Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed-derived highly regenerative tissues

 Highly regenerative tissues of tall fescue and red fescue produced from mature seed-derived embryogenic callus were induced and proliferated on medium containing 2,4-dichlorophenoxyacetic acid (4.5 or 9.0 μM), 6-benzylaminopurine (0, 0.044, 0.44 or 2.2 μM) and cupric sulfate (0.1 or 5.0 μM) under dim-light conditions (10 to 30 μE m^–2 s^–1, 16 h light). Tall fescue tissues were transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA;gus), and red fescue with three plasmids containing hpt, uidA and a synthetic green fluorescent protein gene [sgfp(S65T)]. DNA from T₀ plants of eight independently transformed lines from tall fescue and 11 from red fescue were analyzed by PCR and DNA blot hybridization. The co-expression frequency of all three transgenes [hpt/bar/uidA or hpt/uidA/sgfp(S65T)] in transgenic tall fescue and red fescue plants was 25–27%; for two transgenes [hpt/bar or hpt/uidA for tall fescue and hpt/uidA or hpt/sgfp(S65T) for red fescue], the co-expression frequency was 50–75%. Received: 28 September 1999 / Revision received: 13 March 2000 / Accepted: 16 March 2000     

Transgenic herbicide- and disease-tolerant carrot (Daucus carota L.) plants obtained through Agrobacterium-mediated transformation

. Transgenic carrot (Daucus carota L.) plants expressing a rice thaumatin-like protein (tlp), phosphinothricin acetyltransferase (bar) and the hygromycin phosphotransferase (hpt) genes were obtained by Agrobacterium-mediated transformation. Petiole and hypocotyl segments of three carrot cultivars were used as the explant sources. Following infection, selection was achieved on Murashige and Skoog medium with 1 mg/l phosphinothricin or 25 mg/l hygromycin B, which was increased after 2 weeks to 10 mg/l phosphinothricin and 100 mg/l hygromycin B. The presence of the tlp and bar transgenes was confirmed by polymerase chain reaction and Southern blot analyses, and the expression of the thaumatin-like protein was demonstrated by Western blot analysis. Among 45 primary transformants, 13 were selected for assessment of herbicide and/or disease tolerance. The transgenic plants showed varying levels of tolerance to the herbicide phosphinothricin, depending on the transformation events in different lines. Four transgenic lines also showed significantly enhanced tolerance to the foliar and root pathogen Botrytis cinerea or Sclerotinia sclerotiorum when inoculated under controlled environment conditions. Two lines had significantly enhanced tolerance to the herbicide phosphinothricin as well as to both pathogens. These results demonstrate the feasibility of introducing two potentially useful agronomic traits into carrot through genetic engineering.     

Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event

Elite white maize lines W506 and M37W were transformed with a selectable marker gene (bar) and a reporter gene (uidA) or the polygalacturonase-inhibiting protein (pgip) gene after bombardment of cultured immature zygotic embryos using the particle inflow gun. Successful transformation with this device did not require a narrow range of parameters, since transformants were obtained from a wide range of treatments, namely pre-culture of the embryos for 4-6 days, bombardment at helium pressures of 700-900 kPa, selection-free culture for 2-4 days after bombardment and selection on medium containing bialaphos at 0.5-2 mg l^-1. However, bombardments with helium pressures below 700 kPa yielded no transformants. The culture of immature zygotic embryos of selected elite white maize lines on medium containing 2 mg l^-1 2,4-dichlorophenoxyacetic acid and 20 mM L-proline proved to be most successful for the production of regenerable embryogenic calli and for the selection of putative transgenic calli on bialaphos-containing medium after transformation. Transgenic plants were obtained from four independent transformation events as confirmed by Southern blot analysis. Transmission of the bar and uidA genes to the T₄ progeny of one of these transformation events was demonstrated by Southern blot analysis and by transgene expression. In this event, the transgenes bar and uidA were inserted in tandem.     

Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings

 Genetic transformation using shoot meristematic cultures (SMCs) derived from germinated seedlings is established in commercial varieties of oat cv 'Garry' and barley cv 'Harrington'. Six-month-old SMCs of oat were induced on MPM and bombarded with bar and uidA; 9-month-old SMCs of barley were induced on an improved medium (MPM-MC) containing maltose and high levels of copper and bombarded with bar/nptII and uidA. After 3–4 months on selection, seven independent transgenic lines of oat were obtained, two lines of barley. All transgenic lines produced T₀ plants; five lines of oat and one line of barley were self-fertile, and the other barley line produced T₁ seed when out-crossed. Both Mendelian and non-Mendelian segregation ratios of transgene expression were observed in T₁ and T₂ progeny of transgenic oat. Normal as well as low physical transmission of the transgenes was also seen in T₁ and T₂ progeny of oat. The bar-containing line of barley showed stable transgene expression in all of the T₁ and T₂ progeny tested. Received: 4 January 1999 / Accepted: 14 January 1999     

Development of transgenic rice pure lines by particle bombardment‐mediated transformation and genetic analysis‐based selection

Mature seed‐derived callus from an elite Chinese japonica rice cv. Eyl 105 was transformed with a plasmid containing the selectable marker hygromycin phosphotransferase (hpt) and the reporter β‐glucuronidase (gusA) genes via particle bombardment. After two rounds of selection on hygromycin (30 mg/l)‐containing medium, resistant callus was transferred to hygromycin (30 mg/l)‐containing regeneration medium for plant regeneration. Twenty‐three independent transgenic rice plants were regenerated from 127 bombarded callus with a transformation frequency of 18.1%. All the transgenic plants contained both gusA and hpt genes, revealed by PCR/Southern blot analysis. GUS assay revealed 18 out of 23 plants (78.3%) proliferated on hygromycin‐containing medium had GUS expression at various levels. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation, we identified three independent homozygous transgenic rice lines. The homozygous lines were phenotypically normal and fertile compared to the control plants. We demonstrate that homozygous transgenic rice lines can be obtained via particle bombardment‐mediated transformation and through genetic analysis‐based selection.     

Improvement of rice transformation using bombardment of scutellum-derived calli

Although several reports on rice transformation have been published, producing transgenic plants ofIndica rice varieties is still problematic. We report an improved protocol for transformingIndica rice genotypes. An important agronomic MexicanIndica rice variety, Morelos A-92, was used. Calli derived from scutellum seeds were produced by using auxins and bombarded with 2 vectors, one harboring the reporteruidA gene and the other with thehptII gene conferring hygromycin resistance. The influence of the molar relation of these vectors (uidA-hptIII) in generating callus and plants expressing theuidA reporter gene was analyzed. Selection of bombarded calli was performed under 2 conditions: 50 mg/L of hygromycin with 3 subcultures and 80 mg/L of hygromycin with no subcultures. The best conditions were a 20∶1uidA-hptII molar vector relationship and selection at 80 mg/L of hygromycin, producing 14% of calli expressing GUS. The minimal callus size in regenerating plants was 3 mm. Transformed rice plants were generated with 4.6% efficiency, considering the initial number of bombarded calli. Heredity of theuidA gene behaved as a single locus in transformed rice plants. Homozygous plants were identified in the T₁ generation by means of pollen staining.     

Hygromycin resistance as an efficient selectable marker for wheat stable transformation

Summary A highly efficient method for stable wheat transformation using hygromycin resistance as a selectable marker is described. Young embryogenic calli growing from immature wheat embryos were transformed using a gunpowder-driven microparticle accelerator. Transgenic wheat plants were determined by PCR amplification of transgene fragments and confirmed by Southern hybridization, activity of the transgene expression and by analysis of the progeny. The hpt gene was as good as or a better selectable marker than the bar gene with an average efficiency (number of transgenic plants relative to the number of bombarded calli) of 5.5% compared with 2.6% for the bar gene.     

