Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

Genetic transformation of golden pothos (<Emphasis Type="Italic">Epipremnum aureum</Emphasis>) mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding of this very common indoor plant.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation of a recalcitrant grain legume,lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medik)

A simple and reproducible Agrobacterium-mediated transformation protocol for a recalcitrant legume plant, lentil (Lens culinaris M.) is reported. Application of wounding treatments and efficiencies of three Agrobacterium tumefaciens strains, EHA105, C58C1, and KYRT1 were compared for T-DNA delivery into lentil cotyledonary node tissues. KYRT1 was found to be on average 2.8-fold more efficient than both EHA105 and C58C1 for producing transient β-glucuronidase (GUS) gene (gus) expression on cotyledonary petioles. Wounding of the explants, use of an optimized transformation protocol with the application of acetosyringone and vacuum infiltration treatments in addition to the application of a gradually intensifying selection regime played significant roles in enhancing transformation frequency. Lentil explants were transformed by inoculation with Agrobacterium tumefaciens strain, KYRT1 harboring a binary vector pTJK136 that carried neomycin phosphotransferase gene (npt-II) and an intron containing gusA gene on its T-DNA region. GUS-positive shoots were micrografted on lentil rootstocks. Transgenic lentil plants were produced with an overall transformation frequency of 2.3%. The presence of the transgene in the lentil genome was confirmed by GUS assay, PCR, RT-PCR and Southern hybridization. The transgenic shoots grafted on rootstocks were successfully transferred to soil and grown to maturity in the greenhouse. GUS activity was detected in vegetative and reproductive organs of T₀, T₁, T₂ and T₃ plants. PCR assays of T₁, T₂ and T₃ progenies confirmed the stable transmission of the transgene to the next generations.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Genetic transformation of the medicinal plant <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated method

A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l⁻¹ BAP and 0.5 μmol l⁻¹ NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection medium (co-culture medium supplemented with 60 mg l⁻¹ kanamycin and 200 mg l⁻¹ cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l⁻¹ kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil. The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced in our lab in the past two years.     

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.     

Isolation and characterization of polyphenol oxidase- and peroxidase-producing <Emphasis Type="Italic">Bacillus</Emphasis> strains from fully fermented tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

Sixteen aerobic endospore-forming Bacillus spp. were isolated from fully fermented tea leaf samples from 10 tea factories in Lahijan and Langrod cities (Gillan province, Iran). Bacillus spp. isolates were characterized using phenotypic characteristics, antibiotic susceptibility and cellular fatty acid (CFA) patterns. Based on the data obtained, five isolates of tea Bacillus spp. (TB): TB2, TB4, TB6, TB10 and TB12 belonged to the species B. subtilis. Two isolates, TB1 and TB14 were recognized as B. licheniformis. Two Bacillus spp. isolates, TB9 and TB 16 were identified as B. sphaericus. Two isolates, TB5 and TB13 were shown to be B. pumilus. Two isolates, TB7 and TB15 belonged to B. cereus. Amongst the isolates, Bacillus sp. TB3, Bacillus sp. TB8 and Bacillus sp. TB11 showed different phenotypic traits, distinct antibiotic sensitivity and fatty acid profiles, and they may represent novel species. The isolates showed polyphenol oxidase (tyrosinase) and peroxidase activities. The highest polyphenol oxidase and peroxidase activities were observed for Bacillus sp. TB3 and B. licheniformis TB14, respectively, where values of 5.48 and 3.73 units mL⁻¹ were observed.     

Transformation of a filamentous fungus<Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> using<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1×10⁶ conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the dwarf pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L. var. <Emphasis Type="Italic">nana</Emphasis>)

The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid (NAA), 5 μM N⁶-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed in T₁ generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment of this transformation system is invaluable for investigating fruit-tree-specific phenomena.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.     

Differential expression of <Emphasis Type="Italic">Histone H3</Emphasis> gene in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze) suggests its role in growing tissue

Histone proteins are integral part of chromatin and their expression is typically linked to DNA replication in the S phase of cell cycle. Histone H3 is one of the four histones, along with H2A, H2B and H4, which forms the eukaryotic nucleosome octomer core. Using differential display of mRNA and rapid amplification of cDNA ends (RACE), a full-length Histone H3.1 cDNA (CsH3) was isolated from tea leaves. The open reading frame consisted of 411 nucleotides and deduced amino acid sequence comprised of 136 amino acid residues. CsH3 shared 79-82% and 98% identity at nucleotide and amino acid sequences, respectively with Histone H3 isolated from other plant species. During active-growth period of tea, higher expression was observed in apical buds that decreased gradually with increasing age of the leaf. During dormancy season, the expression of CsH3 was severely down-regulated in all the leaves studied. CsH3 was found to be down regulated in response to drought stress and ABA treatment and up-regulated by GA(3) treatment. A positive association of CsH3 abundance with active cellular growth suggested its role in plant growth and development.     

