virG activation, in the super-transformation ability of this Ti plasmid in citrus.

Little is known about the consequences of releasing genetically engineered plants (GEP) into the environment. Using opine-producing GEP, we show that transgenic plants alter their biological environment, more precisely the root-associated bacterial populations. The alterations were both transgene-specific and target population-specific. Therefore, assessment studies on the introduction of a given transgene into a GEP will be valid on the given transgene. Evidence of any transgene-associated biological effect will depend on the determination of the pertinent target populations, the identification of which is a key step of such studies.

Hairy root formation was induced by inoculating epicotyl, cotyledon and hypocotyl of Allocasuarina verticillata with two strains of Agrobacterium rhizogenes, A4 (agropine type) or 2659 (cucumopine type). Shoot regeneration from hairy roots induced by strain A4, and from control roots excised from untreated seedlings, required a hormone-enriched medium, whereas roots induced by strain 2659 spontaneously regenerated shoots on hormone-free medium. Newly formed shoots were multiplied and rooted to produce transgenic plants that developed unusually extensive, ageotropic root systems. The transformation was demonstrated by the presence of specific opines and by Southern blot analysis.

In octopine-type A. tumefaciens R10, transfer of chromosomal arginine degradation genes (arc genes) was observed under conditions in which Ti plasmid transfer took place. However, transconjugants that had acquired the arc genes but not the Ti plasmid were recovered. During this process, several other chromosomal genes, such as genes encoding phage resistances or genes complementing a galactose utilization mutation or a glycine-serine auxotrophy, were transferred from strain R10 to the recipient.

Independent carrot (Daucus carota) hairy root lines were established by inoculation of discs taken from the same carrot with Agrobacterium rhizogenes 8196 and A. tumefaciens C58C1(pRi8196) carrying pRi8196. Several lines were compared with respect to T-DNA length. One of them was found to have integrated sequences covering more than 50 kbp of the Ri plasmid.

A procedure for transformation and regeneration of the legume species Lotus corniculatus (Bird's-foot trefoil) has been developed. The Agrobacterium rhizogenes 15834 and 8196 strains were used to transform plant cells in wound site infections and transformed roots were propagated in vitro. Transformation was monitored by hybridization with pRi T-DNA sequences and by detection of agropine and mannopine. Transformation frequencies of up to 90% were obtained. Shoots spontaneously formed on hairy root cultures were excised, rooted and inoculated with Rhizobium. Root nodules formed on transformed plants had nitrogenase activities comparable to untransformed nodules. Transcript levels from the nodule-specific leghemoglobin genes and the constitutive ubiquitin genes were similar in transformed and untransformed root nodules. Transformed plants responded to R. loti and Bradyrhizobium sp. (Lotus) strains with phenotypes identical to phenotypes for untransformed plants.

Inoculation of carrot discs and Lotus corniculatus plantlets with mixtures of different Agrobacterium rhizogenes or of A. rhizogenes and A. tumefaciens or with Agrobacterium strains harboring both an Ri and a modified Ti plasmid resulted in frequent multiple (pluribacterial) transformation of cells, as revealed by the mixed opine-type of hairy roots arising from them. Multiple transformation may account for the presence of dispersed T-DNA inserts in crown gall and hairy root lines. A plant genetic engineering strategy based on segregation of T-DNA inserts in the progeny of multiple transformants is proposed.

The crown gall and the hairy root diseases which affect dicotyledonous plants are caused by the pathogenic soil bacteria Agrobacterium tumefaciens and A. rhizogenes. In these organisms genes responsible for pathogenicity are borne on large plasmids (200–400 kb) called respectively Ti plasmids (Tumor inducing) and Ri plasmids (Root inducing) (Zaenen et al., 1974; Van Larebeke et al., 1974, 1975; Watson et al., 1975; White and Nester, 1980 a; Costantino et al., 1981; Chilton et al., 1982; Petit et al., 1983). Both diseases afflict crop plants and produce tumorous or rooty overgrowths which usually develop on the roots or at the crown of the plant, or more rarely on stems. In the laboratory, inoculation of a supsension of virulent bacteria produces typical symptoms (Fig. 1). The molecular basis for pathogenicity is the transfer, integration, and expression of a segment of Ti or Ri plasmid DNA into the nuclear genome of the host cells (Chilton et al., 1977, 1980, 1982; Schell et al., 1979; Lemmers et al., 1980; Thomashow et al., 1980; Willmitzer et al., 1980, 1982; Spano et al., 1982; White et al., 1982). This segment, called T-DNA (Transferred DNA) carries several genes which confer upon the plant cell a specific phenotype (Garfinkel et al., 1981; Willmitzer et al., 1982; Leemans et al., 1982, see also chapter 11).

Primary hairy root tissues as well as aseptic hairy root culture lines contain specific compounds that have been biologically characterized as opines. These substances are agropine, mannopine, mannopinic acid, and agropinic acid; they have been synthesized and their electrophoretic behavior has been studied. Hairy root tissues also contain agrocinopines. According to the opine content of hairy root tissues, two types of Agrobacterium rhizogenes strains have been identified. Agropine-type strains (A4, 15834, HRI) elicit roots containing agropine, mannopine, mannopinic acid, and agropinic acid, whereas mannopine-type strains (8196, TR7, TR101) elicit roots containing only mannopine, mannopinic acid and agropinic acid. A. rhizogenes strains catabolize the opines whose synthesis they induce in the hairy root tissues. However, strain HRI only catabolizes agropine. Except for strain HRI, all A. rhizogenes strains studied contain three plasmids, of which the largest appears to be a cointegrate of the two others. Transconjugants of A. rhizogenes plasmids in A. tumefaciens have been obtained by selection on opines. Their properties have been studied and related to their plasmid content. In the mannopine strain C58C1(pRi8196), the virulence functions and the opine-related functions are located on the same plasmid (pRi8196). In agropine strains the catabolic functions are dissociated: agropine degradation is specified by the virulence plasmid, which also specifies opine synthesis in hairy root tissue, however, mannopine, mannopinic acid and agropinic acid degradation are specified by the smaller plasmid. Strain HRI contains only the virulence plasmid, which explains its inability to degrade mannopine, mannopinic acid, and agropinic acid.

Publisher Summary This chapter explains the opine utilization by Agrobacterium. Utilization of opines by bacterial strains has been assayed in different ways: (1) by measuring the uptake of substrates in the bacterial cells, (2) by following the fate of the substrates in the supernatant of incubation mixtures or culture media, and (3) by assessing the ability of a strain to grow on media containing the opine as the sole carbon and/or nitrogen source. The pathway of octopine and lysopine degradation has been characterized as active transport into the bacterial cell, and cleavage of the molecule to the parent amino acid and α-keto acid. Opine oxidase is the enzyme responsible for the cleavage of opine. The degradation of the other opines of the octopine family as well as that of nopaline and nopalinic acid probably follows the same pathway, as mutants unable to utilize octopine fail to degrade lysopine, octopinic acid, and histopine.

The conjugative behaviour of nopaline and agropine Ti-plasmids has been investigated. Using a technique which avoids enrichment of transconjugants on a mating medium we have shown that preculture in the presence of agrocinopines A or B of donor strains harbouring nopaline Ti plasmids promotes plasmid transfer whereas preculture of the same strains in the presence of nopaline has no such effect. Similarly, preculture in the presence of agrocinopines C or D promotes Ti-plasmid transfer from strains harbouring agropine Ti-plasmids.

Agrobacterium Ti (tumor-inducing) plasmids, the causative agents of crown gall disease, fall into four genetic groups based on the patterns of octopine and nopaline synthesis (by crown gall tumors) and catabolism (by Agrobacterium tumefaciens) for which they are responsible. Two classes of Ti plasmids induce tumors that synthesize neither octopine nor nopaline. The existence of these Ti plasmids challenged the view that opines such as octopine and nopaline play a central role in crown gall biology. We now report the occurrence of an opine in tumors induced by one of these classes of Ti plasmids, the "null-type" plasmids typified by pTi Bo542. The opine was purified by biological enrichment based on its utilization by bacteria containing pTi Bo542 but not by bacteria lacking a Ti plasmid. The mass spectrum and biological properties of this opine are identical to those of agropine, an opine recently discovered in octopine-type tumors. We propose that null-type Ti plasmids now be named for their signal opine, agropine-type Ti plasmids.

Reduction of the Schiff bases formed between glucose, mannose or galactose with glutamic acid yields products related both structurally and biologically to agropine and to a derivative of agropine present in crown gall tumours. The catabolism of these compounds is coded for by octopine and agropine Ti-plasmids of Agrobacterium tumefaciens.

The oncogenic plasmids of Agrobacterium, the Ti-plasmids, carry genes that enable their bacterial host to catabolize opines. Opines are unusual amino acid derivatives that are only produced in crown gall tumours incited by oncogenic strains of Agrobacterium. The 2 opines, octopine and nopaline, are degraded by Agrobacterium strains carrying the octopine or the nopoline Ti-plasmid, respectively, to arginine and pyruvic acid, and to arginine and α-ketoglutaric acid. In this paper it is shown that the Ti-plasmids carry gene(s) involved in the utilisation of arginine as a carbon source. Strains harbouring wild type octopine or nopaline Ti-plasmids in the chromosomal context of strain C58C1 do not grow on arginine as a carbon source. However, they are able to grow on arginine provided that they are induced, or constitutive for opine catabolism. The features of ornithine utilisation are identical. The gene(s) involved in arginine and ornithine utilization in C58C1 (pTi-oct) or C58C1 (pTi-nop) are under the control of the regulator gene that controls octopine or nopaline catabolism. A tentative pathway of octopine utilization is proposed, in which at least two steps are Ti-plasmid coded, and probably belong to the same operon: 1-scission of octopine into arginine and pyruvic acid 2-transformation of an arginine derivative (GSA?) to glutamic acid.