Genetic transformation of a hepatoprotective plant, <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Phyllanthus amarus Schum & Thonn. is a source of various pharmacologically active compounds such as phyllanthin, hypophyllanthin, gallic acid, catechin, and nirurin, a flavone glycoside. A genetic transformation method using Agrobacterium tumefaciens was developed for this plant species for the first time. Shoot tips of full grown plants were used as explants for Agrobacterium-mediated transformation. Transgenic plants were obtained by co-cultivation of shoot tips explants and A. tumefaciens strain LBA4404 containing the pCAMBIA 2301 plasmid harboring neomycin phosphotransferase II (NPT II) and β-glucuronidase encoding (GUS) genes in the T-DNA region in the presence of 200 μM acetosyringone. Integration of the NPT II gene into the genome of transgenic plants was verified by PCR and Southern blot analyses. Expression of the NPT II gene was confirmed by RT-PCR analysis. An average of 25 explants was used, out of which an average of 19 explants produced kanamycin-resistant shoots, which rooted to produce 13 complete transgenic plants.     

Genetic transformation of Medicago truncatula using Agrobacterium with genetically modified Ri and disarmed Ti plasmids

Summary Fertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R₁ progeny.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - NPT neomycin phosphotransferase     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene

Summary Genetically transformed plants of Brassica napus L. (oilseed rape) were obtained from hypocotyl expiants using Agrobacterium tumefaciens vectors. Hypocotyl explants were inoculated with disarmed or oncogenic A. tumefaciens strains, EHA101 and A281, and then cultured on media containing kanamycin. The A. tumefaciens strains harbored a binary vector, which contained a neomycin phosphotransferase II (NPTII) gene driven by the 35S promoter of cauliflower mosaic virus and an engineered napin (seed storage protein) gene with its own promoter (300 nucleotides 5 to the start of translation). Transformation of B. napus plants was confirmed by detection of NPT II enzyme activity, Southern blot analysis and inheritance of the kanamycin-resistance trait (NPT II gene) in the progeny. Expression of the engineered napin gene in embryos but not in leaves of transgenic plants was observed by Northern analysis. These data demonstrate that morphologically normal, fertile transgenic B. napus plants can be obtained using Agrobacterium as a gene vector and that developmentally regulated expression of reintroduced genes can be achieved.     

Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants

Summary Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes.Abbreviations AMV RNA4 Alfalfa mosaic virus RNA4 - BA 6-benzyladenine - CaMV cauliflower mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraacetic acid - FAA formalin-acetic acid-alcohol - GUS ß-glucuronidase - NAA 1-naphthylacetic acid - NPT II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - TE Tris-Cl/EDTA - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl-urea (thidiazuron) - WPM woody plant medium (Lloyd and McCown 1980) - X-GLUC 5-bromo-4-chloro-3-indolyl-ß-glucuronic acid     

Susceptibility of dry bean (Phaseolus vulgaris L.) to Agrobacterium infection: Transformation of cotyledonary and hypocotyl tissues

A germinating-seed assay was developed to determine the susceptibility of dry bean (Phaseolus vulgaris L.) to infection by Agrobacterium tumefaciens. Seedlings infected one to three days after germination were more susceptible to A. tumefaciens infection than seedlings germinated for five to seven days and the galls that formed on the one to three day seedlings were significantly larger. Nineteen genotypes of dry bean were screened with this assay and all were equally susceptible to nopaline, octopine and agropine biotypes of A. tumefaciens. In addition, cotyledonary nodes and hypocotyls of P. vulgaris were inoculated with disarmed strain A. tumefaciens strain C58Z707 and the avirulent A. rhizogenes strain A4RS (pRiB278b), respectively. Both strains contain the binary plasmid pGA482 which has the neomycin phosphotransferase II (NPT II) gene nested between T-DNA borders. From these infected tissues, callus and root tissues, respectively capable of growing in the presence of kanamycin were obtained. These tissues displayed NPT II activity and integrated copies of the NPT II gene were detected from putative transformed root cultures by genomic blot hybridization.     

