Highest Transformation Frequency Research Articles

High-efficient transgenic hairy roots induction in chicory: re-dawn of a traditional herb

Plant roots can be manipulated by Agrobacterium rhizogenes to stimulate the production of heterologous proteins for pharmaceutical applications as green cell-factories. During the present study, four bacterial strains (A4, ATCC15834, ATCC11325 and A13) in combination with three co-cultivation media (MS, B5, LS) were examined to establish an efficient and reliable transformation system for chicory (Cichorium intybus L.) using A. rhizogenes. The maximum chicory hairy roots induction was achieved using A13 strain. The observation confirmed that MS medium was more effective on hairy root growth. Dried biomass accumulation of hairy roots infected by A13 strain was 1.10 g l-1 in MS medium which was significantly higher than those grown in LS and B5 medium (0.88 and 0.72 g l-1, respectively). Beta-glucuronidase (GUS) gene was introduced by A13 strain carrying the pCAMBIA1304 binary vector. The results showed that the highest frequency of transformation (63.15 %) was achieved using A13 strain and MS cultivation medium. Detection of GUS and hptII genes by PCR and GUS histochemical localization confirmed the integrative transformation in hairy roots. In conclusion, the whole process was successfully optimized as a pre-step to manipulate the chicory hairy root cells to improve the unique potential of secondary metabolite production.

A non-antibiotic selection strategy uses the phosphomannose-isomerase (PMI) gene and green fluorescent protein (GFP) gene for Agrobacterium-mediated transformation of Prunus domestica L. leaf explants

We developed an efficient system for agrobacterial transformation of plum (Prunus domestica L.) leaf explants using the PMI/mannose and GFP selection system. The cultivar ‘Startovaya’ was transformed using Agrobacterium tumefaciens strain CBE21 carrying the vector pNOV35SGFP. Leaf explants were placed onto a nutrient medium containing various concentrations and combinations of mannose and sucrose to develop an efficient selection system. Nine independent transgenic lines of plum plants were obtained on a regeneration medium containing 20 g/L sucrose and 15 g/L mannose. The highest transformation frequency (1.40 %) was produced using a delayed selection strategy. Starting from the 1st days after transformation and ending by regeneration of shoots from the transgenic callus, selection of transgenic cells was monitored by GFP fluorescence that allowed avoiding formation of escapes. Integration of the manA and gfp transgenes was confirmed by PCR and Southern blotting. The described transformation protocol using a positive PMI/mannose system is an alternative selection system for production of transgenic plum plants without genes of antibiotic and herbicide resistance, and the use of leaf explants enables retention of cultivar traits of plum plants.

Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation.

While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

Assessment of Balance Disorders in Patients after Ankle Fractures

<p><strong>Purpose</strong>: The objective of the study is to evaluate the balance disorders in patients with surgical treatment of ankle fractures with the use of Sunlight Tetrax. <strong>Methods</strong>:The subjects in the study were 20 patients with ankle fractures treated surgically（after six months of the procedure）and the control group comprised 21 healthy subjects. Two groups compare the general stability index (ST), the high frequency of Fourier transformation (F5-F6),the body weight distribution index (WDI) ,synchronicity of foot under natural standing on solid surface with eyes opened and natural standing on solid surface with eyes closed and the fall index (FI). <strong>Results</strong>: The fall index (FI) and the stability index (ST) under natural standing on solid surface with eyes opened of two groups were statistically significant differences. The balance of patients with ankle fracture after 6 months with surgical procedure is significantly poorer than healthy subjects. </p>

The Effect of Exercise on Balance Function of Patients with Type 2 Diabetes

<p><strong>Purpose: </strong>The objective of the study is to evaluate the effects of exercise on balance function with people who have Type 2 diabetes. <strong>Methods: </strong>The subjects in the study were 26 patients with Type 2 diabetes exercise regularly and 27 patients with Type 2 diabetes who do not exercise. The two groups were compared based on the following: the fall index (FI), the low frequency of Fourier transformation (F1, F2) under natural standing on solid surface with eyes opened,the frequency of Fourier transformation (F1, F2, F5, F6)under Standing on the soft mat with eyes opened（PO），the high frequency of Fourier transformation (F5, F6) under natural standing on solid surface with eyes closed (NC)，the frequency of Fourier transformation (F3, F4)under natural standing on solid surface with eyes closed and head turn to right(HR). <strong>Results</strong>: The fall index (FI), the frequency of Fourier transformation (F1, F5, F6)under Standing on the soft mat with eyes opened（PO）,the high frequency of Fourier transformation（F6) under natural standing on solid surface with eyes closed(NC) were statistically different for the two groups. The other parameters have no significant differences. The balance function of patients who exercise regularly were better than those who don’t exercise.</p>

