Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues

The isopentenyl transferase gene (ipt) fromAgrobacterium tumefaciens was isolated and introduced, via a disarmed binary vector, into tobacco using theAgrobacterium tumefaciens-mediated gene transfer system. The expression of theipt gene was monitored by RNA hybridization, western blotting and cytokinin analysis. The addition of auxin to the media rapidly reduced the level of cytokinins in the transgenic tissues and this was associated with a reduction in IPT mRNA and protein levels. It is concluded that the hormone auxin can regulate expression of a gene involved in biosynthesis of the second hormone cytokinin. Although exogenous benzyladenine did not directly affectipt gene expression, it did antagonize the effect of auxin on levels of cytokinins and IPT mRNA and protein.     

Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542

Summary A 15.2-kb KpnI fragment from the virulence region of pTiBo542, the Ti plasmid harbored by Agrobacterium tumefaciens strain A281, was introduced into binary vectors. The fragment contained the virB, virC and virG genes, and it is known to have the ability to increase the virulence of strains of A. tumefaciens. The strains of A. tumefaciens that carried the resulting plasmids were able to transform cells in a suspension culture of Chenopodium quinoa Willd cells which were not transformable by common vectors. Although the sizes of the plasmids was very large, a foreign segment of DNA was introduced into one of the plasmids by homologous recombination in A. tumefaciens cells, and the segment was subsequently transferred to plant cells.Abbreviations NPT neomycin phosphotransferase - SPT streptomycin/spectinomycin phosphotransferase     

Agrobacterium-mediated DNA transfer in sugar pine

DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptII gene. Expression of the nptII gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.     

Agrobacterium-mediated transformation of commercial mints

Commercial peppermint (P) (Mentha × piperita L. ev. Black Mitcham), native spearmint (NS) (M. spicata L.) and Scotch spearmint (SS) (M. × gracillis Sole cv Baker) petioles and orange mint (OM) (M. citrata Ehrh.) leaf disks were cocultivated with a number of Agrobacterium tumefaciens strains. P, SS and OM initiated tumor-like callus tissue on growth regulator-free MS medium after cocultivation with strain A281, a hypervirulent agropine strain containing Ti plasmid pTiBo542. Callus did not initiate from explants cocultivated with strain C58, a virulent nopaline strain; with A 136, a plasmidless strain, or from uninoculated controls. A281-derived callus was maintained on growth regulator-free medium in the absence of antibiotics for up to two years with no bacterial outgrowth. No shoots regenerated from any of the tumors on regeneration medium. Five of seven OM callus lines assayed gave a positive signal for agropine. DNA extracted from OM tumor tissue hybridized to a DNA probe specific to the T-DNA region of pTi plasmid. Genomic Southern analysis of DNA from tumors of P and SS indicated that one to a few copies of the T-DNA integrated into the mint chromosomes. PCR amplification of genomic DNA with primers specific for one of the T-DNA encoded genes yielded fragments that, when analyzed by restriction enzyme mapping and on Southern blots, corresponded to the cytokinin biosynthesis gene ipt. These results demonstrate transformation of three species of mint and the potential for using A. tumefaciens to transfer economically important genes into commercial mint cultivars.Abbreviations BA benzyladenine - CW coconut water - Cef cefotaxime - P peppermint - SS scotch spearmint - NS native spearmint - OM orange mint - BM basal medium - MS Murashige and Skoog (1962) - PAR photosynthetically active radiation - CTAB hexadecylatrimethylammonium bromide - ipt isopentenyl transferase Received for publication 1994. Published as Miscellaneous Paper No. 1482 of the Delaware Agricultural Experiment Station. Contribution No. 317 of the Department of Plant and Soil Sciences. Mention of trade names in this publication does not imply endorsement by the Delaware Agricultural Experiment Station of products named, nor criticism of similar ones not named.     

Backbone-free transformation of barrel medic (Medicago truncatula) with a Medicago-derived transfer DNA

In the present work, Agrobacterium tumefaciens-mediated genetic transformation of the model legume Medicago truncatula Gaertn. (barrel medic) was carried out using the pSIM843 vector that contains a Medicago-derived transfer DNA, delineated by a 25-bp sequence homologous to bacterial T-DNA borders. The transfer DNA contains an expression cassette for the nptII (neomycin phosphotransferase) gene and is flanked by an expression cassette for the backbone integration marker gene ipt (isopentenyl transferase). Our results demonstrate that the Medicago-derived RB-like elements efficiently support DNA mobilization from A. tumefaciens to M. truncatula. Kanamycin-resistant shoots with normal phenotype and ipt-shooty lines were recovered at a frequency of 11.7 and 7.8%, respectively. Polymerase chain reaction (PCR) analyses demonstrated that 44.4% of the independent transgenic lines were backbone-free and evidenced the occurrence of backbone-transfer events.     

