Effects of 5-azacytidine on transformation and gene expression inNicotiana tabacum

Summary Protoplasts ofNicotiana tabacum var. Xanthi were incubated with liposomes containing the plasmid plGVneo23 encoding kanamycin resistance. Transformed protoplasts and calli and plants derived from transformed protoplasts were treated with the demethylating agent 5-azacytidine. Three lines of evidence indicate that 5-azacytidine can increase NPT II activity in transformed cell lines and plants: a) Addition of azacytidine to the protoplast medium increased the proportion of kanamycin-resistant transformants recovered. b) NPT II activity could not be detected in approximately 50% of calli derived from transformed protoplasts although such calli grew slowly on medium containing kanamycin. Treatment of NPT-negative calli with 5-azacytidine restored detectable gene activity and increased the growth rate of the callus in the presence of kanamycin. c) Shoot tips regenerated from transformed calli were either NPT-positive or NPT-negative. When shoots were NPT-negative, treatment with 5-azacytidine restored detectable gene activity and improved growth in the presence of kanamycin.     

Direct Gene Transfer in Psophocarpus tetragonolobus Resistance to Kanamycin

Epicotyl-derived protoplasts of Psophocarpus tetragonolobuswere isolated and regenerated to plants. These protoplasts weretransformed to kanamycin resistance following uptake of plasmid(pABDl or pHP23) DNA in combination with PEG treatment. Protoplast-derivedtransformed colonies were selected on kanamycin (75 mg l^–1).The transformed calli expressed NPT II activity and also exhibitedthe presence of the plasmid gene integrated into the plant genome.However, none of the transformed clones showed regenerationof shoot buds. Psophocarpus tetragonolobus, winged bean, naked DNA transformation, protoplast culture, regenerated plants     

Transformation of maize (Zea mays L.) protoplasts and regeneration of haploid transgenic plants

Transgenic haploid maize (Zea mays L.) plants were obtained from protoplasts isolated from microspore-derived cell suspension cultures. Protoplasts were electroporated in the presence of plasmid DNA containing the gus A and npt II genes encoding ß-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II), respectively. Transformed calli were selected and continuously maintained on kanamycin containing medium. Stable transformation was confirmed by enzyme assays and DNA. analysis. Stably transformed tissue was transferred to regeneration medium and several plants were obtained. Most plants showed NPT II activity, and some also showed GUS activity. Chromosome examinations performed on representative plants showed that they were haploid. As expected, these plants were infertile.     

Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants

Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton.     

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     

Phenotypic assay for excision of the maize controlling element Ac in tobacco

We describe a phenotypic assay designed to detect excision of the maize controlling element Ac from a selectable marker gene, neomycin phosphotransferase II (NPT II). An NPT II gene which expresses kanamycin resistance in tobacco cells, and contains a unique restriction enzyme site in the untranslated leader region, was constructed. Ac, or a defective Ac element (Ac), was inserted into the leader region of this gene. The transposon insertions inactivated the NPT II gene as determined by transient NPT II expression assays. The three plasmids were inserted into the T DNA of Agrobacterium tumefaciens Ti plasmid vectors, and transferred to tobacco protoplasts. The transformed protoplasts were selected with 100 or 200 µg/ml kanamycin. Protoplasts transformed by the NPT II gene interrupted by Ac formed ˜25% as many calli resistant to 100 or 200 µg/ml kanamycin as protoplasts transformed by the uninterrupted NPT II gene. Protoplasts transformed by the NPT II gene interrupted by Ac did not form any calli resistant to 200 µg/ml of kanamycin when transformed under similar conditions. Southern blot hybridization analyses of seven kanamycin-resistant calli or plants obtained after transformation by the NPT II gene interrupted by Ac revealed that in all cases Ac had excised, restoring the structure of the NPT II gene. This assay is therefore useful to monitor the activity of a transposable element such as Ac and to define the regions of this element involved in transposition activity.     

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.     

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。     

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     

Transformation and regeneration of Brassica oleracea mediated by an oncogenic Agrobacterium tumefaciens

A chimaeric neomycin phosphotransferase II (NPT II) gene was introduced in Brassica oleracea using an oncogenic strain of Agrobacterium tumefaciens harbouring Ti plasmid which contains Nos/NPTII in its T-DNA. The transformation of B. oleracea with the oncogenic Ti plasmid, resulted in regeneration of shoots and roots without any exogenous requirement of phytohormones. The presence of NPT II gene was determined by hybridization of Tn5 encoded NPT II gene with DNA of kanamycin resistant regenerated plants. The expression of NPT II was demonstrated by kanamycin phosphorylation assay. Several regenerated plants were obtained, a few of them were found to be morphological variants and a chlorophyll deficient mutant plant was also obtained.     

