Detection of nopaline dehydrogenase and lysopine dehydrogenase activities in crown gall tumors of different plant species

The LpDH and NpDH activities in crown gall tumors incited byAgrobacterium tumefaciens strain C58 were followed in 7 plant species and 100 tumors were assayed in each experimental variant. The NpDH activity was found in 790 out of 800 crown galls observed. The LpDH activity was tested, after induction of tumors withA. tumefaciens strain B6-806 and 37400, in three experimental variants. The LpDH activity was found in 290 out of 300 crown galls. In the small fraction of the LpDH and NpDH negative tumors, the activity was possibly actually present, but it was below the limits of the sensitivity of the detection technique used.     

Conditioned medium promotes the attachment ofAgrobacterium tumefaciens strain NT 1 to carrot cells

Summary Wild-typeAgrobacterium tumefaciens bind to carrot suspension culture cells. Avirulent strain NT 1 did not bind to carrot cells when they were incubated together in Murashige and Skoog medium. Conditioned medium was prepared by incubatingA. tumefaciens virulent strain C 58 with carrot cells and removing the bacteria and carrot cells using filter sterilization. This conditioned medium promoted the binding of NT 1 to carrot cells. Conditioned medium did not promote the nonspecific attachment ofEscherichia coli to carrot cells. These results suggest that when wild-typeA. tumefaciens are incubated with plant host cells, some substance(s) involved in bacterial attachment are released into the medium. Filter-sterilized medium from the incubation of the nonattachingchvB mutant A 1045 with carrot cells promoted the attachment of strain NT 1 even though A 1045 bacteria did not bind to the carrot cells. However, filter-sterilized medium from the incubation of the non-attachingatt mutant Att-B 123 with carrot cells was unable to promote the binding of strain NT 1. This suggests that nonattaching mutants ofA. tumefaciens can be divided into two groups on the basis of the properties of the substances released into the medium when the bacteria are incubated with carrot cells.Abbreviations MS Murashige and Skoog tissue culture medium Dedicated to the memory of Professor John G. Torrey     

Monoclonal antibodies againstAgrobacterium tumefaciens strain C58

Hybridoma cell lines were derived from the fusion of mouse myeloma cells with spleen cells from a mouse that had been immunized withAgrobacterium tumefaciens strain A759, a flagella-less mutant of strain C58 containing the Ti plasmid of strain B6. All of the 20 antibodies produced by the cloned hybridomas reacted with strain C58 and with other strains derived from C58. The antibodies did not react with 34 other strains ofA. tumefaciens, representing the three biovars, or with strains ofA. radiobacter, A. rubi, Rhizobium leguminosarum, R. meliloti, or other plant-associated bacteria such asErwinia herbicola andPseudomonas syringae. In addition to reacting with whole cells of strain A759, the antibodies reacted with phenol-water extracts of A759, indicating that they may recognize the lipopolysaccharide. These antibodies may be useful for ecological and epidemiological studies ofA. tumefaciens strain C58 in the agroecosystem.     

Localization and restriction maps of the replication origin regions of the plasmids of Agrobacterium rhizogenes strain A4

Summary Banks of cosmids of the plasmids of the agropine-type Agrobacterium rhizogenes strain A₄ were used to transform Agrobacterium tumefaciens strain C58C1, which lacks a Ti plasmid. Hybrid cosmids able to be maintained in this strain were subcloned to localize precisely the origin of replication regions. These regions were mapped with restriction enzymes and compared by hybridization with those of Agrobacterium tumefaciens nopaline pTiC58 and octopine pTiAch5. This led to the characterization of three new plasmids suitable as cloning vectors in Escherichia coli and A. tumefaciens. They are compatible with pTi and pAt plasmids of A. tumefaciens and are maintained stably, even without selection pressure.     

Reduced rhizosphere colonization ability ofAgrobacterium tumefaciens chromosomal virulence (chv) mutants

Mutations in the chromosomal virulence (chv) region ofA. tumefaciens strain A723 reduce virulence, motility, and ability of the bacteria to bind to plant cells. We conducted experiments to assess the ability ofchv mutants to colonize the rhizosphere ofPisum sativum. The mutation had no effect on ability of bacteria to grow with a defined number of root cap cells as the sole carbon and nitrogen source. Ten days after inoculation, there were up to 103-fold more wild type thanchv mutant bacteria present in the rhizosphere of inoculated plants.     