Establishment of a highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation method for <Emphasis Type="Italic">Broussonetia papyrifera</Emphasis>

Broussonetia papyrifera is well-known for its bark fibers, which are used for making paper, cloth, rope etc. This is the first report of a successful genetic transformation protocol for B. papyrifera using Agrobacterium tumefaciens. Callus was initiated at a frequency of about 100% for both leaf and petiole explants. Shoots formed on these calli with a success rate of almost 100%, with 14.08 and 8.36 shoots regenerating from leave-derived and petiole-derived callus, respectively. For genetic transformation, leaf explants of B. papyrifera were incubated with A. tumefaciens strain LBA4404 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, leaf explants were cultured on Murashige and Skoog (Physiol Plant 15:473, 1962) (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA) and 0.05 mg l⁻¹ indole-3-butyric acid (IBA) (CI medium) containing 5 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime, in the dark. Hygromycin-resistant calli were induced from leaf explants 3 weeks thereafter. Regenerating shoots were obtained after transfer of the calli onto MS medium supplemented with 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, and 0.5 mg l⁻¹ gibberellic acid (GA3) (SI medium), 5 mg l⁻¹ hygromycin and 250 mg l⁻¹ cefotaxime under fluorescent light. Finally, shoots were rooted on half strength MS medium (1/2 MS) supplemented with 10 mg l⁻¹ hygromycin. Transgene incorporation and expression was confirmed by PCR, Southern hybridisation and histochemical GUS assay. Using this protocol, transgenic B. papyrifera plants containing desirable new genes can be obtained in approximately 3 months with a transformation frequency as high as 44%.     

Transformation of different barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> infection of in vitro cultured ovules

Most cultivars of higher plants display poor regeneration capacity of explants due to yet unknown genotypic determined mechanisms. This implies that technologies such as transformation often are restricted to model cultivars with good tissue characteristics. In the present paper, we add further evidence to our previous hypothesis that regeneration from young barley embryos derived from in vitro-cultured ovules is genotype independent. We investigated the ovule culture ability of four cultivars Femina, Salome, Corniche and Alexis, known to have poor response in other types of tissue culture, and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary vector pVec8-GFP harboring a hygromycin resistance gene and the green fluorescence protein (GFP) gene, was used for transformation. The results strongly indicate that the tissue culture response level in ovule culture is genotype independent. However, we did observe differences between cultivars with respect to frequencies of GFP-expressing embryos and frequencies of regeneration from the GFP-expressing embryos under hygromycin selection. The final frequencies of transformed plants per ovule were lower for the four cultivars than that for Golden Promise but the differences were not statistically significant. We conclude that ovule culture transformation can be used successfully to transform cultivars other than Golden Promise. Similar to that observed for Golden Promise, the ovule culture technique allows for the rapid and direct generation of high quality transgenic plants.     

High efficiency <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation of <Emphasis Type="Italic">Saponaria vaccaria</Emphasis> L. (Caryophyllaceae) using fluorescence selection

A highly efficient and convenient method for the Agrobacterium rhizogenes-dependent production of transformed roots of Saponaria vaccaria L. (Caryophyllaceae) is described. The parameters tested and optimized include S. vaccaria cultivar, explant type, Agrobacterium rhizogenes strain and culture conditions. For cotransformation using additional recombinant T-DNA-containing A. rhizogenes strains, use of neomycin phosphotransferase and enhanced green fluorescent protein genes as selectable markers were tested alone and in combination. Optimal results, yielding a minimum of one transformed root per explant, were obtained using the cultivar Pink Beauty, the A. rhizogenes strain LBA9402 and internode explants precultured on a phytohormone mixture. Selection of cotransformed roots by observation of enhanced green fluorescent protein fluorescence alone was highly effective and convenient. NRCC Publication No. 48435.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of poinsettia, <Emphasis Type="Italic">Euphorbia pulcherrima</Emphasis>, with virus-derived hairpin RNA constructs confers resistance to <Emphasis Type="Italic">Poinsettia mosaic virus</Emphasis>

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated creeping bentgrass (<Emphasis Type="Italic">Agrostis stolonifera</Emphasis> L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T₁ generation. All independent transformation events carried one to three copies of the transgene, and a majority (60–65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.Abbreviations 2,4-D: 2,4-Dichlorophenoxyacetic acid - bar: Bialaphos resistance gene - GUS: -Glucuronidase - PPT: Phosphinothricin - ubi: Ubiquitin Communicated by J.M. Widholm     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) from stem explants using a two-step kanamycin-hygromycin selection method

Summary To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants, leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types began first with culture on 50 mg l⁻¹ of Km for 6 wk and then transfer to 30 mg l⁻¹ of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mgl⁻¹ cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16h photoperiod of 30 μmol m⁻²s⁻¹. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf dises 11.2%, and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four.