Binary Vector System Research Articles

Production of tropane alkaloids in genetically engineered root cultures

Hyoscyamine 6β-hydroxylase catalyses the oxidative reactions in the biosynthetic pathway leading from hyoscyamine to scopolamine. The hydroxylase gene of Hyoscyamus niger was placed under the control of the cauliflower mosaic virus 35S promoter and introduced to hyoscyamine-rich Atropa belladonna by a binary vector system using Agrobacterium rhizogenes. The presence of the transgene in kanamycin-resistant hairy roots was confirmed by the analysis using polymerase chain reaction. The engineered belladonna hairy roots showed increased amounts and enzyme activities of the hydroxylase, and contained up to five-fold higher concentrations of scopolamine than wild-type hairy roots. The contents of 6β-hydroxyhyoscyamine also increased in the transformed roots. Such genetically engineered hairy roots should be useful for enhancing scopolamine productivity in in vitro root culture systems.

A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome.

A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation. The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene. The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for beta-galactosidase. pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker. The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5' to 3' model of T-DNA transfer. The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ' region. Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.

Effects of agrobacterial oncogenes in kidney vetch (Anthyllis vulneraria L.)

Kidney vetch seedlings were induced to form hairy roots by inoculating their mesocotyls with the wild-type strain 15834 of Agrobacterium rhizogenes or with the A. tumefaciens strain C58C1 containing a binary vector system (the pRiA4b as a helper and the vector pCB1346 bearing a pTiC58-derived isopentenyl transferase gene (ipt, cytokinin biosynthetic gene) under control of its native regulatory sequences). Transgenic lines of three distinct phenotypes were selected: (i) Typically, the pRi15834-transformed tissues were stabilized in vitro and maintained for long periods as aseptic, fast-growing, hormone-independent, plagiotropic hairy root cultures which never regenerated shoots and lost the ability to synthesize opines. Their genomic DNA contained both the TL- and the TR-DNA. (ii) One of the HR-lines transgenic for the T-DNA of pRi15834 (named 52AV34) started to regenerate spontaneously into teratomous shoots. The shoots were found to produce opines and both the TL and TR parts of T-DNA were found to be partly deleted and/or rearranged. They contained phytohormones in similar levels as those found in seed-born shoots. (iii) A practically identical morphogenic response as in the line 52AV34 was observed in the clone 27AV46. However, its shooty, dark-green, slow-growing teratomas were proven to be kanamycin-resistant, opine-producing, and double-transformed by the pRiA4b sequences and the ipt gene. They over-produced auxins as well as cytokinins (mainly indoleacetylaspartic acid and ribosides of zeatin and isopentenyladenine).

INTRODUCTION OF β-GLUCURONIDASE GENE INTO GINSENG (PANAX CINSENG) BY A TI-PLASMID VECTOR SYSTEM AND ITS EXPRESSION

β-Glucuronidase (GUS) gene of Escherichia coli was introduced into ginseng cells by an Agrobacterium binary vector system and expressed in somatic embryos derived from the cells. A binary vector pBI121 carrying CaMV 35S promoter-GUS gene fusion and a neomycin phosphotransferase gene as selection marker was transferred into Agrobacterium tumefaciens LBA4404. Zygotic embryo cotyledonary segments were co-cultivated with A. tumefaciens and transferred to the medium containg 1 mg 2,4-dichlorophenoxyacetic acid/liter, 0.5 mg kinetin/liter, and 100 mg kanamycin/liter. Kanamycin-resistant calli were formed after 3 to 4 weeks of culture. Southern analysis confirmed the resistant calli were transformed with GUS gene. High GUS activities were detected in somatic embryos developed from the calli.

SUPERIOR REGENERATION AND AGROBACTERIUM INFECTABILITY OF BROCCOLI AND CAULIFLOWER TISSUES AND THE IDENTIFICATION OF A PROCEDURE FOR THE GENERATION OF TRANSGENIC PLANTS.