Effect of selectable gene to reporter gene ratio on the frequency of co-transformation and co-expression of uidA and hpt transgenes in protoplast-derived plants of tall fescue

Forty-six independent transformed plants were regenerated under hygromycin selection from cell-suspension derived protoplasts of Festuca arundinacea (Schreb.) after PEG-mediated transformation. Protoplasts were co-transformed with varying molar gene ratios (0.7:1–6:1) of a marker -glucuronidase (uidA) gene and a selective hygromycin (hpt) resistance gene. Logistic regression analysis indicated that, as expected, the proportion of co-transformed plants tended to increase as the proportion of the marker gene was increased. However, although the proportion of plants co-expressing both genes tended to increase up to a molar ratio of 4:1, it appeared to fall at a molar ratio of 6:1. No statistically significant differences were found in the average copy number of the integrated uidA or hpt transgenes, either in GUS expressing, or in non-GUS expressing plants at the different molar ratios. When using naked-DNA gene transformation methods most authors use a molar ratio of 1:1; our data suggest that adding non-selected and selected transgenes at a higher Molar Gene Ratio would probably improve the proportion of plants regenerated which express both transgenes.     

Generation of large numbers of transgenic Kentucky bluegrass (Poa pratensis L.) plants following biolistic gene transfer

A very efficient transformation system, using biolistic bombardment, has been developed for the production of transgenic plants of Kentucky bluegrass (Poa pratensis L.). Embryogenic calli, initiated from immature embryos, were transformed either with pAct1IHPT-4 containing the hygromycin phosphotransferase (hpt) gene or with pDM803 containing the phosphinothricin acetyltransferase (bar) gene and the β-glucuronidase (uidA) gene. In total 119 independent transgenic plants were recovered from 153 hygromycin-resistant lines. Bialaphos selection yielded a total of 99 bialaphos-resistant lines and from these 34 independent transgenic plants were recovered. Southern blot analysis demonstrated the independent nature of the transgenic plants and also revealed a complex transgene integration pattern with multiple insertions. The first two author contributed equally to this work     

Comparison of three selectable marker genes for transformation of tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) plants by particle bombardment

A variety of selection systems have been developed for transformation of forage crops. To compare the most frequently used systems, we tested three selectable marker genes for their selection efficiency under four selection procedures for the production of transgenic tall fescue. Embryogenic calluses initiated from mature embryos were bombarded with three constructs containing either the phosphinothricin acetyltransferase (bar) gene, the hygromycin phosphotransferase (hpt) gene or the neomycin phosphotransferase II (nptII) gene. Transformation efficiency was strongly influenced by the selectable marker gene, selection procedure and genotype. The highest transformation efficiency was observed using the bar gene in combination with bialaphos. Average transformation efficiencies with bialaphos, phosphinothricin (glufosinate), hygromycin and paromomycin selection across the two callus lines used in the experiments were 9.4%, 4.4%, 5.2% and 1.6%, respectively. Southern blot analysis revealed the independent nature of the tested transgenic plants and a complex transgene integration pattern with multiple insertions.     

Variation in the inheritance of expression among subclones for unselected (uidA) and selected (bar) transgenes in maize (Zea mays L.)