The Effect of Exercise on Balance Function of Patients with Type 2 Diabetes

Purpose: The objective of the study is to evaluate the effects of exercise on balance function with people who have Type 2 diabetes. Methods: The subjects in the study were 26 patients with Type 2 diabetes exercise regularly and 27 patients with Type 2 diabetes who do not exercise. The two groups were compared based on the following: the fall index (FI), the low frequency of Fourier transformation (F1, F2) under natural standing on solid surface with eyes opened,the frequency of Fourier transformation (F1, F2, F5, F6)under Standing on the soft mat with eyes opened（PO），the high frequency of Fourier transformation (F5, F6) under natural standing on solid surface with eyes closed (NC)，the frequency of Fourier transformation (F3, F4)under natural standing on solid surface with eyes closed and head turn to right(HR). Results : The fall index (FI), the frequency of Fourier transformation (F1, F5, F6)under Standing on the soft mat with eyes opened（PO）,the high frequency of Fourier transformation（F6) under natural standing on solid surface with eyes closed(NC) were statistically different for the two groups. The other parameters have no significant differences. The balance function of patients who exercise regularly were better than those who don’t exercise.

An efficient shoot regeneration system andAgrobacterium-mediated transformation withcodAgene in a doubled haploid line of broccoli

Broccoli is an important vegetable crop belonging to the genus of Brassica. However, it is often affected by biotic and abiotic stresses that result in large losses in yield and quality. Genetic manipulation has opened the opportunity for germplasm improvement of broccoli. In this study, an efficient shoot regeneration and Agrobacterium-mediated transformation system was established. The optimum medium for shoot induction was selected for three types of explant including hypocotyl, petiole, and peduncle, and up to 90% regeneration frequency was obtained. The transformation procedure was developed with the EHA105 strain, harboring the PUC19 vector, along with the target gene of codA and a hygromycin-resistance gene. Several factors were optimized, including hygromycin concentration for selection and the Agrobacterium density yielding the highest transformation frequency. Among the three types of explants, peduncle explants showed the highest response to this procedure, and the highest frequency of transformation (3.4%) was obtained depending on the analysis of polymerase chain reaction and Southern blot. In conclusion, the present study introduced a reliable system for plant regeneration and genetic transformation with three types of explants for a DH line of broccoli, and peduncle explant resulted in the highest transformation frequency.

Efficient genetic transformation and regeneration system from hairy root of Origanum vulgare.

Origanum vulgare L is commonly known as a wild marjoram and winter sweet which has been used in the traditional medicine due to its therapeutic effects as stimulant, anticancer, antioxidant, antibacterial, anti-inflammatory and many other diseases. A reliable gene transfer system via Agrobacterium rhizogenes and plant regeneration via hairy roots was established in O. vulgare for the first time. The frequency of induced hairy roots was different by modification of the co-cultivation medium elements after infection by Agrobacterium rhizogenes strains K599 and ATCC15834. High transformation frequency (91.3%) was achieved by co-cultivation of explants with A. rhizogenes on modified (MS) medium. The frequency of calli induction with an 81.5% was achieved from hairy roots on MS medium with 0.25mg/L(-1) 2,4-D. For shoot induction, initiated calli was transferred into a medium containing various concentrations of BA (0.1, 0.25, 0.5, 0.75 and 1mg/L(-1)). The frequency of shoot generation (85.18%) was achieved in medium fortified with 0.25mg/L(-1) of BA. Shoots were placed on MS medium with 0.25mg/l IBA for root induction. Roots appeared and induction rate was achieved after 15days.

