Ti Plasmid Homologous Sequences Present in Tissues from Agrobacterium Plasmid-transformed Petunia Protoplasts

Suspension cell protoplasts of albino Petunia hybrida have beentransformed by isolated Agrobacterium tumefaciens Ti plasmid.Uptake of octopine Ti plasmid (pTiACH5) into protoplasts wasstimulated by poly-L-ornithine and polyethylene glycol (PEG).The frequency and efficiency of transformation of protoplaststo phytohormone autotrophy was compared using the two uptakeagents with various concentrations of plasmid. Transformationwas most efficient with PEG-mediated uptake, 5 µg of Tiplasmid per 10⁶ protoplasts giving a frequency of 6?10^–5.Octopine was not synthesised in any of the transformants afterthe second subculture on hormone-free medium. DNA-DNA hybridisationshowed the presence of DNA homologous to the T-DNA region ofpTiACH5 in all clones analysed. (Received November 9, 1981; Accepted January 29, 1982)     

Cultivation of Rice Protoplasts and Their Transformation Mediated by Agrobacterium Spheroplasts

Improvement of the cultivation of rice (Oryza saliva L.) protoplastsisolated from suspension cultures led to their division at afrequency of 5 to 10%. Rapidly growing colonies were obtainedon a hormone-free medium when Agrobacterium tumefaciens spheroplastswere introduced into the protoplasts by polyethylene glycoltreatment. Opines corresponding to the strains of A. tumefaciensused for the spheroplast treatments were detected in some ofthese colonies at a frequency of about 10^–4. Using radioactiveprecursors, [¹⁴C]--ketoglutaric acid and [³H]-arginine, activitiesof nopaline synthase, a marker enzyme of nopaline-type crowngall, were also detected in some of these clones. These resultsshow that the rice cells were transformed by Ti plasmid introducedby the spheroplast method. (Received September 6, 1985; Accepted January 24, 1986)     

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.     

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.     

Fusion of Agrobacterium and E. coli spheroplasts with Nicotiana tabacum protoplasts — Direct gene transfer from microorganism to higher plant

Spheroplasts of Agrobacterium tumefaciens strains and E. coli were fused with protoplasts of Nicotiana tabacum. Fusion products were cultured in the presence of antibiotics to eliminate remaining bacterial spheroplasts. On hormone free medium, tobacco protoplasts treated with wild type Agrobacterium-strains formed colonies with an average frequency of 10^–4. Opine synthesis was detected in the tissues. Some calli derived from protoplasts treated with A. tumefaciens C58C1pRi15834 formed typical hairy roots. Kanamycin resistant calli were obtained after fusion with A. tumefaciens containing pLGVTi23 neo (frequency=10^–3). Fusion of E. coli spheroplasts containing a virulent pTiB6S3::RP4 co-integrate with tobacco protoplasts yielded two hormone independent growing calli producing octopine out of 10⁵ microcalli.Abbreviations PEG Polyethylene glycol - PVA Polyvinyl alcohol     

Re-evaluation of Conditions for Plant Regeneration and Agrobacterium-Mediated Transformation from Tomato (Lycopersicon esculentum)

Conditions for plant regeneration from explants of tomato (Lycopersiconesculentum) cv. UC82B were studied for optimizing transformationprocedure. The best regeneration rate was obtained from cotyledonexplants from 8–10-d-old seedlings on a modified Murashigeand Skoog medium (1962) with 0·5 mg dm^–3 zeatinand 0·5 mg dm^–3 indolylacetic acid. Tomato cultivars(UC82B, Castone, Fl Ferline, Monalbo) and a Lycopersicon peruvkmum‘CMV sel. INRA’ were studied. The cultivarUC82Band the wild Lycopersicon species showed an efficient shootregeneration potential. Early events in the transformation of tomato cotyledons wereanalysed using an Agrobacterium tumefaciens strain carryinga binary vector with an nptII (pnos) gene and a reporter GUS-intron(p35S) chimeric gene. Two days after infection, GUS activityappeared specifically at the cut surface. Subepidermal cellswere more susceptible to transformation than epidermal cells.When selection for kanamycin resistance was applied 2 d afterinoculation, transformed cells were efficiently recovered. Preculturewith feeder cells stimulated cell transformation, but reducedregenerationcapacity from transformed cells. The optimal transformationrate was observed witha time of preculture of 1 and 2 d. Transformationevents for two tomato cultivars (UC82B and Monalbo) occurredat the same rate as 55% of the inoculated explants developedkanamycin resistant calli. However, transformed plants wereobtained at different rates of 8% and 14% for cv. Monalbo andcv. UC82B. Key words: Agrobacterium tumefaciens, ß-glucuronid, Lycopersicon esculentum, plant regeneration, transformation     

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.     

