Transformation of Zea mays L. Using Agrobacterium tumefaciens and the Shoot Apex

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F₁ progeny. Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHA 1, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and β-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F₁ generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F₁ progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes.     

Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants

Summary Transgenic sweet orange (Citrus sinensis L. Osbeck) plants have been obtained by Agrobacterium tumefaciens-mediated gene transfer. An hypervirulent A. tumefaciens strain harboring a binary vector that contains the chimeric neomycin phosphotransferase II (NPT II) and ß-glucuronidase (GUS) genes was cocultivated with stem segments from in vivo grown seedlings. Shoots regenerated under kanamycin selection were harvested from the stem segments within 12 weeks. Shoot basal portions were assayed for GUS activity and the remaining portions were shoot tip grafted in vitro for production of plants. Integration of the GUS gene was confirmed by Southern analysis. This transformation procedure showed the highest transgenic plant production efficiency reported for Citrus.Abbreviations BA benzyladenine - CaMV cauliflowermosaic virus - GUS ß-glucuronidase - LB Luria Broth - MS Murashige and Skoog - NAA naphthalenacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - PEG polyethylene glycol - RM rooting medium - SRM shoot regeneration medium     

Expression and inheritance pattern of two foreign genes in petunia

Transgenic petunia (Petunia hybrida Vilm.) plants were obtained from Agrobacterium-mediated shoot apex transformation. Studies at the phenotypic as well as molecular level established both the presence of the NPT II (neomycin phosphotransferase II) and GUS (-glucuronidase) genes and their level of activity. Twenty-nine primary transformed plants showed varying patterns of phenotype expression of both genes. NPT II and GUS expression in 7 primary plants over a 4-month interval showed varying levels of gene expression within and among individual plants. All primary transgenic plants were self-pollinated and backcrossed to establish the inheritance patterns of both genes. Mendelian and non-Mendelian inheritance patterns for both genes were observed. Analysis of the progeny showed poor transmission of the foreign genes through the pollen especially when two or more bands were present in the Southern hybridization. Most plants whose progeny segregated in Mendelian ratios for either the NPT II or GUS gene had just one copy of the gene. In this study where both foreign genes were examined in both self and test crosses, no transgenic plant showed Mendelian patterns of inheritance for both foreign traits.Department of Plant Pathology and Microbiology     

Transgenic sweet potato plants obtained byAgrobacterium tumefaciens-mediated transformation

Stable expression of foreign genes was achieved in sweet potato (Ipomoea batatas (L.) Lam) plants using anAgrobacterium tumefaciens mediated system. Embryogenic calluses produced from apical meristems of cultivar White Star were multiplied and cocultivated withA. tumefaciens strain EHA101 harboring a binary vector containing the -glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes. The calluses were transferred to selective regeneration medium and kanamycin resistant embryos were recovered which developed into morphologically normal plants. Histochemical and fluorimetric GUS assays of plants developed from the kanamycin resistant embryos were positive. Amplified DNA fragments were produced in polymerase chain reactions using GUS-specific primers and DNA from these plants. Transformation was confirmed by Southern analysis of the GUS gene. With the developed method, transgenic sweet potato plants were obtained within 7 weeks. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.Florida Agricultural Experiment Station Journal Series N-02231. This research was partially supported by CNP_q/RHAE (Brazil).     

Expression of foreign genes in transgenic yellow-poplar plants

Cells of yellow-poplar (Liriodendron tulipifera L.) were transformed by direct gene transfer and regenerated into plants by somatic embryogenesis. Plasmid DNA bearing marker genes encoding β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) were introduced by microprojectile bombardment into single cells and small cell clusters isolated from embryogenic suspension cultures. The number of full-length copies of the GUS gene in independently transformed callus lines ranged from approximately 3 to 30. An enzyme-linked immunosorbent assay for NPT II and a fluorometric assay for GUS showed that the expression of both enzymes varied by less than fourfold among callus lines. A histochemical assay for GUS activity revealed a heterogeneous pattern of staining with the substrate 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid in some transformed cell cultures. However, cell clusters reacting positively (blue) or negatively (white) with 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid demonstrated both GUS activity and NPT II expression in quantitative assays. Somatic embryos induced from transformed cell cultures were found to be uniformly GUS positive by histochemical analysis. All transgenic plants sampled expressed the two marker genes in both root and shoot tissues. GUS activity was found to be higher in leaves than roots by fluorometric and histochemical assays. Conversely, roots expressed higher levels of NPT II than leaves.     