Optimisation of a somatic embryogenesis and transformation protocol for farmer-preferred cassava cultivars in Kenya

Cassava (Manihot esculenta Crantz) is a major food crop in developing countries, and holds potential for industrial use. It is, however, affected by various biotic and abiotic stresses that greatly affect its production. The existing regeneration and transformation protocols are not compatible with all cassava cultivars, thus efficient and robust transformation and regeneration protocols for farmer-preferred cultivars need to be optimised for ease of transfer of novel genes. The objective of this study was to develop an efficient transformation and regeneration protocol for a farmer-preferred Kenyan cassava cultivar. We cultured immature leaf lobe and stem explants on Murashige and Skoog (MS) medium, supplemented with varying concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram and á-naphthalene acetic acid (NAA). Plants were recovered on media with 6- Benzylaminopurine (BAP) and GA3 under a 16 hour light/8 hour darkness photoperiod regime. Results showed high regeneration and transformation frequencies for both cultivars. High frequencies of callus induction (>98%) for both cultivars, were obtained when 2,4-D and Picloram were used. Similarly, both auxins initiated somatic embryogenesis, with Picloram producing the highest frequency of somatic embryos (>92%) in TMS 60444, using stem explants. Gus assays revealed high frequencies of transformation of >77% (TMS 60444) and 60% (Kibanda meno mkubwa). This protocol offers promising perspectives for rapid improvement of these cultivars and, therefore, provides a platform for cleaning planting materials, as well as cassava genetic improvement programmes such as control of viral diseases.Key Words: Cytokinin, Manihot esculenta, regeneration protocol

Development of a Candida glabrata dominant nutritional transformation marker utilizing the Aspergillus nidulans acetamidase gene (amdS).

The gene encoding Aspergillus nidulans acetamidase (amdS) was placed under control of Candida albicans ACT1 promoter and terminator sequences and then cloned into a plasmid containing C. glabrata ARS10,CEN8 or ARS10+CEN8 sequences. All plasmids transformed C. glabrata wild-type cells to acetamide+, with the ARS-only containing plasmid transforming cells at the highest frequencies (>1.0 × 10(4) transformants μg(-1)). Plasmids were rapidly lost under non-selective conditions with the frequency dependent on chromosomal element, thus recycling the acetamide- phenotype. The amdS plasmid was used to transform a set of clinical isolates resistant to a variety of antifungal drugs. All strains were successfully transformed to the acetamide+ phenotype at high frequency, confirming that this plasmid construct could be used as a simple dominant marker on virtually any strain. Gap repair experiments demonstrated that just as in Saccharomyces cerevisiae, gap repair functions efficiently inC. glabrata, suggesting that C. glabrata has numerous similarities toS. cerevisiae with regard to ease of molecular manipulation. The amdS system is inexpensive and efficient, and combined with existing C. glabrata plasmid elements, confers a high transformation frequency for C. glabrata with a phenotype that can be easily recycled.

Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera.

The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar – Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development.

High Frequency of Malignant Transformation of Ovarian Mature Teratoma into Squamous Cell Carcinoma in Young Patients in Northeast Brazil.

The malignant behavior of an ovarian teratoma is related to immaturity, or rarely to the malignant transformation of a somatic component in a mature teratoma (MT). The aim of this work was to review 189 consecutive ovarian teratomas diagnosed between 2006 and 2010 at a public referral center for cancer in Brazil, focusing on cases of MT with malignant transformation. MTs with transformation to squamous cell carcinoma (SCC) were further analyzed by immunohistochemistry for p16 staining. The median age of all patients was 36 yr (mean age, 39.6 yr; SD±4.9). Mature and immature teratomas represented 95.7% (181/189) and 4.2% of the cohort, respectively. Immature teratoma occurred mainly in adolescents under 18 yr. Malignant transformation of the somatic component in MT was observed in 10 of 181 patients (5.5%). SCC was the most common subtype (4/10), followed by differentiated thyroid carcinoma in struma ovarii(3/10), adenosquamous carcinoma (1/10), mucinous intestinal-type adenocarcinoma (1/10), and a well-differentiated neuroendocrine tumor/carcinoid (1/10). Two of 4 SCC cases were strong and diffusely positive for p16, and 2 were negative. In 5 further patients, MT was synchronously observed with other benign and malignant ovarian neoplasms in the ipsilateral ovary (3 mucinous cystadenomas and 1 Brenner tumor) and 1 cystadenocarcinoma in the contralateral ovary. MTs with malignant transformation were larger than those without transformation (P<0.001), but did not demonstrate any association with age. Indeed, our patients with SCC in MT were much younger [median and mean age, 37 and 38 yr (SD±4.9), respectively] than those described previously. As p16 is considered a surrogate marker for HPV infection, the malignant transformation of MT into SSC in young patients raises the possibility of HPV infection as a risk factor in some of these cases. However, molecular studies are needed to clarify the possible role of HPV in the malignant transformation of MT to SCC.

