Generation of low copy number and stably expressing transgenic creeping bentgrass plants using minimal gene cassette bombardment

A minimal gene cassette comprised of the ubiquitin (Ubi) promoter + green fluorescent protein (Gfp) gene + Nos terminator DNA sequences, derived from the plasmid vector pPZP201-Gfp was utilized for transformation of creeping bentgrass using particle bombardment. Bentgrass calli bombarded individually with equivalent amounts of the cassette or whole plasmid DNA were compared for Gfp expression and the GFP-positive calli were subsequently regenerated into plants. Percentage of GFP expressing calli and the number of GFP spots/calli were significantly higher in calli that were bombarded with the minimal gene cassette when compared to the whole plasmid. The Gfp expression was stable up to the T₂ generation in minimal gene cassette transformants and there was a lower degree of gene silencing. Southern blot analysis of transgenic plants derived from minimum gene cassette bombardment revealed the presence of single or few copy of the transgene and fairly simple integration patterns. In comparison, whole plasmid transformants had multiple copies and complex integration patterns of the transgene. These results illustrate the advantages of using simple gene cassette for stable plant transformation in bentgrass with possible applications to other plant species.     

基因枪轰击谷子幼穗获得转基因植株

以ＪＱ－７００型国产基因枪轰击豫谷２号谷子幼穗,在１５０ｍｇ／Ｌ卡那霉素选择培养基上筛选到９０８块抗性愈伤组织,其中,绿芽块愈伤５块,共分化出１６株绿苗,组织化学检测ＧＵＳ表达,获得３株阳性植株,Ｓｏｕｔｈｅｒｎ杂交证明１株为阳性。以轰击总外植体计算的转基因植株频率为０．０５％。     

Efficient production of transgenic Alstroemeria plants by using Agrobacterium tumefaciens

A highly efficient and reproducible protocol was developed to obtain transgenic Alstroemeria plants by combining Agrobacterium tumefaciens with friable embryogenic callus (FEC). To develop this transformation method, factors such as infection time, cocultivation period, effect of acetosyringone (AS), different dilution concentrations of the bacterium and temperature during cocultivation were evaluated. A protocol was developed in which transient GUS expression activity was observed ranging from 25% to 55% out of the cocultivated FEC cultures, when FEC cultures were infected for 30 min with 50 μM AS, 1:10 dilution of bacteria, and then cocultivated at 24°C in the dark for 7 days with Agrobacterium strain LBA4404 (pTOK233) that carried gus, nptII and hpt genes. Seven independent experiments produced a total of 1300 transformed somatic embryos with shoots from 3.5 g of FEC. Of these germinated embryos, 50% developed into plants in vitro. Thus, on average, 500 mg of FEC infected with A. tumefaciens produced approximately 80–100 transgenic plants within 6–8 months via a selection process with 2.5–20 mg L^?1 hygromycin. Additionally, transformation was also performed with Agrobacterium strain AGL1 (containing the uidA and ppt genes), and this showed that luciferase‐based selection was less detrimental to the transgenic lines than was herbicide‐based selection. The transformation efficiency was 18.6% for the luciferase‐based selection and 7.6% for the PPT‐based selection, although with luciferase‐based selection, more false positives were obtained (about a quarter of the lines were escapes). The nptII and uidA genes were detected by polymerase chain reaction analysis in nine of the 19 tested lines. The results indicate that the system developed here can be used as an alternative to particle bombardment of Alstroemeria.     

Production of fertile transgenic lentil (Lens culinaris Medik) plants using particle bombardment

Summary A reproducible system for gene transfer in lentil through particle bombardment is presented. Lentil cotyledonary nodes excised from germinated seedlings were bombarded with a plasmid containing a mutant acetolactate synthase gene (ALS) from tobacco conferring resistance to sulfonylurea herbicides. Putative transgenic shoots regenerated on Murashige and Skoog medium supplemented with 6-benzylaminopurine (BA) and chlorsulfuron (5 nM for first 4 wk followed by 2.5 nM for the remainder of the culture period) were micrografted and successfully transferred to soil. T₀ and selfed progeny plants were screened using metsulfuron herbicide leaflet painting. The non-transformed escapes died and transformed plants survived the test. The surviving plants were phenotypically normal and produced viable seeds. The presence and stable transmission of the transgene into genomic DNA of screened T₁ transformants was confirmed by PCR and Southern hybridization. This method for producing transformed plants will allow new opportunities for lentil breeding to produce improved cultivars.     

