Transformation of soybean via particle bombardment of embryogenic suspension culture tissue

Summary Embryogenic suspension culture tissue of soybean (Glycine max Merrill.) was bombarded with particles coated with plasmid DNAs encoding hygromycin resistance andβ-glucuronidase (GUS). One to two weeks after bombardment, embryogenic tissue was placed in a liquid proliferation medium containing hygromycin. Four to six weeks after bombardment, lobes of yellow-green, hygromycin-resistant tissue, which began as outgrowths on brown clumps of hygromycin-sensitive tissue, were isolated and cultured to give rise to clones of transgenic embryogenic material. In vivo GUS assays of hygromycin-resistant clones showed that the early outgrowths could be negative, sectored, or positive for GUS activity. Transgenic, fertile plants could be routinely produced from the proliferating transgenic embryogenic clones. Southern hybridization analyses confirmed stable transformation and indicated that both copy number and integration pattern of the introduced DNA varied among independently transformed clones. Hybridization analysis of DNA from progeny plants showed genetic linkage of multiple copies of introduced DNA. An average of three transgenic clones were obtained per bombardment making this procedure very suitable for transformation of soybean.     

Herbicide resistant transgenic papaya plants produced by an efficient particle bombardment transformation method

A system for the production of transgenic papaya (Carica papaya L.) plants using zygotic embryos and embryogenic callus as target cells for particle bombardment is described. Phosphinothricin (bar ) and kanamycin (npt II) resistance genes were used as selectable markers, and the gus gene (uidA) as a reporter gene. Selection with 100 mg/l kanamycin and 4 mg/l phosphinothricin (PPT) yielded a total of over 90 resistant embryogenic colonies from three independent experiments using embryogenic callus as a target tissue. This represents an efficiency of 60 transgenic clones per gram of fresh weight callus bombarded. The efficiency of genetic transformation using zygotic embryos was lower, as only 8 independent resistant clones were recovered out of 645 bombarded zygotic embryos, giving a efficiency of 1.24%. Subsequent subculture of transgenic somatic embryos both from zygotic embryos and embryogenic callus led to the development of plants with apparently normal morphology. Histological, fluorimetric assay for GUS, NPT II assay and DNA analysis (Southern hybridization) showed that kanamycin /PPT resistant plants carried and expressed the transgenes.Abbreviations Gus -glucuronidase - NPTII neomycin phophotransferase II - bar phophinothricin acetyl transferase gene - Pat phosphinothricin acetyl transferase - PPT phosphinothricin - Km kanamycin - 2,4-D 2,4-dichlorophenoxyacetic acid - K kinetin - BAP benzylaminopurine - IBA indolbutyric acid     

Inheritance of transgenes in transgenic tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> schreb.)

Summary Tall fescue (Festuca arundinacea Schreb.) is the most important forage species worldwide of the Festuca genus. Single genotype-derived embryogenic suspension cultures were established from tall fescue cultivar Kentucky-31, and were used as target cells for biolistic transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker, and a chimeric β-glucuronidase (gusA) gene was co-transformed with hph. Transgenic plants were recovered after microprojectile bombardment of suspension cells and subsequent selection in the presence of a high concentration of hygromycin. Fertile transgenic plants were obtained after vernalization under field conditions. T1 and T2 progenies were obtained after reciprocal crosses between transgenic and untransformed control plants. PCR and Southern hybridization analyses revealed a 1∶1 segregation ratio for both transgenes in the T1 and T2 generations. Southern hybridization patterns were identical for T0, T1, and T2 plants. The results demonstrated for the first time the stable meiotic transmission of transgenes following Mendelian rules in transgenic tall fescue.     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation using embryogenic suspension cultures in sweetpotato, <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.

Efficient Agrobacterium tumefaciens-mediated transformation was achieved using embryogenic suspension cultures of sweetpotato (Ipomoea batatas (L.) Lam.) cv. Lizixiang. Cell aggregates from embryogenic suspension cultures were cocultivated with the A. tumefaciens strain EHA105 harboring a binary vector pCAMBIA1301 with gusA and hygromycin phosphotransferase II gene (hpt II) genes. Selection culture was conducted using 25 mg l⁻¹ hygromycin. A total of 2,218 plants were regenerated from the inoculated 1,776 cell aggregates via somatic embryogenesis. β-glucuronidase (GUS) assay and PCR, dot blot and Southern blot analyses of the regenerated plants randomly sampled showed that 90.37% of the regenerated plants were transgenic plants. The number of integrated T-DNA copies varied from 1 to 4. Transgenic plants, when transferred to soil in a greenhouse and a field, showed 100% survival. No morphological variations were observed in the ex vitro transgenic plants. These results exceed all transformation experiments reported so far in the literature in quantity of independent events per transformation experiment in sweetpotato.     