Optimization of Factors Influencing Agrobacterium mediated Genetic Transformation in Eclipta alba (L.) Hassk.

An efficient and reproducible protocol for in vitro plant regeneration and Agrobacterium-mediated genetic transformation was developed for Eclipta alba (L.) Hassk. using nodal explants. Several parameters affecting Agrobacteriummediated gene delivery were investigated and optimized. These include antibiotic concentration, preculture period, infection time, use of acetosyringone, co-cultivation period and temperature and increased level of copper in induction, co-cultivation and regeneration medium. Incorporation of elevated level of CuSO4 led to significant improvements in plant regeneration and recovery of transformed plants. The highest frequency of transformation was observed when explants were infected and co-cultivated at 23oC. Higher transformation frequency was attained by augmenting the medium with 1.0 ?M CuSO4 which was ten times the normal MS level. Addition of acetosyringone in the infection and co-cultivation media was very beneficial and resulted in greater transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected explants and leaves of regenerated transformants, respectively. Molecular analysis of transformed plants with nptII and gus A specific primers revealed the amplification of nptII and gus gene thereby demonstrated the efficacy of optimized protocol for A. tumefaciens-mediated genetic transformation in Eclipta alba for the first time.Plant Tissue Cult. &amp; Biotech. 25(2): 155-164, 2015 (December)

Evaluation of Factors Impacting Agrobacterium-Mediated Indica Rice Transformation of IR58025B - a Public Maintainer Line

n this study, we report our efforts to develop an efficient Agrobacterium-mediated transformation protocol for the transformation of Indica rice IR58025B with mannose as a selection agent. Various factors, such as basal medium, culture temperature, strains for infection, air-drying duration of calli after infection, and salt concentration of coculture medium, were systematically optimized so that the highest transformation frequency (TF) reached to 74%. About 94% of mature embryos were induced to generate calli of high quality on modified NB medium. Both callus quantity and quality were improved when callus was induced from endosperm-free embryos in comparison to intact mature seeds. Higher temperature (32°C) showed improvements when compared to lower temperatures at (25°C and 28°C) for both callus induction and transformation. Agrobacterium strains of EHA105 produced the best TF in comparison with EHA101, LBA4404 and AGL1. However, LBA4404 produced the highest single copy frequency. Prior to co-cultivation, air-drying of inoculated calli was performed in a laminar flow hood for 4-7 hours and produced a higher TF. Lower salt concentration for co-cultivation medium enhanced both DNA delivery and TF.

Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae

The endophytic filamentous fungus Harpophora oryzae is a beneficial endosymbiont isolated from the wild rice. H. oryzae could not only effectively improve growth rate and biomass yield of rice crops, but also induce systemic resistance against the rice blast fungus, Magnaporthe oryzae. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was employed and optimized to modify the H. oryzae genes by either random DNA fragment integration or targeted gene replacement. Our results showed that co-cultivation of H. oryzae conidia with A. tumefaciens in the presence of acetosyringone for 48h at 22°C could lead to a relatively highest frequency of transformation, and 200μM acetosyringone (AS) pre-cultivation of A. tumefaciens is also suggested. ATMT-mediated knockout mutagenesis was accomplished with the gene-deletion cassettes using a yeast homologous recombination method with a yeast–Escherichia–Agrobacterium shuttle vector pKOHo. Using the ATMT-mediated knockout mutagenesis, we successfully deleted three genes of H. oryzae (HoATG5, HoATG7, and HoATG8), and then got the null mutants ΔHoatg5, ΔHoatg7, and ΔHoatg8. These results suggest that ATMT is an efficient tool for gene modification including randomly insertional mutagenesis and gene deletion mutagenesis in H. oryzae.

