Efficient transformation and regeneration of carnation cultivars usingAgrobacterium

We have developed an efficient method for transformation and regeneration of plants from carnation,Dianthus caryophyllus L. Whole leaves fromin vitro shoot cultures were mixed withAgrobacterium, cocultivated for 5 days and then plated on 2 µg/l chlorsulfuron (CS). Regenerated shoots and shoot clusters were divided into smaller sections and plated on 3 µg/l CS for selection to produce fully transformed shoots. Geneticin (G418) and kanamycin used were not as effective selective agents as CS. All regenerated shoots were vitrified. These were normalized, rooted and transferred to the greenhouse. 100% of regenerated plants were transformed based on rooting assay, GUS assay, PCR and Southern analysis.     

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.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of four lupin species

The complete protocols for long-term micropropagation of some cultivars of four lupin species: Lupinus luteus, L. albus, L. angustifolius and L. mutabilis were elaborated. The shoots were regenerated in vitro via induction of axillary buds development. Plantlets were multiplicated on lowered salts MS-derived media containing BAP in diverse and generally low concentrations. Significant differences in regeneration capacity between species and cultivars were observed. The highest multiplication ratio revealed L. mutabilis and L. luteus. Regenerated shoots were rooted in vitro on low-salts MS-derived media with B5 vitamins. Media were supplemented with different auxins that affected roots formation of particular species and cultivars. Rooting ability of regenerated shoots decreased rapidly through in vitro culture. For that reason, grafting was applied as an alternative method of transfer of shoots to in vivo conditions. This method turned out to be successful for the majority of studied species and cultivars. Complete rooted or grafted plantlets were cultivated in pots with perlit in greenhouse. An erratum to this article is available at .     

Agrobacterium-mediated transformation and plant regeneration of buckwheat (Fagopyrum esculentum Moench.)

Genetic transformation of buckwheat (Fagopyrum esculentum Moench.) and regeneration of transgenic plants were obtained by using Agrobacterium tumefaciens strains as vectors. Buckwheat cotyledons were excised from imbibed seeds, co-cultivated with A. tumefaciens and subjected to previously reported protocols for callus and shoot regeneration. The transformation with oncogenic strains was confirmed by opine and DNA analyses of tumour tissue extracts. Plants were regenerated on cotyledon fragments incubated with strain A281, harboring pGA472, which carries the neomycin phosphotransferase II gene for kanamycin resistance. The transformation of resistant shoot clones was confirmed by NPTII enzyme assay and DNA hybridization. A large number of transformed shoots were rooted and fertile plantlets were raised in the greenhouse. Transgenic plants comprised pin and thrum clones, which were allowed to cross-pollinate. In about 180 R₂ seeds tested for kanamycin resistance, the ratio of resistant to sensitive seedlings was roughly 3:1.Abbreviations BAP 6-benzylaminopurine - 2,4-D dichloro-phenoxyacetic acid - 2iP 6-(, ,-dimethylallyl-amino)-purine - IBA indole-3-butyric acid - IAA indole-3-acetic acid - Km kanamycin - NPTII neomycin phosphotransferase II     

Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.)

Summary Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding.     

Regeneration of transgenic vegetable brassicas (Brassica oleracea andB. campestris) via Ri-mediated transformation

A procedure for the production of fertile transgenic brassicas via Ri-mediated transformation is reported in this paper. Transgenic hairy root lines were selected for 12 vegetable brassica cultivars and lines representing six varieties: broccoli, Brussels sprouts, cabbage, cauliflower, rapid-cycling (allBrassica oleracea) and Chinese cabbage (B. campestris). Leaf explants or petioles of intact cotyledons were co-cultivated withAgrobacterium strain A4T harbouring various binary vectors. The T-DNA region of all binary vectors contained a neomycin phosphotransferase II gene for kanamycin resistance, in addition to other genes. Hairy root lines grew prolifically on hormone-free medium containing kanamycin. Transgenic shoots were regenerated from all cultivars either spontaneously or after transfer of hairy roots to a hormone-containing medium. Southern analysis confirmed that the plants were transgenic. Plants from all brassica types were successfully transferred to greenhouse conditions. Plants were fertile and segregation analysis confirmed transmission of traits to progeny.Abbreviations BA 6-Benzylaminopurine - GUS -Glucuronidase - LS Linsmaier and Skoog medium - NAA I-Naphthaleneacetic acid - NPTII Neomycin phosphotransferase II - TDZ thidiazuron     

Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens — mediated gene transfer

Summary A transformation system that allows regeneration of transgenic pea plants from calli selected for antibiotic resistance was developed. Explants from axenic shoot cultures and seedling epicotyls were cocultivated with nononcogenic Agrobacterium tumefaciens strains, and transformed callus could be selected on callus-inducing media containing either 15 mg/l hygromycin or 75 mg/l kanamycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on hygromycin-resistant calli, but not on the calli selected for kanamycin resistance. Regenerated shoots could subsequently be rooted and transferred into the greenhouse. In addition, the effects of different callus-inducing and growth media on organogenesis were investigated. The transformation of the calli and regenerated plants was confirmed by DNA analysis.     

Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors

A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.     

Transformation of Solanum brevidens using Agrobacterium tumefaciens

Leaf pieces of in vitro-cultured plantlets of the wild potato species Solanum brevidens Phil. were cocultivated with Agrobacterium tumefaciens that contained nptII and uidA genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from solidified and liquid medium that contained 50 mg l^–1 kanamycin. Two Agrobacterium strains were investigated for transformation efficiency. GV2260, which contained p35SGUSINT, resulted in a 11% transformation frequency, compared with 1% using LBA4404. Transformation rates were 7% in liquid culture and 3% on solidified medium. All kanamycinresistant, putatively transformed plantlets were confirmed positive by histochemical GUS assays. GUS activity in 22 independently transformed plants was quantified by fluorometric assay. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the uidA gene.Abbreviations GUS ß-glucuronidase - MS Murashige-Skoog medium - BA 6-benzylaminopurine - 2ip 6-(, -dimethylallylamino)purine - IAA indole-3-acetic acid - GA₃ gibberellic acid - npt II neomycin phosphotransferase II - NOS nopaline synthase - MUG 4-methyl umbelliferyl glucuronide - MU 7-hydroxy-4-methylcoumarin - X-gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid     

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     

Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting transformation efficiency are discussed.     

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.     

Genetic transformation of Centaurium erythraea Rafn by Agrobacterium rhizogenes and the production of secoiridoids

Hairy roots of Centaurium erythraea were obtained by infection with Agrobacterium rhizogenes strain LBA 9402. They spontaneously regenerated adventitious shoots in Woody Plant liquid medium without growth regulators. The shoots were grown continuously in Murashige and Skoog (MS) liquid or agar solidified media supplemented with 0.1 mg l⁻¹ indole-3-acetic acid and 1.0 mg l⁻¹ 6-benzylaminopurine. These shoots produced roots 4 weeks after transfer into agar-solidified MS medium without phytohormones. Regenerated plants grown and flowered under greenhouse conditions. The transgenic value of the regenerated plants was confirmed by the polymerase chain reaction amplification. Transformation by Agrobacterium rhizogenes alters plant morphology and production of secoiridoid glucosides. The level of secoiridoids was also modified by development stage of transformed plants. The total content of the compounds (expressed as the sum of gentiopicroside, sweroside and swertiamarin) in 10-week old pRi-transformed regenerants was 280 mg g⁻¹ dry weight and was 8-times the content in the sample of commercially available C. erythraea herb.     

Spontaneous extensive chromosome elimination in somatic hybrids between somatically congruent species Nicotiana tabacum L. and Atropa belladonna L.

Summary Mesophyll protoplasts of the kanamycin-resistant nightshade, Atropa belladonna, were fused with mesophyll protoplasts of the phosphinothricin resistant-tobacco, Nicotiana tabacum. A total of 447 colonies resistant to both inhibitors was selected. Most of them regenerated shoots with morphology similar to one of the earlier obtained and described symmetric somatic hybrids Nicotiana + Atropa. However, three colonies (0.2%) regenerated vigorously growing tobacco-like shoots; they readily rooted, and after transfer to soil, developed into normal, fertile plants. Unlike their tobacco parental line, BarD, the obtained plants are resistant to kanamycin [they root normally in the presence of kanamycin (200 mg/1)] and possess activity of neomycin phosphotransferase (NPT II) with the same electrophoretic mobility as the one of the nightshade line. According to Southern blot hybridization analysis carried out with the use of radioactively labeled cloned fragments of the Citrus lemon ribosomal DNA repeat, as well as with Nicotiana plumbaginifolia genus-specific, interspersed repeat Inp, the kanamycin-resistant plants under investigation have only species-specific hybridizing bands from tobacco. Cytological analysis of the chromosome sets shows that plants of all three lines possess 48 large chromosomes similar to Nicotiana tabacum ones (2n = 48), and one small extra chromosome (chromosome fragment) similar to Atropa belladonna ones (2n = 72). Available data allow the conclusion that highly asymmetric, normal fertile somatic hybrids with a whole diploid Nicotiana tabacum genome and only part (not more than 2.8%) of an Atropa belladonna genome have been obtained without any pretreatment of a donor genome, although both these species are somatically congruent.     

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 26⁰C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4dichlorophenoxy acetic acid - IAA Indole acetic acid - IBA Indole butaric acid - NAA Naphthalene acetic acid     

Haploid transformation in Brassica napus using an octopine-producing strain of Agrobacterium tumefaciens

Summary Microspore-derived embryos of Brassica napus were transformed using the disarmed octopine-producing LBA4404 strain of Agrobacterium tumefaciens containing the binary vector pBin19. Octopine-producing strains have previously been reported to be ineffective in transforming Brassica. Four actively growing yellow/ green sectors were selected from the embryos on 50 mg/l kanamycin and plants regenerated. Analysis for NPT-II activity in these young plants initially indicated no expression of the bacterial NPT-II gene. The plants were nevertheless grown to maturity, selfed and S₁ seed was collected. Three of the S₁ plants produced microspores which were from 4 to 20 times more tolerant to kanamycin than the original parent. Southern analysis revealed that one plant (EC-1) had a single site of insertion and the other two plants (EC-2 and EC-6) had two sites of insertion with sequence homology to the bacterial NPT-II gene. Microspores from the EC-2 and EC-6 transgenics produced embryos on approximately five times the level of kanamycin tolerated by microspores from untransformed plants, while the EC-1 transgenic produced microspores with more than 20 times the tolerance to kanamycin. Analysis of S₁ progeny of the EC-1 transgenic indicated that 100% of the progeny exhibited the trait through both Southern analysis and by expressing tolerance to kanamycin in microspore-derived embryos.     

