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 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     

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     

A combined use of microprojectile bombardment and DNA imbibition enhances transformation frequency of canola (Brassica napus L.)

Efforts to increase the frequency of recovered homozygous transgenic B. napus plants from direct DNA transformation treatments led to the development of a method of combined microprojectile bombardment and desiccation/DNA imbibition. The combined method was compared to individual treatments in two experiments utilizing microspore-derived embryo hyocotyls as targets for the -glucuronidase (GUS) and NPT II genes. Both the transient gene expression of -GUS and the stable transformation by NPT II demonstrated that the combined use of microprojectile bombardment and desiccation/DNA imbibition yielded more transgenic plants (at least three-times more) than either individual transformation protocol. In a histochemical analysis for -GUS activity, an average of 37% of the hypocotyls receiving the combined treatment displayed a positive response, whereas only 8% of the hypocotyls showed a positive response following microprojectile bombardment alone. The hypocotyls obtained by the joint treatment also showed more multisite expression of the -GUS gene per hypocotyl than those treated only with microprojectile bombardment. Southern analysis of NPT II gene integration into subsequently-derived secondary embryos indicated that the transformation efficiency of the combined treatment was 2% in comparison to 0.6% for that of the singular microprojectile bombardment. The number of inserts integrating per transformation event appears to be independent of the transformation methods. Neither of the marker genes was expressed in hypocotyls treated only with desiccation/DNA imbibition. Utilization of hypocotyl regeneration from microspore-derived embryos via a secondary embryogenesis system provided a reliable method for producing transgenic plants. The combined use of microprojectile bombardment and desiccation/DNA imbibition proved to be an efficient approach to obtain homozygous transgenic canola plants.     

Mitochondrial DMA variation in plants regenerated from embryogenic callus cultures of CMS triticale

The mitochondrial DNA (mtDNA) organization of primary hexaploid cytoplasmic male-sterile (CMS) triticale regenerants containing Triticum timopheevi cytoplasm was analysed by hybridization experiments and compared with the mitochondrial genome organization of the corresponding regenerants with maintainer cytoplasm. Callus cultures had been derived from immature embryos, and 623 triticale plants were regenerated via somatic embryogenesis after three to four subcultures. The chondriome of 159 regenerants was investigated with regard to somaclonal variation. Six different mitochondrial gene probes and four different restriction enzymes were used for Southern blot analyses by the non-radioactive digoxigenin labeling technique. Alloplasmic regenerants showed a gain or loss of hybridization signals up to a high percentage, while euplasmic ones revealed only minor variability with respect to band stoichiometries. In 24 cases rearrangements in the mtDNA were proved. We suppose that recombination processes and selective amplification events are responsible for these findings.     

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.     

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 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.     

Efficient plant regeneration through somatic embryogenesis from callus induced on mature rice embryos (Oryza sativa L.)

To obtain a reproducible efficient procedure for regeneration of rice plants through somatic embryogenesis from callus four published methods of callus induction and regeneration were compared. Callus was initiated from mature embryos of the Japonica cultivar Taipei 309 of rice (Oryza sativa L.). The number, mass and morphology of the callus formed on the scutellum were dependent on the medium used. A limited humidity and an optimal aeration of the culture vessels enhanced the frequency of embryogenesis and plant regeneration. A method described by Poonsapaya et al. (1989) was found to be the most efficient and was slightly modified. As a result 98% of the T309 embryos formed callus, of which 63% regenerated into plants. Each callus yielded an average of 6 plants. Plant morphology, fertility and seed set of the regenerants were found to be normal.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - IAA 3-indole-acetic acid - BA 6-benzyladeninepurine - S.E.M. standard error of mean     

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.     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

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.     

Regeneration Capacity of Calli Derived from Immature Embryos in Spring Barley Cultivars

Callogenesis, somatic embryogenesis and regeneration capacity in twenty-three agronomically important spring barley (Hordeum vulgare L.) cultivars on induction media with 2,4-dichlorophenoxyacetic acid (2,4-D) or 3,6-dichloro-o-anisic acid (dicamba) and on modified regeneration media were studied. The frequency of zygotic embryos exhibiting callogenesis varied from 88 to 100 % according to genotype. Dicamba was more suitable for somatic embryogenesis induction and exhibited a higher frequency of regenerants than did 2,4-D. Green regenerants were obtained in all cultivars, and there were no albino plants. Except for cv. Victor all cultivars used in the experiment showed lower regeneration capacity as compared to the model cv. Golden Promise. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variability in regenerated plants of Ungernia victoris