Genotype independent and efficient Agrobacterium-mediated genetic transformation of the medicinal plant Withania somnifera Dunal

Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD600 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36 ± 13.2 to 277.6 ± 22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T0 transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.

Genetic Transformation of Plumbago zeylanica with Agrobacterium rhizogenes Strain LBA 9402 and Characterization of Transformed Root Lines

High frequency transformation (73.80 ± 2.24%) has been obtained in Plumbago zeylanica using nodes and internodes of axenic whole plants infected with Agrobacterium rhizogenes strain LBA 9402. The root lines could be distinguished morphologically into two types : Root lines of morphotypes I and II. While morphotype I showed profuse branching with short (&lt; 1 cm), highly dense hairy laterals, the roots of morphotype II roots were characterized also by profuse branching with long hairy laterals (&gt; 3 ? 4 cm). Only four of the ten root lines showed integration of four rol genes (rolA, rolB, rolC and rolD) of TL?DNA. None of the root lines showed presence of any of the five genes of TR?DNA. It is noteworthy that the root morphotypes (I and II) showed a clear distinction in the nature of integration and expression of rol genes. The transformed root lines varied significantly (p ? 0.05) with respect to DW (GI DW basis, 2.19 ± 0.24 ? 5.31 ± 0.6) after 4 weeks of culture on solid modified MS; and plumbagin contents in root lines (4.81 ± 0.16 ? 6.69 ± 0.34 mg/g DW) were higher than that reported earlier. Transformed root lines of P. zeylanica maintained in vitro on phytohormone devoid medium for over 2 years can be used for scale up studies for the production of plumbagin in bioreactors.Plant Tissue Cult. &amp; Biotech. 25(1): 21-35, 2015 (June)

An efficient genetic manipulation protocol for Ustilago esculenta.

Ustilago esculenta grows within the flowering stem of the aquatic grass Zizania latifolia, resembling a fungal endophyte. The fungus colonizes Z. latifolia and induces swelling which results in the formation of galls near the base of the plant. Due to their unique flavor and textures these galls are considered as a delicacy in southern China. Efficient genetic manipulation is required to determine the relationship between U. esculenta and Z. latifolia. In this study, we report a protoplast-based transformation system for this unique fungal species. We have explored various factors (enzyme digesting conditions, osmotic pressure stabilizers, vectors and selection agents) that might impact protoplast yield and high frequencies of transformation. A haploid strain (UeT55) of U. esculenta was found to produce higher yields of protoplasts when treating with 15 mgmL(-1) lywallzyme in a sucrose-containing solution at 30°C for 3 h. The transformation frequencies were higher when fungal strain was transformed with a linear plasmid harboring hygromycin or carboxin resistance gene and regenerated on a sucrose-containing medium. A UeICL gene (coding isocitrate lyase) was disrupted and an EGFP (coding enhanced green fluorescent protein) gene was overexpressed successfully in the UeT55 strain using the developed conditions. The genetic manipulation system reported in this study will open up new opportunities for forward and reverse genetics in U. esculenta.

A New Method of Design and Verification of Digital Silicon Gyroscopes Closed-Loop Driving System Based on MicroBlaze

The article brings out a digital silicon gyroscopes closed-loop driving system using the phase relationship of the input driving signal and the signal of the forced motion of the mass block in sensor structure based on analyzing the working principle and phase characters of digital silicon gyroscopes. We make a low-cost, simple and high precision measurement technique for resonant frequency of silicon micro-machinery gyroscopes by the whole digital locked loop system designed with MicroBlaze method. Dichotomy and Fourier transform spectrometry method constitute the algorithm of the system and the solution precision can be determined by the number of the times of approximation. Under the control of the MicroBlaze softcore, the system realizes the realization of the digital signal, the high speed frequency transformation, the acquisition of mass block response signal and the calculation of phase difference. Lastly, each module of the system and the whole system are simulated, the whole system is approved on the test broad.

Complete Genome Sequence of Mycoplasma yeatsii Strain GM274B (ATCC 43094).

Mycoplasma yeatsii is a goat mycoplasma species that, although an obligate parasite, accommodates this lifestyle as an inapparent commensalist. High-frequency transformation has also been reported for this species. The complete 895,051-bp genome sequence of strain GM274B has been determined, enabling an analysis of the features of this potential cloning host.