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 of carrot tissues derived from proembryogenic suspension cells: A useful model system for gene expression studies in plants

A method is described for the high frequency transformation of carrot proembryogenic suspension culture cells by a non-oncogenic Ti-plasmid vector (pGV3850::1103) which carried a chimaeric kanamycin resistance gene (nos-NPT-II). Plants were regenerated efficiently from transformed material by somatic embryogenesis in the presence of kanamycin. Transformed tissues expressed readily detectable levels of both NPT-II and nopaline. NPT-II could be detected in total protein extracts by Western blotting. This analysis indicated that NPT-II was produced as a single, full length polypeptide. The T-DNA copy number in individually selected transformants was analysed by Southern blotting and ranged from 1–8 per diploid genome. The copy number and organization of the T-DNA was retained in plants regenerated from these transformants by somatic embryogenesis. These data suggested a clonal origin for the selected kanamycin resistant colonies. NPT-II expression levels appeared to be directly related to gene dosage.     

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.     

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     

Optimization of Agrobacterial (<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>) Transformation of Maize Embryogenic Callus

The method for genetic transformation of maize (Zea mays L.) via embryogenic callus infection with Agrobacterium tumefaciens was developed. Calli were co-cultivated with the overnight culture of A. tumefaciens strain LBA4404 harboring the pBI121 plasmid with the nptII and uidA genes. Thereafter, the sensitivity of calli and regenerated plantlets to kanamycin (Km) was determined. It was shown that kanamycin selection was more efficient at the stage of regenerated plantlets than in callus culture. Both vacuum infiltration at the infection step and preliminary activation of Agrobacterium by acetosyringone or by tobacco leaves exudate increased the frequency of Km-resistant plants. The frequency of Km-resistant plants also varied depending on the morphogenic ability of calli. Polymerase chain reaction confirmed the presence of the nptII gene in the genome of regenerated plants and their progeny. β-Glucuronidase gene expression was observed in roots of T1 plants.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 600–607.Original Russian Text Copyright © 2005 by Danilova, Dolgikh.     

Transfer of a 4.3-kb fragment of the TL-DNA of Agrobacterium rhizogenes strain A4 confers the pRi transformed phenotype to regenerated tobacco plants

Two strategies were used to transfer into tobacco a 4.3-kb fragment of the TL-DNA of the Ri plasmid of Agrobacterium rhizogenes strain A4. In the liposome-mediated procedure a plasmid containing a neomycin phosphotransferase II (NPT II) gene conferring kanamycin resistance and another plasmid containing the 4.3-kb Eco RI fragment (pRiA4 Eco RI-15) were co-transferred into the tobacco genome. In the Agrobacterium transformation procedure, a micro-Ri vector containing a kanamycin resistance gene and the same pRiA4 fragment was used to transform tobacco leaf fragments. Kanamycin resistant plants were regenerated in both cases. They present a phenotype similar to that of plants regenerated from hairy roots induced by A. rhizogenes, that is wrinkled leaves, reduced apical dominance and ability to form hairy root on leaf fragments. In one plant (Ka158), the organization, expression and transmission to the progency of the inserted foreign DNA were analyzed more precisely.     

Transgenic Tobacco plants with T-DNA Phytohormone synthesis genes

Agrobacterium tumefaciens binary vectors carrying kanamycin resistance gene and either C58 T-DNA gene 4 for cytokinin synthesis or genes 1 and 2 for auxin synthesis were constructed and used for transformation of a short-day tobacco Maryland Mammoth. Kanamycin resistant plants were regenerated from a small fraction of transformed tissue and the presence of T-DNA in their genome was verified by Southern blotting. The level of endogenous cytokinin in plants transgenic for gene 4 and the level of endogenous IAA in those transgenic for genes 1 and 2 increased by more than 100 %. A number of morphological characteristics distinguish them from untransformed controls.     

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.     

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     

Agrobacterium-mediated transformation and regeneration of guayule

In vitro grown shoot tissue of facultative apomictic lines of guayule (Parthenium argentatum Gray), a rubber producing desert shrub, were transformed by Agrobacterium-mediated DNA transfer and regenerated into complete plants. Guayule shoots of lines 11591, UC101 and UC104 were inoculated with A. tumefaciens strains LBA4404 or PC2760 harboring the binary vector pCGN1557. Axillary shoots were regenerated from transformed cells and rooted in vitro in the presence of kanamycin. Genetic transformation in all cases was verified by Southern blot analysis. Transgenic plants were grown to maturity in the greenhouse and, as predicted for apomictic species, all seed produced possessed kanamycin resistance. Because apomicts have limitations for gene transfer by normal sexual crosses, this method offers a new means of transferring genes into this species.Abbreviations BA benzyladenine - EDTA ethylene diamine tetraacetate - kan_R kanamycin resistance - MS salts salts of Murashige and Skoog medium (1962) - NAA naphthalene acetic acid - NPT-II neomycin phosphotransferase - SDS sodium dodecyl sulfate     

Genetic transformation of prickly-pear cactus (Opuntia ficus-indica) by Agrobacterium tumefaciens

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l⁻¹ kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus.     

Agrobacterium-mediated transformation of cultivated tomato with construct carrying the nucleoprotein (N) gene from tomato spotted wilt virus (TSWV)

Significant yield losses in commercial tomato production caused by tomato spotted wilt virus (TSWV) are the reason why we have undertaken studies on resistance to this pathogen. One of the possible sources of resistance can be the incorporation of the nucleoprotein N viral gene by Agrobacterium transformation. The N gene was introduced into three Lycopersicon esculentum forms. Out of the total of 3044 cotyledon explants 14.7% regenerated shoots, but only a few were rooted on medium containing kanamycin. The preliminary analysis indicated that 18 plants are putative transformants.     

A protocol for efficient biolistic transformation of mothbean<Emphasis Type="Italic">Vigna aconitifolia</Emphasis> L. Jacq. Marechal

A protocol for efficient direct gene transfer by using particle gun bombardment was developed for mothbeanVigna aconitifolia L. Jacq. Marechal. Hypocotyl explants from 2 cultivars of mothbean were transformed with 3 plasmids: pBI121, pHS101, and pHS102. Stable transformants were regenerated on MS medium supplemented with benzyladenine, α-naphthaleneacetic acid, and kanamycin. The helium pressure, plasmid type, and cultivar that were used determined the stable transformation frequency. Complete plants were regenerated and transferred to soil. The integration of the stable transgenes and reporter genes in plant genomes was shown by means of PCR amplification of these genes from plant genomic DNA and Southern blot hybridization with gene-specific probes. This method allows high-efficiency production of transgenic plants in mothbean. Suchita Kamble and Hari S. Misra contributed equally to this work.     

Uptake, integration, expression and genetic transmission of a selectable chimaeric gene by plant protoplasts

Summary Genetic transformation of Nicotiana tabacum protoplasts was achieved by incubation of protoplasts with a plasmid DNA-calcium phosphate coprecipitate, followed by fusion of the protoplasts in the presence of polyvinyl alcohol and subsequent exposure to high pH. A derivative of the plasmid pBR322 containing a chimaeric gene, consisting of the nopaline synthase promoter, the coding region of the aminoglycoside phosphotransferase gene of Tn5 and the polyadenylation signal region of the octopine synthase gene, was used for these transformation experiments. This chimaeric gene confers resistance of transformed plant cells to kanamycin. This novel transformation procedure reproducibly yielded transformants at frequencies of approximately 0.01%. Aminoglycoside phosphotransferase II activity was detected in both transformed calli and in regenerated plants. DNA from some of the transformed clones was analyzed by Southern blot hybridization. The input DNA appears to be integrated into high molecular weight cellular DNA. Genetic analysis of one of the kanamycin resistant plants shows that the chimaeric gene is transmitted to the progeny as a single dominant trait in a Mendelian fashion. As a comparison the input DNA was also introduced into tobacco protoplasts using Agrobacterium tumefaciens and Ti-plasmid derived gene vectors.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

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-mediated transformation of pepino and regeneration of transgenic plants

Regeneration of pepino (Solanum muricatum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS (-D-glucuronidase) reporter gene carried on pKIWI110. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation.     

Cre-mediated site-specific gene integration for consistent transgene expression in rice

To minimize expression variability amongst transgenic lines, we have utilized the strategy of Cre/lox-mediated site-specific gene integration. This method allows the precise integration of a transgene in a lox site previously placed in the genome. Using the biolistic method for DNA delivery, we have generated several site-specific integrant lines, derived from three different target lines. About 80% of the selected lines contain precise integration of the gusA reporter gene and fall into two categories: single-copy (SC) lines that contain site-specific integration without additional random integrations, and multicopy (MC) lines that contain random integrations in addition to the site-specific integration. The expression of the gusA gene was studied in callus cells and regenerated plants. The isogenic SC lines displayed significantly lower expression variation, whereas much higher expression variation was observed in MC lines. Furthermore, stable inheritance of the gusA gene was observed in T1 plants derived from a subset of SC lines. This demonstrates that consistent gene expression can be obtained in rice by Cre-mediated site-specific integration.