Production of transgenic gentian plants by particle bombardment of suspension-culture cells

 Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l^–1 hygromycin in liquid MS medium that contained 10 mg l^–1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l^–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l^–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l^–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA. Received: 3 June 1999 / Revision received: 21 September 1999 / Accepted: 20 September 1999     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Stable genetic transformation of garlic plants using particle bombardment

The improvement of garlic plants (Allium sativum L.) via biotechnological approaches is currently limited by the lack of an applicable direct gene transfer system. In this paper, we present the development of a genetic transformation system using particle bombardment for gene delivery and immature clove-derived callus as the gene target. Plasmid DNA (pBI221.23), containing the selectable "hpt" gene for hygromycin resistance and the reporter "gus" gene, was delivered into callus tissue that had been previously treated with aurintricarboxylic acid as an endogenous nuclease inhibitor. The transformed calli were selected using hygromycin B, regenerated, and analysed at the molecular level using DNA hybridization, transgenome rescue and histochemical beta-glucuronidase assay. The results indicated that biolistic transformation can lead to the transfer, expression and stable integration of a DNA fragment into garlic chromosomal DNA. The relative simplicity of this system is a good recommendation for its future use in the production of genetically modified garlic plants.     

Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l^–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants. Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998     

Recovery of transgenic orchid plants with hygromycin selection by particle bombardment to protocorms

Protocorms of orchid (Dendrobium hybrid) were transformed by microprojectile bombardment with a helium-pressured PDS 1000 particle gun. Gold particles coated with plasmid DNA containing ß-glucuronidase (GUS) and hygromycin phosphotransferase (Hpt) marker genes were used. Potentially transformed tissues were identified by active growth on MS medium supplemented with 50mg l^-1 hygromycin. After 4–6 months of continuous selection, 15 hygromycin-resistant lines were recovered. Integration of transgenes into the genome of the transformed protocorms and plantlets were confirmed by GUS histochemical assay and Southern blot hybridization. The transgenic protocorms have gone through propagation for more than 8 months and maintained their transgenic characters. These results indicate that we have established a system for orchid transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants.     

Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation

Particle bombardment and Agrobacterium-mediated transformation are two popular methods currently used for producing transgenic maize. Agrobacterium-mediated transformation is expected to produce transformants carrying fewer copies of the transgene and a more predictable pattern of integration. These putative advantages, however, tradeoff with transformation efficiency in maize when a standard binary vector transformation system is used. Using Southern, northern, real-time PCR, and real-time RT-PCR techniques, we compared transgene copy numbers and RNA expression levels in R₁ and R₂ generations of transgenic maize events generated using the above two gene delivery methods. Our results demonstrated that the Agrobacterium-derived maize transformants have lower transgene copies, and higher and more stable gene expression than their bombardment-derived counterparts. In addition, we showed that more than 70% of transgenic events produced from Agrobacterium-mediated transformation contained various lengths of the bacterial plasmid backbone DNA sequence, indicating that the Agrobacterium-mediated transformation was not as precise as previously perceived, using the current binary vector system.     

An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens.

