Independent integration and seed-transmission of the TR-DNA of the octopine Ti plasmid pTi Ach5 in Nicotiana plumbaginifolia

Summary After co-cultivation of diploid Nicotiana plumbaginifolia protoplasts with an octopine-type Agrobacterium tumefaciens strain (LBA 4013) putative transformants were selected for hormone-independent growth, and were tested for T-DNA markers. The number of transformants expressing only T_L-DNA markers, i.e. phytohormone autotrophy and octopine synthase, was an order of magnitude higher than that of the cell lines which were simultaneously positive for both T_L- and T_R-DNA markers (the latter being mannopine and agropine). In one transformant, line no. 101, only the T_R-DNA markers were found. Not each of the T_L-, or T_R-DNA markers were expressed in each transformant resulting in a variety of phenotypes. It included the unorganized or the shoot-teratoma type of growth combined with the presence or absence of opines; e.g. agropine was absent from some of the transformants containing its precursor, mannopine. 5-Azacytidine did not induce agropine synthesis in these lines. Southern blot analysis showed that the T_R-DNA region coding for agropine synthesis was rearranged or absent in one of these lines. Similar variation in the expression of agropine and mannopine production was observed in transformants obtained with the leucinopine-type strain A281.From line 101 plants could be easily regenerated with the ability to synthesize agropine and mannopine. The segregation in the self-progeny fitted to a 3:1 ratio, indicating that the T_R-DNA was carried by a single chromosome. The Southern blot analysis showed that only opine-positive plants contained T_R-DNA. It also confirmed the absence of the T_L-DNA, demonstrating the independent integration of the T_R-region of the octopine-type Ti plasmid pTi Ach5.     

Silent T-DNA genes in plant lines transformed by Agrobacterium tumefaciens are activated by grafting and by 5-azacytidine treatment

Summary A shooty tumor induced by a shooter mutant of an octopine strain of Agrobacterium tumefaciens was cloned. One clone obtained (TS038) behaved aberrantly in that it grew as a shooty tumor tissue on phytohormone free medium, but did not contain octopine synthase activity. In line TS038 the genes for octopine synthase and for the enzymes involved in agropine and mannopine synthesis were present, but were not transcribed. However, the above genes became active in TS038 tumor shoots after grafting as well as after treatment with the hypomethylating agent 5-azacytidine. After an unusually long incubation period in the growth cabinet shoot cultures appeared to have developed small shoots from the top of the leaves. This unusual form of differentiation was found to be accompanied by the induction of octopine synthase activity.     

Agrobacterium tumefaciens TR-DNA encodes a pathway for agropine biosynthesis

Summary A pathway for biosynthesis of the crown-gall opine, agropine is proposed and three potential new precursors characterised. The location of genes involved in the three steps of this pathway was determined by site directed insertions and deletions in the T_R region of the octopine Ti plasmid, pTiB6Tra^c. The proposed biosynthetic pathway for agropine which involves three T-DNA genes is in contrast to the biosynthesis of octopine and nopaline where single T-DNA genes are involved.     

Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization,T-DNA methylation and conditional expression of opine genes

Summary Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut⁺), shoot regeneration (Reg⁺) and octopine synthesis (Ocs⁺)), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut⁺Reg⁺ but shows little or no octopine synthesis activity (Ocs^-). Subclones of TSO38, however, are either Ocs^- or Ocs⁺. Ocs^- shoots become Ocs⁺ under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs^- subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs⁺ and an Ocs^- subclone of TSO38, which were negative for the presence of mannopine (Mas^-) and agropine (Ags^-), became Mas⁺Ags⁺ after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs^- line and in the Ocs⁺ line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs^- and an Ocs⁺ TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs⁺ and an Ocs^- subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs^- and the Ocs⁺ TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.     