Characteristics of the interspecific somatic hybrids <Emphasis Type="Italic">Solanum pinnatisectum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis> H-8105

The morphological, cytological and molecular analyses of the plants regenerated after PEG-induced fusion between mesophyll protoplasts from the dihaploid potato clone H-8105 and the wild tuberous disease-resistant species S. pinnatisectum, were performed. A single fusion experiment yielded 313 calli, although only two calli produced shoots. From the rooted shoots, two stable clones (PT-01-1 and PT-01-2) exhibiting different vigor and habitat, were developed. The plants of PT-01-1 clone grew slowly in vitro, produced tubers after transfer to soil but did not set flowers. In contrast, the plants of the vigorous clone PT-01-2 produced both tubers and flowers after transfer to soil. The flower and tuber morphology of PT-01-1 and PT-01-2 regenerants was intermediate in comparison to the parental species. Cytological analysis revealed that the PT-01-1 clone was pentaploid and the PT-01-2 clone was tetraploid. The molecular (RAPD) analysis confirmed hybridity of both clones. The preliminary tests on late blight resistance of the hybrids showed no differences with a potato parent.     

Somatic embryogenesis of Prunus subhirtella autumno rosa and regeneration of transgenic plants after Agrobacterium-mediated transformation

Embryogenic lines of Prunus subhirtella autumno rosa were established on a modified MS medium supplemented with 1 mg/l NAA, 0.06 mg/l IBA and 0.04 mg/l BA from petioles of axenically grown shoots of adult origin. To induce normal development of plantlets we compared a range of approaches on solid culture media as well as in suspension cultures including treatments with ABA, GA3, zeatin, darkness, and cold. A series of experiments were conducted to follow the temporal pattern of somatic embryo development.Separation of embryos at different stages of development was carried out by sieving the suspension cultures through nylon nets. While the embryogenic masses were used for further subcultures, well formed embryos were used for germination experiments.Transformed Prunus subhirtella plants were regenerated from somatic embryos by inoculating an embryogenic callus with Agrobacterium strain LBA 4404 containing the ß-glucuronidase (GUS) gene on plasmid pBinGUSint. Several putative transformed embryogenic calli were selected for continued proliferation on kanamycin containing media. Finally transgenic plants were regenerated on shoot multiplication medium containing kanamycin. Embryos and plants were shown to express the GUS gene by histochemical assays and northern blot analysis. With a yield of 110 transgenic lines from a single transformation experiment this approach appears ideal for the study of the influence on level of expression caused by different copy number, site of insertion etc. This will be helpful in establishing parameters according to which the best transgenic line for a chosen purpose should be selected.Abbreviations BA 6-benzylaminopurine - IBA 3-indolebutyric acid - GA₃ gibberellic acid - NAA 1-naphthylacetic acid - ABA abscisic acid - GUS ß-glucuronidase - NPTII neomycin phosphotransferase II - SDS sodium dodecyl sulphate - SSC standard saline citrate - PEM proembryogenic masses Dedicated to Franticek Novak     

Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus

Factors influencing transformation frequencies using the Agrobacterium-mediated protocol developed for Citrus seedling internodal stem segments in this laboratory were evaluated, with particular emphasis on decreasing the numbers of ``escape' shoots produced. Although the use of a wild-type ``shooty' Agrobacterium strain allowed relatively high frequencies of β-glucuronidase positive (GUS+) shoots to be produced, none of the shoots were free of wild-type T-DNA and would not root. Both use of a liquid medium/kanamycin overlay and horizontal placement of stem segments increased the efficiency of kanamycin selection. Wounding via particle bombardment prior to Agrobacterium inoculation did not increase transformation frequencies. The concentration of benzyladenine (BA) in the regeneration/selection medium inversely influenced the numbers of shoots that regenerated and the subsequent ability of the shoots to root. Regeneration in the presence of kanamycin also influenced the ability of shoots to root. Many of the shoots that regenerated on selection medium were chimeric for GUS expression, and plants established from such shoots ranged from non-staining to solidly staining for GUS. However, solidly transformed plants with integrated T-DNA were obtained, and these plants have maintained the expression of transgenes over several years. The transgenic plants include ones of sour orange (C. aurantium L.) and Key lime (C. aurantifolia (Christm.) Swing.), two species not previously transformed, and have integrated and express the coat protein gene of citrus tristeza virus. This is the first report of a potentially agriculturally important transgene being expressed in Citrus. Received: 8 October 1996 / Revision received: 1 April 1997 / Accepted: 18 April 1997