Efficient Transformation Method Research Articles

Direct leaf-peeling method for areca protoplasts: a simple and efficient system for protoplast isolation and transformation in areca palm (Areca catechu)

BackgroundAreca palm (Areca catechu) is a woody perennial plant of both economical and medicinal importance grown in tropical and subtropical climates. Yet, the molecular biology study of areca palm is extremely impeded by its unavailability of a transformation method. An efficient protoplast isolation and transformation system could be highly desirable to overcome this barrier.ResultsHere, we described a simple and efficient method for protoplast isolation and transformation from the perennial plant areca palm. A high yield of protoplasts (2.5 × 107 protoplasts per gram of fresh leaf tissues) was obtained from the fresh light green leaflet from the newly-emerged leaf digested overnight in the enzyme solution [2% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, 0.7 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] by the direct leaf-peeling method. The isolated areca protoplasts maintain viability of 86.6% and have been successfully transformed with a green fluorescent protein (GFP)-tagged plasmid (pGreen0029-GFP, 6.0 kb) via the polyethylene glycol (PEG)-mediated transformation. Moreover, the mannitol concentration (optimal: 0.7 M) was determined as a key factor affecting areca protoplast isolation. We also demonstrated that the optimal density of areca protoplast for efficient transformation was at 1.0–1.5 × 106 cells/ml. With the optimization of transformation parameters, we have achieved a relatively high transformation efficiency of nearly 50%.ConclusionWe have established the first efficient protocol for the high-yield isolation and transformation of areca palm protoplasts. This method shall be applied in various biological studies of areca palm, such as gene function analysis, genome editing, protein trafficking and localization and protein–protein interaction. In addition, the protoplast system offers a great genetic transformation approach for the woody perennial plant-areca palm. Moreover, the established platform may be applied in protoplast isolation and transformation for other important species in the palm family, including oil palm and coconut.

Non Sub-Sampled Contourlet with Joint Sparse Representation Based Medical Image Fusion

Medical Image Fusion is the synthesizing technology for fusing multimodal medical information using mathematical procedures to generate better visual on the image content and high-quality image output. Medical image fusion represents an indispensible role in fixing major solutions for the complicated medical predicaments, while the recent research results have an enhanced affinity towards the preservation of medical image details, leaving color distortion and halo artifacts to remain unaddressed. This paper proposes a novel method of fusing Computer Tomography (CT) and Magnetic Resonance Imaging (MRI) using a hybrid model of Non Sub-sampled Contourlet Transform (NSCT) and Joint Sparse Representation (JSR). This model gratifies the need for precise integration of medical images of different modalities, which is an essential requirement in the diagnosing process towards clinical activities and treating the patients accordingly. In the proposed model, the medical image is decomposed using NSCT which is an efficient shift variant decomposition transformation method. JSR is exercised to extricate the common features of the medical image for the fusion process. The performance analysis of the proposed system proves that the proposed image fusion technique for medical image fusion is more efficient, provides better results, and a high level of distinctness by integrating the advantages of complementary images. The comparative analysis proves that the proposed technique exhibits better-quality than the existing medical image fusion practices.

Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen Sphaerulina musiva.

CRISPR-Cas9 is a versatile genome editing system widely used since 2013 to introduce site-specific modifications into the genomes of model and non-model species. This technology is used in various applications, from gene knock-outs, knock-ins, and over-expressions to more precise changes, such as the introduction of nucleotides at a targeted locus. CRISPR-Cas9 has been demonstrated to be easy to establish in new species and highly efficient and specific compared to previous gene editing strategies such as Zinc finger nucleases and transcription activator-like effector nucleases. Grand challenges for emerging CRISPR-Cas9 tools in filamentous fungi are developing efficient transformation methods for non-model organisms. In this paper, we have leveraged the establishment of CRISPR-Cas9 genome editing tool that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) in the model species Trichoderma reesei and developed the first protocol to efficiently transform the non-model species, Sphaerulina musiva. This fungal pathogen constitutes a real threat to the genus Populus, a foundational bioenergy crop used for biofuel production. Herein, we highlight the general considerations to design sgRNAs and their computational validation. We also describe the use of isolated protoplasts to deliver the CRISPR-Cas9 RNP components in both species and the screening for targeted genome editing events. The development of engineering tools in S. musiva can be used for studying genes involved in diverse processes such as secondary metabolism, establishment, and pathogenicity, among many others, but also for developing genetic mitigation approaches. The approach described here provides guidance for potential development of transformation systems in other non-model spore-bearing ascomycetes.