Multiple copies of virG enhance the transient transformation of celery,carrot and rice tissues by Agrobacterium tumefaciens

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical -glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.Current address: Department of Agronomy, Purdue University     

Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation

Summary The 200 kb Agrobacterium Ti-plasmid pTiT37 carries a 25 kb segment of T-DNA which it transfers to plant cells during crown-gall tumorigenesis. We have previously engineered into this T-DNA a pBR322-derived cloning vector which enabled us to rescue-clone full length T-DNA from the Ti-plasmid into a 36 kb MINI-Ti plasmid. We report here the deletion of oncogenes from MINI-Ti to produce Micro-Ti containing the nopaline synthase gene and the ampicillin resistance gene and origin of replication of pBR322, flanked by left and right T-DNA borders. Micro-Ti was recloned into the wide host range plasmid pRK290 and transformed into an A. tumefaciens strain carrying a helper plasmid that could supply Virulence (VIR) genes in trans. Using the octopine Ti-plasmid pTiB6-806 as a helper, transformed tobacco cells were obtained which produced both nopaline and octopine. Two cloned cell lines producing both opines were found to be hormone dependent and to produce fertile tobacco plants. We selfed one of these plants and found that the two opine markers segregated in the F1 progeny in a Mendelian fashion. This showed that the T-DNAs were not linked in the transformed plant genome. Southern blot analysis of the genomic DNA from the regenerated plant showed that only part of the (oncogenic) octopine T-DNA was present indicating that it had suffered a deletion in the auxin producing locus (tms region). Presence of the cytokinin autonomy locus presumably accounts for the abnormal rooting behavior of the F1 progeny seedlings containing this T-DNA.Abbreviations NAA Naphtalene acetic acid - IAA Indole-3-acetic acid - BA 6-benzylaminopurine - pCPA para-chlorophenoxyacetic acid Part of this work was presented for her doctoral thesis by A. JdF at the National Institute of Agronomy of Paris-Grignon, January 1983     

Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens

A chimaeric neomycin phosphotransferase II (NPT II) gene was introduced in Brassica oleracea using an oncogenic strain of Agrobacterium tumefaciens harbouring Ti plasmid which contains Nos/NPTII in its T-DNA. The transformation of B. oleracea with the oncogenic Ti plasmid, resulted in regeneration of shoots and roots without any exogenous requirement of phytohormones. The presence of NPT II gene was determined by hybridization of Tn5 encoded NPT II gene with DNA of kanamycin resistant regenerated plants. The expression of NPT II was demonstrated by kanamycin phosphorylation assay. Several regenerated plants were obtained, a few of them were found to be morphological variants and a chlorophyll deficient mutant plant was also obtained.     

Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens

Cell wall regenerating protoplasts from soybean cells kept in suspension culture were cocultivated with bacteria which were derived from the nopaline strain C58 of Agrobacterium tumefaciens. When the bacteria carried an oncogenic Ti-plasmid, about 5% of the surviving protoplasts were able to form calli on hormone-free agar in contrast to controls, where bacteria without Ti-plasmid were applied, and where no calli were formed. After isolation of DNA from hormone-independently growing cells further evidence for transformation was obtained by hybridization to Ti-plasmid specific RNA and by rescue of a segment with a bacterial resistance gene which had been inserted before into the T-DNA. Transfer of T-DNA harboring a neomycin-resistance gene activated by the nos-promoter resulted in calli growing on kanamycin. Verification of segments located at the left and the right part of the T-DNA indicated the presence of its entire length in transformed soybean cells. Expression of T-DNA genes was measured by the assay of nopaline-synthase. Cells cultured on agar had a much higher level of nopaline-synthase than fast growing cells in suspension culture. Transferring them to agar or treatment with azacytidine strongly increased synthesis of nopaline-synthase indicating a reversible repression presumably via a methylation mechanism.     

