Transformation of Sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens

A method has been developed for the regeneration of transformedplants of the commercially important crop sugarbeet (Beta vulgarisL.), using Agrobacterium tumefaciens. Binary vectors were used,carrying both screenable and selectable genes. Plant regenerationfrom shoot-base tissues was found to be relatively rapid andfrequent compared with petioles or leaf tissue. Inoculationof cultured shoot-base tissues resulted in the production oftransformed plants, as determined by (1) introduced resistanceto kanamycin, (2) introduced CAT or GUS activity, and (3) Southernblot analysis to show the integration of foreign DNA. The transformationfrequency was found to be dependent upon explant source, plantgenotype and selection conditions used. Key words: Agrobacterium tumefaciens, sugarbeet (Beta vulgaris L.), transformation.     

Evidence for the Presence of Bacteria-specific Proteins in Sterile Crown Gall Tumor Tissue

Cross-reacting antigens were found in bacteria-free crown gall tumor tissue tested with serum prepared against Agrobacterium tumefaciens (Smith and Towns.) Conn., but no such antigens were detected in callus tissue. Soluble proteins from tumor tissue, callus tissue, and the crown gall bacteria were fractionated on a DEAE-Sephadex (A-50) column. The diethylaminoethyl-Sephadex elution profile for tumor tissue showed three protein fractions that were not detected in the callus tissue. Two of these protein fractions were shown to be exclusively bacteria specific. Besides these qualitative differences between the two tissues, significant quantitative differences in the amount of protein fractions were also observed. The diethylaminoethyl-Sephadex column fractions from tumorigenic strain of A. tumefaciens corresponding in position to the three additional peaks in the tumor tissue also showed cross-reacting antigens when tested with serum prepared against sterile tumor tissue. It is suggested that tumor formation by A. tumefaciens involves integration of the bacterial genome into the host-cell genome.     

Crown Gall on Castor Bean Leaves: II. FORMATION OF SECONDARY TUMOURS

Secondary tumours were formed on the cotyledonary leaf petiole,the hypocotyl, and first true leaf of castor bean seedlingsafter inoculating the blades of the cotyledonary leaves withAgrobacterium tumefaciens. Depending on the strain of bacteriaemployed, 0 to 80 per cent of the plants developed secondarytumours. The ability of different strains to initiate secondarytumours was not obviously correlated with their relative effectivenessin initiating primary tumours. Though all produced primary tumours,five out of ten auxotrophic strains failed to yield secondarytumours, whereas only one out of 14 prototrophic strains failedto do so. Both the number of plants developing secondary tumoursand the frequency with which these tumours occurred on differentparts of the plant were positively correlated with the concentrationof the primary inoculum. Tumours also developed on the cotyledonaryleaf petiole and on the hypocotyl after vacuum infiltrationof A. tumefaciens into the blade of cotyledonary leaves. Inmost instances (9 out of 11 plants) no tumours were formed onthe blade of the infiltrated leaf. Thus, tumour formation equivalentto secondary tumours can occur in the absence of a primary tumouror an overt primary wound. Excision of inoculated leaves showedthat the stimulus for secondary tumour formation moves fromthe blade to the hypocotyl within 24 h. Attempts to demonstratethe presence of a sub-cellular tumour-initiating agent in homogenatesof inoculated leaves were unsuccessful. A. tumefaciens, however,was found in the petiole of the cotyledonary leaf and in thehypocotyl within 24 h of inoculation. The migrating agent responsiblefor secondary tumour formation in castor beans is concludedto be intact bacteria.     

Quantitative Evaluation by Reassociation Kinetics of Agrobacterium DNA Sequences residing in the Genome of a Crown-gall Tissue 2

The amount of homologous Agrobacterium tumefaciens DNA carriedin the genome of Scorzonera hispanica Crown-gall tissue cultureshas been determined from the reassociation kinetics of ³²P-labelledbacterial DNA in the presence of unlabelled Crown-gall DNA.Unrepeated DNA extracted from Crown-gall tissues was found tocontain sequences homologous to the bacterial DNA; quantitativeestimation shows the equivalent of about one Agrobacterium genomeper copy of unrepeated Crown-gall DNA. It is concluded thatan entire single genome of Agrobacterium is integrated intoeach haploid complement of a Crown-gall cell.     