Significance of Pyruvate Carboxylase in Sugar Metabolism of Agrobacterium tumefaciens

Mutants, which fail to grow on glucose medium but can grow on succinate medium, were isolated by treatment with N-methyl-N′-nitro-N-nitrosoganidine from the wild-type strain of Agrobacterium tumefaciens, and were found to lose growth on several hexoses and three-carbon intermediates. The revertant mutants, which recovered the ability to grow on glucose medium, simultaneously regained the ability to grow on hexoses and three-carbon intermediates. By comparison of biochemical properties of the wild-type, the mutants and the revertant mutants, two mutant strains were characterized to be pyruvate carboxylase-deficient. Then, we concluded that these mutants might be induced by a single mutation at a genetic locus of pyruvate carboxylase and that the deficiency in the enzyme gave a pleiotropic effect on the ability to grow on hexoses and three-carbon intermediates. Some properties of pyruvate carboxylase of this bacterium were also presented.     

Lysogeny among mycobacteria

Our investigations to detect naturally lysogenic strains of mycobacteria were limited to 1 strain ofMycobacterium smegmatis, 4 strains ofMycobacterium borstelense var.niacinogenes, and to 5 strains ofMycobacterium marinum (Syn:Mycobacterium balnei), all together 10 strains. They were chosen because as a sign of lysis they secrete a large quantity of cytoplasmatic components (nucleic acids proteins, amino acids etc.) into the fluid medium (for instance phosphate buffer), in which they are suspended. In a first series of experiments culture filtrates were tested on 84 strains of slowly and rapidly growingMycobacterium species as indicator strains. Using this method free phage particles were only found in the culture filtrate of 1 strain,Mycobacterium smegmatis SN 46, isolated from a patient with achalasia. Phage particles could not be found in the filtrates of the other 9 probably lysogenic strains. In a second series of experiments more closely related indicator strains were used. The 10 probably lysogenic strains were cultured in bovine serum or antiphage-antiserum containing medium and single selected colony cultures a small part of which showed sensitivity to the filtrates. The released and adapted phages, designated as B₂₄, B₃₀, B₃₂, B₃₃, B₃₄ and B₃₅ have a very narrow host range. The plaques are very small and turbid. On electron micrographs the temperate phages B₂₄, B₃₀ and B₃₅ exhibit the typical head-tail morphology. The head of the temperateborstelense var.niacinogenes phage B₃₀ is 45 nm in diameter, the tength of tail is about, 120nm. The average dimensions of the long head ofsmegmatis phage B₂₄ are 40 × 80 nm, the tail is about 160 nm long. The balnei phage B₃₅ is very similar morphologically to phage B₃₀. The head is about 50 nm in diameter, the length of tail about 160 nm. The phage sensitive variants are not “carrier” strains. Their phage sensitivity is not a stable property. After several culture passages in serum-free medium the variants regain their phage immunity completely and release phages like the lysogenic parent strains. The sensitive variants must therefore be considered to be also lysogenic. TheMycobacterium borstelense var.niacinogenes phages are serologically very related. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

Transgenic broccoli expressing aBacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F₁ hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F₁ larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.     

Genetic transformation ofPelargonium X hortorum

Summary TransgenicPelargonium X hortorum have been producedvia Agrobacterium tumefaciens-mediated transformation. The regeneration protocol used provided a regeneration frequency approximately to 95 percent. Clumps of regenerants, from cotyledons and hypocotyls ofPelargonium X hortorum seedlings, were inoculated with the disarmed strain EHA101 ofAgrobacterium tumefaciens. This strain contains a binary vector carrying neomycin phosphotransferase II, hygromycin B phosphotransferase and ß-glucuronidase genes. Selection on the regeneration medium supplemented with hygromycin allowed production of transgenic plants in up to 20% of the inoculated explants. The insertion of foreign DNA was demonstrated by Southern and polymerase chain reaction analysis: these experiments indicated that the inserted T-DNA is not full length for most of the plants. All RO transgenic plants exhibited a normal phenotype and are fertile.Abbreviations GUS ß-glucuronidase coding sequence - PCR polymerase chain reaction - CaMV cauliflower mosaic virus - NPTII neomycin phosphotransferase coding sequence - NOS nopaline synthase gene promoter and terminator - HPH hygromycin B phosphotransferase coding sequence - SDS sodium dodecyl sulphate - EDTA (ethylenedinitro trilo)tetra-acetic acid disodium salt     