Production of transgenic <Emphasis Type="Italic">Podophyllum peltatum via Agrobacterium tumefaciens</Emphasis>-mediated transformation

Transgenic Podophyllum peltatum plants were successfully produced by Agrobacterium tumefaciens-mediated transformation. Embryogenic callus was co-cultivated with Agrobacterium tumefaciens harboring a binary vector pBI 121 carrying β-glucuronidase (GUS) and neomycinphosphotransferase (NPT II) gene. GUS-histochemical analysis revealed that, 50 μM acetosyringone treatments during Agrobacterium infection and 3 d co-cultivation with Agrobacterium showed enhanced transformation efficiency. Percentage of GUS positive callus increased rapidly as the subculture time proceeded on selection medium containing 100 mg dm⁻³ kanamycin. Kanamycin resistant somatic embryos were formed from embryogenic callus after cultivation with 11.35 μM abscisic acid (ABA) for 3 weeks and then on hormone-free selection medium. Somatic embryos were germinated and converted into plantlets on medium containing 2.89 μM gibberellic acid (GA₃). The integration of GUS and NPT II gene into transgenic plants was confirmed by polymerase chain reaction and Southern analysis.     

Agrobacterium tumefaciens-mediated transformation of embryogenic tissues of tea (Camellia sinensis (L.) O. Kuntze)

Embryogenic tissues of tea were cocultivated withAgrobacterium tumefaciens LBA4404. The plasmid pBi121, which contains the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the β-glucuronidase (uidA) reporter gene, was used as binary vector. The highest transformation frequency (12 transformants/g fresh weight [FW] of treated embryogenic tissue) was obtained with 5-day-old tissues grown in liquid medium and cocultivated withAgrobacterium for 2 d in the same medium but containing 50 μM acetosyringone. There was improvement in the recovery of kanamycin-resistant tissues when tissues were first grown for 10 d on a medium containing 350 mg/L Timentin to prevent bacterial overgrowth, before application of the selection pressure. Resistant tissues obtained after 6 wk on kanamycin-selection medium showed stableuidA expression. Polymerase chain reaction demonstrated the presence of the transgenes, while Southern hybridization confirmed their integration into the genome. Transgenic plants were regenerated from transformed tissues within 4 mo after coculture.     

Stable genetic transformation of embryogenic cultures of <Emphasis Type="Italic">Abies nordmanniana</Emphasis> (nordmann fir) and regeneration of transgenic plants

Summary Stable genetic transformation of embryogenic cultures of Abies nordmanniana (Nordmann fir or Caucasian fir) was achieved using the Biolistic^? transformation technology, followed by regeneration of transgenic plants. Selection of the transgenic tissue was based on the antibiotic resistance induced by the neomycin phosphotransferase II gene (npt II), in combination with the antibiotic geneticin. Six transclones were recovered from a total of 215 bombardments. Expression of the β-glucuronidase gene (uidA) was confirmed by histochemical analysis, and expression of npt II was verified by quantification of NPTII protein by enzyme linked immunosorbent assay (ELISA). Both genes were still expressed in the embryogenic tissue after 5 yr of in vitro culture and in mature somatic embryos and plants produced from these cultures. The integration of npt II was confirmed by Southern hybridization in embryogenic tissue after 5 yr of culture. After 5 yr of growth, uidA was still expressed in needles from the transformed trees.     

Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants

Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T₁ progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T₂ progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations.     

Regeneration of transgenic cassava plants (Manihot esculenta Crantz) through Agrobacterium-mediated transformation of embryogenic suspension cultures

A protocol was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. The bacterial strain ABI containing the binary vector pMON977 with the nptII gene as selectable marker and an intron-interrupted uidA gene (encoding β-glucuronidase) as visible marker was used for the experiments. Selection of transformed tissue with paromomycin resulted in the establishment of antibiotic-resistant, β-glucuronidase-expressing lines of friable embryogenic callus, from which embryos and subsequently plants were regenerated. Southern blot analysis demonstrated stable integration of the uidA gene into the cassava genome in five lines of transformed embryogenic suspension cultures and in two plant lines.     

Optimizing Agrobacterium-mediated transformation of grapevine

Summary A translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTH) genes was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTH fusion gene allowed for a single promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and climinating potential promoter-promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S) promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos, and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 gl⁻¹ of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic lines than did the control. A 48h co-cultivation period combined with 75 mgl⁻¹ kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome: 63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr.     