Altered development of Arabidopsis thaliana carrying the Agrobacterium tumefaciens ipt gene is partially due to ethylene effects

Transgenic Arabidopsis thaliana plants containingthe Agrobacterium tumefaciens cytokinin-biosynthesis geneipt were produced to study the effect of increasedcytokinin (CK) levels on the development of this rosette plant species. Inthreeindependently transformed lines (ipt-156, 158 and 161),Arabidopsis plants had smaller leaves, an underdevelopedroot system and decreased apical dominance in inflorescence stems. The smallertransgenic leaves were highly serrated along the margins, pale green and hadpointed leaf tips. In cross section, transgenic leaves had smaller cells andirregularly shaped epidermal cells. In the ipt-161 line,leaves and hypocotyls frequently exhibited purple color due to anthocyaninproduction. The most severe phenotype was observed in tissue cultureconditions,while growth in soil reduced or eliminated some phenotypic effects. Compared toC24 wild type plants, ipt-161 plants accumulated zeatinandzeatin riboside with an approximate 10-fold increase in the total pool of CKs.Astudy of the progeny resulting from crosses between theipt-161 transgenic line and the ethylene insensitivemutants ein1, ein2 andeti5 suggested that part of the altered developmentexhibited by the ipt transgenic plants was caused byincreased ethylene levels.     

Crown-gall-resistant transgenic apple trees that Silence Agrobacterium tumefaciens oncogenes

Crown gall disease is an economically significant problem in fruit and nut orchards, vineyards, and nurseries worldwide. Tumors on stems and leaves result from excessive production of the phytohormones auxin and cytokinin in plant cells genetically transformed by Agrobacterium tumefaciens. High phytohormone levels result from expression of three oncogenes transferred stably into the plant genome from A. tumefaciens: iaaM, iaaH, and ipt. The iaaM and iaaH oncogenes direct auxin biosynthesis, and the ipt oncogene causes cytokinin production. In contrast to other tissues, roots do not respond to high cytokinin levels, and auxin overproduction is sufficient to cause tumor growth on roots. Inactivation of iaaM abolished gall formation on apple tree roots. Transgenes designed to express double-stranded RNA from iaaM and ipt sequences prevented crown gall disease on roots of transgenic apple trees.these authors contributed equally to this workthese authors contributed equally to this work     

Mapping and genetic organization of pTiChry5, a novel Ti plasmid from a highly virulent Agrobacterium tumefaciens strain

Agrobacterium tumefaciens Chry5, a wild-type strain originally isolated from chrysanthemum, is unusually tumorigenic, particularly on soybean. We have mapped the Chry5 Ti plasmid by genomic walking and restriction endonuclease analysis, and have located its virulence, T-DNA, plasmid incompatibility, and l,l-succinamopine utilization loci. Southern analysis has revealed that about 85% of the Chry5 Ti plasmid is highly homologous to another Ti plasmid, pTiBo542. Although all the functions that we have located on pTiChry5 are encoded by pTiBo542-homologous regions, the two Ti plasmids differ in their genetic organization. The overall patterns of restriction sites in the plasmids also differ, with the exception of an approximately 12 kb segment of the virulence region, where the BamHI sites appear to be conserved. Complementation analysis has shown that deletion of a DNA segment which flanks the oncogenic T-DNA results in severe attenuation of virulence. This region also contains a sequence that is repeated in the Chry5 genome outside the Ti plasmid, and that is widely distributed in the Rhizobiaceae.     