Variation in the inheritance of expression among subclones for an unselected (uidA) and a selected (bar) transgene was analyzed in two individual transformation events in maize. The unselectable gene (uidA) and the selectable gene (bar), on two separate plasmids, were transferred to maize (Hi-II derivative) by particle bombardment of embryogenic calli or suspension cells. A total of 188 fertile T1 plants were obtained from one transformant (transformation event BG which integrated uidA and bar). A total of 98 fertile T1 plants were obtained from a second transformant (transformation event B which integrated bar). Through self-pollination and/or cross-pollination in the greenhouse, approximately 10 000 T2 progeny were obtained from event BG, and more than 1000 T2 progeny were obtained from event B. Segregation of transgene expression was analyzed statistically in a total of 2350 T2 progeny from 40 T1 subclones of event BG and in 217 T2 progeny from six T1 subclones from event B. Variation in the inheritance of expression among subclones for the two transgenes (uidA and bar) was observed in the two transformants. A significant difference was observed between the use of the female or male as the transgenic parent in the inheritance of expression for the two transgenes in event BG. No inheritance through the pollen was observed in two of four T1 subclones analyzed in event B. Co-expression analysis of event BG showed that both transgenes were co-expressed in 67.7% of the T2 plants which expressed at least one of the two transgenes. Of the T2 expressing plants, 30.4% expressed only bar, and 1.9% expressed only uidA. Inactivation of the unselected (uidA) and the selected (bar) transgenes was observed in individual T2 plants.     

Production of Transgenic Rice Homozygous Lines with Enhanced Resistance to the Rice Brown Planthopper

Mature seed‐derived callus from an elite Chinese japonica rice (Oryza sativa L.) cv. Eyi 105 was cotransformed with two plasmids, pWRG1515 and pRSSGNA1,containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β‐glucuronidase gene (gusA) and the snow‐drop (Galanthus nivalis) lectin gene (gna) via particle bombardment. After two rounds of selection on hygromycin‐containing medium, resistant callus was transferred to hygromycin‐containing regeneration medium for plant regeneration. Twenty‐six independent transgenic rice plants were regenerated from 152 bombarded calli with a transformation frequency of 17%. Seventy‐three percent of transgenic plants contained all three genes, which was revealed by PCR/Southern blot analysis. Thirteen out of 19 transgenic plants containing the gna gene expressed GNA (68%) at various levels with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parentplants showing 3:1 Mendelian segregation patterns, we identified three independent homozygous lines containing and expressing all three transgenes.Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to the rice brown planthopper (Nilaparvata lugens, BPH) by decreasing BPH survival and overall fecundity, retarding BPH development and reducing BPH feeding.This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis‐based selection, conferred enhanced resistance to BPH, one of the most damaging insect pests in rice.     

Development of transgenic rice pure lines with enhanced resistance to rice brown planthopper

Summary Mature seed-derived callus from an elite Chinese japonica rice cv. Ewan 5 was cotransformed with two plasmids, pWRG1515 and pRSSGNAl, containing the selectable marker hygromycin phosphotransferase gene (hpt), the reporter β-glucuronidase gene (gusA) and the snowdrop (Galanthus nivalis) lectin gene (gna) via particle bombardment. Thirty-five independent transgenic rice plants were regenerated from 177 bombarded calluses. Eighty-three percent of the transgenic plants contained all three genes, as revealed by Southern blot analysis. Western blot analysis revealed that 23 out of 29 gna-containing transgenic plants expressed Galanthus nivalis agglutinin (GNA) (79%) at various levels, with the highest expression being approximately 0.5% of total soluble protein. Genetic analysis confirmed Mendelian segregation of all three transgenes (gna, hpt and gusA) in the R2 progeny. Amongst the R2 generation two independent homozygous lines were identified that expressed all three transgenes. Insect bioassay and feeding tests showed that these homozygous lines had significant inhibition to rice brown planthopper (Nilaparvata lugens, BPH) by decreasing the survival, overall fecundity of BPH, retarding development, and decreasing the feeding of BPH. These BPH-resistant lines have been incorporated into a rice insect resistance breeding program. This is the first report that homozygous transgenic rice lines expressing GNA, developed by genetic transformation and through genetic analysis-based selection, conferred enhanced resistance to BPH.     