Isolation of Agrobacterium Ti-plasmid regulatory mutants

Summary Crown gall tumors incited by Agrobacterium tumefaciens synthesize basic amino acid derivatives called opines. Opine production in tumours and opine catabolism by A. tumefaciens are coded by Ti-plasmids which confer oncogenicity on this bacterium. Catabolism of opines is inducible, and a method for isolation of regulatory mutants is described. From octopine-type bacteria, by plating on non-inducing substrates (noroctopine, noroctopine acid, D-histopine) we have isolated regulatory mutants of three types: constitutive, partially constitutive, and fully inducible by the analogue. From nopaline-type bacteria, by plating on octopine (a non inducing substrate) we have isolated analogous regulatory mutants.Synthetic opines, in which the amino acid moiety has been replaced by toxic arginine analogues, are toxic for these regulatory mutants. We isolated mutants resistant to such synthetic opines, and found that some had lost the capacity to utilize octopine. A survey of a large number of such mutants revealed that all of them still incited octopine synthetizing tumors.Mutants constitutive for octopine catabolism are in some instances also constitutive for Ti-plasmid transfer. A simple method for screening regulatory mutants for constitutive Ti-plasmid transfer is described.This work has been supported in part by grants from the Centre National de la Recherche Scientifique (contrats ATP 2814 and 3363).     

A study of some factors affectingAgrobacterium transformation and plant regeneration ofDendranthema grandiflora Tzvelev (syn.Chrysanthemum morifolium Ramat.)

In an attempt to develop a system for producing transformed plants from explants ofDendranthema grandiflora, the susceptibility of the cultivar Super White to various wild-type strains ofAgrobacterium tumefaciens andA. rhizogenes was investigated. Tumour formation was not a reliable indicator of the ability of a related disarmed strain to mediate transformation. Following inoculation of explants with disarmedAgrobacterium strains, a number of shoots developed on selective media. However, none of these shoots were transformed. By co-cultivating stem internode explants with a mixed inoculum of wild-type and disarmed strains, it was possible to obtain a callus stably transformed withAgrobacterium carrying a disarmed T-DNA. Histological analysis of explants revealed that shoot regeneration initially occurred from the cells of the epidermis and subsequently from the cortex. However, the cells which were susceptible to T-DNA transfer were confined to the vascular tissue.     

Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr^-) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr^- controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr^- against the sensitive strain but was similar against the resistant strain.     

DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions

Summary DNA transfer fromAgrobacterium tumefaciens, a soil bacterium, to the non-host graminaceous monocotyledonous plantZea mays, was analysed using the recently developed technique of agroinfection. Agroinfection ofZ. mays with maize streak virus using strains ofA. tumefaciens carrying mutations in the pTiC58 virulence region showed an almost absolute dependence on the products of the bacterialvirC genes. In contrast, agroinfection of the control hostBrassica rapa with cauliflower mosaic virus was less dependent on thevirC gene products. In other respects, the basic mechanism of the plant-bacterium interaction was found to be similar. While intactvirA, B, D and G functions were absolutely necessary, mutants invirE were attenuated. Agroinfection of maize was effective in the absence of an exogenously suppliedvir gene inducer, and indeed woundedZ. mays tissues were found to produce substance(s) which induced the expression ofA. tumefaciens vir genes. These findings are discussed in the light of current knowledge about the function ofAgrobacterium vir genes.     

落叶型大丽轮枝菌T-DNA插入突变体库的构建

采用ATMT技术建立大丽轮枝菌落叶型菌株XJ2008菌株的T-DNA插入突变体文库,共获得6 043个突变体。从中随机挑选104个突变体,以野生型XJ2008菌株为参照,评价其致病性、菌落生长速率、分生孢子及微菌核的产生能力等。结果表明,有12.5%的突变体丧失产孢能力,4.8%的突变体的生长速率显著减慢,8.7%的突变体的生长速率显著加快,12.5%的突变体丧失产生微菌核的能力,47.1%的突变体的致病性显著低于野生型菌株XJ2008,且突变体2-736、2-740、2-745的病情指数分别约为野生型菌株XJ2008的0.184、0.168和0.197倍。该突变体库突变体遗传稳定性好,性状多样性丰富。     

Tissue culture and transformation ofOenothera biennis

Five cultivars ofOenothera biennis have been tested for callogenesis and organogenesis on different media. The cultivar CV3 has been transformed byAgrobacterium tumefaciens strain which introduces into the plant genome kanamycin resistance gene and the T-DNAipt gene which causes increased levels of cytokinins. Transformed tissues showed elevated levels of cytokinins and grew as teratomas forming clumps of short, branched shoots with small modified leaves. Roots appeared rarely in later subcultivations of some teratomous clones.     