Transformation of broccoli (Brassica oleracea var. italica) with isopentenyltransferase gene via Agrobacterium tumefaciens for post-harvest yellowing retardation

Transgenic plantlets with a retarding effect on post-harvest yellowing in broccoli have been generated via Agrobacterium tumefaciens-mediated transformation of cytokinin synthesizing ipt (isopentenyltransferase) gene. The ipt gene is constructed under the control of senescence-associated gene promoters from Arabidopsis in the forms of pSG529(+) and pSG766A, which were the gifts from Dr R.M. Amasino at University of Wisconsin, Madison. Evidence of transgene integration was confirmed by assays on neomycin phosphotransferase II (NPTII) activity of selection markers, PCR and Southern hybridization. Based on the chlorophyll retention rate (>50%) after 4 days of post-harvest storage at 25 °C, it was found that 31% of transformants exhibited the effect of retarding yellowing in detached leaves, with 16% having the effect on florets and 7.2% on both leaves and florets. RT-PCR revealed that ipt gene expression occurred early on the day of detachment. Factors such as vacuum aid infiltration, plasmid differences, explant types, seedling ages and kanamycin concentrations were also studied. Putative transformation frequencies tended to vary with plasmids and explant types. The advantage of vacuum aid infiltration depended on explant types. The optimal kanamycin concentration should be determined experimentally for each study to avoid the high escape rate of kanamycin selection. Flow cytometric analysis of explant nuclear DNA phases was found to be helpful for selecting suitable explants for transformation and minimizing the polyploid transformants. A reproducible transformation protocol without any pre-culture was established for explants of hypocotyl, cotyledon, and peduncle. Most of the ipt transformants with a retarding effect on yellowing had a chimeric nature but showed little or no serious morphological abnormality in comparison with their parental line. Through proper selection, transformation lines with the capability of retarding post-harvest yellowing in broccoli should be feasible.     

Production of marker-free transgenic Nierembergia caerulea using MAT vector system

Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Nierembergia caerulea cv. Mont Blanc employing ipt gene as the selectable marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. The MAT vector system is a positive selection system that gives the advantage of regeneration to the transgenic cells without killing the non-transgenic cells. Infected explants were cultured on hormone- and antibiotic-free MS medium, and 65% of the regenerated shoots developed ipt shooty phenotype-morphologically abnormal shoots, within approximately 3 months after co-cultivation. Twenty morphologically normal shoots were produced from 12 transgenic ipt shoots 7 months after co-cultivation. The normal shoots rooted well on hormone-free MS medium. Ninety percent of the normal shoots were ipt ⁻, GUS⁺ and excision⁺ as determined by PCR and Southern blot analyses. These results indicate that ipt-type MAT vector system can be used successfully in Nierembergia to produce marker-free transgenic plants without using phytohormones and selective chemical agents.     

Nucleotide sequence, evolutionary origin and biological role of a rearranged cytokinin gene isolated from a wide host range biotype III Agrobacterium strain

Summary A DNA fragment with homology to the cytokinin (ipt) gene from biotype I Agrobacterium tumefaciens strain Ach5 was cloned from the Ti plasmid of the wide host range biotype III Agrobacterium strain Tm-4 and sequenced. The fragment contains an intact ipt coding sequence. However, the 3 non-coding region of this ipt gene is rearranged due to a 0.9 kb deletion fusing it to the 3 coding region of the neighbouring gene 6a, most of which was found to be deleted. The Tm-4 ipt gene is strongly related to the partially deleted ipt gene of the limited host range biotype III strain Ag162. To test its biological activity, the Tm-4 ipt gene was inserted into a specially constructed, disarmed Ti vector lacking tzs and tested on tobacco, where the rearranged ipt gene induced shoot formation. The cloned Tm-4 ipt gene was mutated with Tn5 and the intact gene on the wild-type Tm-4 Ti plasmid was replaced by the mutated gene. The resulting strain was avirulent on tobacco but normally virulent on the natural host of the wild-type strain Tm-4, grapevine. As the biotype 1 6b gene diminishes the effect of a corresponding ipt gene, a larger Tm-4 fragment carrying both the ipt gene and an adjacent 6b-like gene was also tested on tobacco and compared with the Tm-4 ipt fragment alone and with an ipt and 6b/ipt fragment derived from Ach5. The Tm-4 6b gene diminishes the effect of the Tm-4 ipt gene, showing the Tm-4 6b gene to be active as well. The Tm-4 6b/ipt combination is less effective than the Ach5 combination. These results provide further insight into the molecular basis of the host range differences between limited host range and wide host range biotype III Agrobacterium strains and show that the WHR cytokinin gene, although active, does not significantly contribute to tumour formation on the natural host of the WHR biotype III strains, grapevine.Abbreviations LHR limited host range - WHR wide host range - onc oncogenicity genes - iaaH indoleacetamide hydrolase gene - iaaM tryptophan monooxygenase gene - ipt isopentenyl transferase gene - tzs transzeatin secretion gene - NAA -naphthalene acetic acid - BAP 6-benzylaminopurine - Km kanamycin - Neo neomycin - Cm chloramphenicol     

