Highest Transformation Frequency Research Articles

Complete Genome Sequence of Bacillus thuringiensis Mutant Strain BMB171

Bacillus thuringiensis has been widely used as a biopesticide for a long time. Here we report the finished and annotated genome sequence of B. thuringiensis mutant strain BMB171, an acrystalliferous mutant strain with a high transformation frequency obtained and stocked in our laboratory.

Comparative Analysis of Rice Transformation Using Agrobacterium tumefaciens and Rhyzobium leguminosarum

This study was aimed to study the effectiveness of Rhizobium transformation system compared to the most widely used Agrobacterium mediated transformation system on three rice cultivars, Ciherang (Indica), Nipponbare (Japonica), and Rojolele (Javanica). Six day old calli induced from immature embryos were inoculated with Rhizobium leguminosarum bv trifolii ANU845 and Agrobacterium tumefaciens LBA288 that harbored with vector pCAMBIA 5106. This plasmid contained a minimum set of transfer machinery genes and had a gusplus and an hptII gene driven by 35S CaMV promoter in the T-DNA. The results showed that the transformation frequencies (number of PCR positive plants per number of calli inoculated) ranging from 0 to 12.05 % depend on the genotype and transfer agent used. The highest transformation frequency (12.05%) was obtained in Ciherang transformed with R. leguminosarum. Most of the transgenic rice obtained by Rhizobium transformation were normal in morphology and fertile similar to those obtained by Agrobacterium transformation. Integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis in T0 and T1 generations. Key words : Rhizobium leguminosarum, immature embryos, Agrobacterium tumefaciens

Agrobacterium-mediated transformation of protocorm-like bodies in Cattleya

Protocorm-like bodies (PLBs) of Cattleya orchid CM2450 were cocultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harboring genes coding for neomycin phosphotransferase II, hygromycin phosphotransferase, and β-glucuronidase (GUS) or plasmid pBBRacdS harboring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. PLBs were maintained in a liquid New Dogashima (ND) medium and then added to a bacterial suspension culture (OD600 ≈ 0.6) yielding dilution ratios of either 1:10 or 1:40, and these were incubated for either 30 min, 3 h, or 6 h. Hygromycin-resistant secondary PLBs were induced after 4 weeks of culture on an ND medium containing 1.0 mg L−1 naphthaleneacetic acid (NAA), 0.1 mg L−1 benzyladenine (BA), 10 mg L−1 hygromycin, 20 mg L−1 meropenem, 10 g L−1 sucrose, and solidified with 2.5 g L−1 gellan gum. The highest frequency of transformation was obtained when PLBs were inoculated with 1:10 Agrobacterium liquid culture for 3 h. The transformation of hygromycin-resistant plantlets regenerated from different sites of inoculated PLBs was confirmed by histochemical GUS assay, polymerase chain reaction (PCR) analysis, and Southern blot hybridization.

Comparison of transformation frequencies among selected Streptococcus pneumoniae serotypes

Although there are over 90 serotypes of Streptococcus pneumoniae, antimicrobial resistance is predominantly found in a limited number of serotypes/serogroups, namely 6, 9, 14, 19 and 23. There is no compelling mechanism to account for this restriction. We aimed to determine whether serotypes commonly associated with drug resistance have higher transformation frequencies than those that are susceptible to antimicrobial agents. An in vitro investigation of the genetic transformation frequency of drug-resistant serotypes compared with that of susceptible serotypes under the influence of synthetic competence-stimulating peptides was performed. The transforming DNA was genomic DNA carrying a Tn 916-like transposon containing the mefE gene that confers resistance to erythromycin. It was observed that serotypes 6, 9, 14, 19 and 23, which are highly associated with drug resistance, do not exhibit a higher degree of transformation efficiency than other serotypes. These findings suggest that the association of serotype with drug resistance is likely due to prolonged exposure to transforming DNA resulting from longer nasopharyngeal carriage and to a greater selective pressure from antimicrobials, particularly in children. This is the first study to compare the transformation frequencies of pneumococcal clinical isolates using genomic DNA that carries the composite Tn916-like element.