Optimization of callus induction and proliferation of Paeonia lactiflora Pall. and Agrobacterium-mediated genetic transformation.

Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with P. lactiflora. Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D), 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as Agrobacterium cell density, infection time and co-culture duration were studied to optimize transformation efficiency. Agrobacterium strains EHA105 and pBI121 binary vector harboring the β-glucuronidase (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L-1 NAA, 0.5 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was optimal for callus induction and MS medium containing 0.5 mg·L-1 NAA, 1.0 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L-1 and 200 mg·L-1, respectively. Among various combinations analyzed, the best transformation result was obtained via the 25 min of infection of Agrobacterium at 0.6 OD600 and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in P. lactiflora.

Protoplast-Based Transient Expression and Gene Editing in Shrub Willow (Salix purpurea L.).

Shrub willows (Salix section Vetrix) are grown as a bioenergy crop in multiple countries and as ornamentals across the northern hemisphere. To facilitate the breeding and genetic advancement of shrub willow, there is a strong interest in the characterization and functional validation of genes involved in plant growth and biomass production. While protocols for shoot regeneration in tissue culture and production of stably transformed lines have greatly advanced this research in the closely related genus Populus, a lack of efficient methods for regeneration and transformation has stymied similar advancements in willow functional genomics. Moreover, transient expression assays in willow have been limited to callus tissue and hairy root systems. Here we report an efficient method for protoplast isolation from S. purpurea leaf tissue, along with transient overexpression and CRISPR-Cas9 mediated mutations. This is the first such report of transient gene expression in Salix protoplasts as well as the first application of CRISPR technology in this genus. These new capabilities pave the way for future functional genomics studies in this important bioenergy and ornamental crop.

Markerless Genome Editing in Competent Streptococci.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.

Generation of Composite Rosa roxburghii Plants with Transgenic Roots by Agrobacterium-Mediated Transformation

Rosa roxburghii Tratt. is an emerging fruit endemic to China, which has the reputation of being the “King of Vitamin C” because of its abundance of vitamin C. However, it is also a recalcitrant species that imposes severe limitations on the transformation and whole-plant regeneration processes, restricting the verification of the functional genes. Therefore, developing a feasible and efficient genetic transformation method for R. roxburghii is an urgent requirement. Herein, K599 with eGFP was used as the Agrobacterium strain to optimize the genetic transformation from four factors: bacterial concentration, seedling age, infection site, and method. First, the original roots of 5-day-old seedlings were excised, and then the slant cuts of the remaining hypocotyls with 0.5 cm length were placed in K599 at an OD600 of 0.4. Subsequently, the explants were planted in a moistened sterile vermiculite after the beveled site was stained with a clump of bacteria. The results showed that the transformation efficiency of this cutting method was almost 28% at 30 days post-inoculation, while the transformation efficiency obtained by injecting 5-day-old seedlings 0.5–1.0 cm away from the primary root with K599 at an OD600 of 0.4 was only about 7%. Taken together, the current findings provide evidence that Agrobacterium-mediated transformation is a simple, fast, and efficient approach for generating composite R. roxburghii plants. Thus, this method has a broad application to analyze the gene functions in R. roxburghii and other related plant species.

A Decision Probability Transformation Method Based on the Neural Network

When the Dempster-Shafer evidence theory is applied to the field of information fusion, how to reasonably transform the basic probability assignment (BPA) into probability to improve decision-making efficiency has been a key challenge. To address this challenge, this paper proposes an efficient probability transformation method based on neural network to achieve the transformation from the BPA to the probabilistic decision. First, a neural network is constructed based on the BPA of propositions in the mass function. Next, the average information content and the interval information content are used to quantify the information contained in each proposition subset and combined to construct the weighting function with parameter r. Then, the BPA of the input layer and the bias units are allocated to the proposition subset in each hidden layer according to the weight factors until the probability of each single-element proposition with the variable is output. Finally, the parameter r and the optimal transform results are obtained under the premise of maximizing the probabilistic information content. The proposed method satisfies the consistency of the upper and lower boundaries of each proposition. Extensive examples and a practical application show that, compared with the other methods, the proposed method not only has higher applicability, but also has lower uncertainty regarding the transformation result information.

Establishment of a Protoplasts-Based Transient Expression System in Banana (Musa spp.)