Changes in cyclic-AMP phosphodiesterase activity during the formation of crown-gall tumors on potato discs

Cyclic-AMP phosphodiesterase activity was found in crown-galltumor tissue obtained from the inoculation of potato discs withAgrobacterium tumefacicns strain B6. Consistently more enzymeactivity was found, in this tumor tissue than in the correspondingnormal tissue. This difference in enzyme activity was observedwhen strains other than B6 were used to induce tumors, or whennormal and tumor potato tissue grown in culture were examinedfor phosphodiesterase activity. (Received January 27, 1978; )     

Dynamics of Endogenous IAA and Cytokinins during the Growth Cycle of Soybean Crown Gall and Untransformed Callus

The dynamics of the endogenous IAA and cytokinin levels duringthe growth cycle of two soybean crown gall lines (green andpale) induced by a nop⁺ Agrobacterium tumefaciens C58 strain,were compared with an untransformed soybean callus line. In both transformed tumor lines maximum cytokinin (essentiallyglucosyl-trans-zeatin) levels were attained in the beginningof the exponential growth phase, followed by a drastic decreasejust before the stationary phase was reached. Quantitativelythe green tumor line showed a 2–3 times higher cytokinincontent compared with the pale line. In the untransformed soybeancallus hardly any significant levels of cytokinin could be detected. Analysis of endogenous IAA levels showed no difference betweenthe two tumor lines and the untransformed callus tissue, allshowing a low and constant level throughout the entire growthcycle. The relevance of the endogenous accumulation of phytohormonesin relation to the hormone autotrophic growth of transformedsoybean tissue is discussed. ³ Senior Research Associate Nationaal Fonds WetenschappelijkOnderzoek (N.F.W.O.). ⁴ Recipient of an Instituut voor Wetenschappelijk Onderzoekin Nijverheid en Landbouw (I.W.O.N.L.) grant.     

The Galls on Forsythia: Inoculation Studies

Two fungi, Fusarium lateritium and Phomopsis dominici and twobacteria (S3) and (G1) resembling Agrobacterium radiobactervar. tumefaciens and Corynebacterium fascians isolated fromgalls on Forsythia intermedia failed to produce galls upon reinoculation.Similarly, cultures of C. fascians, Pseudomonas savastanoi andP. savastanoi var. fraxini failed to induce galls. Both A. radiobactervar. tumefaciens and tumerous carrot tissue infected with A.radiobacter var. rhizogenes produced tumerous tissue in inoculatedplants but only the latter induced an overgrowth resemblinga natural gall on Forsythia.     

Agrobacterium-mediated Transformation of Sunflower (Helianthus annuusL.): A Simple Protocol

A simple and reproducibleAgrobacterium tumefaciens-mediatedtransformation system was developed for sunflower (HelianthusannuusL.) ‘KBSH-1’. The objective was to substantiallyeliminate thein vitroregeneration component from the transformationprotocol. Two-d-old seedlings with one cotyledon detached wereinfected withAgrobacterium tumefaciensstrain LBA 4404/pKIWI105harbouring genes for ß-glucuronidase (GUS) and neomycinphosphotransferase (NPT II). Following co-cultivation, seedlingswere grown aseptically for 5 d on a growth regulator-free basalmedium supplemented with 250 µg ml^-1cefotaxime (a bacteriostatused to control gram negative bacteria). Seedlings were screenedfor transient GUS expression and the shoot portions of the putativetransformants were utilized for propagation as transgenic plants.The excised shoots that initiated roots following selectionwere subsequently transferred to a glasshouse. Molecular analysisof transgenic plants confirmed concordance between the presenceof foreign genes and enzyme activity. The transformation regimefacilitated rapid generation of up to 2% phenotypically normalfertile plants containing functional transgenes. The transmissionand integration of the marker genes in the progeny is demonstrated.Copyright1999 Annals of Botany Company Sunflower (Helianthus annuusL.), transformation,Agrobacterium,simple protocol.     