Stable wheat transformation obtained without selectable markers

Transgenic wheat plants without the selectable marker gene were obtained either in the presence or in the absence of selective pressure during the transformation protocol. When using hygromycin as selective agent in a co-transformation experiment involving a mixture of plasmids pGL2, containing the hpt gene, and pAI₁Gus, containing the uidA gene, 3 out of 19 transgenic wheat plants had the uidA gene alone as shown by Southern blots. The gene was transmitted to the progeny following Mendelian rules. Segregation and loss of the selectable marker gene was also found in three out of six events from other experiments where high-molecular-weight glutenin genes were expressed or over-expressed. On the other hand, in 7 experiments where no selective pressure was applied and that involved 1016 bombarded explants, 23 transgenic wheat plants were obtained. The uidA gene was stably integrated as suggested by its transmission to the progeny.these authors contributed equally to the work     

Conifer genetic engineering: transgenic Pinus radiata (D. Don) and Picea abies (Karst) plants are resistant to the herbicide Buster

A biolistic transformation procedure was applied to co-transform embryogenic tissue of Pinus radiata and Picea abies with two plasmid DNAs. The first vector contained the bar gene, specifying resistance to the herbicide glufosinate, under the control of the maize ubiquitin promoter. This plasmid also contained the Pinus radiata germin cDNA sequence, in either sense or antisense orientation, driven by the ubiquitin promoter. The second vector contained both the nptII gene under control of the CaMV 35S promoter for selection of transgenic tissue on geneticin and the uidA reporter gene under control of the double CaMV 35 promoter. Polymerase chain reaction analysis of selected geneticin-resistant tissue showed that the transformation rates for the co-bombarded plasmid were high in both Pinus radiata (75%) and Picea abies (86%). A combination of phenotypic analysis and Northern hybridisation demonstrated that a number of the transgenic lines expressed all four transgenes. Regenerated plantlets from Pinus radiata and Picea abies transgenic lines were spray-tested with commercial rates of Buster (glufosinate at 0.5, 1.0, 2.0 and 4.0 kg active ingredient per hectare). Transgenic plants survived and continued to grow with minor or no damage to their needles, whereas non-transgenic plants regenerated from the same cell lines died within 8 weeks of spraying. To our knowledge, this is the first report on genetically engineered herbicide resistance in conifers, and the results demonstrate that this trait is a feasible option for plantation forestry.     

Stable transformation of a recalcitrant kentucky bluegrass (Poa pratensis L.) cultivar using mature seed-derived highly regenerative tissues

Summary An efficient method to produce highly regenerative tissues from seeds of a previously recalcitrant cultivar of Kentucky bluegrass (Poa pratensis L. ev. Kenblue) was established under dim-light conditions (10–30 μE m⁻²s⁻¹, 16-h light) using media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5 or 9.0 μM), 6-benzylaminopurine (BA; 0.44 or 2.2 μM), and a high level of cupric sulfate (5.0 μM). The tissues were co-transformed with three plasmids containing the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA; gus), and a synthetic green fluorescent protein gene [sgfp(S65T)]. From 463 individual explants bombarded, 10 independent transgenic events (2.2%) were obtained after a 3–4-month selection period for hygromycin resistance using 30–100 mg l⁻¹ hygromycin B; of the 10 independent events, seven (70%) were regenerable. Stable integration of the transgene(s) in transgenic plants was confirmed by polymerase chain reaction and DNA blot hybridization analyses. Coexpression frequency of all three genes was 20%; for two transgenes, either hpt/uidA or hpt/sgfp(S65T), coexpression frequency was 30–40%.     