An efficient and reproducible procedure for the transformation of white spruce (Picea glauca [Moench] Voss) embryogenic tissues was developed using A. tumefaciens-mediated gene transfer. Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A. tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47. The plasmid pBi121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the beta-glucuronidase (uidA) reporter gene, was used as binary vector. The highest frequency of transformation (15 transformed tissues g(-1) FW of treated embryogenic tissue) was obtained with 5-d-old tissues grown in liquid medium and co-cultivated with Agrobacterium for 2 d in the same medium but containing 50 microM acetosyringone. Recovery of kanamycin-resistant tissues was improved when tissues were first grown for 10 d on a timentin-containing medium (400 mg l(-1)), to prevent bacterial overgrowth, before application of the selection pressure. After 6 weeks on kanamycin-selection medium, resistant tissues were obtained and showed stable uidA expression. The presence of the transgenes was demonstrated by PCR analysis and their integration into the genome was confirmed by Southern hybridization. Transgenic plants were regenerated from transformed tissues within 4 months after co-culture.     

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.     

Generation of chlorsulfuron-resistant transgenic garlic plants (Allium sativum L.) by particle bombardment

We established an effective biolistic transformation procedure fortransferring foreign genes into garlic (Allium sativumL.),which we demonstrated by generating transgenic plants resistant tochlorsulfuron, a sulfonylurea herbicide. We subcultured callus tissue from theapical meristem of garlic cloves and repeatedly selected calli with brittle,non-mucilaginous surfaces for over six months, to increase transformationefficiency. We then constructed recombinant DNA that contained the acetolactatesynthase (ALS) gene from a chlorsulfuron-resistantArabidopsis mutant, the cauliflower mosaic virus 35Spromoter, the -glucuronidase (GUS) reporter gene, and the hygromycinphosphotransferase (HPT) selectable marker gene. The garlic calli werebombarded twice with tungsten particles coated with the DNA constructs. Transformed calliwere efficiently selected by embedding them in solid agar medium containing 50mg l^–1 hygromycin B. Selected propagules wereregenerated into 12 independent plants. We confirmed that the transgenes wereintegrated and expressed in the plants using PCR-Southern and Northern blotanalyses and by -glucuronidase expression assay forGUS. The regenerated plants survived in the presence of 3mg l^–1 chlorsulfuron, demonstrating that theirALS was insensitive to this herbicide. These results illustrate the successfultransformation of foreign genes into garlic plants. The set of proceduresdeveloped in this study is applicable to the generation of transgenic garlicplants with other agronomically beneficial traits. These authors contributed equally to this work     

Production of transgenic cassava (Manihot esculenta Crantz) plants by particle bombardment using luciferase activity as selection marker

Cassava embroids derived from friable embryogenic callus of the genotype TMS60444 were bombarded with DNA of the constructs pJIT100 or pJIT64. Both constructs contain the non-invasive reporter gene luciferase from firefly driven by the CaMV 35S promoter. The influence of several particle gun machine parameters and pretreatment of plant material on transient luciferase activity were studied to determine the most essential conditions for stable transformation. Two weeks after bombardment pieces of friable calli with luciferase activity were selected. In total, 67 independent selected calli with luciferase activity (spots), derived from five different experiments, were further cultured either in liquid or on solid medium. Per plate or flask one spot was cultured. In subsequent selection rounds all spots of one individual plate or flask were cultured as one individual group. In this way different transformation events were separated and multiplied.Eight weeks after bombardment 34 cultures still contained luciferase activity. The mean number of luciferase spots per culture had increased from 1 to 4.6 spots in liquid and to 2.5 spots on solid medium. After two more months of subsequent culture and luciferase selection presence of the construct in these cultures was confirmed at the molecular level using the polymerase chain reaction assay and Southern analysis.Friable embryos derived from four transformation events were cultured for maturation. Between 3% and 21% of the mature embryos of the different transformation events were luciferase-positive. After multiplication of the luciferase-positive mature embryos by secondary somatic embryogenesis they were germinated. The plantlets analysed contained one to several copies of the inserted DNA. The method presented enables the transformation of this particular cassava genotype, thus allowing the genetic improvement of this important tropical crop by transgenesis.     

