Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens: regeneration of transformed shoots via a callus phase

Genetic transformation of flax (Linum usitatissimum) has been achieved using an A. tumefaciens strain carrying a non-oncogenic Ti plasmid-derived vector containing a chimaeric npt-II gene and a wild type nopaline synthase gene. Fertile, transformed shoots were most easily obtained from Km^r callus developing on hypocotyl sections. The totipotency of the Km^r callus was dependent upon its origin. T-DNA was visualised by Southern blotting in all Km^r tissues. Efficient expression of nopaline synthase and the chimaeric npt-II gene was found in transformed Km^r callus and regenerated shoots.Abbreviations npt-II neomycin phosphotransferase II gene - NPT-II neomycin phosphotransferase II - nos nopaline synthase gene promoter - Km^r kanamycin resistant - BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MSD4×2 medium D4×2 based on Murashige & Skoog medium (see Scott & Draper, 1987)     

Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens

Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens harbouring pGV 38501103 neo (dimer) containing chimaeric kanamycin resistance reporter genes. Transformed protoplasts were regenerated to fertile and phenotypically normal plants. Transformation was confirmed by kanamycin resistance, nopaline production, neomycinphosphotransferase II activity, and Southern blot hybridization. Seed progeny from self-pollinated transformants expressed the introduced kanamycin resistance as a Mendelian trait.Abbreviations BAP 6-benzylaminopurine - Cf Claforan^R - 2.4D 2,4-dichlorophenoxy acetic acid - Km kanamycin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - NPT II neomycinphosphotransferase - npt II neomycinphosphotransferase II gene - NOS nopaline synthase - nos nopaline synthase gene - ocs octopine synthase gene - IAA indole-3-acetic acid     

Transformation of Brassica napus with Agrobacterium tumefaciens based vectors

A reproducible system to produce transgenic Brassica napus plants has been developed using stem segments. Stem segments from 6–7 week old plants were inoculated with an Agrobacterium tumefaciens strain containing a disarmed tumor-inducing plasmid pTiT37-SE carrying a chimeric bacterial gene encoding kanamycin resistance (pMON200). Stem explants were cocultured for 2 days before transfer to kanamycin selection medium. Shoots regenerated directly from the explant in 3–6 weeks and were excised, dipped in Rootone®, and rooted in soil. Transformation was confirmed by opine production, kanamycin resistance, and DNA blot hybridization in the primary transformants. Final proof of transformation was demonstrated by the co-transfer of opine production and kanamycin resistance to progeny in a Mendelian fashion. Over 200 transgenic Brassica napus plants have been produced using this system.Abbreviations BA 6-benzyladenine - NAA -naphthalene-acetic acid - T-DNA transferred DNA into plants - IBA indole butyric acid - IAA indole acetic acid - TXD Tobacco Xanthi diploid suspension cells     

Celery transformation by Agrobacterium tumefaciens: cytological and genetic analysis of transgenic plants

Transgenic celery plants were obtained following co-cultivation of petiole explants with Agrobacterlum tumefaciens containing pMON200, a cointegrate vector carrying genes for kanamycin resistance and nopaline synthase. Transformants were selected by ability of callus to grow in the presence of 50mg/l kanamycin. Transformation was confirmed either by the presence of nopaline or by Southern blots. Cytological analysis of 14 transformed plants revealed chromosomal aberrations, both in structure and number. Only 20% of the regenerated plants had the normal karyotype. Kanamycin resistance behaved as a monogenic, dominant trait, segregating in a 3:1 ratio in three families derived from plants with normal karyotypes.Abbreviations KB Kilobases - 2-4D 2,4-diphenoxyacetic acid     

A set of novel Ti plasmid-derived vectors for the production of transgenic plants

Summary We describe in this paper the construction and use of a set of novel Ti plasmid-derived vectors that can be used to produce transgenic plants. These vectors are based on one of two strategies: 1) double recombination into the wild-type Ti plasmid of genetic information flanked by two T-DNA fragments on a wide-host range plasmid; 2) the binary vector strategy. The vector based on the double recombination principle contains a kanamycin resistance gene for use as a plant selectable marker, a polylinker for the insertion of foreign genes, and a nopaline synthase gene. The vector was constructed such that a disarmed T-DNA results from the double recombination event. The binary vector combines several advantageous features including an origin of replication that is stable in Agrobacterium in the absence of selection, six unique sites for insertion of foreign genes, an intact nopaline synthase gene, and a kanamycin resistance marker for selection of transformed plant cells. All of these vectors have been used to produce tobacco plants transformed with a variety of foreign genes.     

Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome

Summary The genomic distribution and genetic behavior of DNA sequences introduced into the tomato genome by Agrobacterium tumefaciens were investigated in the backcross progeny of 10 transformed Lycopersicon esculentum x L. pennellii hybrids. All transformants were found to represent single locus insertions based on the co-segregation of restriction fragments corresponding to the T-DNA left and right border sequences in the backcross progeny. Isozyme and restriction fragment length polymorphism (RFLP) markers were used to test linkage relationships of the insertion in each backcross family. The T-DNA inserts in 9 of the 10 transformants were mapped in relation to one or more of these markers, and each mapped to a different chromosomal location. Because only one insertion did not show linkage with the markers employed, it must be located somewhere other than the genomic regions covered by the markers assayed. We conclude that Agrobacterium-mediated insertion in the Lycopersicon genome appears to be random at the chromosomal level. No discrepancies were found between the T-DNA genotype and the nopaline phenotype in the 322 backcross progeny of the nopaline positive transformants. Backcross progeny of two nopaline negative transformants showed incomplete correspondence between the T-DNA genotype and the kanamycin resistance phenotype. No alteration of T-DNA was observed in progeny showing a discrepancy between T-DNA and kanamycin resistance. However, two kanamycin resistant progeny plants of one of these two transformants possessed altered T-DNA restriction patterns, indicating genetic instability of the T-DNA in this transformant.Journal article no. 1223 of the New Mexico Agricultural Experiment Station     

Genetic analysis of T-DNA insertions into the tobacco genome

A genetic test was performed on seeds from 283 transgenic tobacco plants obtained by T-DNA transformation. Seeds from self-fertilized transgenic plants were germinated on kanamycin-containing medium, and the percentage of seeds which germinated, as well as the ratio of kanamycin-resistant to kanamycin-sensitive seedlings were scored. Nine categories of transformants could be distinguished according to the number of loci into which T-DNA had inserted, and according to the effects of T-DNA integration on seed or seedling development. In most of the plants, T-DNA was inserted into a single site; others contained multiple independent copies of T-DNA. The number of T-DNA integration sites was found to be independent of whether a binary vector system or a cointegrate Ti plasmid had been used to obtain the transgenic plant. Loss of marker genes or marker gene expression from generation to generation appeared to be a quite frequent event. Plants which appeared to be insertional recessive embryo-lethal mutants did not exhibit this trait in the next generation.Abbreviations Kan^R kanamycin resistant - Kan^S kanamycin sensitive - NOP nopaline - NOS nopaline synthase - NPT II neomycin phosphotransferase II     

Agrobacterium-mediated transformation of commercial melon (Cucumis melo L., cv. Amarillo Oro)

Cotyledon explants of muskmelon (Cucumis melo L., cv. Amarillo Oro) seedlings were co-cultivated with disarmed Agrobacterium tumefaciens strain LBA4404 that contained the binary vector plasmid pBI121.1. The T-DNA region of this binary vector contains the Nopaline synthase/neomycin phosphotransferase II (NPTII) chimeric gene for kanamycin resistance and the Cauliflower Mosaic Virus 35S/-glucuronidase (GUS) chimeric gene. After infection, the cotyledon pieces were placed in induction medium containing 100 mg/l kanamycin. Putative transformed shoots were obtained, followed by the development of morphologically normal plantlets. The transgenic nature of regenerants was demonstrated by polymerase chain reaction, Southern blot analysis, plant growth on medium selective for the transgene (NPTII) and expression of the co-transformed GUS gene. Factors affecting the transformation procedure are discussed.Abbreviations CaMV Cauliflower Mosaic Virus - Cf Cefotaxime - GUS -glucuronidase - Km Kanamycin - MS Murashige and Skoog - NOS nopaline synthase - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

Fertile transgenic Indica rice plants obtained by electroporation of the seed embryo cells

We have obtained fertile transgenic plants of Indica rice variety IR36, by using electroporation to transfer the neomycin phosphotransferase II (nptII) gene into cells of mature embryos. Resistant calli were selected in the presence of 30 g/ml G418. Nearly thirty transgenic plants were regenerated within three months after transformation. Many of them yielded seeds following self-pollination. Data from molecular analysis and enzyme assay proved that the foreign gene was stably integrated into the genome of resistant calli, R0 and R1 plants, and also expressed. Mendelian segregation of the nptII gene was observed in R1 progeny plants.Abbreviations NOS nopaline synthase - NPTII and nptII neomycin phosphotransferase II - OCS octopine synthase - Km kanamycin     

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     

Regeneration of transgenic plants from the commercial apple cultivar Royal Gala

A transformation system was developed for the commercial apple (Malus X domestica Borkh.) cultivar Royal Gala. Leaf pieces from in vitro-grown shoots were cocultivated for 2 days with Agrobacterium tumefaciens strain LBA4404 containing the binary vectors pKIWI105 or pKIWI110. Shoots were produced on a shooting medium containing kanamycin (50 mg·L^–1). A 2-day incubation period on kanamycin-free medium prior to antibiotic selection enhanced the regeneration of kanamycin-resistant shoots. The majority of the kanamycin-resistant shoots also expressed GUS (-glucuronidase) activity. The putatively transformed shoots were rooted on a medium containing kanamycin (50 mg·L^–1). Rooted plants were established in a greenhouse, and plants transformed with pKIWI110, which contains a mutant Arabidopsis acetolactate synthase gene, were shown to be resistant to the herbicide Glean. Integration of T-DNA into the apple genome was confirmed by PCR and Southern hybridization analyses.Abbreviations NAA -naphthaleneacetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine     

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid     

Monohybrid and dihybrid segregations in the progenies of tobacco transformed for kanamycin resistance with a Ti-vector system

A chimeric DNA construction having nopaline synthase promoter, coding sequences of neomycin phosphotransferase gene conferring resistance to antibiotic kanamycin and OCS (octopine synthase) polyadenylation sequences bracketed by T-DNA ends was transferred to tobacco. Leaf discs were infected withA. tumefaciens containing disarmed, cointegrate plasmid pGV3850:: 1103 and allowed to form a callus in the presence of kanamycin. Shoots regenerated from infected leaf discs either through the callus or arising directly were further selected for their ability to root in kanamycin-containing media. Among the nine transgenic plants that were progeny tested, the transferred bacterial gene segregated as monohybrid ratio (3 Kan^R: 1 Kan^s) in seven. Segregation data of two plant progenies indicated the presence of two independent loci of Kan^R DNA insertion (15 Kan^R: 1 Kan ^s ). Back-cross segregation data were consistent with the monohybrid or independent assortment of duplicate factors. Thus in the two cases, a minimum independent integration of two copies of T-DNA each with a Kan^R marker is inferred.     

Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts

Summary Mesophyll protoplasts of a kanamycin-resistant, nopaline-positive Nicotiana plumbaginifolia seed line were inactivated by -irradiation and electrically fused with unirradiated mesophyll protoplasts of N. tabacum. Hybrids were selected on kanamycin and regenerated. Genetic material from N. plumbaginifolia was detected in these plants by the following criteria: (1) morphology, (2) esterase isozyme profiles, and (3) the presence of nopaline in leaf extracts. All of the plants regenerated were morphologically more similar to N. tabacum than to N. plumbaginifolia, and many were indistinguishable from N. tabacum. It was found that 37 plants displayed one or two esterases characteristic of N. plumbaginifolia in addition to a full set of esterases from N. tabacum. Based on their esterases, we have classified these plants as somatic hybrids. However, irradiation has clearly reduced the amount of N. plumbaginifolia genetic material that they retain; 24 plants were found that had only N. tabacum esterases but that produced nopaline and were kanamycin resistant. Genomic DNA from several of these plants was probed by Southern blotting for the presence of the authentic neomycin phosphotransferase gene (kanamycin-resistance gene) — all were found to contain the gene. These plants were classified as asymmetric hybrids. Finally, 25 plants were regenerated that were kanamycin sensitive, negative for nopaline, and contained only N. tabacum esterases. All of the regenerated plants, including this final category, were male sterile. As transferring the N. plumbaginifolia cytoplasm to an N. tabacum nuclear background results in an alloplasmic form of male sterility, all of the plants regenerated in this study appear to be cybrids irrespective of their nuclear constitution. Chromosome analysis of the asymmetric hybrids showed that most of them contained one more chromosome than is normal for N. tabacum. The somatic hybrids examined all had several additional chromosomes. Although male sterile, the asymmetric hybrids were female fertile to varying degrees and were successfully backcrossed with N. tabacum. Analysis of the resultant F₁ progeny indicated that the kanamycin-resistance gene from N. plumbaginifolia is partially unstable during meiosis, as would be expected for factors inherited on an unpaired chromosome.Abbreviations Km ^r kanamycin resistant - Km ^s kamacysin sensitive - Nop ⁺ nopaline positive - Nop ^– nopaline negative     

Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 g/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 g/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.NRCC No. 31491During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94).     

Agrobacterium tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.)

Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring -glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - SDS Lauryl sulfate     

The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector

Summary A plant gene vector cassette to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.Abbreviations OCS octopine synthase (gene) - NOS nopaline synthase (gene) - NPT-II neomycin phosphotransferase (gene) of transposon Tn5 - vir Ti-plasmid region encoding virulence functions - Cb carbenicillin - Gm gentamycin - Km kanamycin - Cm chloramphenicol - Sm streptomycin - Sp spectinomycin - Rif rifampicin - Ery erythromycin - bom basis of mobilization - ori _r origin of conjugational plasmid transfer - Tra, Mob functions required for conjugational transfer of plasmids - BAP N⁶-benzylaminopurine - NAA -naphthaleneacetic acid - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide     

Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

Agrobacterium tumefaciens-mediated transformation of severalRubus genotypes and recovery of transformed plants

Experiments in shoot regeneration and virulentAgrobacterium tumefaciens-mediated transformation were used to develop a protocol forRubus transformation. This protocol was then used to produce transformedRubus plants fromin vitro internodes inoculated with anAgrobacterium tumefaciens encoding neomycin phosphotransferase on its disarmed T-DNA. Two transformed plants were selected from 800 inoculations on a medium containing 10 µg ml^–1 kanamycin. Results indicated that this level of kanamycin successfully selected against non-transformed cells but did not reduce the number of transformed, kanamycin-resistant, shoots formed. Enzyme assays and Southern blot analysis verified the presence of the -glucuronidase gene in the plant genome.