The breeding of triploid banana cultivars with improved traits, such as yield and disease resistance, remains a major challenge for breeders. One reason is that the molecular study and functional gene analysis in bananas fall behind due to the difficulties of its genetic manipulation. The plant protoplast-based transient transformation has been documented and widely used as a versatile and convenient system for functional gene analysis in many plant species. However, an efficient high-quality protoplast isolation and transformation system is still lacking for bananas. Here, we established an efficient protoplast isolation and transformation method for bananas by selecting proper source materials, optimizing conditions for enzymatic hydrolysis and PEG-mediated transfection. We found the best source materials for banana protoplasts’ isolation are young suckers, which give a yield of protoplasts ranging from 2.5 × 106 to 10.1 × 107 g−1 fresh weight after 5 to 6 h of enzymolysis. The yield is sufficient for most assays that have been established in protoplasts-based systems, such as protein subcellular localization and protein interaction assays. Moreover, using the established transient gene expression system in banana protoplasts, we validated the subcellular localization of Arabidopsis VESICLE SORTING RECEPTOR 1 (VSR1) and the protein self-interaction of Arabidopsis CNGC20 on the cell membrane. The results indicated this system works well and could be routinely used for the functional characterization of banana genes.

A novel ultrasonic wave-based analog Fourier transform computing system

The Fourier transform is a powerful tool that has manifold applications in various disciplines. With the rise of new applications, such as real-time image processing, there is a need for more efficient Fourier transformation methods. This has led to a number of advancements in the field of wave-based analog computing, with the development of the ultrasonic Fourier transform most recently. This paper presents an alternative design to existing ultrasonic Fourier transform analog computing systems, with its relative compactness being the main advantage. In this study, its capabilities and limitations are extensively examined.

Establishment of an efficient genetic transformation system: A case study of RNAi-induced silencing of the transcription factor MeGI in Diospyros oleifera Cheng seedlings

Persimmon (Diospyros) is an important genus of temperate fruit trees that originated in East Asia. High-efficiency genetic transformation in Diospyros spp. remains challenging, limiting studies of gene function and the use of modern technology, such as gene editing, in breeding programs. In this study, we established an efficient genetic transformation method based on the infection of hypocotyls and radicles obtained from immature D. oleifera seeds. In particular, we focused on optimising methods for infection and induction of calluses and adventitious buds. RNA interference vectors targeting the transcription factor MeGI, which has been reported to have a feminising effect, were transformed into the hypocotyls and radicles of D. oleifera, producing five positively transformed seedlings. Compared with the wild-type controls, the positively transformed seedlings exhibited dwarfed adventitious buds and narrower leaves. The phenotypic characteristics of the flowers of these positively transformed seedlings will be observed at a later date; this will not only confirm the function of MeGI in Diospyros spp. but also potentially induce the development of male floral buds in gynoecious species and further promote the development of breeding methods. The proposed genetic transformation protocol will be valuable for investigations into gene function and the development of persimmon breeding programs.

Establishment of an efficient transformation method of garden stock (Matthiola incana) using a callus formation chemical inducer.

Matthiola incana is an important floricultural plant that blooms from winter to spring, and had been desired to be established a transformation system. This study successfully obtained stable transgenic plants from M. incana. We used Agrobacterium tumefaciens harboring a binary vector containing the β-glucuronidase gene (GUS) under the control of cauliflower mosaic virus 35S promoter to evaluate the transformation frequency of M. incana. We observed that cocultivation with the A. tumefaciens strain GV3101 for 5 days effectively enhanced the infection frequency, assessed through a transient GUS expression area in the seedling. Furthermore, the addition of 100 µM acetosyringone was necessary for Agrobacterium infection. However, we could not obtain transgenic plants on a shoot formation medium supplemented with 1 mg l-1 6-benzyladenine (BA). For callus formation from the leaf sections, a medium supplemented with 1-50 µM fipexide (FPX), a novel callus induction chemical, was employed. Then, the callus formation was observed after 2 weeks, and an earlier response was detected than that in the BA medium (4-6 weeks). Results also showed that cultivation in a selection medium supplemented with 12.5 µM FPX obtained hygromycin-resistant calli. Thus, this protocol achieved a 0.7% transformation frequency. Similarly, progenies from one transgenic line were observed on the basis of GUS stains on their leaves, revealing that the transgenes were also inherited stably. Hence, FPX is considered a breakthrough for establishing the transformation protocol of M. incana, and its use is proposed in recalcitrant plants.

A Neural Network Model for Color Element Data Analysis for Urban Spatial Environment.