Somatic Hybrids between the Forage Legumes Lotus corniculatus L. and L. tenuis Waldst et Kit

A cell suspension of Lotus tenuis was established, as a sourceof protoplasts, from kanamycin resistant callus derived fromroots transformed by Agrobacterium rhizogenes LBA9402 (pRil855-pBinl9).Such protoplasts were treated with a sublethal dose of sodiumiodoacetate prior to their electrofusion with green cotyledonprotoplasts of L. corniculatus. Putative somatic hybrid colonieswere selected on medium containing kanamycin sulphate. The hybridityof plants regenerated from these selected colonies was confirmedby their morphology, esterase banding patterns, the presenceof condensed tannins in leaves and stems, and chromosome complements.The latter approximated to the expected allohexaploid numberof 2n = 6x = 36. Key words: Forage legumes, Lotus corniculatus, L. tenuis, protoplasts, electrofusion, kanamycin resistance, sodium iodoacetate, somatic hybridization     

Transformation of Vinca protoplasts mediated by Agrobacterium spheroplasts

Summary Vinca rosea protoplasts and Agrobacterium tumefaciens spheroplasts harboring octopine-type Ti plasmid were mixed and treated with polyethylene glycol or polyvinyl alcohol, which facilitated the introduction of spheroplasts into plant protoplasts. After the protoplasts had been kept at 40° C for 4 days, bacteria were found to be completely eliminated from the medium. Among treated protoplasts 1–2 per 1,000 formed colonies on the Murashige and Skoog medium (1962) lacking hormones. When the colonies were isolated and subcultured, they could be maintained as clones. Octopine, an amino acid specific to crown gall, was detected in half of these clones. The phenotypic features of these putative transformants were compared but did not show any coincidental tendencies in relation to color, hardness, form, growth rate, or octopine production. The significance of this system in transformation of higher plant cells is discussed.     

Embryo Transformation, A Practical Approach for Realizing Transgenic Plants of Safflower (Carthamus tinctorius L.)

A gene transfer system that ensured recovery of whole planttransformants was developed for safflower (Carthamus tinctoriusL.). Embryo axes of germinating seeds with one of the cotyledonsremoved were pricked with a sterile sewing needle at the cotyledonarynode and infected by gentle agitation for 10 min in a suspensionofAgrobacterium tumefaciens . Following a 24 h co-cultivationand decontamination with cefotaxime for 1 h, they were placedon soilrite moistened with water to allow germination to progress.Later, the seedlings were transferred to soil in pots wherethey grew into normal healthy plants in the greenhouse. Thehistochemical assay of an uid A gene that expresses only inplant tissues and PCR amplification of uid A and npt II markergenes were used for early determination of putative transformants,whereas Southern analysis of T₀and T₁plant DNA was used to confirmintegration of the transgenes. The combined results indicatedthat the frequency of transformation was 5.3% in safflower ‘A-1’and 1.3% in ‘A-300’. Four T₀plants of ‘A-1’yielded transformed T₁progeny. The strategy, in principle, shouldbe applicable to all cultivars and genotypes of safflower whichare susceptible to Agrobacterium tumefaciens infection. Thusfar, this is the only procedure available for safflower thatcould successfully be used to generate whole plant transformants.Copyright 2000 Annals of Botany Company Safflower (Carthamus tinctorius L.), transformation, non-tissue culture method, embryo axes,Agrobacterium , transgene expression     

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.     

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).     