Transformation and regeneration of the self-incompatible species Nicotiana alata Link & Otto

A transformation and regeneration system has been developed for Nicotiana alata, a plant which is being intensively studied as a model of gametophytic self-incompatibility. Plantlets can be regenerated efficiently from seedling hypocotyls. Kanamycin-resistant, transformed plants have been obtained by cocultivation of regenerating hypocotyls with Agrobacterium tumefaciens strain LBA4404 containing a binary vector. The transformation frequency was low with <1% of tissue explants regenerating transformed plants. The transformed plants contained from one to three copies of the introduced DNA. In most cases, the kanamycin resistance phenotype was transmitted to the offspring as a normal Mendelian factor. In one unusual case, none of the offspring inherited the kanamycin resistance of the transformed maternal parent. This plant may have been chimeric or the kanamycin resistance gene may have been inactivated.     

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。     

Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation

Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5mg/L benzylamino purine (BAP), 100mg/L kanamycin and 250mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR.     

Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus

In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.     

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.     

Biolistic Transformation of Rose (Rosa hybridaL.)

A reproducible method has been developed for the Biolistic transformationand regeneration of transgenic plants from embryogenic callusof rose (Rosa hybridaL.) cv. Glad Tidings. DNA delivery wasoptimized using the ß-glucuronidase (gus) gene. Thedistance between the stopping screen and target explants andsupplementation of pre-and post-bombardment culture media with0.25Mmyo-inositol influenced the transformation efficiency.Prior to culture on selection medium containing 250 mg l^-1kanamycinsulphate, embryogenic calli were bombarded, using optimizedgene delivery parameters, with a plasmid carrying the neomycinphosphotransferase (nptII) gene. Somatic embryo-derived kanamycin-resistantplants were regenerated and subsequently transferred to glasshouseconditions. Transformation was confirmed by kanamycin resistanceof calli and plants, NPT II ELISA assay and Southern analysis.All transgenic plants were morphologically normal (true-to-type).Copyright1998 Annals of Botany Company Biolistic; genetic engineering; rose;Rosa hybridaL.; transformation.     

Factors influencing Agrobacterium tumefaciens mediated transformation and expression of kanamycin resistance in pickling cucumber

Cucumber (Cucumis sativus L.) petiole and leaf segments of two pickling genotypes were transformed with Agrobacterium tumefaciens strain LBA 4404, an octopine Ti-plasmid deletion mutant that is avirulent (disarmed plasmid), but to which were added T-DNA inserts on binary plasmids (pBIN 19, ca. 10 kb, and pCGN 783, ca. 25 kb). Expression of neomycin phosphotransferase (NPT II) encoding resistance to the aminoglycoside kanamycin was used as a selectable marker. Factors which influenced the frequency of callus development on medium containing kanamycin (75 mg l^-1) were explant size, bacterial concentration and length of exposure, cocultivation period, and presence of acetosyringone. The optimal procedure involved exposing segments of petiole (4–6 mm) or leaf (0.5 cm²) segments to a bacterial suspension (10⁸ cells ml^-1) containing 20 M acetosyringone for 5 min, followed by a 48 h cocultivation period on a tobacco feeder layer. Explants were placed on MS medium containing 500 mg l^-1 carbenicillin, 75 mg l^-1 kanamycin, and NAA/BA (5.0/2.5 M) or 2,4-d/BA (5.0/5.0 M) and subcultured twice, each after a 2–3 week period, onto fresh media. The overall frequency of transformed callus was 20–50%; the frequency of plantlet regeneration from transformed callus was 8–15%. Twenty-one out of 23 individual plants recovered from two genotypes of pickling cucumber were NPT II positive (transformation frequency of 9%). Copy number of the NPT II gene insert (35S-NPT II-3 fragment, ca. 2.2 kb) in three transformed plants was estimated at ten per haploid genome, indicative of multiple insertions within the cucumber genome. Multimers of the gene (visible as 4.4 and 6.6 kb fragments in Southern analysis) were detected in one plant, suggestive of tandem duplications or repeats. Progeny from a cross between this transformed plant and a nontransformed control showed segregation for the NPT II gene in dot-blot assays; at least 24 plants out of 32 were kanamycin positive. Copy number in the progeny was variable, and ranged from none to ten.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA- napthaleneacetic acid - BA benzyladenine     

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 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