Inhibition of agrobacterial oncogene expression by means of antisense RNAs

Infection of plants by soil bacterium Agrobacterium tumefaciens induces tumors referred to as crown galls. Tumor development is determined by the introduction of agrobacterial genes governing phytohormone (auxin and cytokinin) production into the plant genome. The most important of these genes are iaaM and ipt. Development of transgenic plants inhibiting the expression of these genes allows a raise of varieties resistant to crown gall disease. For this purpose, single and double tobacco transformants with antisense copies of iaaM and ipt fused with single and double promoters for the 35S RNA of the cauliflower mosaic virus (CaMV 35S and CaMV 35SS) were obtained. Inoculation of transgenic plants harboring the antisense oncogene copies with virulent A. tumefaciens strains C58 (pTiC58) and A6 (pTiA6) revealed significant, but still incomplete, inhibition of these genes. Agrobacterium-mediated transformation of transgenic plants gave rise to weakened tumors, which varied in morphology and allowed regeneration of whole plants. Analysis of the inhibition of the iaaM and ipt expression in tumor cells demonstrated that the RNA interference strategy is promising for developing plant varieties resistant to agrobacterial infection.     

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Erratum

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco     

Genetic characterization of a double-flowered tobacco plant obtained in a transformation experiment

Summary A leaf-disk transformation experiment was performed with tobacco (Nicotiana tabacum L.) using a binary vector and a strain of Agrobacterium tumefaciens that carried a wild-type Ti-plasmid, pTiBo542. Although the majority of kanamycin-resistant, transgenic plants was morphologically normal, one of the plants was double-flowered and had a slightly wavy stem and leaves whose edges were bent slightly upwards. The abnormal morphology was controlled by a single, dominant Mendelian gene. Young plants that carried this gene were distinguishable from normal plants at the stage of cotyledons. The homozygotes, with respect to this gene, were more seriously deformed than the heterozygotes. DNA segments derived from the binary vector and from the T_L-and T_R-DNA of pTiBo542 were detected in the double-flowered plant, but the T-DNA genes involved in biosynthesis of phytohormones were absent from the plant. The abnormal morphology, the resistance to kanamycin, and the segments of foreign DNA were genetically linked, and the linkage was very tight, at least between the abnormal morphology and the resistance to kanamycin; the meiotic recombination frequency was less than 0.02%, if recombination occurred at all.     

Two Agrobacterium tumefaciens genes for cytokinin biosynthesis: Ti plasmid-coded isopentenyltransferases adapted for function in prokaryotic or eukaryotic cells

Summary Tzs and ipt are two Ti plasmid genes coding for proteins with isopentenyltransferase (IPT) activity in vitro. We cloned both genes for protein expression in Escherichia coli and in Agrobacterium tumefaciens, and we investigated differences between the two genes by analysing the properties of the proteins in vitro and in vivo. In vitro, extracts with tzs or ipt-coded proteins had high IPT activity, and the enzymes were identical in most properties. The most important difference was detected in vivo: the tzs-encoded protein was very active in cytokinin production, while the ipt protein required overexpression in order to obtain measurable activity in bacteria. In both cases, rans-zeatin was the major product of the gene activity. Formation of this cytokinin requires a hydroxylase function in addition to the IPT reaction. No such activity could be ascribed to tzs or ipt-encoded proteins in vitro or in vivo, but cytokinin hydroxylase activity was detected in cells and extracts of E. coli, regardless of the presence or absence of the cytokinin genes. Based on these results it is proposed that both genes code for a single enzyme activity (isopentenyltransferase), that the genes and proteins are adapted for function either in bacteria (tzs) or in transformed plant cells (ipt), and that in both prokaryotic and eukaryotic cells hydroxylation to trans-zeatin is a function contributed by host enzymes.Abbreviations DMAPP dimethylallylpyrophosphate - iP isopentenyladenine - iPA isopentenyladenosine - iPMP isopentenyladenosine 5-monophosphate - IPT isopentenyltransferase - trans-Z trans-zeatin     

Agrobacterium tumefaciens 6bgenes are strain-specific and affect the activity of auxin as well as cytokinin genes