Transient transfection of mammalian cells with DNA of the plant-pathogenic Ti-plasmid and expression of marker and resident sequences

Summary The large Ti-plasmid from Agrobacterium tumefaciens strain C58 has been used for transfection experiments with mammalian cells. In DNA from Tupaia baby fibroblasts Ti-plasmid sequences could be identified by filter hybridization as long as four weeks after transfection including two cell passages. The hybridization signals decreased rapidly after addition of the Ti-plasmid DNA-coprecipitate to the cells. The signals were often not detected any more after the first day, but were visible one week after transfection. Nuclei prepared from Ti-plasmid-transfected cells hybridized to pTi-specific RNA. With the chloramphenicol acetyl transferase-gene as marker no discrimination in DNA uptake was found between the Ti-plasmid and much smaller plasmids. According to the number of nuclei with homology to pTi-sequences it is assumed that about 0.2% of the cells carry Ti-plasmid DNA in the nucleus. Analysis of RNA isolated from cells transfected with cloned segments of the Ti-plasmid revealed that the TDNA region of the Ti-plasmid was predominantly transcribed.Abbreviations CAT Chloramphenicol Acetyl Transferase - NPT Neomycin Phosphotransferase - SDS Sodium Dodecylsulfate - TK Thymidine Kinase     

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.     

High-efficiency transformation of Arabidopsis thaliana with a selectable marker gene regulated by the T-DNA 1' promoter

Two selectable marker genes harbouring the bar coding region but differing in their promoters were compared in an Arabidopsis thaliana transformation assay using in planta infiltration with Agrobacterium tumefaciens. Surprisingly, in four Arabidopsis ecotypes examined, the 1′ promoter from the right T-DNA was superior to the most commonly used 35S promoter of cauliflower mosaic virus (CaMV). The ecotype Wassilewskija gave the highest transformation frequencies, with an average of between 5.3 and 6.3 % of the seedlings subjected to the selection. This is approximately 30-fold higher than previously reported results. Analysis of T-DNA integration patterns in single transformed plants or pooled populations revealed independent T-DNA integration events in each case. Results show that the 1′ promoter is an attractive alternative to the 35S promoter for the generation of T-DNA insertion lines. The 1′ promoter may be especially beneficial for the secondary transformation of transgenic strains containing the 35S promoter to exclude homology-mediated gene silencing.     

Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1, D2 endonuclease

During the initial stages of crown gall tumorigenesis, the T-DNA region of the Agrobacterium tumefaciens Ti-plasmid is processed, resulting in the production of T-DNA molecules that are subsequently transferred to the plant cell. Processing of the T-DNA in the bacterium involves the nicking of T-DNA border sequences by an endonuclease encoded by the virD locus, and the subsequent tight (possibly covalent) association of the VirD2 protein with the 5′ end of the processed single-stranded or double-stranded T-DNA molecule. To investigate the interaction of the VirD1,D2 endonuclease with a right T-DNA border, a set of plasmids containing both the border and virD sequences on the same high-copy-number replicon has been constructed and introduced into Escherichia coli. In this model system a tight nucleoprotein complex is formed between the relaxed double-stranded substrate plasmid and the VirD2 protein. This putative T-DNA processing complex may be analogous to the covalent relaxation complex formed between the pilot protein and plasmid DNA during bacterial conjugation. VirD2 attachment to the relaxed substrate plasmid was resistant to denaturing agents but sensitive to S1 nuclease digestion, indicating a single-stranded region near the site of protein attachment. We speculate that this structure may be an intermediate formed prior to T-strand unwinding from the substrate plasmid in a host bacterium.     

Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens

Introduction of foreign genes into plant tissues via Agrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L.), five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors.Abbreviations GUS ß-glucuronidase (EC 3.2.1.31) - NPT-II neomycin phosphotransferase II (EC 2.7.1.95) - EDTA ethylene-diamine-tetracetic acid     

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     

Transformation of Cucumis sativus tissue by Agrobacterium tumefaciens and the regeneration of transformed plants

Cotyledons of cucumber seedlings (Cucumis sativus L. cv. Poinsett 76) were co-cultivated with disarmed Agrobacterium strain C58Z707. The Agrobacterium strain contained the Agrobacterium-derived binary vector plasmid pGA482, its T-DNA region contains a plant expressible bacterial derived neomycin phosphotransferase II (NPT II) gene which upon transfer, genome integration, and expression in plant tissues confers resistance to the antibiotic kanamycin. After growth of inoculated cotyledon sections on selective medium containing 100 mg/l kanamycin, transformed embryogenic calli were obtained followed by the development of embryos and plant regeneration. Transformed R₀ and R₁ cucumber plants appeared normal and tested positive for NPT II enzyme activity. Genomic DNAs isolated from the NPT II positive plants all showed hybridization to the characteristic 2.0 kb (BamHI to HindIII) NPT II gene-containing fragment. These results show that the Agrobscterium-mediated gene transfer system and regeneration via somatic embryogenesis is an effective method for the transfer of genetic material into plant species belonging to the family Cucurbitaceae.Abbreviation Cb carbenicillin - 2,4-D 2,4-dichlorophenoxyacetic acid - Km kanamycin - KN kinetin - MS Murashige and Skoog - NAA naphthaleneacetic acid - NPT II neomycin phosphotransferase II     

Agrobacterium mediated transformation and regeneration of Populus

Summary A plant transformation and regeneration system has been developed for Populus species. Leaf explants, from stabilized shoot cultures of a Populus hybrid NC-5339 (Populus alba x grandidentata), were co-cultivated with Agrobacterium tumefaciens on a tobacco nurse culture. Both oncogenic and disarmed strains of A. tumefaciens harboring a binary vector which contained two neomycin phophotransferase II (NPT II) and one bacterial 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (aroA) chimeric gene fusions were used. Shoots did not develop when leaf explants were co-cultivated with the binary disarmed strain of A. tumefaciens. However, transformed plants with and without the wild type T-DNA were obtained using an oncogenic binary strain of A. tumefaciens. Successful genetic transformation was confirmed by NPT II enzyme activity assays, Southern blot analysis and immunological detection of bacterial EPSP synthase by Western blotting. This is the first report of a successful recovery of transformed plants of a forest tree and also the first record of insertion and expression of a foreign gene of agronomic importance into a woody plant species.     

Genetic studies on the role of octopine T-DNA border regions in crown gall tumor formation

Summary Crown gall tumors result from transfer and integration of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant nuclear DNA. In the present study, recombinant plasmids containing deletion and rearrangement deriviatives of the T-DNA region of the octopine Ti plasmid pTiA6 were tested in a binary tumorigenesis system (Hoekema et al. 1983) to determine the requirements for T-DNA border regions in tumor formation. Since two defined segments of the T-DNA region of octopine Ti plasmids can be detected in tumor DNA (the left (T_L-) and right (T_R-) DNA), four border regions exist in this Ti plasmid. Agrobacteria harboring plasmid constructs which contain a T-DNA gene capable of inciting tumors (gene 4, the tmr gene, which is involved in cytokinin biosynthesis) and various T-DNA border regions were tested for ability to cause tumors on Nicotiana glauca and other host plants. Such tmr constructs containing as their only border region the right border of either the T_L-DNA or the T_R-DNA are fully tumorigenic. Analogous tmr constructs containing only the T_L-DNa left border region are not tumorigenic. These results do not depend on the orientation or position of the single border with respect to the tmr gene; furthermore, the T_R-DNA right border can confer tumor-forming ability despite the presence of an intervening copy of the T_L-DNA left border.These results for relatively small plasmids are contrasted with previously determined requirements for border regions in tumorigenesis by intact Ti plasmids. A model previously proposed by Wang et al. (1984) for the role of border regions in DNA transfer to plant cells is extended in order to explain the tumor-forming ability of plasmid constructs containing a single border region. The results of this study interpreted according to the model suggest that the octopine T_L-DNA left border is defective in this DNA-transfer process.