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。     

Transgenic papaya plants from Agrobacterium-mediated transformation of petioles of in vitro propagated multishoots

Summary In order to establish a model system for introduction of foreign genes into papaya (Carica papaya L.) plants by Agrobacterium-mediated transformation, petioles from multishoots were used as explant source and bacterial neomycin phosphotransferase II (NPT II) gene and -glucuronidase (GUS) gene were used as a selection marker and a reporter, respectively. Cross sections of papaya petioles obtained from multishoots micropropagated in vitro were infected with A. tumefaciens LBA4404 containing NPTII and GUS genes and co-cultured for 2 d. The putative transformed calluses were identified by growth on the selective medium containing kanamycin and carbenicillin, and consequently regenerated to plants via somatic embryogenesis. Thirteen putative transgenic lines were obtained from a total of 415 petiole fragments treated. Strong GUS activity was detected in the selected putative transgenic calli or plants by fluorogenic assay. Western blot analysis using GUS antiserum confirmed that the GUS protein was expressed in putative transformed papaya cells and transgenic plants. The presence of the GUS gene in the papaya tissues was detected by PCR amplification coupled with Southern blot.     

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     

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     

Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens

Summary Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed B. rapa cvs. Tobin and Emma. Transformed plants were obtained within three months of cocultivation. Transformation frequencies for the cultivars Tobin and Emma were 1–9%. Evidence for transformation was shown by NPT II dot blot assay, the GUS fluorometric assay, Southern analysis, and segregation of the kanamycin-resistance trait in the progeny. The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivars of B. napus.     

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.     

Stable transformation of lettuce cultivar South Bay from cotyledon explants

Transgenic plants of lettuce cultivar (cv.) South Bay were produced by using Agrobacterium tumefaciens vectors containing the -glucuronidase (GUS) reporter gene and the NPT II gene for kanamycin resistance as a selectable marker. High frequency of transformation, based on kanamycin resistance and assays for GUS expression, was obtained with 24 to 72-h-old cotyledon explants cocultivated for 48 h with Agrobacterium tumefaciens. After the cocultivation period, the explants were placed in selection medium containing 50 or 100 mg l^–1 of kanamycin, 100 mg l^–1 cefotaxime and 500 mg l^–1 carbenicillin for 10 days. Surviving explants were transferred every 14 days on shoot elongation medium. Progenies of R₀ plants demonstrated linked monogenic segregation for kanamycin resistance and GUS activity.Florida Agricultural Experiment Station Journal Series R-02231. This research was partially supported by CNPq/RHAE (Brazil).     

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     

Transformation of soybean via particle bombardment of embryogenic suspension culture tissue

Summary Embryogenic suspension culture tissue of soybean (Glycine max Merrill.) was bombarded with particles coated with plasmid DNAs encoding hygromycin resistance andβ-glucuronidase (GUS). One to two weeks after bombardment, embryogenic tissue was placed in a liquid proliferation medium containing hygromycin. Four to six weeks after bombardment, lobes of yellow-green, hygromycin-resistant tissue, which began as outgrowths on brown clumps of hygromycin-sensitive tissue, were isolated and cultured to give rise to clones of transgenic embryogenic material. In vivo GUS assays of hygromycin-resistant clones showed that the early outgrowths could be negative, sectored, or positive for GUS activity. Transgenic, fertile plants could be routinely produced from the proliferating transgenic embryogenic clones. Southern hybridization analyses confirmed stable transformation and indicated that both copy number and integration pattern of the introduced DNA varied among independently transformed clones. Hybridization analysis of DNA from progeny plants showed genetic linkage of multiple copies of introduced DNA. An average of three transgenic clones were obtained per bombardment making this procedure very suitable for transformation of soybean.     

Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants

Summary Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.Abbreviations GUS ß-glucuronidase - X-Gluc 5-bromo-4-chloro-3indolyl ß-D-glucuronic acid - NPT II neomycin phosphotransferase II     

Regeneration of transgenic plants of grapevine (Vitis vinifera L.) via Agrobacterium rhizogenesmediated transformation of embryogenic calli

Genetically transformed roots and calli were induced from leafsegments of grapevine (Vitis vinifera L. cv. Koshusanjaku) afterco-cultivation with wild-type Agrobacterium rhizogenes strains,but plant regenera tion from them was not achieved. On the otherhand, transgenlc grapevine plants were obtained via somaticembryogenesis after co-cultivation of embryogenic calli withan engineered A. rhizogenes strain including both the neomycinphosphotransferase II (NPT II) and the ß-glucuronidase(GUS) genes, followed by selection of secondary embryos forkanamycin resistance. All these plants showed GUS gene expressionrevealed by histochemical assay. Southern blot analysis revealedthe stable integration of the GUS cording region in their genome.Transformants containing Ri T-DNA exhibited various phenotypes:most of them showed a typical Ri-transformed phenotype suchas wrinkled leaves, while the others looked normal. Key words: Agrobacterium rhizogenes, grapevine, transgenic plants, Vitis vinifera     

A rapid method for qualitative assay of both neomycin phosphotransferase II and β-glucuronidase activities in transgenic plants

A rapid and efficient method for assaying both NPT II and GUS activities was developed. In this method, which is modified from that of McDonnell et al. (1987), and Jefferson (1987), no sample processing procedures such as grinding and centrifugation are necessary. Cut plant tissues (leaves) or intact calli or cells expressing the genes of interest are placed in wells of a microtiter plate containing reaction mixture, and after incubation the reaction mixture is directly used for both NPT II and GUS assays. For the NPT II assay, aliquots of the reaction mixture are blotted onto Whatman P81 paper through a manifold, and the product of the reaction is detected by autoradiography. For GUS activity, aliquots or the rest of the reaction mixture are observed for fluorescent emission under a hand-held UV light or read in a fluorimeter after adding stop buffer to the reaction mixture. This method is the simplest, cheapest, and quickest assays for NPT II and GUS reported to date, and is extremely efficient and suitable for assaying small amounts of samples (as little as 0.3 mg tissue), such as in transient expression assays, or for the quick screening of large numbers of samples, such as in studies of gene inheritance in transgenic plants. In our laboratory, it has been used successfully in assaying NPT II activities for transient and stable gene expression in transformed protoplasts, calli, and leaf tissues of various transgenic plants. It has also been used for detecting both NPT II and GUS activities in transgenic rice plants, in which more than 400 samples could be assayed per day per person.     

Fertile transgenic barley generated by direct DNA transfer to protoplasts

We report the generation of transgenic barley plants via PEG-mediated direct DNA uptake to protoplasts. Protoplasts isolated from embryogenic cell suspensions of barley (Hordeum vulgare L. cv Igri) were PEG-treated in a solution containing a plasmid which contained the neomycin phosphotransferase (NPT II) gene under the control of the rice actin promoter and the nos terminator. Colonies developing from the treated protoplasts were incubated in liquid medium containing the selective antibiotic G418. Surviving calli were subsequently transferred to solid media containing G418, on which embryogenic calli developed. These calli gave rise to albino and green shoots on antibiotic-free regeneration medium. NPT II ELISA revealed that approximately half of the morphogenic calli expressed the foreign gene. In total, 12 plantlets derived from NPT-positive calli survived transfer to soil. Southern hybridization analysis confirmed the stable transformation of these plants. However, the foreign gene seemed to be inactivated in plants from one transgenic line. Most of the transgenic plants set seed, and the foreign gene was transmitted and expressed in their progenies, which was ascertained by Southern hybridization and NPT II ELISA.     

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).