Diversity of Opines and Opine-Catabolizing Bacteria Isolated from Naturally Occurring Crown Gall Tumors

The diversity of opines from 43 naturally occurring crown gall tumors on several plant species was analyzed for the presence of agropine, chrysopine, iminodiacid, an unidentified leucinopine-like iminodiacid (IDA-B), mannopine, octopine, nopaline, DL- and LL-succinamopine, leucinopine and heliopine. Opine utilization patterns of agrobacteria and fluorescent pseudomonads resident in a tumor were then analyzed and compared for agreement with the opine isolated from that tumor. Nopaline was the most common opine found and was detected in tumors from cherry, blackberry, grape, and plum. Octopine was not found, although octopine-catabolizing bacteria were isolated from several tumors. A new, previously undescribed iminodiacid of the succinamopine-leucinopine type (provisionally designated IDA-B) was isolated from tumors of wild blackberry. Field tumors from apple, blueberry and grape yielded no detectable opines, even though opine-utilizing bacteria were present. Bacterial isolates from plum and cherry showed the best correspondence between the opine in tumors (nopaline) and the presence of bacteria that catabolized that opine. However, several unusual opine catabolic combinations were identified, including isolates that catabolized a variety of opines but were nonpathogenic. More variability was observed among isolates from field tumors on the remaining plant species. We isolated novel mannopine-nopaline type agrobacteria from field tumors of cherry, plum and blackberry that induced tumors containing either mannopine (plus agropine) or nopaline, but not both. Epidemiologically, the galled plants from an area were not of clonal origin (same Ti plasmid), indicating that the field tumors from a small area were incited by more than one type of Ti plasmid.     

Crown gall transformation of lentil (Lens culinaris Medik.) with virulent strains of Agrobacterium tumefaciens

Summary Four diverse strains of Agrobacterium tumefaciens (C58, Ach5, GV3111, and A281) were capable of inducing tumors at a high frequency on inoculated stems of lentil (Lens culinaris Medik. cultivar Laird) in vivo, and on excised shoot apices in vitro. GV3111 and Ach5 produced the largest and heaviest tumors in vivo, while A281 produced the heaviest tumors in vitro. Tumor formation and opine production are indicative of plant cell transformation and tumors produced appropriate opines: nopaline (C58), octopine (Ach5 and GV3111), and agropine and mannopine (A281). Southern analysis of DNA from a tumor line produced by strain C58 showed that a T-DNA fragment had been transferred into the lentil genome.     

Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4

Summary The vitopine Ti plasmid pTiS4 of Agrobacterium vitis has an unusual T-DNA organization. The pTiS4 oncogenes, localized by screening selected pTiS4 clones for growth-inducing activity, are localized on three T-DNAs, whereas in all other characterized Ti plasmids one or two T-DNAs are found. The nucleotide sequences and predicted amino acid sequences of the pTiS4 oncogenes set them apart from the corresponding genes from other Ti or Ri plasmids. The oncogenes induce the same type of reaction on various test plants as the well-known pTiAch5 oncogenes but the pTiS4 ipt gene induces considerably more shoots than its Ach5 homologue. We have also identified the gene coding for vitopine synthase as well as a vitopine synthase pseudogene. Both sequences show homology to the octopine synthase gene. In terms of both nucleotide sequence and overall organization, the pTiS4 T-DNAs appear to be only distantly related to previously characterized T-DNAs.Abbreviations Ap ampicillin - IS insertion sequence - iaaH indole acetamide hydrolase - iaaM tryptophan monooxygenase - ipt isopentenyl transferase - Km kanamycin - LB Luria broth - m/a mannopine/agropine - o/c octopine/cucumopine - ocs octopine synthase     

The opine synthase genes carried by Ti plasmids contain all signals necessary for expression in plants

Signals necessary for in vivo expression of Ti plasmid T-DNA-encoded octopine and nopaline synthase genes were studied in crown gall tumors by constructing mutated genes carrying various lengths of sequences upstream of the 5' initiation site of their mRNAs. Deletions upstream of position -294 did not interfere with expression of the octopine synthase gene while those extending upstream of position -170 greatly reduced the gene expression. The estimated size of the octopine synthase promoter is therefore 295 bp. The maximal length of 5' upstream sequences involved in the in vivo expression of the nopaline synthase gene is 261 bp. Our results also demonstrated that Ti plasmid-derived sequences contain all signals essential for expression of opine synthase genes in plants. Expression of these genes, therefore, is independent of the direct vicinity of the plant DNA sequences and is not activated by formation of plant DNA and T-DNA border junction.     