Broccoli and cauliflower are among the most regeneratively intractable genotypes found in the brassicaceae. To develop a method for transfer of the gene encoding S-adenosylmethionine hydrolase (SAMase) into inbred broccoli and cauliflower germplasm, we investigated the morphogenic competence and Agrobacterium susceptibility of a wide range of tissues of varied source. Appropriately controlled expression of the SAMase gene should, theoretically, reduce the plant's capacity for ethylene biosynthesis and extend the post harvest shelf life of the flower head. Through examination of the in vitro response of a wide range of tissues we identified procedures which support caulogenesis from 100% of explants, each producing more than 30 shoots which readily convert to plantlets. Studies with several wild type and disarmed Agrobacterium strains, and utilization of the binary vector system and appropriate marker and reporter genes, led to the identification of methods for high frequency T-DNA transfer to explant tissues and the flow frequency of transgenic plants containing SAMase gene.

Expression of cowpea mosaic virus coat protein precursor in transgenic tobacco plants

Tobacco, Nicotiana tabacum L., supports cowpea mosaic virus (CPMV) replication and cell-to-cell movement, and thus may serve as a model system to study coat protein-mediated protection against CPMV. A chimeric gene consisting of the cauliflower mosaic virus 35S promoter, CPMV 60K coat proteins-precursor (CP-P) coding region, and the nopaline synthase polyadenylation signal was transferred to tobacco cv. Burley 21 via the Agrobacterium tumefaciens binary vector system. Gene integration and expression in the transgenic tobacco plants were confirmed by Southern and RNA dot blot analyses. Accumulation of CPMV 60K CP-P in transgenic plants, up to 2 micrograms/g of wet weight tissue, was detected by ELISA and Western blots. The results of Western blots and immunosorbent electron microscopy further indicated that CPMV CP-P neither undergoes autoproteolysis to generate the mature viral coat proteins nor assembles into virus-like capsids, suggesting that processing of the CP-P may be required for virus assembly. Because CPMV neither induces symptoms in tobacco nor moves systemically, evaluation of the reactions of the transgenic plants to virus inoculation was based on virus accumulation in the inoculated leaves. Results from such infectivity experiments did not differentiate between CP-P expressers and vector-transformed plants. The transgenic tobacco plants expressing CP-P should provide valuable material for investigating comovirus polyprotein processing and capsid assembly in vivo.

Stability of a Cucumber Mosaic Virus Satellite RNA Sequence Integrated into Tobacco Nuclear DNA Using Agrobacterium tuefaciens.

A monomeric cDNA copy of cucumber mosaic virus (CMV) satellite RNA which is non-necrogenic to tomato was introduced into nuclear DNA of tobacco plants with the aid of the binary vector system of Agrobacterium tumefaciens. In order to check the stability of the introduced genes, kanamycin resistance and expression of biologically active satellite RNA upon infection with CMV were assayed up to the fifth generation of the homozygous progeny obtained from a primary transformant. The inserted cDNA in the progeny of the fifth generation was sequenced after cloning of the sequence amplified with the polymerase chain reaction. Neither loss nor mutations of the inserted genes were detected.

Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants

Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5' upstream sequence of the granule-bound starch synthase gene from potato and the beta-glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.

Tissue-specific and stress-enhancing expression of the TR promoter for mannopine synthase in transgenic medicinal plants

Transgenic tobacco (Nicotiana tabacum L.), licorice (Glycyrrhiza uralensis Fisher) and foxglove (Digitalis purpurea L.) were obtained with binary vector systems based on a disarmed Agrobacterium tumefaciens and on a virulent A. rhizogenes. The chimeric neomycin phosphotransferase II (NPT-II) gene (kan) and the ß-glucuronidase (GUS) gene (uidA) were under the control of the TR1' and 2' promoters, respectively, on a binary vector, pGSGluc1. Tissue-specific expression of the chimeric TR2' -uidA gene was studied using regenerants of N. tabacum from transformed callus and hairy roots, and also using the transformed roots of G. uralensis and D. purpurea. In all these transformed tissues, the phloem and surrounding tissues were stained with 5-bromo-4-chloro-3-indolyl-ß-d-glucuronide, showing the specific expression of uidA. The enzymatic assays of NPT-II and GUS indicated that the expression of both TR1' -kan and TR2'- uidA were coordinately enhanced by wounding and by the addition of plant growth regulators. These results indicate that the gene expression by the dual TR promoters is regulated in the several plant species studied in a tissue-specific manner and enhanced by physiological stresses.

