<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.     

Production of transgenic lily plants by<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation

A system for the production of transgenic plants was developed for the Oriental hybrid lily, Lilium cv. Acapulco, by Agrobacterium-mediated genetic transformation. Filament-derived calli were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm, which harbored a binary vector carrying the neomycin phosphotransferase II, hygromycin phosphotransferase, and intron-containing -glucuronidase genes in the T-DNA region. Six hygromycin-resistant (Hyg^r) culture lines were obtained from 200 calli by scratching them with sandpaper prior to inoculation and using NH₄NO₃-free medium for co-cultivation and a hygromycin-containing regeneration medium for selection. Hyg^r culture lines regenerated shoots, which developed into plantlets following transfer to a plant growth regulator-free medium. All of these plantlets were verified to be transgenic by GUS histochemical assay and inverse PCR analysis.Abbreviations AS Acetosyringone (3,5-dimethoxy-4-hydroxy-acetophenone) - BA Benzyladenine - CaMV Cauliflower mosaic virus - GUS -Glucuronidase - HPT Hygromycin phosphotransferase - Hyg^r Hygromycin-resistant - NOS Nopaline synthase - NPTII Neomycin phosphotransferase II - PGR Plant growth regulator - PIC Picloram (4-amino-3,5,6-trichloropicolinic acid)Communicated by H. Ebinuma     

<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.     

Stable <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

London plane tree (Platanus acerifolia Willd.) is an important tree in urban landscaping but it suffers from a number of negative traits which genetic engineering could be used to address. As with many woody species, P. acerifolia has appeared recalcitrant to genetic transformation. However, the recent development of a method for regenerating shoots from P. acerifolia leaf explants suggests that such material could be a target for gene-transfer. Using an Agrobacterium tumefaciens strain in which the T-DNA carries the histochemically detected reporter gene β-glucuronidase (GUS), we have followed the transfer of genes from Agrobacterium to leaf explants of Platanus acerifolia. Using this system, we have identified a set of inoculation and co-cultivation conditions (notably: the pre-treatment of leaf explants with 0.4 M mannitol, an inoculation period of 10 min, a bacterial OD₆₀₀ of 0.8–1.0 and a co-cultivation period of 5 days) that permit a good frequency and reliability of transient gene-transfer. Optimum levels of antibiotics for bacterial elimination and kanamycin-resistant shoot regeneration were also established. By applying these parameters, we recovered eight independent stably transformed shoots that were kanamycin-resistant and contained the nptII T-DNA gene, as confirmed by PCR analysis. Furthermore, Southern blot analysis confirmed that, in at least five of these lines, the transgene was associated with high molecular weight DNA, so indicating integration into the plant genome.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of European chestnut embryogenic cultures

An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material. When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded. Murashige and Skoog culture medium containing 150 mg/l of kanamycin was used as the selection medium. The addition of acetosyringone was detrimental to the transformation efficiency. Transformation was confirmed by a histochemical -glucuronidase (GUS ) assay, PCR and Southern blot analyses for the uidA (GUS) and nptII (neomycin phosphotransferase II) genes. At present, 93 GUS-positive chestnut embryogenic lines are being maintained in culture. Low germination rates (6.3%) were recorded for the transformed somatic embryos. The presence of the transferred genes in leaves and shoots derived from the germinated embryos was also verified by the GUS assay and PCR analysis.     

An effective method of sonication-assisted <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of chickpeas

An efficient and reproducible transformation method of sonication- assisted Agrobacterium-mediated transformation (SAAT) was developed for chickpea (Cicer arietinum L.). Agrobacterium tumefaciens (LBA4404) harboring pCAMBIA1305.2 was used to transform decapitated embryo explants of two cultivars of chickpeas. By using a series of co-cultivation, callus induction, shoot initiation and root inducing media, a large number of transgenic plants were recovered. Transient expressions of GUS gene were detected by X-Gluc histochemical assay in transformed tissues. DNA analysis of T0 and T1 plants by PCR and Southern hybridization confirmed the integration of transgenes in initial and next generation transformants in different transgenic lines. The transformation efficiency was more than two times higher in SAAT treatment than simple Agrobacterium without sonication.     

Evaluation of parameters affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of citrus

An improved protocol for genetic transformation of juvenile explants of Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.), Duncan (Citrus paradisi Macf.), Hamlin (Citrus sinensis (L.) Osbeck) and Mexican Lime (Citrus aurantifolia Swingle) cultivars using a vector containing a bifunctional egfp-nptII fusion gene is described. Several parameters were investigated to optimize genetic transformation of these four cultivars. It was determined that a short preincubation in hormone rich liquid medium and subculture of Agrobacterium for 3 h in YEP medium containing 100 μM acetosyringone were required for improvement of transformation efficiency. Co-cultivation duration as well as addition of acetosyringone to co-cultivation medium also played an important role in transformation efficiency as did OD₆₀₀ value of the Agrobacterium suspension used for transformation. We regenerated numerous EGFP expressing transgenic lines from all four cultivars. Based on these results, we conclude that genetic transformation of citrus is cultivar specific and optimization of conditions for maximum transgenic production are required for each individual cultivar.     