Octopine Accumulation Early in Crown Gall Development is Progressive

A purification of octopine from crown gall tissue was developed to quantitate conversion of precursor [³H]arginine into [³H]octopine. Plant wound tissue which was sterile or infected with an avirulent strain of Agrobacterium tumefaciens did not accumulate detectable quantities of octopine, consistent with opine synthesis not being induced by wounding or infection. Octopine was only recovered from tissue infected with virulent tumor-inducing strains of A. tumefaciens. In every case tested, the morphological appearance of tumors preceded the accumulation of octopine by at least 1 week, and in some instances 3 weeks. Thus, what was necessary and sufficient for the expression of plant hormones (auxin and cytokinin) required for tumor growth was not sufficient for the accumulation of octopine. The possible nature of the temporal difference in the expression of hormone autotrophy and octopine synthesis is discussed.     

Octopine- and Nopaline-Inducible Proteins in Agrobacterium tumefaciens Are Also Induced by Arginine

Octopine induced the synthesis of 83, 76, 62, 58, 44, 42, 31, and 22 kDa proteins in Agrobacterium tumefaciens strains harboring the tumor-inducing (Ti) plasmids pTiA6 and pTiAch5. Nopaline induced the synthesis of 83, 76, 62, 58, 56, 44, 42, 31, and 22 kDa proteins in A. tumefaciens strains harboring the Ti plasmids pTiC58 and pTiT37. The molecular masses of proteins induced by octopine and nopaline were very similar. In accordance with the ‘opine concept’, octopine and nopaline were found to induce protein synthesis only in strains harboring the respective Ti plasmids. Arginine, a common catabolic product of octopine and nopaline, induced the synthesis of most of the proteins induced by the two opines. Our results show that only the initial step(s) of octopine and nopaline catabolism are induced by specific opines in the respective strains. The subsequent steps are likely to be regulated by arginine in both strains. Received: 5 January 1996 / Accepted: 21 February 1996     

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

Summary 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.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Brassica grown gall tumourigenesis and in vitro of transformed tissue

A number of Brassica species and cultivars were tested and found to be highly susceptible to crown gall induction by both nopaline and octopine strains of Agrobacterium tumefaciens. Only B. napus did not form tumours when inoculated with octopine strains. Seedlings of very young plants were poor hosts but efficient infection occurred after 8–10 weeks of growth. Teratomas arising on tumours in planta were relatively frequent on induction with nopaline strains. Axenically cultured tumour calli of Brassicas were very active in opine synthase activity and stably maintained this transformed phenotype; however, transformed plants could not be regenerated. These results suggest that disarmed nopaline Ti plasmid vectors are well suited for the genetic engineering of this important crop family.     

Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation

Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 μM) to an OD₆₆₀ ≅ 0.6, diluted to a density of 10⁹ cells ml⁻¹, followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 μg ml⁻¹). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T _L -DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T _R -DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation.     

Restriction map of an agropine-type Ri plasmid and its homologies with Ti plasmids

The Ri plasmid of an agropine-type Agrobacterium rhizogenes, strain HRI, was cloned in a cosmid and mapped with the restriction endonucleases BamHI, EcoRI, KpnI, SmaI, and XbaI. This plasmid is almost identical to pRi1855 and pRiA₄b. A study by Southern hybridizations of the homologies with octopine pTiB₆806 and nopaline pTiC58 makes it possible to propose the localization of certain functions on this plasmid, such as virulence, agropine catabolism, agropine synthesis, and the origin of replication.     