Using high competent shoot apical meristems of cockscomb as explants for studying function of ASYMMETRIC LEAVES2-LIKE11 (ASL11) gene of Arabidopsis

Though shoot apical meristems (SAMs) commonly exhibit low or no competence for transformation, the potent regeneration of this tissue merits further research. Especially, when shoot regeneration is recalcitrant using other tissues as explants, SAM probably is an excellent selection. In cockscomb plants, using SAMs from seedlings obtained from MS medium with 0.5 mg l(-1) 6-BA as explants, high frequency of transformation (approximate 20%) is obtained; whereas control SAMs performed poorly for transformation (approximate 3%). These SAMs are malformed in morphology compared to control SAMs. Further observation found that, in these SAMs, cell proliferation and/or TE formation are seen; which are not found in control SAMs. GUS assays indicated that GUS-positive blue spots at TE zones are obvious; whereas the case was contrary in control SAMs. All these data suggest that cell proliferation and/or TE formation might cause high effective transformation. This transformation system should facilitate the use of this species for studies on gene manipulation and expression. Therefore, we introduced 35S:ASL11-GFP to cockscomb via Agrobacterium tumefaciens. ASYMMETRIC LEAVES2-LIKE11 (ASL11) gene of Arabidopsis is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family, and its function is largely unclear. By confocal laser scanning microscopy, we found that in most over 35S:ASL11-GFP cockscomb plants, ASL11-GFP fusion protein was in discrete nuclear location. These results indicate that the T-DNA contains within the construct inserted into the host chromosomes in an integral form, and also suggest that ASL11 might be a nuclear protein and function as a potential transcription factor. Moreover, SAMs of the over 35S:ASL11-GFP plants show needle-like patterns that lack organ primordial; suggesting ASL11 might be involved in sustaining indeterminate cell fate of SAMs.

Agrobacterium-mediated transformation of shoot apices of Kentucky bluegrass (Poa pratensis L.) and production of transgenic plants carrying a betA gene

Apical meristems of multiple shoots produced from axenic seedlings of Kentucky bluegrass (Poa pratensis L.) were used for Agrobacterium tumefaciens-mediated transformation. Transformation parameters were optimized for concentration of bacterial cells, duration of infection, and vacuum infiltration. The highest transformation frequency (1.42%) was obtained by infection with Agrobacterium suspension of OD600 = 0.6 for 5 min, under a negative pressure of 0.5 × 105 Pa. After co-cultivation, the herbicide-resistant plants were rooted and transplanted into flowerpots. Transgenic plants were confirmed by polymerase chain reaction (PCR) assay and Southern blot analysis. Using this transformation system, the betA gene encoding choline dehydrogenase and mutant als gene encoding the enzyme acetolactate synthase were introduced into three Kentucky bluegrass cultivars.

Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium

A highly efficient Agrobacterium-mediated transformation system for Lilium × formolongi was established by modifying the medium used for inoculation and co-cultivation. Meristematic nodular calli of Lilium were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm harboring an intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase, and neomycin phosphotransferase II genes. The effects of ten different types of media and carbohydrates (sucrose, d-glucose, and l-arabinose) in both inoculation and co-cultivation media were evaluated. Interestingly, a dramatic increase in the frequency of transformation (25.4%) was observed when Murashige and Skoog (MS) medium containing sucrose and lacking KH2PO4, NH4NO3, KNO3, and CaCl2 was used. Hygromycin-resistant transgenic calli were obtained only in medium supplemented with sucrose. The effects of this modified medium were also investigated for Lilium cultivars ‘Acapulco’, ‘Casa Blanca’, and ‘Red Ruby’. The highest frequency of transformation (23.3%) was obtained for cv. Acapulco. Hygromycin-resistant calli were successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were confirmed by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot analyses.

