Evaluation of microprojectile-mediated DNA delivery and reporter genes for genetic transformation of the Mediterranean cypress (Cupressus sempervirens L.)

Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and 21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria. Received: 18 November 1996/ Revision received: 19 February 1997/ Accepted: 20 November 1997     

Agrobacterium tumefaciens mediated transformation and regeneration of muskmelon plants

Transgenic muskmelon (Cucumis melo L.) plants were produced efficiently by inoculating cotyledon explants with Agrobacterium tumefaciens strain LBA4404 bearing a Ti plasmid with the NPT II gene for kanaymcin resistance. After co-cultivation for three days, expiants were transferred to melon regeneration medium with kanamycin to select for transformed tissue. Shoot regeneration occurred within 3–5 weeks; excised shoots were rooted on medium containing kanamycin before transferring to soil. Morphologically normal plants were produced in three months. Southern blot analysis confirmed that ca. 85% of the regenerated plants contained the NPT gene. Dot blot analysis and leaf callus assay of progeny of transgenic plants verified transmission of the introduced gene(s) to the next generation. Factors affecting transformation efficiency are discussed.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - IAA indole 3 acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NPT II neomycin phosphotransferase II     

Use of paromomycin as a selective agent for oat transformation

Summary Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with two different plasmids containing the E. coli tn5 neomycin phosphotransferase II gene (npt II). Selection was accomplished using the antibiotic paromomycin sulfate following microprojectile bombardment. From two independent experiments, 88 paromomycin-resistant tissue cultures were shown to be transgenic based on Southern blot analysis and detection of the neomycin phosphotransferase (NPT II) protein using ELISA. Copy numbers of the npt II gene ranged from one to eight copies per haploid oat genome integrated into high molecular weight DNA of the paromomycin-resistant cultures. Plants were regenerated from 32 of the 88 transgenic tissue cultures. Plants from 17 of the 32 regenerable cultures exhibited fertility. Stable transformation was shown by segregation patterns of the NPT II protein in R₁ seedlings produced from 16 fertile culture lines that were tested. The overall results demonstrate that the combination of the npt II gene and paromomycin provides efficient selection of transgenic oat tissue cultures. Oat plants transformed with the npt II gene present reduced ecological risk compared to the previously used herbicide-resistance selection system.Abbreviations GUS beta-glucuronidase - uid A E. coli gene coding for GUS - NPT II neomycin phosphotransferase II of Tn 5 - npt II gene for NPT II - 2,4-D 2,4-dichlorophenoxy acid - X-gluc 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid cyclohexyl-ammonium salt - NOS nopaline synthase - NAA naphthalene acetic acid - BAP benzylaminopurine - ELISA enzyme-linked immunosorbant assay     

Use of a TR T-DNA promoter to express genes in plants and bacteria

Summary A series of plasmids have been constructed in which a promoter from the T_R region of the Agrobacterium tumefaciens Ti-plasmid has been fused to genes encoding neomycin phosphotransferase II (NPT II) or a cDNA clone encoding a 22 kd zein protein. After recombination into the Ti-plasmid pTiA6, A. tumefaciens strains harboring these plasmids were used to incite tumors on sunflower hypocotyl sections. Tumors containing the NPT II gene in the correct orientation relative to the promoter were able to grow on normally inhibitory concentrations of the antibiotic G418. Tumors with the NPT II gene in the incorrect orientation, or with the zein gene in either orientation, were killed by the antibiotic. S1 nuclease protection experiments indicated that for both the NPT II and zein genes, the T-DNA promoter was functioning correctly. The T-DNA fragment containing the promoter active in plants also contained promoter sequences active both in E. coli and in A. tumefaciens.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens

High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 g/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants.     

Genetic transformation of Medicago truncatula using Agrobacterium with genetically modified Ri and disarmed Ti plasmids

Summary Fertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R₁ progeny.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - NPT neomycin phosphotransferase     

Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants

Summary Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes.Abbreviations AMV RNA4 Alfalfa mosaic virus RNA4 - BA 6-benzyladenine - CaMV cauliflower mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraacetic acid - FAA formalin-acetic acid-alcohol - GUS ß-glucuronidase - NAA 1-naphthylacetic acid - NPT II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - TE Tris-Cl/EDTA - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl-urea (thidiazuron) - WPM woody plant medium (Lloyd and McCown 1980) - X-GLUC 5-bromo-4-chloro-3-indolyl-ß-glucuronic acid     

Promotion of flowering and morphological alterations in Atropa belladonna transformed with a CaMV 35S-rolC chimeric gene of the Ri plasmid