Segregation in the progeny of transformed rapeseed (Brassica napus)

The primary transformant of spring rapeseed cv. HM-81 contained TL- and TR-DNA of agropine plasmid pRi ofAgrobacterium rhizogenes 15834. The presence of TL-DNA corresponds to visible transformed phenotype in its progeny; the leaves are wrinkled and the plants are shorter than normal plants. R₁ R₂ and R₃ generations have mostly transformed phenotype. The normal phenotype appears in a low frequency in F₁ generation. Autogamised F₁ plants segregate in F₂ transformed and normal phenotype in 3:1 ratio. It is possible to suppose that TL-DNA is present in two differentloci of one pair of homologic chromosomes. The recombination frequency is 12 % (microsporogenesis) or 6 % (microsporogenesis and macrosporogenesis). In some crosses the transformed phenotype has a maternal type of inheritance. Maternal inheritance influences also several growth characteristics,e.g. length of plants and number of seeds/pods.     

Specificity of Octopine Uptake by Rhizobium and Pseudomonas Strains

The octopine-utilizing strain Agrobacterium tumefaciens B6S3 and three nonagrobacteria which had the capacity to utilize this opine were compared for octopine uptake. The characteristics of uptake by Rhizobium meliloti A3 and strain B6S3 were similar. In both bacteria, uptake activity was inducible by octopine and by the related opine octopinic acid, and competition assays showed that these two opine substrates were accepted by the same uptake system with an equivalent affinity. Cells of Pseudomonas putida 203 accumulated octopine against a concentration gradient, and this activity was induced specifically by octopine. While strain 203 did not utilize octopinic acid, a spontaneous mutant with a combined capacity for octopine and octopinic acid utilization was obtained. Both opines induced octopine uptake by this mutant, but octopinic acid was not a substrate for the induced system. Thus, the Pseudomonas uptake system exhibited a different specificity for octopine than the corresponding Agrobacterium system. The nonfluorescent pseudomonad GU187j, which utilized the three related opines octopine, octopinic acid, and nopaline, was constitutive for octopine uptake. Strain GU187j possessed a system which accepted these three opines, but not arginine or ornithine, with a similar affinity.     

Genetic studies on the role of octopine T-DNA border regions in crown gall tumor formation

Summary Crown gall tumors result from transfer and integration of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant nuclear DNA. In the present study, recombinant plasmids containing deletion and rearrangement deriviatives of the T-DNA region of the octopine Ti plasmid pTiA6 were tested in a binary tumorigenesis system (Hoekema et al. 1983) to determine the requirements for T-DNA border regions in tumor formation. Since two defined segments of the T-DNA region of octopine Ti plasmids can be detected in tumor DNA (the left (T_L-) and right (T_R-) DNA), four border regions exist in this Ti plasmid. Agrobacteria harboring plasmid constructs which contain a T-DNA gene capable of inciting tumors (gene 4, the tmr gene, which is involved in cytokinin biosynthesis) and various T-DNA border regions were tested for ability to cause tumors on Nicotiana glauca and other host plants. Such tmr constructs containing as their only border region the right border of either the T_L-DNA or the T_R-DNA are fully tumorigenic. Analogous tmr constructs containing only the T_L-DNa left border region are not tumorigenic. These results do not depend on the orientation or position of the single border with respect to the tmr gene; furthermore, the T_R-DNA right border can confer tumor-forming ability despite the presence of an intervening copy of the T_L-DNA left border.These results for relatively small plasmids are contrasted with previously determined requirements for border regions in tumorigenesis by intact Ti plasmids. A model previously proposed by Wang et al. (1984) for the role of border regions in DNA transfer to plant cells is extended in order to explain the tumor-forming ability of plasmid constructs containing a single border region. The results of this study interpreted according to the model suggest that the octopine T_L-DNA left border is defective in this DNA-transfer process.     