Associative effect of inoculation of different strains of Azotobacter and homologous rhizobium on the yield of moong (Vigna radiata), soybean (Glycine max) and pea (Pisum sativum)

Summary It has been observed that in the case ofVigna radiata andGlycine max incorporation of suitable strain of Azotobacter gave higher yield than obtained by the use of Rhizobium as inoculant. In the case ofVigna radiata even a strain of Azotobacter isolated from the rhizosphere of berseem gave similar yields as Rhizobium. In the case ofPisum sativum association of Rhizobium with a strain ofAzotobacter chroococcum isolated from the rhizosphere of pea gave numerically higher yield than Rhizobium alone. It may be possible that statistically higher yield may be obtained when a suitable strain of Azotobacter is used after screening a large number of strains of Azotobacter from the rhizosphere of pea.     

Transmission genetics of Nicotiana hybrids produced by protoplast fusion

Somatic cell hybrids were produced by fusing protoplasts isolated from callus cells of a tobacco line transformed by Agrobacterium tumefaciens (octopine synthesizing strain B6S3), and mesophyll protoplasts from haploid plants of Nicotiana plumbaginifolia. Hybrids were selected by using differential medium (hormone-independent growth plus greening capacity), or by mechanical isolation and cloning of individual heterokaryocytes. The analysis of hybrid cell lines included the determination of lysopine dehydrogenase activity (encoded by the T-region of Agrobacterium tumefaciens plasmid), examination of isozymes of esterase, and study of chromosome number and morphology. All eight cell lines selected on the screening medium were identified as nuclear hybrids, while only three of the eight evaluated clones obtained by mechanical isolation and cloning were found to be nuclear hydrids; the rest of them were nuclear segregants of tobacco [1] or N. plumbaginifolia [4] type. These data give independent evidence for the occurrence of non-fusion and segregation of nuclei in fusion products, that can be revealed only by using nonselective methods for hybrid screening. In this paper we demonstrate the value of microisolation for the recovery of cytoplasmic hybrids.     

Efficient protoplast regeneration ofBacillus thuringiensis andAgrobacterium tumefaciens

Summary Treatment ofBacillus thuringiensis andAgrobacterium tumefaciens taken from the early growth phase (8 h) with lysozyme at 1 mg/ml gave 90–99% protoplast formation and 10–12% protoplast regeneration on the minimal medium in absence of plasma expander (Bovine serum albumin). Enhanced fusion frequency was obtained when protoplasts from 8 h grown cells were used for fusion experiments.     

Correlation between binding of Agrobacterium tumefaciens by root cap cells and susceptibility of plants to crown gall

We compared the binding of Agrobacterium tumefaciens by freshly isolated root cap cells with susceptibility of plants to crown gall tumorigenesis. A high binding reaction was strongly correlated with susceptibility to tumorigenesis in a survey of the binding of strain B6 to cells from 48 species in 17 families. In reciprocal experiments with nine virulent A. tumefaciens strains, tumors developed in plant-bacteria combinations that gave a high binding response in the root cap cell assay. Binding was quantified by direct measurement of the number of bacteria bound to the periphery of individual cells. Root cap cells from six susceptible species bound significantly more bacteria than did cells from five resistant species.     

The effect of Sa and MiniSa plasmids on the induction of plant crown galls and hairy roots byAgrobacterium tumefaciens andAgrobacterium rhizogenes

The Inc-W group plasmid Sa or its derivative MiniSa were introduced into two strains ofAgrobacterium tumefaciens with Ti plasmids, one strain ofA. tumefaciens with the Ri plasmid and oneA. rhizogenes strain with the Ri plasmid. The effect was similar in allAgrobacterium strains. The pSa suppressed fully the virulence ofAgrobacterium strains (i.e. their ability to induce tumor growths - crown galls or hairj⁷ roots) inKalanchoe plants and carrot root slices. The MiniSa plasmid caused only a slight decrease of the frequency and size of tumor growths induced. The mechanism of suppression of virulence by the Sa plasmid inAgrobacterium tumefaciens andAgrobacterium rhizogenes seems to be similar.