The possible involvement of adenyl cyclase and cyclic-AMP phbsphodiesterase in the formation of crown-gall tumors on the the primary leaves of Pinto beans

Adenyl cyclase activity was found in crown-gall tumor tissueobtained from the inoculation of the primary leaves of Pintobeans with Agrobacterium tumefaciens strain B6. Consistentlymore enzyme activity was found, however, in normal leaf tissueor wounded leaf tissue inoculated with sterile broth than inthe tumor tissue. On the other hand, cyclic-AMP phosphodiesteraseactivity increased with tumor age. These two enzymes were also found in extracts of both infectiousand non-infectious strains of Agrobacterium tumefaciens, althoughno obvious correlation between the production of these enzymesand infectivity could be established. Some of the implicationsof these results will be discussed. (Received May 25, 1976; )     

Improved protoplast yield and cell wall regeneration in Gracilaria verrucosa (Huds.) Papenfuss (Gracilariales, Rhodophyta)

Protoplasts were isolated enzymatically from the red alga Gracilariaverrucosa using only two enzymes: agarase prepared from marinebacteria and commercial cellulase. Yields of protoplasts weredependent on the donor material and by choosing young bladesor algae in a state of higher growth rate, the production ofprotoplasts reached a maximum of 10⁷ protoplasts per gram offresh tissue. Cell viability was better with NaCI used as osmoticumthan with sorbitol in the culture medium and on reducing culturemedia to normal osmolarity in 4 d. 25% of the cultured protoplastswere able to regenerate a cell wall (i.e. cellulose) within7 d as confirmed by staining with calcofluor white althoughonly a few protoplasts were able to divide. During the first24 h of culture, the synthesized agar contained higher amountsof L-galactose-6-sulphate than the cell wall of thalli. Theamount of agar in the protoplasts, however, did not increase,indicating that the protoplasts synthesize a qualitatively differentcellwall. Key words: Agarase, agar, cell wall regeneration, Gracilaria verrucosa, protoplasts     

Transformation of protoplast-derived cell colonies and suspension cultures by Agrobacterium tumefaciens

In this paper we describe procedures for transforming micro colonies derived from mesophyll protoplasts of Petunia hybrida with Agrobacterium tumefaciens. The method is efficient, up to 70% of the colonies were transformed, and we used a similar method to transform cells from a suspension culture of haploid Nicotiana plumbaginifolia.Abbreviations RH Relative humidity - MES 2 (N - Morpholino) ethane sulphonic acid - NAA 1 - Naphthylacetic acid - BAP 6 - Benzyl amino purine - B5 Gambourg's B5 medium (Gambourg 1970) - MS Murashige and Skoog Medium (1962) - PZ saline 0.9% NaCl pH 7.0 - 2, 4-D 2,4, Dichlorophenoxyacetic acid - Ty tryptone yeast     

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.     

Growth responses of sunflower hypocotyl disks inoculated with Escherichia coli and Agrobacterium tumefaciens I. Morphological observations

Disks of sunflower hypocotyls 1 mm thick grown in light anddarkness on agar containing mineral salts and sucrose to whichIAA was added in varying concentrations, were inoculated withE. coli, A. tumefaciens or sterile synthetic medium. Light-growndisks inoculated with E. coli proliferated from the lower surfaceand formed numerous long roots but dark-grown disks were usuallyinhibited in comparison with uninfected disks. Inoculation withA. tumefaciens induced proliferation mainly from the upper surfaceand a few short roots were formed. Uninfected disks grown with0.01 ppm IAA proliferated in a manner similar to that of E.coli-infected light-grown disks on simple medium but in thedark similar treatments produced quite different morphologicaleffects. The form of proliferation induced by E. coli underall conditions of growth could not be equated with that inducedby A. tumefaciens or by different concentrations of IAA. (Received December 5, 1967; )     

Observation by SEM of the attachment ofAgrobacterium tumefaciens to the surface of vinca,asparagus and rice cells

Transformation of vinca cells was performed by the co-cultivation of cell-wall regenerated vinca protoplasts withAgrobacterium tumefaciens. Using thisin vitro and single cell system, attachment of the bacteria to the surface of vinca cells was observed by scanning electron microscopy (SEM). Figures of the bacteria polarly binding to the plant cell wall were often observed. AsEscherichia coli does not attach to the plant cells at all, the observed attachment ofA. tumefaciens is suggested as a characteristic feature in crown gall induction. Even though no evidence of transformation was obtained by the co-cultivation methods, a similar attachment was observed in the cell-wall regenerated protoplasts of rice. The bacteria also attached to the surface of isolated mesophyll cells of asparagus and root hairs of rice. From these observation, we concluded that the attachment is not the limiting step of crown gall induction byA. tumefaciens in monocotyledonous plants. Extracellular fibrils like pili were observed with a few strains of A.tumefaciens for the first time. These fibrils were observed regardless of their ability of attachment and infectivity.     

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