Regeneration and transformation of Ribes

Transformation of the black currant cv. Ben More was achieved by utilising the binary vector system of Agrobacterium tumefaciens. This system involved the inoculation of peeled internodal stem segments with A. tumefaciens strain LBA4404 containing the binary vector PBI121.X with the marker genes Betaglucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Shoot regeneration occurred on nutrient media based on M&S salts. Transformation was confirmed by the fluorogenic assay procedure which determined that the GUS gene had been transferred into the plant material and was being expressed. Concurrent transfer of the NPTII gene into the plant material was also confirmed with a ‘dot blot’ assay on selected GUS positive plantlets.

Transformation of Japanese potato cultivars with the β-glucoronidase gene fused with the promoter of the pathogenesis-related 1a protein gene of tobacco

A fragment of the pathogenesis-related protein 1a (PR1a) gene of tobacco was introduced into Japanese potato cultivars by Agrobacterium-mediated gene transfer using the binary vector system. A PR1a promoter/β-glucoronidase (GUS) fusion gene was used to study the expression of the gene after salicylic acid treatment. Transformed shoots were recovered from tuber discs using a kanamycin resistant gene as a selective marker. Transformants were obtained from 11 cultivars including leading Japanese varieties. Southern blot analysis of transformants revealed that a DNA probe for the GUS coding region hybridized with nuclear DNA of transformants and the copy number of introduced gene was different among transformants. The transformants having the GUS reporter gene fused with the PR1a promoter showed high GUS activity after salicycic acid treatment. The level of GUS gene expression was almost as high as in transformed potatoes having the GUS gene fused with the CaMV-35S promoter. Wide variation in the level of gene expression among regenerated transformants was observed. The inducible expression of tobacco PR1a gene in transformed potato cultivars showed that the promoter of the PR1a gene is valuable for potato improvement and is capable of driving the expression of target genes under stress conditions.

Expression of a Chicken Ovalbumin Gene in Three Lucerne Cultivars

Routine procedures have been developed for the transformation of lucerne (Medicago sativa cv. Rangelander) with foreign genes using the Agrobacterium tumefaciens binary vector system and for the regeneration of transgenic plants from tissue culture, via somatic embryogenesis. Lucerne transformation was carried out with a gene encoding neomycin phosphotransferase (npt), which conferred resistance to the antibiotic kanamycin, together with a cDNA clone encoding chicken ovalbumin which was modified for expression in plant cells. The ovalbumin cDNA protein coding sequence was combined with the cauliflower mosaic virus 35S promoter and the nopaline synthase 3' flanking sequence to make a chimeric ovalbumin gene. A DNA construct containing both these genes was transferred to lucerne, and ovalbumin was detected in leaves of regenerated plants using protein immunoblots. Pulse-chase labelling experiments and analysis of leaves from the top to bottom of the transformed plants indicated that ovalbumin, once formed, was stable in the leaves of transgenic lucerne. A wide variation in ovalbumin level was frequently observed in plants regenerated from multiple embryos on a single transformed callus. This variation correlated with changes in the restriction enzyme digestion pattern of the ovalbumin DNA from the transgenic plants. These results indicate that each transformed callus may have arisen from more than one transformation event. An alternative interpretation is that the callus may have arisen from a single transformed cell but during cell proliferation the DNA in some cells may have undergone rearrangement prior to embryogenesis. Transformation and regeneration procedures were also developed for two Australian commercial cultivars of lucerne. Although the frequency of recovery of transformed plants was lower than with cv. Rangelander, these protocols open the way for a relatively rapid

The non-conserved region of cucumopine-type Agrobacterium rhizogenes T-DNA is responsible for hairy root induction

The T-DNA regions of three strains of Ri plasmids 1855, 8196, 2659 (agropine, mannopine and cucumopine type respectively) share two highly conserved regions flanking a non-homologous central part. We have cloned segments of the cucumopine Ri plasmid 2659 T-DNA in the binary vector system Bin 19 and infected carrot discs with recombinant Agrobacterium strains. We show here that the central non-conserved region is crucial in hairy root induction as it is sufficient to induce rooting on the apical (auxin-rich) surface of carrot discs; in order to observe rooting on the basal (auxin-depleted) side of the discs, a longer T-DNA fragment, also encompassing part of the right conserved region, had to be utilized in conjunction with a Agrobacterium strain carrying aux genes. Differences of growth properties in culture are exhibited by roots transformed with different fragments of pRi 2659 T-DNA, although all transformed roots show the plagiotropic behaviour typical of hairy roots.

Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid

A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two of the constructs these multiple cloning sites are flanked by a strong promoter to activate transcription of inserted DNA in planta. High-efficiency transformation was prompted by a high-copy, stable virulence helper plasmid pUCD2614, which contains a cloned virulence region of pTiC58 and tandem copies of the par locus of plasmid pTAR. Southern blot hybridization and genetic analyses of the progeny of transformed plants showed that the hygromycin resistance gene was stably inherited.

Genetic transformation of foxglove (Digitalis purpurea) by chimeric foreign genes and production of cardioactive glycosides

The chimeric neo and gus genes on a mini Ti vector are efficiently transferred into the genome of fox glove (Digitalis purpurea L.) using a binary vector system based on a rootinducing Ri plasmid, pRi15834. The transgenic state of established transformed roots was confirmed by Southern blot analysis and by detection of agropine and mannopine. The expression of the chimeric genes controlled by the promoters from TR 1'-2' genes, nos gene and cauliflower mosaic virus 35S RNA was demonstrated by enzymatic and histochemical assays of neomycin phosphotransferase II and ß-glucuronidase. Enzyme-linked immunosorbent assay (ELISA) was carried out using polyclonal antibody reactable against digitoxin to investigate the production of cardenolides. The results of ELISA indicated that the cardioactive glycosides were highly produced in the green transformed hairy roots.

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.

Stable transfer and expression of chimeric genes in licorice (Glycyrrhiza uralensis) using an Ri plasmid binary vector

The pharmaceutically important plant, licorice (Glycyrrhiza uralenesis Fisher), was transformed with a binary vector system of an Ri plasmid, pRi15834, and a mini Ti vector, pGSGluc1, containing chimeric neo and gus genes. The transgenic state of transformed roots was confirmed by detection of agropine and mannopine and by Southern blot hybridization with T-DNA of pGSGluc1. One to three copies of T-DNA of pGSGluc1 was integrated into the genomic DNA of G. uralensis. The expression of chimeric neo and gus genes driven by TR 1' and 2' promoters, respectively, was demonstrated by enzymatic assays. Histochemical analysis showed that the chimeric TR2'-gus gene was expressed specifically in phloem and pericycle tissues of the transformed licorice roots.

Use of the GUS gene as a selectable marker for Agrobacterium-mediated transformation of Rubus

A transformation system was established for red raspberry, blackberry and blackberry x raspberry hybrids, utilizing the binary vector system of Agrobacterium tumefaciens. Leaf discs or internodal stem segments were inoculated with Agrobacterium strain LBA4404 containing the binary vectors PBI121.X, which has the β-glucuronidase (GUS) marker gene, or Bin 19, which has the neomycin phosphotransferase II (NPT II) gene. Regenerants were produced on media containing MS salts, 20 gl-1 sucrose, 7 gl-1 agar, 100 mgl-1 inositol, 0.5 mgl-1 nicotinic acid, 0.5 mgl-1 pyridoxine-HCl, 0.1 mgl-1 thiamine, and either 0.1 mgl-1 IBA and 2 mgl-1 BAP for leaf discs, or 0.2 mgl-1 BAP and 0.2 mgl-1 2,4-D for stem segments. Kanamycin sulphate, which was used as a selective agent for the NPT II gene, inhibited organogenesis at 50 mgl-1 and was therefore unsuitable for use as a selectable marker gene in Rubus. All regenerants were assayed utilizing the fluorogenic assay procedure to determine if the GUS gene had been transferred into the material and could therefore cleave the substrate 4-methyl-umbelliferyl-β-D-glucuronide. Seven GUS-positive plantlets were obtained which confirmed that this marker gene had been transferred into Rubus. A ‘dot blot’ assay was carried out on GUS-positive plant material to establish if the NPT II gene had also been transferred to the plant material.

Genetic Transformation of Nicotiana Tabacum cv. Nevrokop A24

ABSTRACTThe efficiency of genetic transformation of N. tabacum cv.Nevrokop A24 by the binary vector system of Agrobacterium tumefaciens has been studied. By gene expression assays the transgenic nature of the obtained plants has been determined. The influence of the integration of chimeric genes into the genome of tobacco has been investigated by following the changes in the ultrastructure of chloroplasts.

Transformation of white spruce and other conifer species byAgrobacterium tumefaciens

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0-80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved.