Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated -glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting -glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.     

Effects of an antisense napin gene on seed storage compounds in transgenic Brassica napus seeds

To manipulate the quantity and quality of storage components in Brassica napus seeds, we have constructed an antisense gene for the storage protein napin. The antisense gene was driven by the 5-flanking region of the B. napus napin gene to express antisense RNA in a seed-specific manner. Seeds of transgenic plants with antisense genes often contained reduced amounts of napin. In some transgenic plants, no accumulation of napin was observed. However, the total protein content of transgenic and wild-type seeds did not differ significantly. Seeds lacking napin accumulated 1.4 to 1.5 times more cruciferin than untransformed seeds, although the oleosin content was not affected. Fatty acid content and composition in the seeds of transgenic plants were also analyzed by gas chromatography. Though the total fatty acid content of the transformants was the same as that of non-transformants, there was a reduction in 18:1 contents and a concomitant increase of 18:2 in seeds with reduced napin levels. This observed change in fatty acid composition was inherited in the next generation.     

Heavy metal tolerant transgenic Brassica napus L. and Nicotiana tabacum L. plants

Summary A chimeric gene containing a cloned human metallothionein-II (MT-II) processed gene was introduced into Brassica napus and Nicotiana tabacum cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens. Transformants expressed MT protein as a Mendelian trait and in a constitutive manner. Seeds from self-fertilized transgenic plants were germinated on media containing toxic levels of cadmium and scored for tolerance/ susceptibility to this heavy metal. The growth of root and shoot of transformed seedlings was unaffected by up to 100 M CdCl₂, whereas control seedlings showed severe inhibition of root and shoot growth and chlorosis of leaves. The results of these experiments indicate that agriculturally important plants such as B. napus can be genetically engineered for heavy metal tolerance/sequestration and eventually for partitioning of heavy metals in non-consumed plant tissues.     

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     

Developmentally regulated site-specific marker gene excision in transgenic <Emphasis Type="Italic">B. napus</Emphasis> plants

We have developed a self-excision Cre-vector to remove marker genes from Brassica napus. In this vector cre recombinase gene and bar expression cassette were inserted between two lox sites in direct orientation. These lox-flanked sequences were placed between the seed-specific napin promoter and the gene of interest (vstI). Tissue-specific cre activation resulted in simultaneous excision of the recombinase and marker genes. The vector was introduced into B. napus by Agrobacterium-mediated transformation. F1 progeny of seven lines with single and multiple transgene insertions was subjected to segregation and molecular analysis. Marker-free plants could be detected and confirmed by PCR and Southern blot in all transgenic lines tested. The recombination efficiency expressed as a ratio of plants with complete gene excision to the total number of investigated plants varied from 13 to 81% dependent on the transgene copy number. Potential application of this system would be the establishment of marker-free transgenic plants in generatively propagated species.     

Use of a TR T-DNA promoter to express genes in plants and bacteria

Summary A series of plasmids have been constructed in which a promoter from the T_R region of the Agrobacterium tumefaciens Ti-plasmid has been fused to genes encoding neomycin phosphotransferase II (NPT II) or a cDNA clone encoding a 22 kd zein protein. After recombination into the Ti-plasmid pTiA6, A. tumefaciens strains harboring these plasmids were used to incite tumors on sunflower hypocotyl sections. Tumors containing the NPT II gene in the correct orientation relative to the promoter were able to grow on normally inhibitory concentrations of the antibiotic G418. Tumors with the NPT II gene in the incorrect orientation, or with the zein gene in either orientation, were killed by the antibiotic. S1 nuclease protection experiments indicated that for both the NPT II and zein genes, the T-DNA promoter was functioning correctly. The T-DNA fragment containing the promoter active in plants also contained promoter sequences active both in E. coli and in A. tumefaciens.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

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.     

Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants

Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA benzyladenine - CaMV cauliflowermosaic virus - GUS ß-glucuronidase - LB Luria Broth - MS Murashige and Skoog - NAA naphthalenacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PEG polyethylene glycol - RM rooting medium - SRM shoot regeneration medium     

A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and -glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R₂ progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.     

Transgenic sweet potato plants obtained byAgrobacterium tumefaciens-mediated transformation

Stable expression of foreign genes was achieved in sweet potato (Ipomoea batatas (L.) Lam) plants using anAgrobacterium tumefaciens mediated system. Embryogenic calluses produced from apical meristems of cultivar White Star were multiplied and cocultivated withA. tumefaciens strain EHA101 harboring a binary vector containing the -glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes. The calluses were transferred to selective regeneration medium and kanamycin resistant embryos were recovered which developed into morphologically normal plants. Histochemical and fluorimetric GUS assays of plants developed from the kanamycin resistant embryos were positive. Amplified DNA fragments were produced in polymerase chain reactions using GUS-specific primers and DNA from these plants. Transformation was confirmed by Southern analysis of the GUS gene. With the developed method, transgenic sweet potato plants were obtained within 7 weeks. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.Florida Agricultural Experiment Station Journal Series N-02231. This research was partially supported by CNP_q/RHAE (Brazil).     

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.     

Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: transgenic plants with normal phenotypes harbor binary vector T-DNA,but no Ri-plasmid T-DNA

Summary Cultivated tomato was genetically transformed using two procedures. In the first procedure, punctured cotyledons were infected with disarmed Agrobacterium tumefaciens strain LBA4404 or with A. rhizogenes strain A4, each containing the binary vector pARC8. The chimeric neomycin phosphotransferase (NPT II) gene on pARC8 conferred on transformed plant cells the ability to grow on medium containing kanamycin. Transformation reproducible yielded kanamycin-resistant transformants in different tomato genotypes. NPT II activity was detected in transformed calli and in transgenic plants. All of these plants were phenotypically normal, fertile and set seeds. Using the second procedure, inverted cotyledons, we recovered transformed tomato plants from A. rhizogenes-induced hairy roots. In this case, all of the transgenic plants exhibited phenotypes similar to hairy root-derived plants reported for other species. Southern blot analysis on these plants revealed that the plant DNA hybridized with both probes representing pARC8-T-DNA, and the T-DNAs of the A4 Ri-plasmid. However, southern analysis on those phenotypically normal transgenic plants from the first procedure revealed that only the pARC8-T-DNA was present in the plant genome, thus indicating that the pARC8-T-DNA integrated into the plant genome independently of the pRi A4-T-DNA. Genetic analysis of these phenotypically normal transgenic plants for the kanamycin-resistance trait showed Mendelian ratios, 31 and 11, for selfed (R1) and in crossed progeny, respectively.     

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofβ-glucuronidase in transgenicBrassica plants

A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS -Glucuronidase     

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     

Agrobacterium-mediated transformation of thin cell layer explants from Brassica napus L.

Summary Agrobacterium-mediated transformation of thin cell layer explants (Klimaszewska and Keller 1985) yielded large numbers of transgenic plants of a major Canadian rapeseed cultivar Brassica napus ssp. oleifera cv Westar. The morphology and fertility of these plants were indistinguishable from controls. The Ti plasmid vector, pGV3850 (Zambryski et al. 1983) was used as a cis vector and as a helper plasmid for the binary vector pBin19 (Bevan 1984). Selectable marker genes that conferred resistance to high levels of kanamycin (Km) on Nicotiana tabacum were less efficient in the selection of transgenic B. napus. At low levels of Km (15 g/ml) large numbers of transgenic plants (50%) were identified among the regenerants by nopaline synthase activity and several of these were confirmed by Southern blot analyses. Only a small number were resistant to higher levels of Km (80 g/ml). Preliminary analyses indicated that resistance to Km was transmitted to the selfed progeny. Chimeric chloramphenicol acetyl transferase genes were ineffective biochemical markers in transgenic B. napus.Contribution No. 1092 Plant Research Centre, Ontario, Canada     

Fertile transgenic barley generated by direct DNA transfer to protoplasts

We report the generation of transgenic barley plants via PEG-mediated direct DNA uptake to protoplasts. Protoplasts isolated from embryogenic cell suspensions of barley (Hordeum vulgare L. cv Igri) were PEG-treated in a solution containing a plasmid which contained the neomycin phosphotransferase (NPT II) gene under the control of the rice actin promoter and the nos terminator. Colonies developing from the treated protoplasts were incubated in liquid medium containing the selective antibiotic G418. Surviving calli were subsequently transferred to solid media containing G418, on which embryogenic calli developed. These calli gave rise to albino and green shoots on antibiotic-free regeneration medium. NPT II ELISA revealed that approximately half of the morphogenic calli expressed the foreign gene. In total, 12 plantlets derived from NPT-positive calli survived transfer to soil. Southern hybridization analysis confirmed the stable transformation of these plants. However, the foreign gene seemed to be inactivated in plants from one transgenic line. Most of the transgenic plants set seed, and the foreign gene was transmitted and expressed in their progenies, which was ascertained by Southern hybridization and NPT II ELISA.     

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter--glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.     

Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens

High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 g/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants.     

Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens

Summary Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed B. rapa cvs. Tobin and Emma. Transformed plants were obtained within three months of cocultivation. Transformation frequencies for the cultivars Tobin and Emma were 1–9%. Evidence for transformation was shown by NPT II dot blot assay, the GUS fluorometric assay, Southern analysis, and segregation of the kanamycin-resistance trait in the progeny. The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivars of B. napus.