Genetic transformation of duckweed Lemna gibba and Lemna minor

Summary We developed efficient genetic transformation protocols for two species of duckweed, Lemna gibba (G3) and Lemna minor (8627 and 8744), using Agrobacterium-mediated gene transfer. Partially differentiated nodules were co-cultivated with Agrobacterium tumefaciens harboring a binary vector containing β-glucuronidase and nptII expression cassettes. Transformed cells were selected and allowed to grow into nodules in the presence of kanamycin. Transgenic duckweed fronds were regenerated from selected nodules. We demonstrated that transgenic duckweed could be regenerated within 3 mo. after Agrobacterium-mediated transformation of nodules. Furthermore, we developed a method for transforming L. minor 8627 in 6 wk. These transformation protocols will facilitate genetic engineering of duckweed, ideal plants for bioremediation and large-scale industrial production of biomass and recombinant proteins.     

Agrobacterium-mediated transformation of Lupinus mutabilis L. using shoot apical explants

A procedure for regenerating plants of Lupinus mutabilis from shoot apices, from which the leaf primordia and initial cell layer(s) of the apical meristem were removed, has been used to generate transgenic plants following Agrobacterium tumefaciens-mediated gene delivery. Transformation competent cells, from which buds developed, were located at the periphery of the apical meristem. Kanamycin resistant plants were obtained which expressed β-glucuronidase activity. Integration of the neomycin phosphotransferase II and β-glucuronidase genes into the genomes of transgenic plants was confirmed by non-radioactive DNA-DNA hybridisation. This is the first report of the generation of transgenic plants in L. mutabilis.     

Production of herbicide-resistant sweet potato plants transformed with the <Emphasis Type="Italic">bar</Emphasis> gene

Herbicide-resistant sweet potato plants were produced through biolistics of embryogenic calli derived from shoot apical meristems. Plant materials were bombarded with the vectors containing the β-glucuronidase gene (gusA) and the herbicide-resistant gene (bar). Selection was carried out using phosphinothricin (PPT). Transformants were screened by the histochemical GUS and Chlorophenol Red assays. PCR and Southern-blot analyses indicated the presence of introduced bar gene in the genomic DNA of the transgenic plants. When sprayed with Basta, the transgenic sweet potato plants was tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to sweet potato.     

Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.)

 Transgenic plants of grapefruit cv. Rio Red (Citrus paradisi Macf.) have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and kanamycin as the selective agent. The transformation procedure includes a shoot elongation phase with a liquid medium overlay, which provides additional selection against non-transgenic shoots. Transformed shoots are invigorated and multiplied on a non-selective medium prior to grafting, thus assuring that plants can be recovered from transgenic shoots. We have constructed a binary vector, pBin34SGUS, with an intron-containing β-glucuronidase gene (uidA) under the control of the Figwort mosaic virus 34S promoter. The 34S promoter efficiently drives uidA gene expression both in transient assays and in transgenic Rio Red leaf tissue, although at levels five- to sevenfold lower than the Cauliflower mosaic virus 35S promoter. An untranslatable coat protein gene (uncp) of the Citrus tristeza virus strain SY568 and the Galanthus nivalis agglutinin gene (gna) were inserted into pBin34SGUS and transgenic plants have been obtained. Stable integration of the uncp and gna genes was confirmed by Southern hybridization and gna gene expression was confirmed by Western blot analysis. Received: 2 February 2000 / Revision received: 21 June 2000 / Accepted: 29 June 2000     

Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation

Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated for two years. Four independent transgenic plants produced flowers 1–2 years after acclimatization, and all of them contained one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation.     

Construction of a GFP-BAR plasmid and its use for switchgrass transformation

 A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by replacing the uidA (β-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l^–1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red to yellow. Basta tolerance was observed in T₁ plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene. Received: 11 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000     

Transgenic grapefruit plants expressing the PAPETALA3-IPT gp gene exhibit altered expression of PR genes

Transgenic grapefruit plants (Citrus paradisi cv. ‘Duncan’) with the isopentenyltransferase (ipt) gene under the control of APETALA3 promoter have been produced using Agrobacterium-mediated transformation. The relative expression level of the ipt gene was between 2.3 and 7 times higher in transformed plants than in the wild-type but despite the presence of a tissue-specific promoter, the expression was not limited only to flower tissue. Increased levels of trans-zeatin riboside between 9.4 and 32-fold found in transgenic grapefruit were considered the consequence of ectopic expression of the ipt gene. Chlorophyll levels in fully expanded uppermost leaves were also about 30% higher in transgenic than in wild-type plants. Involvement of cytokinins in control of expression of three pathogenesis-related protein genes: β-1,3-glucanase, a stress related PR gene 24P220, and an acidic chitinase, 24P262 was examined. Expression of β-1,3-glucanase, and 24P220 gene were significantly enhanced in transgenic plants while the expression of chitinase was reduced to low levels. Our results confirm the effect of cytokinins on expression of genes implicated in the response of grapefruit plants to pathogen attack and suggest a possible role of cytokinins in pathogen resistance.     

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.