Efficient Agrobacterium-mediated transformation of commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry

Many economically important species of Populus, especially those in sections Aigeiros and Tacamahaca, remain recalcitrant to genetic transformation. In this study, a simple and reliable protocol was developed for the efficient Agrobacterium-mediated transformation of a difficult-to-transform, but commercially viable, hybrid poplar Populus nigra L. x P. maximowiczii A. Henry (NM6). A plant transformation vector designed to express the beta-glucuronidase (GUS) gene was used to detect transformation events at early stages of plant regeneration and to optimize parameters affecting poplar transformation. The use of zeatin riboside in shoot-induction medium, regeneration of shoots via indirect organogenesis, and early selection pressure were the major modifications that drastically improved the efficiency of poplar transformation and minimized the number of untransformed regenerants. Transgenic shoots were routinely obtained 4-10 weeks after co-culture with A. tumefaciens, with a greater than 90% rate of plant recovery. Stable transgene integration, ranging from a single insertion to ten copies per genome, was confirmed by Southern blot analysis. The mean transformation frequency was 36.3% and about two-thirds of the lines had 1-2 transgene copies. Among the explants, petioles and leaves had a higher transformation frequency than did stem segments. Growth characteristics and the morphology of transgenic poplar plants were identical to untransformed controls. These findings will accelerate the development of P. nigra x P. maximowiczii plants with novel traits, and may also be useful to improve transformation procedures for other Populus species.

Genetic transformation of Harpagophytum procumbens by Agrobacterium rhizogenes: iridoid and phenylethanoid glycoside accumulation in hairy root cultures

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types of explants and acetosyringone (AS) (200 μM). The highest frequency of transformation (over 50% of explants forming roots at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal stem explants and A. rhizogenes strain A4. The addition of 200 μM AS to root induction medium was found to enhance hairy root induction but its effect varied depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC–ESI-MS as the major compounds from all analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g−1 dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g−1 dry wt. and 9.97 mg g−1 dry wt., respectively) comparable to those found in root tubers.

Agrobacterium-mediated genetic transformation of Cattleya with an Odontoglossum ringspot virus replicase gene sequence

Protocorm-like bodies (PLBs) of Cattleya orchid CM2450 cultured either under constant illumination with cool-white-fluorescent lamps or in the dark were cocultivated with Agrobacterium tumefaciens strain EHA105 carrying plasmid pSMAHdN627-ORSV harboring genes coding for Odontoglossum ringspot virus (ORSV) replicase and hygromycin phosphotransferase. PLBs were maintained in liquid New Dogashima (ND) medium and then added to a bacterial suspension culture (OD600�0.6) yielding medium dilution ratio of 1 : 10 and incubated for either 30 minutes or 3 h. Hygromycin-resistant secondary PLBs were induced after 4 weeks of culture on 2.5 g l �1 gellan gum-solidified ND medium containing 1 mg l �1 naphthaleneacetic acid (NAA), 0.1 mg l �1 benzyladenine (BA), 10 mg l �1 hygromycin, 20 mg l �1 meropenem, 10 g l �1 sucrose in both light- and dark-cultured PLBs. The number of resistant PLBs generated using dark-cultured PLBs was higher than those cultured under constant illumination. Presence of acetosyringone (AS) in the pre-culture medium was also effective for the transformation. The highest frequency of transformation was obtained when dark-cultured PLBs were pre-cultured with 100 mM AS for 3 days and inoculated with Agrobacterium liquid culture for 3 h. Transformation of hygromycin-resistant plantlets regenerated from different sites of inoculated PLBs was confirmed by Southern blot hybridization. Transcription of ORSV replicase gene in transgenic lines was successfully confirmed by Northern blot hybridization.

Evaluation of properties of several cheese-ripening fungi for potential biotechnological applications

Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast production, and protoplast regeneration abilities. Transformation of these strains with a plasmid containing gene encoding phleomycin resistance showed that they also have a high transformation frequency. In addition, both strains showed low extracellular proteolytic activity. Thus, these strains have all the characteristics to make them suitable for future genetic improvement, recombinant protein production, and other potential biotechnological applications.

Evaluation of properties of several cheese-ripening fungi for potential biotechnological applications

Strains of Penicillium camemberti and Penicillium roqueforti were tested for properties that could be important for future biotechnological applications of these fungi. Penicillium camemberti CECT 2267 and P. roqueforti NRRL 849 showed the most promising performances in terms of growth, protoplast production, and protoplast regeneration abilities. Transformation of these strains with a plasmid containing gene encoding phleomycin resistance showed that they also have a high transformation frequency. In addition, both strains showed low extracellular proteolytic activity. Thus, these strains have all the characteristics to make them suitable for future genetic improvement, recombinant protein production, and other potential biotechnological applications.