Summary Kanamycin-resistant plants of belladonna (Atropa belladonna) were obtained after Agrobacterium mediated transformation. When a rolC gene, which is one of the loci located on Ri plasmid of Agrobacterium rhizogenes, was co-introduced with a kanamycin resistant (NPT II) gene under control of a cauliflower mosaic virus 35S promoter, the rolC gene was expressed strongly in leaves, flowers, stems and roots. The transformed plants exhibited dramatic promotion of flowering, reduced apical dominance, pale and lanceolated leaves and smaller flowers. On the other hand, when native rolC gene was co-introduced with NPT II, the transgenic plants obtained did not exhibit the altered phenotypes observed in 35S-rolC transformants, and the expression level of the rolC gene was much lower than in 35S-rolC transformants. These results suggest that the morphological changes in transgenic Atropa belladonna were related to the degree of expression of the rolC gene.Abbreviations native rolC rolC gene under control of its own promoter - 35S-rolC rolC gene under control of a cauliflower mosaic viras 35S promoter     

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (NPT II). Transformed calluses lost neomycin phosphotransferase II activity after several subcultures. Treatment of calluses with 5-azacytidine, a demethylating agent, restored enzyme activity, suggesting that methylation of npt II sequences might be responsible for loss of NPT II activity. Studies presented here were designed to test that hypothesis. Results indicated that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity. There was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased activity of NPT II. In contrast, the auxin 2,4-D increased both the NPT II protein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism. In vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine- or hydroxyurea-treated calluses showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calluses treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor (or factors) in callus extracts which affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment. These results suggested that one effect of 5-azacytidine in increasing NPT II activity was posttranslational.Abbreviations ELISA enzyme-linked immunosorbent assay - NOS nopalene synthase - nos DNA segment encoding NOS - NPT II neomycin phosphotransferase - npt II DNA segment encoding NPT II - PAGE polyacrylamide gel electrophoresis     

Transgenic plants of rutabaga (Brassica napobrassica) tolerant to pest insects

Cotyledons cut from axenic seedlings were immersed inAgrobacterium tumefaciens suspension which was treated with acetosyringone and nopaline at low pH overnight. The infected cotyledon explants were cultured on MSB medium (MS salts + B₅ Vitamins) containing 6-BA 3mg/1 for 2–3 days, and transferred onto selective medium (MSB with kanamycin 50–100 mg/l). Kanamycin-resistant shoots were selected. More than 60 regenerated plants were obtained. About 60% of the plants showed high NPT II activity. Southern blot hybridization showed that some of the plants gave a positive signal with the insecticidal crystal protein gene (cry IA gene) probe, and exhibited tolerant to insects such asPieris rapae (cabbage caterpillar) in leaf feeding experiments. Kanamycin-resistance and insect-resistance were maintained in the progeny.Abbreviations 6-BA 6-benzylaminopurine - IBA indole-3-butyric acid - CryIA gene bacillus thuringiensis insecticidal crystal protein genecryIA - NPT II neomycin phosphotransferase II     

Recovery of transgenic rice plants expressing the rice dwarf virus outer coat protein gene (S8)

 The coding region of the eighth largest segment (S8) of the rice dwarf virus (RDV) was obtained from a RDV Fujian isolate. It was then cloned into pTrcHisA for expression in E. coli and into vector pE3 for plant transformation. By using callus derived from mature rice embryos as the target tissue, we obtained regenerated rice plants after bombardment of the former with plasmid pE3R8 containing the RDV S8 gene and the marker gene neomycin phosphotransferase (NPT II). Southern blotting confirmed the integration of the RDV S8 gene into the rice genome. The expression of the outer coat protein in both E. coli and rice plants was confirmed by western blotting. The recovery of transgenic rice plants expressing S8 gene is an important step towards studying the function of the RDV genes and obtaining RDV-resistant rice plants. Received: 1 March 1996 / Accepted: 2 August 1996     

Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene

Summary Genetically transformed plants of Brassica napus L. (oilseed rape) were obtained from hypocotyl expiants using Agrobacterium tumefaciens vectors. Hypocotyl explants were inoculated with disarmed or oncogenic A. tumefaciens strains, EHA101 and A281, and then cultured on media containing kanamycin. The A. tumefaciens strains harbored a binary vector, which contained a neomycin phosphotransferase II (NPTII) gene driven by the 35S promoter of cauliflower mosaic virus and an engineered napin (seed storage protein) gene with its own promoter (300 nucleotides 5 to the start of translation). Transformation of B. napus plants was confirmed by detection of NPT II enzyme activity, Southern blot analysis and inheritance of the kanamycin-resistance trait (NPT II gene) in the progeny. Expression of the engineered napin gene in embryos but not in leaves of transgenic plants was observed by Northern analysis. These data demonstrate that morphologically normal, fertile transgenic B. napus plants can be obtained using Agrobacterium as a gene vector and that developmentally regulated expression of reintroduced genes can be achieved.     

Agrobacterium rhizogenes mediated transformation of grapevine (Vitis vinifera L.)

