Agrobacterium tumefaciens－mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer

Immature embryos of rice varieties "Xiushuill" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant calli were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56% approximately 72% measured as numbers of Geneticin (G418)-resistant calli produced and 36% approximately 60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38% approximately 61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16% approximately 75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.     

Transgenic Pinus radiata from Agrobacterium tumefaciens–mediated transformation of cotyledons

A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed. Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere. Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos. The main drawback to the method is the small number of genotypes that are amenable to transformation and regeneration. Since more than 80% of genotypes of radiata pine can be regenerated using cotyledons from mature seed, cotyledon explants were cocultivated with A. tumefaciens strain AGL1 containing a plasmid coding for the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (GUS) gene (uidA). Transformed shoots were selected using either geneticin or kanamycin. Critical factors for successful transformation were survival of the cotyledons after cocultivation and selection parameters. Of the 105 putative transformants that were recovered from selection media, 70% were positive for integration of the nptII gene when analysed by PCR. GUS histochemical assay for uidA expression was unreliable because of reaction inhibition by unidentified compounds in the pine needles. Further, only 4 of the 26 independent transformants characterised by PCR and Southern analysis contained an intact copy of both genes. The remaining 22 transformants appeared to have a truncated or rearranged copy of the T-DNA. It is possible that the truncation/rearrangements are due to the Cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the T-DNA junction sites and sequencing of the introduced DNA will help elucidate the nature of T-DNA insertion so that genetic modification of radiata pine can be targeted effectively.Communicated by P. Debergh     

Agrobacterium tumefaciens–mediatated transformation of shoot-buds of chicory

Vegetative propagation of chicory via axillary shoot proliferation is one of the best ways to obtain an offspring with complete genetic stability. These shoot buds were used in transformation experiments using Agrobacterium tumefaciens strains containing binary plasmids carrying the neomycin phosphotransferase gene (nptII) and the β-glucuronidase gene (uidA). Selection was carried out on basal medium containing 100 mg l⁻¹ kanamycin. Transformed plantlets were recovered at a frequency of about 10% within four weeks after co-cultivation. The presence of the uidA gene was demonstrated by transient gene expression experiments using the histochemical GUS staining procedure. Evidence for stable transformation was shown by subculturing leaf discs on kanamycin selection medium, and Southern blot analysis confirmed the integration of the nptII and the uidA genes in the plant genome. Analysis of the progenies showed that kanamycin resistance was inherited as a single dominant trait. This method for obtaining transgenic chicory plants represents an alternative to leaf disc transformation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Agrobacterium tumefaciens–mediated genetic transformation and overexpression of the flavonoid 3′5′-hydroxylase gene increases the flavonoid content of the transgenic Aconitum carmichaelii Debx. plant

In Vitro Cellular & Developmental Biology - Plant - Aconitum carmichaelii Debx. is a medicinal plant that contains a variety of valuable medicinal substances, including flavonoids, alkaloids,...     

Agrobacterium tumefaciens– mediated transformation of embryogenic avocado cultures and regeneration of somatic embryos

Embryogenic avocado cultures were genetically transformed with the uidA (GUS) and nptII genes, and transformed somatic embryos were recovered from these cultures. Embryogenic avocado cultures derived from zygotic embryos of `Thomas' and consisting of proembryonic masses were gently separated and co-cultivated with disarmed, acetosyringone-activated Agrobacterium tumefaciens strain A208, which contained the cointegrative vector pTiT37-ASE::pMON9749 (9749 ASE). Kanamycin-resistant embryogenic suspension cultures were selected in two steps: (1) initial selection in maintenance medium, consisting of MS basal medium, supplemented with 0.1 mg l^–1 picloram and 50 mg l^–1 kanamycin sulfate for 2–4 months and (2) subsequent selection in maintenance medium with 100 mg/ml kanamycin sulfate for 2 months in order to eliminate chimeras. Somatic embryo maturation was initiated by subculture onto semisolid maturation medium (without picloram) followed by transfer to maturation medium with 100 mg l^–1 kanamycin sulfate. Genetic transformation of embryogenic cultures and somatic embryos was confirmed by the X-gluc reaction, and integration of nptII and uidA into the avocado genome was confirmed by PCR and Southern hybridization, respectively. Received: 2 June 1997 / Revision received: 26 September 1997 / Accepted: 11 October 1997     