High-efficiency <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of citrus via sonication and vacuum infiltration

An improved method for the Agrobacterium infiltration of epicotyl segments of ‘Pineapple’ sweet orange [Citrus sinensis (L.) Osbeck] and ‘Swingle’ citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method (‘dipping’ method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for ‘Pineapple’ sweet orange and 11.2% for ‘Swingle’ citrumelo) are the highest ones reported for both cultivars.     

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.     

Regeneration and<Emphasis Type="Italic"> Agrobacterium-</Emphasis>mediated transformation of hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.)

An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - GUS -Glucuronidase - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - TDZ 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)Communicated by H. Lörz     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the genome-sequenced poplar clone,nisqually-1 (<Emphasis Type="Italic">Populus trichocarpa</Emphasis>)

The US Department of Energy recently released a 6.8X draft of the genome sequence for Nisqually-1, a genotype of black cottonwood (Populus trichocarpa). To improve its utility for functional genomics research, having an efficient means for transformation and regeneration is necessary. To examine several parameters known to affect the transformation rate, we cocultivated leaf disc and stem explants with a strain ofAgrobacterium tumefaciens harboring a binary plasmid vector containing genes for both neomycin phosphotransferase (NPTII) and β-glucuronidase (GUS). Shoot regeneration from stem explants was observed in the presence of kanamycin when thidiazuron was incorporated in the selection medium. Transformation efficiency was influenced by the level of thidiazuron to which explants were exposed during the early stages of shoot induction. Histochemical assays revealed expression of theGUS gene in leaf, stem, and root tissues of transgenic plants. Polymerase chain reaction confirmed the presence of both selectable marker and reporter genes in all lines that stained positive for β-glucuronidase activity. By use of our modified protocol, transgenic plants were recovered within 6 mo at an efficiency of 6%, adequate to produce a large number of transgenic events with modest effort.     

An improved procedure for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of trifoliate orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.) via indirect organogenesis

Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical assay, PCR, and Southern blot analysis.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation of Common Bermudagrass (<Emphasis Type="Italic">Cynodon dactylon</Emphasis>)

Common bermudagrass, Cynodon dactylon, is a widely used warm-season turf and forage species in the temperate and tropical regions of the world. We have been able to transform the species using Agrobacterium-mediated approach. In seven experiments reported here, a total of 67 plates of calluses and suspensions were infected with Agrobacterium tumefaciens strains, and nine hygromycin B resistant calluses were obtained after selection. Among them two green independent transgenic plants were recovered. The plants growing in pots looked relatively compact at the beginning, but the ploidy level of the plants, as determined by nuclear DNA content, was not altered.     

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.     

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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and development of herbicide-resistant sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> species hybrids) using axillary buds

Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies. Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing -glucuronidase (gus-intron) genes in the T-DNA region. A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l^–1) was the most effective selection agent for axillary bud transformation. Repeated proliferation of shoots in the selection medium eliminated chimeric transformants. Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l^–1 6-benzyladenine (BA) and 5.0 mg l^–1 PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l^–1 BA, 1.0 mg l^–1 kinetin (Kin), 0.5 mg l^–1 -napthaleneacetic acid (NAA) and 5.0 mg l^–1 PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l^–1 NAA and 5.0 mg l^–1 PPT. About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse. Transformation was confirmed by a histochemical -glucuronidase (GUS) assay and PCR amplification of the bar gene. Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants. Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain. A 3-day co-cultivation with 50 M acetosyringone considerably increased the transformation efficiency. Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars. Depending on the variety, 50–60% of the transgenic plants sprayed with BASTA (60 g l^–1 glufosinate) grew without any herbicide damage under greenhouse conditions. These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%.Abbreviations BA 6-Benzyladenine - CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kin Kinetin - NAA -Naphthaleneacetic acid - Nos Nopaline synthase - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - PPT Phosphinothricin - YEP Yeast extract and peptone     

Hypocotyl-based <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) and application for RNA interference

An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of GNA transgenic sugarcane

Six plasmids carrying a snowdrop lectin (Galanthus nivalis agglutinin, GNA) and one of three selection markers were successfully transferred into two sugarcane cultivars (FN81–745 and Badila) via Agrobacterium-mediated transformation. Agrobacterium strains LBA4404, EHA105 and A281 that harboured a super-binary vector were used for sugarcane transformation. The use of the hygromycin (Hyg) resistance gene (hpt II), phosphinothrincin (PPT) resistance gene (bar) or G418 resistance gene (npt II) as a screenable marker facilitated the initial selection of GNA transgenic sugarcane callus with different efficiencies and helped the rapid segregation of individual transformation events. All the three selective marker genes were controlled by CaMV 35S promoter, while GNA gene was controlled by promoter of RSs-1 (rice sucrose synthase-1) or Ubi (maize ubiquitin). Factors important to successful transformation mediated by Agrobacterium tumefaciens were optimized, which included concentration of A. tumefaciens, medium composition, co-cultivated methods with plant tissue, strain virulence and different selective marker genes. An efficient protocol for sugarcane transformation mediated by A. tumefaciens was established. The GNA gene has been integrated into sugarcane genome as demonstrated by PCR and Southern dot blotting detections. The preliminary results from bioassay demonstrated a significant resistance of the transgenic sugarcane plants to woolly aphid (Ceratovacuna lanigera Zehnther) indicating thus the possibility for obtaining a transgenic sugarcane cultivar with resistance to woolly aphid.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> Wright,an important pharmaceutical crop

Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated from initial infected callus explants.