To determine the suitability of micropropagation techniques developed for conserving rare medicinal herb Ungernia victoris we estimated the genetic fidelity of plants produced through direct regeneration from the bulb scale segments and organogenesis from long-term callus culture. Average value of the Jaccard’s distances between explant-derived regenerants and maternal plants calculated from RAPD data was 0.5 %, while that of estimated between callus-derived regenerants and maternal cell line was 4.2 %; average distances between the objects among the explant-derived and callus-derived regenerants were 0.7 % and 2.5 %, respectively. The data obtained suggest that conditions for in vitro culture applied in this work provide relatively high genetic stability of the species upon the direct regeneration in vitro and regeneration from the long-term cultured callus.     

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.     

In Vitro Micropropagation of Freesia hybrida and the Assessment of Genetic and Epigenetic Stability in Regenerated Plantlets

Freesia hybrida is an important worldwide cut flower, especially in America and Europe. For efficient regeneration of this flower from young inflorescence and rachillae in tetraploid, we developed a simple in vitro micropropagation protocol. Explants of Freesia hybrida can regenerate plantlets through somatic embryogenesis via two kinds of pathways, that is, directly from the epidermal cells or indirectly from an embryonic callus, depending on the exogenous plant growth regulators (PGRs) used in the culture media. In direct embryogenesis, when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 11.43 μM indole acetic acid (IAA) and 4.44 μM 6-benzylaminopurine (6-BA), the induction rate was 84% for young inflorescence and 100% for rachillae. After the multishoots were subcultured on the rooting MS medium containing 1.08 μM α-naphthalene acetic acid (NAA), the rooting rate was close to 100%. In indirect embryogenesis, embryonic calluses were formed when the culture medium contained 22.22 μM 6-BA and 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), and the induction rate was 92.4% for young inflorescence and 100% for rachillae. After the embryonic calluses were transferred to the medium supplemented with 11.43 μM IAA and 13.33 μM 6-BA, they could develop into plantlets with roots. In assessing the two regeneration pathways in terms of genetic and epigenetic fidelity of the regenerants, two kinds of molecular markers [amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP)] were employed. The AFLP analysis used 20 primer pairs that yielded 916 scorable bands among the donor plant and 11 regenerants from direct embryogenesis, of which 8 (0.87%) were polymorphic. The regenerants from indirect embryogenesis had 1075 clear bands of which 3 (0.27%) were polymorphic scorable bands from 18 primer pairs. Moreover, the variant band patterns included two types, that is, loss-of-original and gain-of-novel bands. MSAP analysis revealed that tissue culturing of the flower induced DNA cytosine methylation alterations in both CG and CNG levels and patterns compared with the donor plant. The variation rate was 1.1 and 1.3% for the direct and indirect embryogenesis pathways, respectively. The findings show that tissue culture of flowering plants is a form of stress which can induce some heritable epigenetic variations and should be considered in future long-term genotype preservation programs of Freesia hybrida.     

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.     

Transformation of barley by microinjection into isolated zygote protoplasts

Barley zygote protoplasts were mechanically isolated, embedded in agarose droplets, and microinjected with a rice actin promoter Act1–gusA-nos gene construct. On average 62% of the cells survived the injection and of these 55% continued development into embryo-like structures and eventually to plants. PCR screening for the presence of a 307-bp fragment in the middle of the gusA gene showed that on average 21% of the derived structures contained this fragment. However, among the hundreds of injected zygotes, derived structures and regenerants we only found significant GUS expression in two cases (embryo-like structures nine days after injection). Two lines of green plants, derived from zygotes microinjected with linearized plasmid (line A147-1) or an isolated Act1–gusA-nos gene cassette (line A166-h) proved to be transgenic. Line A147-1 appeared to contain a single and intact copy of the expression cassette but a PCR based progeny analysis indicated the presence of additional shorter fragments of the cassette. Line A166-h appeared to contain a single fragment of the gusA gene that was transferred to the progeny as a single Mendelian trait. One additional fragment of the gusA gene was identified in this line. The present data show that transformation of barley by microinjection of DNA into isolated zygotes is feasible but also that gene expression rarely is achieved, possibly due to degradation of the introduced DNA.     

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