Summary The T-region located 6b gene of Agrobacterium tumefaciens has been found to interfere with cytokinin effects produced by the cotransferred ipt gene. We have compared the biological activity of three different 6b genes: A-6b from Ach5 (octopine, biotype 1), C-6b from C58 (nopaline, biotype 1) and T-6b from Tm4 (octopine, biotype III) by using different biological assays. Each 6b gene was inserted into a disarmed vector and tested on tobacco stems in coinfection experiments with the Ach5 cytokinin (ipt) gene (A-ipt). A-ipt/C-6b coinfections produced tumours with shoots, A-ipt/A-6b coinfections green tumours and A-ipt/T-6b coinfections tumours with a necrotic surface. The tumour phenotypes obtained were independent of the 6b/A-ipt coinfection ratios, indicating that the strain-specific 6b effects result from the activity of a non-diffusible 6b encoded product. Studies with ipt-less Tm4 mutants showed that 6b genes affect other tumour genes besides the ipt gene and pointed to an influence of T-6b on auxin effects resulting from the Tm4 iaa system. T-iaa/T-6b coinfection experiments showed that T-6b did indeed strongly increase tumour formation by the Tm4 iaa genes. The three 6b genes also have effects which do not require other T-region genes. The complex role of the 6b gene in crown gall induction and Agrobacterium host range will be discussed.     

Asexual production of selectable marker-free transgenic woody plants, vegetatively propagated species

We describe here a practical system for generating selectable marker-free transgenic woody plants independent of sexual crossing. We previously reported that the GST-MAT vector system could produce marker-free transgenic tobacco plants containing a single-copy transgene at high frequency. The GST-MAT vector system consists of a DNA excision cassette of the R/RS site-specific recombination system from Zygosaccharomyces rouxii into which the isopentenyltransferase gene from Agrobacterium tumefaciens has been inserted. In this study, we applied this new GST-MAT vector to hybrid aspen (Populus Sieboldii X Populus grandidentata), a model of vegetatively propagated plant species, to produce selectable marker-free transgenic woody plants. In the new GST-MAT vector, the chimeric ipt gene fused with a light-inducible rbcS promoter efficiently produced transgenic ipt-shooty with GUS activity from 38.0% of infected stems. Upon excision of the R and ipt genes between RS sites, regulated by the inducible promoter of the maize glutathione-S-transferase (GST-II-27) gene, 3 (21.4%) transgenic hybrid aspen plants with marker-free and normal phenotype were generated from 14 ipt-shooty lines within 2 months after cutting induction. These results suggest that the MAT-vector system might be useful for removing a selectable marker gene independent of sexual crossing in vegetatively propagated species.     

The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI)

 A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the problems related to the use of antibiotics diminished. Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000     

Expression of isopentenyl transferase gene (<Emphasis Type="Italic">ipt</Emphasis>) in leaf and stem delayed leaf senescence without affecting root growth

A cytokinin biosynthetic gene encoding isopentenyl transferase (ipt) was cloned with its native promoter from Agrobacterium tumefaciens and introduced into tobacco plants. Indolebutyric acid was applied in rooting medium and morphologically normal transgenic tobacco plants were regenerated. Genetic analysis of self-fertilized progeny showed that a single copy of intact ipt gene had been integrated, and T₂ progeny had become homozygous for the transgene. Stable inheritance of the intact ipt gene in T₂ progeny was verified by Southern hybridization. Northern blot hybridization revealed that the expression of this ipt gene was confined in leaves and stems but undetectable in roots of the transgenic plants. Endogenous cytokinin levels in the leaves and stems of the transgenic tobaccos were two to threefold higher than that of control, but in roots, both the transgenic and control tobaccos had similar cytokinin levels. The elevated cytokinin levels in the transgenic tobacco leaves resulted in delayed leaf senescence in terms of chlorophyll content without affecting the net photosynthetic rate. The root growth and morphology of the plant were not affected in the transgenic tobacco.     

Regeneration of flax plants transformed by Agrobacterium rhizogenes

Regeneration of flax (Linum usitatissimum) following transformation by either Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector, or Agrobacterium rhizogenes carrying an unmodified Ri plasmid, was examined. Hypocotyl and cotyledon explants inoculated with A. tumefaciens formed transformed callus, but did not regenerate transformed shoots either directly or via callus. However, cotyledon explants inoculated with A. rhizogenes formed transformed roots which did regenerate transformed shoots. Ri T-DNA encoded opines were detected in the transformed plantlets and Southern hybridization analysis confirmed the presence of T-DNA from the Ri plasmid in their DNA. Transformed plantlets had curled leaves, short internodes and some had a more developed root system characterized by plagiotropic behaviour.