Efficient in vitro plant regeneration and generation of transgenic plants in barley (Hordeum vulgare L.) using particle bombardment

Plant regeneration from immature embryos of 15 Indian barley genotypes (Hordeum vulgare L.) was examined. Highest percent callus induction and number of regenerated plantlets were obtained in cultivars BL 2 (94.4 %; 12.1), RD 2668 (92.6 %; 9.1) and RD 2552 (90.8 %; 7.8). The highly responding cultivar BL 2 was selected for further development of transformation protocol. The plasmid DNA isolated from pCAMBIA1381 harbouring hptII gene as selectable marker and gusA gene as reporter was used. Particle bombardment was used for transformation of immature embryos and regeneration of transgenic plants in Indian barley genotype for the first time. Transformation experiments were carried out using different parameters and optimum conditions for DNA delivery was standardized. The transient expression of gusA gene was investigated as a preliminary test of optimum DNA delivery and for selecting the most appropriate bombardment parameters. The optimum conditions were: gold microparticles (diameter 1.0 μm) shot with 1,100 psi rupture disc pressure. The 3 cm distance between rupture disk and macrocarrier and 9 cm target tissue distance yielded high transient GUS expression. The immature embryos were bombarded twice to increase area for efficient gene delivery. Osmotic medium optimization with 0.4 M sorbitol and preculture of immature embryos for 5 days prior to bombardment resulted into efficient gene transfer in barley. Selection of transformed tissue was performed after 7 days resting step on selection medium containing 50 mg?l^?1 hygromycin. After two more selection steps, green shoots were rooted on MSB₅ medium with 50 mg?l^?1 hygromycin. PCR analysis using primers specific for hptII and gusA genes and Southern blot analysis with hptII probes confirmed the stable integration of transgene in barley genome. Molecular analysis of T₁ generation plantlets revealed the amplification of selectable marker hptII gene in the progeny.     

Molecular analysis of the genome of transgenic rice (Oryza sativa L.) plants produced via particle bombardment or intact cell electroporation

In the present work we utilised some of the most discriminative molecular tools, such as RAPD, AFLP, AFRP and RAMP, to analyse the genome of independently derived transgenic plants from three elite Italian cultivars (cv. Lido, Carnaroli and Thaibonnet) and found that two methods for direct gene transfer, namely particle bombardment and intact cell electroporation (the latter being a procedure set up in this work), result in transgenic rice (Oryza sativa L.) plants that exhibit negligible genomic changes. This is in contrast with recently published results showing relevant changes in the DNA of transgenic rice plants generated through protoplasts electroporation and of transgenic poplar plants engineered through Agrobacterium tumefaciens infection. Implications of these findings are discussed in the context of selecting appropriate gene transfer methodologies to produce transgenic plants expressing genes of interest while retaining their genomic integrity and, thus, their superior agronomic and/or industrial traits.     

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     

Production of transgenic sugarbeet plants (Beta vulgaris L.) using Agrobacterium rhizogenes

Normal phenotype sugarbeet plants transformed with Agrobacterium rhizogenes were produced using direct regeneration from explants without hairy root phase. Kanamycin resistant plants and Ri-roots carrying the genes of neomycin phosphotransferase II and b-glucuronidase have been obtained. Integration of transgenes into sugarbeet genome was confirmed with GUS-assay and PCR using primers for the introduced genes.     

Production of transgenic rape plants (Brassica napus L.) using Agrobacterium tumefaciens

The procedure for genetic transformation of two spring and one winter rapeseed cultivars was developed. No-paline strains of Agrobacterium tumefaciens GV3101 and EHA105 were shown to be preferable for gene transfer, as compared to the octopine strain GV2260. With two types of plant explants, the segments of hypocotyls and cotyledons, transformation was successful; however, its efficiency was somewhat higher with the fragments of hypocotyls. Analysis of regenerated plants by PCR and Southern blotting confirmed the presence of the nptII and nisA genes in transformants. RNA analysis by Northern blotting showed expression of the nisA gene in transformed shoots. The transgenes were inherited in T2 as Mendelian traits. The effect of biotic and abiotic factors on the efficiency of genetic transformation in rapeseed is discussed.