Agrobacterium‐mediated transformation of lavandin (Lavandula x intermedia Emeric ex Loiseleur)

Lavandin (Lavandula x Emeric ex Loiseleur) is an aromatic plant, the essential oil of which is widely used in the perfume, cosmetic, flavouring and pharmaceutical industries. The qualitative or quantitative modification of its terpenescontaining essential oil by genetic engineering could have important scientific and commercial applications. In this study, we report the first Agrobacterium tumefaciensmediated gene transfer into lavandin. The transformation protocol was optimized by lengthening precultivation and cocultivation periods and by testing five different bacterial strains. We obtained transformed callus lines at a frequency of 40–70 with strains AGL1/GI, EHA105/GI and C58/GI. Transgenic shoots were regenerated from these kanamycin resistant calli and rooted on selective medium with 150mg l_-1 kanamycin. The final percentage of transgenic plants obtained varied from 3 to 9, according to the strain used, within 6 months of culture. The presence of the introduced glucuronidase and neomycin phosphotransferase II genes was shown both by PCR and Southern blot analysis. Transgene expression was investigated using histoenzymatic glucuronidase assays, leaf callus assays and RTPCR. Results showed that both glucuronidase and neomycin phosphotransferase II genes were expressed at a high level in at least 41 of the transgenic plants regenerated. This efficient transformation strategy could be used to modify some genetic traits of lavandin (flower colour, pathogens resistance) and to study the biosynthesis of the major monoterpene components of its essential oil (linalool, linalyl acetate, camphor and 1,8cineole).     

Adventitious bud regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Eucalyptus urophylla × E. tereticornis interspecific hybrid

In Vitro Cellular & Developmental Biology - Plant - A high-efficiency regeneration and genetic transformation system is indispensable for generating desirable traits in important trees such as...     

Targeting of extracellular protein–protein interactions with macrocyclic peptides

Targeting of extracellular protein–protein interactions (PPI) is emerging as a major application for de novo discovered macrocyclic peptides. Modern discovery platforms can routinely identify macrocyclic peptide ligands capable of highly selective modulation of extracellular signaling pathways; amenability to chemical synthesis and natural modularity of peptides additionally provides an avenue for their further structural elaboration, while the challenge of cell internalization can be minimized. Here, we discuss the recent progress in targeting extracellular PPIs with macrocyclic peptides by focusing on a number of recent case studies. We analyze the scope and potential limitations of the discovery systems in identifying functional macrocyclic ligands. We also highlight the recent technical advancements allowing for a more streamlined discovery pipeline and our brief perspective in this field.     

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B₅ basal medium supplemented with 5×10⁻⁶ M BAP, 2.5×10⁻⁶ M each of 2,4-D and NAA and 50 mg l⁻¹ kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes. Transformed calli were found resistant to kanamycin up to 1000 mg^.l⁻¹. Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B₅ or B₅ containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B₅ medium containing 6-benzylaminopurine (5×10⁻⁷ M) and 75 mg l⁻¹ kanamycin. The putative transformed shoots were rooted on B₅+indole-3-butyric acid (5×10⁻⁶ M) within 10–14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T₀ seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T₀ plants and their seeds.     

Mapping the protein–protein and genetic interactions of cancer to guide precision medicine

Massive efforts to sequence cancer genomes have compiled an impressive catalogue of cancer mutations, revealing the recurrent exploitation of a handful of ‘hallmark cancer pathways’. However, unraveling how sets of mutated proteins in these and other pathways hijack pro-proliferative signaling networks and dictate therapeutic responsiveness remains challenging. Here, we show that cancer driver protein–protein interactions are enriched for additional cancer drivers, highlighting the power of physical interaction maps to explain known, as well as uncover new, disease-promoting pathway interrelationships. We hypothesize that by systematically mapping the protein–protein and genetic interactions in cancer—thereby creating Cancer Cell Maps—we will create resources against which to contextualize a patient’s mutations into perturbed pathways/complexes and thereby specify a matching targeted therapeutic cocktail.     

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.     