High-frequency genetic transformation of Phalaenopsis amabilis orchid using tomato extract-enriched medium for the pre-culture of protocorms

SummaryThe development of an efficient methodology for the genetic transformation of orchids is needed in order to support thegenetic engineering of orchids. It is therefore important to identify those factors affecting the transformation process.Previously, we reported a convenient method for the transformation of Phalaenopsis amabilis using Agrobacterium tumefaciens, in which intact protocorms were used. We also found that embryos cultured on a medium containing tomato extract grew more rapidly than those cultured on a medium with coconut water. When we used protocorms grown on a medium containing tomato extract, we obtained regenerated shoots that had been transformed with a kanamycin resistance gene at relatively high frequencies (7 – 17%). These results suggest that the rate of growth of pre-cultured protocorms may be important for the successful regeneration of transformed shoots. We also obtained regenerated shoots that had been transformed with the green fluorescent protein (GFP) gene at a high frequency (10 – 14%). Both the presence and expression of these transgenes were confirmed in transformed plants by molecular analyses and by the detection of green fluorescence following excitation with blue light.

Cognitive Impairments in Progression of Parkinson’s Disease

A total of 88 patients with progression of Parkinson's disease (PD) were studied. Cognitive impairments (CI) in PD were in most cases progressive in nature, predominantly because of increases in the severity of dysregulatory and neurodynamic disorders, impairments to visuospatial functions, and, in some cases, deficits in nominative speech function. A high frequency of transformation of moderate cognitive impairments to dementia was demonstrated over periods of 2-5 years. Predictors of the progression of CI in PD were identified: elderly age, later onset of disease, and the severity of PD. The greatest rate of progression of CI was seen in patients with initially more severe impairments of regulatory and visuospatial functions.

The RBE of 3.4 MeV -particles and 0.565 MeV neutrons relative to 60Co -rays for neoplastic transformation of human hybrid cells and the impact of culture conditions

The neoplastic transformation of human hybrid CGL1 cells is affected by perturbations from external influences such as serum batch and concentration, the number of medium changes during the 21-day expression period and cell seeding density. Nevertheless, for doses up to 1.5 Gy, published transformation frequencies for low linear energy transfer (LET) radiations (gamma-rays, MeV electrons or photons) are in good agreement, whereas for higher doses larger variations are reported. The (60)Co gamma-ray data here for doses up to 1.5 Gy, using a low-yield serum batch and only one medium change, are in agreement with published frequencies of neoplastic transformation of human hybrid cells. For 3.4 MeV alpha-particles (LET = 124 keV/mum) and 0.565 MeV monoenergetic neutrons relative to low doses of (60)Co gamma-rays, a maximum relative biological effectiveness (RBE(M)) of 2.8 +/- 0.2 and 1.5 +/- 0.2, respectively, was calculated. Surprisingly, at higher doses of (60)Co gamma-rays lower frequencies of neoplastic transformation were observed. This non-monotonic dose relationship for neoplastic transformation by (60)Co gamma-rays is likely due to the lack of a G2/M arrest observed at low doses resulting in higher transformation frequencies per dose, whereas the lower frequencies per dose observed for higher doses are likely related to the induction of a G2/M arrest.

Transgenic ramie [Boehmeria nivea (L.) Gaud.]: factors affecting the efficiency of Agrobacterium tumefaciens-mediated transformation and regeneration

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for ramie [Boehmeria nivea (L.) Gaud.] based on the examinations of several factors affecting plant transformation efficiency. The effects of Agrobacterium cell density, acetosyringone, co-cultivation temperature, co-cultivation duration, co-cultivation photoperiod and pH on stable transformation were evaluated. Agrobacterium at a concentration of OD = 0.5-0.8 improved the efficiency of transformation. Concentration of acetosyringone at 50 mg/L during co-cultivation significantly increased transformation efficiency. Co-cultivation at 20 degrees C, in comparison to 15, 25 and 28 degrees C, consistently resulted in higher transformation frequencies. A relatively short co-cultivation duration (3 days) was optimal for ramie transformation. Co-cultivation medium at pH 5.9 and co-cultivation in darkness both improved the transformation efficiencies of ramie. An overall scheme for producing transgenic ramie is presented, through which an average transformation rate from 10.5 to 24.7% in five ramie varieties was obtained. Stable expression and integration of the transgenes were confirmed by histochemical GUS assay, kanamycin painting assay, PCR and Southern blotting. This optimized transformation system should be employed for efficient Agrobacterium-mediated transformation of ramie.