Rapid,in situ detection of Agrobacterium tumefaciens attachment to leaf tissue

Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging‐based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum‐infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post‐infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

The concentration of selected cations in normal and crown-gall tumors obtained from potato discs

Crown-gall tumor tissue obtained from potato discs inoculatedwith virulent strains of Agrobacterium tumefadens containedhigher concentrations of K⁺, Mg²⁺ and Ca²⁺ than the correspondingnormal tissue. These tumors also contained higher concentrationsof these cations than normal tissue inoculated with an avirulentstrain of Agrobacterium tumefadens, or than normal tissue adjacentto the crown-gall tumors on the same potato disc. The concentrationof these cations remained significantly higher than controltissue regardless of the tumor age. 2,4-Dinitrophenol and myo-inositol,while affecting the concentration of Ca²⁺ in these tissues,had no effect on the Mg²⁺ and K⁺ concentrations. These resultssuggest increased concentrations of certain cations may be aspecific property of crown-gall tumors. (Received August 16, 1978; )     

Callus Induction from Mesophyll and Hypocotyl Protoplasts of Mungbean (Vigna radiata (L))

Mesophyll and hypocotyl protoplasts were isolated from Vignaradiata using a combination of Cellulase Onozuka RIO (1%) andMacerozyme (0·2%). On a modified V-47 medium, 60% ofthe cultured protoplasts divided and developed into colonies.Protoclones differentiated roots on MS medium supplemented withdifferent auxins and cytokinins; however, shoot differentiationwas not obtained. Co-cultivation of protoplasts with wild andshooter mutants of Agrobacterium tumefaciens did not lead todifferentiation of plantlets. Lysopine and nopaline dehydrogenasewere not detected in any of the selected protoclones. Vigna radiata, Mungbean, Agrobacterium tumefaciens, co-cultivation, protoplasts     

Transformation of Stylosanthes spp. using Agrobacterium tumefaciens

Tumours were incited on leaf sections of Stylosanthes humilis, S. hamata, S. guianensis and S. scalra following infection by Agrobacterium tumefaciens. The suitability of 2 binary vectors (pGA472, BIN6) for gene transfer in S. humilis was tested and kanamycin-resistant tumour tissue was obtained from infected leaf pieces. The presence and expression of the neomycin phosphotransferase (NPT II) gene in the plant cells was demonstrated by hybridization of the coding region of the NPT II gene of the transposon Tn5 to DNA and RNA of kanamycin resistant tumours and by detection of significant NPT II activity in tissue extracts. Tumours also produced teratomatous shoots expressing the NPT II gene, but these could not be rooted.     

Crown-gall tumors possess tumor-specific antigenic sites on their cell walls

Rabbits were injected with cell walls obtained from crown-gall tumor tissue or the corresponding cell walls from normal potato tissue. The serum obtained from rabbits 53 days after they were injected with tumor cell walls contained immunoglobins that reacted with both tumor and normal cell walls as well as with the cells from the inciting strain of Agrobacterium tumefaciens. When this serum was repeatedly absorbed against normal cell walls and the cells of the inciting strain of Agrobacterium tumefaciens, only tumor-specific immunoglobins remained. These immunoglobins did not react with cell walls obtained from meristematic (nontumorous) potato tissue. Yet this same serum reacted with crown-gall tumor cell walls obtained from turnip and carrot discs.     

In Vivo Modulation of T-DNA Encoded Amidohydrolase Activity in Transformed Tobacco Cells

Auxin autonomous growth of most crown gall tumor cells requires the expression of two auxin biosynthesizing genes (tms 1 and tms 2) from the T-DNA of Agrobacterium tumefaciens. The potential role of the tms 2 locus to affect auxin accumulation was studied by measuring the activity of its gene product, indoleacetamide hydrolase (AH), in cloned cells of tobacco (Nicotiana tabacum) transformed by the A6 strain of Agrobacterium tumefaciens. AH activity followed a consistent pattern over a 30 day culture cycle with a peak at 10 to 14 days. This same pattern was observed in a number of independently isolated clones as well as in uncioned tumor tissue, suggesting that AH activation is a regular process in wounded, transformed cells. Transfer of unwounded tissue to fresh media resulted in a similar pattern of AH activation, but with the peak activity only about 50 percent of the cut tissues. These results show that the tms 2 encoded AH activity is modulated over the culture cycle, and that the modulation is affected by wounding and supplying fresh nutrients in the medium. AH activity correlated closely with free indoleacetic acid levels which suggests that it can be an important determinant in controlling free IAA levels in transformed cells.     

Trans-Kingdom Conjugation Between Agrobacterium tumfaciens and Saccharomyces cerevisiae, a Bacterium and a Yeast

For conjugation between prokaryotic Agrobacterium tumefaciensand eukaryotic Saccharomyces cerevisiae, we constructed twonovel conjugative plasmids. A. tumefaciens transmitted the plasmidsto S. cerevisiae with the aid of tra genes on a helper plasmid.The transmitted plasmids retained their original structure andfunction in transconjugant yeasts. The presence of Ti plasmidbarely affected the trans-kingdom conjugation. ¹A preliminary report of this work was presented in Japaneseby Yoshida et al. (1993).     