Efficient Method of Agrobacterium-mediated Transformation for Triticale (x Triticosecale Wittmack)

Transgenic plants of triticale cv. Wanad were obtained after transformation using three combinations of strain/vectors. Two of them were hypervirulent Agrobacterium tumefaciens strains (AGL1 and EHA101) with vectors containing bar under maize ubiquitin 1 promoter (pDM805), and both hpt under p35S and nptII under pnos (pGAH). The third one was a regular LBA4404 strain containing super-binary plasmid pTOK233 with selection genes the same as in pGAH. The efficiency of transformation was from 0 to 16% and it was dependent on the selection factor, auxin pretreatment, and the strain/vector combination. The highest number of transgenic plants was obtained after transformation with LBA4404(pTOK233) and kanamycin selection. Pretreatment of explants with picloram led to the highest number of plants obtained after transformation with both Agrobacterium/vector systems LBA4404(pTOK233) and EHA101(pGAH) and selected with kanamycin. Transgenic character of selected plants was examined by PCR using specific primers for bar, gus, nptII, and hpt and confirmed by Southern blot hybridization analysis. There was no GUS expression in T₀ transgenic plants transformed with gus under p35S. However the GUS expression was detectable in the progeny of some lines. Only 30% of 46 transgenic lines showed Mendelian segregation of GUS expressing to GUS not expressing plants. In the remaining 70% the segregation was non-Mendelian and the rate was much lower than 3:1. Factors that might effect expression of transgenes in allohexaploid monocot species are discussed.     

Transformation of potato (Solanum tuberosum) using particle bombardment

Internodes, leaves and tuber slices from potato (Solanum tuberosum), genotype 1024-2, were subjected to particle bombardment. Transient expression was optimized using the uidA and the luc reporter genes that encode #-glucuronidase (GUS) and luciferase, respectively. Stable transformation was achieved using the neomycin phosphotransferase (nptII) gene, which confers resistance to the antibiotic kanamycin. The influence of biological parameters (tissue type, growth period before bombardment, pre- and post-bombardment osmoticum treatment) and physical parameters (helium pressure, tissue distance) that are known to possibly affect stable transformation were investigated. Putative transgenic plants, which rooted in media containing kanamycin, were obtained from all of the tissues tested although there were large differences in the efficiency: internodes (0.77 plants per bombarded explant), microtuber slices (0.10 plants per bombarded explant) and leaves (0.02 plants per bombarded explant). Southern blot analysis of putative transgenic plants confirmed the integration of the transgenes into plant DNA. The results indicate that an efficient particle bombardment protocol is now available for both transient and stable transformation of potato internodal segments, thus contributing to an enhanced flexibility in the delivery of transgenes to this important food crop.     

Development of wheat scab symptoms is delayed in transgenic wheat plants that constitutively express a rice thaumatin-like protein gene

The possibility of controlling wheat scab (caused by Fusarium graminearum Schw.) was explored by engineering wheat plants for constitutive expression of pathogenesis-related (PR) protein genes. A rice thaumatin-like protein (TLP) gene (tlp) and a rice chitinase gene (chi11) were introduced into the spring wheat cultivar ’Bobwhite’ by co-transformation of the plasmids pGL2ubi-tlp (ubiquitin/tlp//CaMV 35S/hpt) and pAHG11 (CaMV 35S/chi11//ubiquitin/bar). The transformation was by biolistic bombardment. Bialaphos was used as the selection reagent. The integration and expression of the tlp, bar, chi11 and hpt genes were analyzed by Southern, Northern and Western blot analyses. The four transgenes co-segregated in the T₁ progeny of the transgenic plant and were localized at the telomeric region of the chromosome 6A long arm by sequential N-banding and fluorescent in situ hybridization (FISH) using pAHG11 or pGL2ubi-tlp as the probes. Only the transgenes tlp and bar, under the control of the ubiquitin promoter-intron, were expressed. No expression of the chi11 and hpt genes, controlled by the CaMV 35S promoter, was detected in T₁ plants. After inoculation with conidia of F. graminearum, the symptoms of scab developed significantly slower in transgenic plants of the T₁, T₂ and T₃ generations expressing the tlp gene than in non-transformed control plants. This is the first report of enhanced resistance to F. graminearum in transgenic wheat plants with constitutive expression of TLP. Received: 15 December 1998 / Accepted: 30 January 1999