Structure and characterization of the crown gall opines heliopine, vitopine and ridéopine

The crown gall opines heliopine from tumors induced by octopine type Agrobacterium tumefaciens strains A6, A136(pTiB6-806), E9, A652 and 1590-1 and vitopine from tumor induced by grapevine strains S4 and T2 are identical to synthetic N2-(1'R-carboxyethyl)-L-glutamine. Tumors produced by strains S4 and T2 do not contain octopine or lysopine, but they do contain heliopine and the new opine ridéopine identified as N-(4'-aminobutyl)-D-glutamic acid. Grapevine strains S4 and T2 grow normally on tumor heliopine or synthetic heliopine and on tumor and synthetic ridéopine as well as on ridéopine lactam as sole carbon source. While octopine strains A6 and A136(pTiB6-806) do not grow on heliopine, mutant colonies do appear after a few weeks. Heliopine catabolism by octopine strains is not induced by octopine.     

Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation

Summary The 200 kb Agrobacterium Ti-plasmid pTiT37 carries a 25 kb segment of T-DNA which it transfers to plant cells during crown-gall tumorigenesis. We have previously engineered into this T-DNA a pBR322-derived cloning vector which enabled us to rescue-clone full length T-DNA from the Ti-plasmid into a 36 kb MINI-Ti plasmid. We report here the deletion of oncogenes from MINI-Ti to produce Micro-Ti containing the nopaline synthase gene and the ampicillin resistance gene and origin of replication of pBR322, flanked by left and right T-DNA borders. Micro-Ti was recloned into the wide host range plasmid pRK290 and transformed into an A. tumefaciens strain carrying a helper plasmid that could supply Virulence (VIR) genes in trans. Using the octopine Ti-plasmid pTiB6-806 as a helper, transformed tobacco cells were obtained which produced both nopaline and octopine. Two cloned cell lines producing both opines were found to be hormone dependent and to produce fertile tobacco plants. We selfed one of these plants and found that the two opine markers segregated in the F1 progeny in a Mendelian fashion. This showed that the T-DNAs were not linked in the transformed plant genome. Southern blot analysis of the genomic DNA from the regenerated plant showed that only part of the (oncogenic) octopine T-DNA was present indicating that it had suffered a deletion in the auxin producing locus (tms region). Presence of the cytokinin autonomy locus presumably accounts for the abnormal rooting behavior of the F1 progeny seedlings containing this T-DNA.Abbreviations NAA Naphtalene acetic acid - IAA Indole-3-acetic acid - BA 6-benzylaminopurine - pCPA para-chlorophenoxyacetic acid Part of this work was presented for her doctoral thesis by A. JdF at the National Institute of Agronomy of Paris-Grignon, January 1983     

Selection of somaclonal variants with low-temperature germinability in melon (Cucumis melo L.)

Summary Plants were regenerated from adventitious buds and somatic embryos (R₀) of melon (Cucumis melo L.), the cultivar Andes. Somaclonal variants of melon with low temperature germinability were selected from the progenies (R₁) of R₀ plants. Among 5,618 R₁ seeds harvested from 23 R₀ plants that were regenerated from adventitious buds 4 seeds germinated after 5 days of culture at 15 °C (selection rate; 0.07%). However, among 374 R₂ seeds harvested from 2 R₁ plants no seed germinated after 7 days of culture at 14 °C. Among 9,181 R₁ seeds harvested from 50 R₀ plants regenerated from somatic embryos 110 seeds germinated after 5 days of culture at 15 °C (selection rate; 1.20%). Among 3,717 R₂ seeds harvested from 17 R₁ plants 113 seeds germinated after 7 days of culture at 14 °C (selection rate; 3.04%). R₃ seeds were collected from these R₂ plants following self-pollination. Forty-five of the 47 lines (R₃) originated from 10 R₀ plants showed higher germination rates than that of the original cultivar. Selected lines with low-temperature germinability showed greater fruit growth rate than the original cultivar during the middle stage when they were cultivated in a greenhouse under low-temperature conditions. Of fruits harvested from 31 lines, 15 lines showed greater fruit volume than the original cultivar.