RETRACTED ARTICLE: Transgenic ramie [Boehmeria nivea (L.) Gaud.]: factors affecting the efficiency of Agrobacterium tumefaciens-mediated transformation and regeneration

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for ramie [Boehmeria nivea (L.) Gaud.] based on the examinations of several factors affecting plant transformation efficiency. The effects of Agrobacterium cell density, acetosyringone, co-cultivation temperature, co-cultivation duration, co-cultivation photoperiod and pH on stable transformation were evaluated. Agrobacterium at a concentration of OD = 0.5–0.8 improved the efficiency of transformation. Concentration of acetosyringone at 50 mg/L during co-cultivation significantly increased transformation efficiency. Co-cultivation at 20°C, in comparison to 15, 25 and 28°C, consistently resulted in higher transformation frequencies. A relatively short co-cultivation duration (3 days) was optimal for ramie transformation. Co-cultivation medium at pH 5.9 and co-cultivation in darkness both improved the transformation efficiencies of ramie. An overall scheme for producing transgenic ramie is presented, through which an average transformation rate from 10.5 to 24.7% in five ramie varieties was obtained. Stable expression and integration of the transgenes were confirmed by histochemical GUS assay, kanamycin painting assay, PCR and Southern blotting. This optimized transformation system should be employed for efficient Agrobacterium-mediated transformation of ramie.

The role of single subunits of the DNA transport machinery of Thermus thermophilus HB27 in DNA binding and transport

Thermus thermophilus HB27 is well known for its extraordinary trait of high frequencies of natural transformation, which is considered a major mechanism of horizontal gene transfer. We show that the DNA translocator of T. thermophilus binds and transports DNA from members of all three domains. These results, together with the data obtained from genome comparisons, suggest that the DNA translocator of T. thermophilus has a major impact in adaptation of Thermus to thermal stress conditions and interdomain DNA transfer in extreme hot environments. DNA transport in T. thermophilus is mediated by a macromolecular transport machinery that consists of at least 16 subunits and spans the cytoplasmic membrane and the entire cell periphery. Here, we have addressed the role of single subunits in DNA binding and transport. PilQ is involved in DNA binding, ComEA, PilF and PilA4 are involved in transport of DNA through the outer membrane and PilM, PilN, PilO, PilA1-3, PilC and ComEC are essential for the transport of DNA through the thick cell wall layers and/or through the inner membrane. These data are discussed in the light of the subcellular localization of the proteins. A topological model for DNA transport across the cell wall is presented.

Agrobacterium-mediated genetic transformation of mint with E. coli glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l−1 BAP, 0.2 mg l−1 NAA, 50 mg l−1 kanamycin and 125 mg l−1 cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l−1 NAA and 50 mg l−1 kanamycin. The presence and expression of transgenes in transgenics (T0) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.

Agrobacterium rhizogenes-mediated Transformation and Regeneration of the Apocynum venetum

A system for the Agrobacterium rhizogenes-mediated transformation and plant regeneration of A. venetum has been developed. The highest transformation frequency was 100%, achieved using strain LBA9402 with root explants. The highest density of hairy roots reached 22 when root explants transformed by R1000 cultured in the dark. Adventitious shoots were obtained from profusely branched, fast-growing (type PBF) hairy roots, and the adventitious shoot induction frequency was 20%. Regenerated shoots rooted easily on hormone-free ½ MS solid medium in 2 weeks. Approximately 1/3 regenerated plants derived from hairy roots exhibited prolific roots with shortened internodes, whereas other regenerated plants showed another phenotype: long internodes, strong stems, and fleshy blades. However, all regenerated plants displayed a relatively fast development procedure and stronger than the aseptic seedlings. Polymerase chain reaction (PCR) analyses confirmed that the hairy root lines and regenerated plants were induced by A. rhizogenes.