Agrobacterium-mediated transformation of chickpea with α-amylase inhibitor gene for insect resistance

Chickpea is the world’s third most important pulse crop and India produces 75% of the world’s supply. Chickpea seeds are attacked byCallosobruchus maculatus andC. chinensis which cause extensive damage. The α-amylase inhibitor gene isolated fromPhaseolus vulgaris seeds was introduced into chickpea cultivar K850 throughAgrobacterium- mediated transformation. A total of 288 kanamycin resistant plants were regenerated. Only 0.3% of these were true transformants. Polymerase chain reaction (PCR) analysis and Southern hybridization confirmed the presence of 4.9 kb α-amylase inhibitor gene in the transformed plants. Western blot confirmed the presence of α-amylase inhibitor protein. The results of bioassay study revealed a significant reduction in the survival rate of bruchid weevilC. maculatus reared on transgenic chickpea seeds. All the transgenic plants exhibited a segregation ratio of 3:1.     

Improved methods in Agrobacterium–mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols

A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 μM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.     

181 Integrative analysis of gene expression and protein–protein interaction networks in Glioblastoma

Glioblastoma multiforme (GBM) is the most malignant of all the brain tumors with very low median survival time of one year, as per Central Brain Tumor Registry of the USA, 2001. Efforts are ongoing to understand this disease pathogenesis in complete details. Global gene expression changes in GBM pathogenesis have been studied by several groups using microarray technology (e.g. Carro et al., 2010). One of the many approaches to ‘understand the control mechanisms underlying the observed changes in the activity of a biological process’ (Cline et al., 2007) is integration of gene expression and protein–protein interactions (PPI) datasets. Among several examples, aberrant activation of Wnt/β-catenin signaling pathway as well as sonic hedgehog (SHH) signaling pathway is reported in GBMs (Klaus & Birchmeier, 2008). Further, these two pathways are also involved in proliferation and clonogenicity of glioma cancer stem cells (Li et al., 2009), which are thought to play a role in glioma initiation, proliferation, and invasion, and are one of the important points of intervention. Hedgehog–Gli1 signaling is also found to regulate the expression of stemness genes. In this paper, analyses of the relationship between the significant differential expression of these and other genes and the connectivity as well as topological features of a PPI network would be discussed. This way, genes potentially overlooked when relying solely on expression profiles may be identified which can be biologically relevant as possible drug target/s or disease biomarker/s.     

SHARP: genome-scale identification of gene–protein–reaction associations in cyanobacteria

Genome scale metabolic model provides an overview of an organism’s metabolic capability. These genome-specific metabolic reconstructions are based on identification of gene to protein to reaction (GPR) associations and, in turn, on homology with annotated genes from other organisms. Cyanobacteria are photosynthetic prokaryotes which have diverged appreciably from their nonphotosynthetic counterparts. They also show significant evolutionary divergence from plants, which are well studied for their photosynthetic apparatus. We argue that context-specific sequence and domain similarity can add to the repertoire of the GPR associations and significantly expand our view of the metabolic capability of cyanobacteria. We took an approach that combines the results of context-specific sequence-to-sequence similarity search with those of sequence-to-profile searches. We employ PSI-BLAST for the former, and CDD, Pfam, and COG for the latter. An optimization algorithm was devised to arrive at a weighting scheme to combine the different evidences with KEGG-annotated GPRs as training data. We present the algorithm in the form of software “Systematic, Homology-based Automated Re-annotation for Prokaryotes (SHARP).” We predicted 3,781 new GPR associations for the 10 prokaryotes considered of which eight are cyanobacteria species. These new GPR associations fall in several metabolic pathways and were used to annotate 7,718 gaps in the metabolic network. These new annotations led to discovery of several pathways that may be active and thereby providing new directions for metabolic engineering of these species for production of useful products. Metabolic model developed on such a reconstructed network is likely to give better phenotypic predictions.     

Agrobacterium－mediated transformation and assessment of factors influencing transgene expression in loblolly pine（Pinus taeda L.）

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.     

Agrobacterium tumefaciens– mediated transformation of soybean [Glycine max (L.) Merrill.] using immature zygotic cotyledon explants

Agrobacterium tumefaciens -mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus. Received: 6 December 1999 / Revised: 11 February 2000 / Accepted: 14 March 2000