Genetically transformed grapevine (Vitis vinifera L.) roots were obtained after inocultation of in vitro grown whole plants (cv. Grenache) with Agrobacterium rhizogenes. The strain used contains two plasmids: the wild-type Ri plasmid pRi 15834 and a Ti-derived plasmid which carries a chimaeric neomycin phosphotrans-ferase gene (NPT II) and the nopaline synthase gene. Expression of the NPT II gene can confer kanamycin resistance to transformed plant cells. Slowly growing axenic root cultures derived from single root tips were obtained. Opine analysis indicated the presence of agropine and/or nopaline in established root cultures. For one culture, the presence of T-DNA was confirmed by dot-blot hybridization with pRi 15834 TL-DNA. Callogenesis was induced by subculturing root fragments on medium supplemented with benzylaminopurine and indoleacetic acid.Transformation of in vitro cultured grapevine cells has recently been reported (baribault T.J. et al., Plant Cell Rep (1989) 8: 137–140). In contrast with the results presented here, expession of the NPT II gene Conferred kanamycin resistance to Vitis vinifera calli that was sufficient for selection of trasformed cells.Abbreviations BAP benzylaminopurine - IAA indoleacetic acid - NAA naphtaleneacetic acid - NPT II neomycin phosphostransferase II - EDTA ethylenediaminetetraacetic acid     

Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 g/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 g/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.NRCC No. 31491During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94).     

Transformation of Cucumis sativus tissue by Agrobacterium tumefaciens and the regeneration of transformed plants

Cotyledons of cucumber seedlings (Cucumis sativus L. cv. Poinsett 76) were co-cultivated with disarmed Agrobacterium strain C58Z707. The Agrobacterium strain contained the Agrobacterium-derived binary vector plasmid pGA482, its T-DNA region contains a plant expressible bacterial derived neomycin phosphotransferase II (NPT II) gene which upon transfer, genome integration, and expression in plant tissues confers resistance to the antibiotic kanamycin. After growth of inoculated cotyledon sections on selective medium containing 100 mg/l kanamycin, transformed embryogenic calli were obtained followed by the development of embryos and plant regeneration. Transformed R₀ and R₁ cucumber plants appeared normal and tested positive for NPT II enzyme activity. Genomic DNAs isolated from the NPT II positive plants all showed hybridization to the characteristic 2.0 kb (BamHI to HindIII) NPT II gene-containing fragment. These results show that the Agrobscterium-mediated gene transfer system and regeneration via somatic embryogenesis is an effective method for the transfer of genetic material into plant species belonging to the family Cucurbitaceae.Abbreviation Cb carbenicillin - 2,4-D 2,4-dichlorophenoxyacetic acid - Km kanamycin - KN kinetin - MS Murashige and Skoog - NAA naphthaleneacetic acid - NPT II neomycin phosphotransferase II     

外源甜蛋白thaumatin II基因转入烟草的研究

利用土壤农杆菌系统,将高甜度的外源甜蛋白thaumatin II基因转入烟草细胞,并得到大量转基因植株及其后代。经分子杂交分析确证thaumatin II基因已整合到烟草植株的基因组中,并在转录水平检测到表达。标记基因胭脂碱合成酶(NOS)基因及新霉素磷酸转移酶(NPT II)基因也在转基因植株中正常表达。     

低能离子束介导外源基因转化烟草的研究

以烟草ＮＣ－８９种子为材料,用显微扫描电镜（ＥＳＭ）和电子自旋共振（ＥＳＲ）波谱仪研究氮离子束对烟草种子表面的刻蚀作用及能量沉积产生自由基的间接效应,为离子束介导转移外源基因提供了形态结构依据。将烟草种子用２０Ｋｅｖ的氮离子束处理后,浸入含有ＰＢⅠ１２１质粒的缓冲介质中,在含有卡那霉素１００ｍｇ／Ｌ的ＭＳ０培养基上继代筛选,得到３株抗性植株。取抗性植株的叶片,经组织培养后得到再生抗性植株。经过ＰＣＲ及ｓｏｕｔｈｅｒｎ杂交分析,证明外源基因已转入烟草。     

A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and -glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R₂ progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.     

Genetic transformation of a hepatoprotective plant, <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Phyllanthus amarus Schum & Thonn. is a source of various pharmacologically active compounds such as phyllanthin, hypophyllanthin, gallic acid, catechin, and nirurin, a flavone glycoside. A genetic transformation method using Agrobacterium tumefaciens was developed for this plant species for the first time. Shoot tips of full grown plants were used as explants for Agrobacterium-mediated transformation. Transgenic plants were obtained by co-cultivation of shoot tips explants and A. tumefaciens strain LBA4404 containing the pCAMBIA 2301 plasmid harboring neomycin phosphotransferase II (NPT II) and β-glucuronidase encoding (GUS) genes in the T-DNA region in the presence of 200 μM acetosyringone. Integration of the NPT II gene into the genome of transgenic plants was verified by PCR and Southern blot analyses. Expression of the NPT II gene was confirmed by RT-PCR analysis. An average of 25 explants was used, out of which an average of 19 explants produced kanamycin-resistant shoots, which rooted to produce 13 complete transgenic plants.