Combined Effects of Auxin Transport Inhibitors and Cytokinin: Alterations of Organ Development in Tobacco

We have examined the effects of the auxin transport inhibitors1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid(TIBA) on leaf morphogenesis of transgenic Nicotiana tabacum(cv. Xanthi) plants expressing the Agrobacterium tumefacienscytokinin biosynthetic gene, ipt. We have observed the formationof saucer-shaped leaf-like organs at the shoot apex and at lateralbuds. The formation of apical saucer-shaped leaf-like organscan be duplicated by the application of exogenous NPA and cytokininto wild-type tobacco seedlings. We have also observed adventitiousleaf-like organs with altered petiole and blade morphology inthe transgenic plants treated with auxin transport inhibitors.These results suggest that the combination of diminished auxintransport and elevated cytokinin can lead to alterations inleaf development in tobacco. ⁴Present address: Genesis Research and Development Corporation,P.O. Box 50, Auckland, New Zealand     

Transformation of Brassica napus L. with a 'Disarmed' Octopine Plasmid of Agrobacterium tumefaciens: Molecular Analysis and Inheritance of the Transformed Phenotype

Phenotypically normal and fertile transgenic Brassica napuscv. Westar plants were obtained following co-cultivation ofstem epidermal explants with an Agrobacterium tumefaciens straincontaining a disarmed octopine tumour-inducing plasmid pTiB6S3-SE.The A. tumefaciens cells also contained pMON316, a cointegratevector carrying genes for kanamycin resistance and a scorablemarker nopaline synthase. Transformants were selected by theirability to grow in the presence of 100 µg cm^-3 kanamycin.Transformation was confirmed by the activities of neomycin phosphotransferaseII and nopaline synthase enzymes and by Southern blots. Thekanamycin resistance trait was transferred to the progeny ofthe self-fertilized plants. Key words: Transformation, octopine Ti-plasmid, oilseed rape     

Gibberellin Production in Cultured Cells of Nicotiana tabacum

Endogenous gibberellins (GAs) in several kinds of crown gallcells and cultured cells derived from normal tissue of Nicotianatabacum were systematically analyzed by gas chromatography-selectedion current monitoring (GC-SICM) after chromatographic purifications,and GA₁, GA₉, GA₁₉ and GA₂₀ were identified. Agrobacterium tumefaciens,a pathogen of crown gall, was confirmed not to produce GAs inits culture. We also investigated endogenous GAs of mother plant,tobacco, and found the same kinds of GAs as in cultured cells. ³ Present address: College of Agriculture, Chonnam NationalUniversity, Kwangju 500, Korea. (Received May 19, 1982; Accepted July 22, 1983)     

Identification of a crown-gall tumor growth factor as GABA

A crown-gall tumor growth factor (TGF-II) isolated from bean leaves inoculated with Agrobacterium tumefaciens strain 13333 is shown to be γ-aminobutyric acid (GABA). This identification is based on the comparative behavior of purified TGF-II and authentic GABA with respect to elution from preparative ion exchange and molecular sieve columns, ninhydrin reaction, TLC, co-chromatography on an automated amino acid analyzer, MS analysis and biological activity. GABA is detected by bioassay only in bean leaves infected with the bacterium and is in growth limiting supply when only a few tumors are present per leaf. GABA promotes tumor growth when as little as 1 ng is applied per leaf.     

Transgenic Grapevines: Regeneration of Shoots Expressing {beta}-Glucuronidase

Genetic transformation has been studied in fragmented shootapex cultures of Vitis vinifera L. following co-cultivationwith Agrobacterium tumefaciens. Transgenic shoots of the cultivarCabernet Sauvignon, tolerant to low levels of kanamycin, havebeen produced. Proliferation of transgenic cells in the presumptivebud forming area of cultured fragments has been observed usingthe enzyme activity of ß-glucuronidase (GUS) as amarker. The distribution of GUS stained cells both in this tissueand in transgenic shoots, the presence of low copy numbers ofthe neomycin phosphotransferase II gene in transgenic shootsand the relatively low levels of kanamycin resistance suggestthat these shoots contain both transformed and untransformedcells. Key words: Grapevine, transformation, Agrobacterium, GUS, NPTII