In this paper, a CNN model for color element data analysis of the urban spatial environment is constructed through an in-depth study of color element data analysis. This paper investigates a high-order structure formed by a few nodes; it proposes a motif-based graph autoencoder MODEL, combining redefined first- and second-order similarities and perfectly integrating motif structure and autoencoder. The algorithm first proposes an efficient graph transformation method to add the influence of central nodes. It then offers a primary awareness mechanism to aggregate the information of noncentral neighbors. Cen GCN_D and Cen GCN_E outperform the latest algorithms in node classification, link prediction, node clustering, and network visualization. As the number of network layers increases, the advantages of these two variants become progressively more prominent. This paper uses a support vector machine to implement classification validation based on CNN. The experimental results show that when 450 images are randomly selected as training data, the classification accuracy obtained by using the features of different CNN output layers is distributed between 91.4% and 95.2%. When the training set of the experiment reaches more than 300, the accuracy can exceed 90%, and the experimental results corresponding to different training sets a more stable trend. Finally, the trained classifier model is obtained in this thesis, which achieves the purpose of fast classification prediction based on CNN for color element data analysis of urban spatial environments.

A Rapid and Efficient Method for Isolation and Transformation of Cotton Callus Protoplast.

Protoplasts, which lack cell walls, are ideal research materials for genetic engineering. They are commonly employed in fusion (they can be used for more distant somatic cell fusion to obtain somatic hybrids), genetic transformation, plant regeneration, and other applications. Cotton is grown throughout the world and is the most economically important crop globally. It is therefore critical to study successful extraction and transformation efficiency of cotton protoplasts. In the present study, a cotton callus protoplast extraction method was tested to optimize the ratio of enzymes (cellulase, pectinase, macerozyme R-10, and hemicellulase) used in the procedure. The optimized ratio significantly increased the quantity and activity of protoplasts extracted. We showed that when enzyme concentrations of 1.5% cellulase and 1.5% pectinase, and either 1.5% or 0.5% macerozyme and 0.5% hemicellulase were used, one can obtain increasingly stable protoplasts. We successfully obtained fluorescent protoplasts by transiently expressing fluorescent proteins in the isolated protoplasts. The protoplasts were determined to be suitable for use in further experimental studies. We also studied the influence of plasmid concentration and transformation time on protoplast transformation efficiency. When the plasmid concentration reaches 16 µg and the transformation time is controlled within 12–16 h, the best transformation efficiency can be obtained. In summary, this study presents efficient extraction and transformation techniques for cotton protoplasts.

Standardization of an efficient protocol for transformation in Indian cauliflower (Brassica oleracea) var. botrytis

Cauliflower is an important Cole crop grown all over the world for its nutritional benefits. But it is encountered by a serious problem caused by the insect- pest attack. Plutella xylostella is one such insect which causes huge losses. An efficient and simple method for genetic transformation and regeneration of Indian cauliflower, Brassica oleracea var. botrytis has to be developed. Hence, optimization of the plant transformation is a pre- requisite for successful transformation process. Therefore, we worked on this objective at the Indian Agricultural Research Institute, New Delhi, India, and optimized the Agrobacterium-mediated transformation protocol in Indian cauliflower, Pusa Snowball K1 and Pusa Snowball KT25 varieties for developing insect resistance against Plutella xylostella using Bt genes. The Agrobacterium tumefaciens mediated transformation process was utilized for carrying out the transformation and the explant was obtained from the seedlings germinated from the seeds of the respective variety and used for genetic transformation process. The transgene integration and expression were later confirmed by PCR and RT-qPCR, respectively. Moreover, insect bioassay showed that transgenic plants effectively controlled the Diamond Back moth infestation concerning to the control plants indicating the presence of resistance developed against the insect. Hence, this protocol efficiently regenerated transgenic plants that lead to the resistance against biotic stress, specific to the insect Diamond back moth.

State Feedback Stabilization for a Class of Upper-Triangular Stochastic Nonlinear Systems with Time-Varying Control Coefficients

The problem explored in this article concerns the stability of the state feedback control of the upper-triangular stochastic nonlinear systems whose control coefficients are time-varying. First, the state feedback control of the corresponding nominal system is carried out by utilizing the backstepping technique combined with the appropriate Lyapunov function. Then, low-gain homogeneous domination technology and the efficient coordinate transformation method are adopted to realize the state feedback control of the original system and ensure the global asymptotic stability (GAS) in probability of the system. Finally, an example is given to illustrate the feasibility and correctness of the method.

Analysis of the Development of IT Outsourcing Companies in The Era of Cloud Computing--Take Infosys as an Example

With the development of information technology and mobile Internet, especially with the impact of the COVID-19 pandemic, cloud computing, which emphasizes distributed patterns rather than the centralized pattern is gradually known and applied. More and more enterprises have to adopt remote work, which greatly promotes the process of enterprise digital transformation, the digitization is greatly changing the world. The emergence of cloud computing is a huge change in traditional enterprises’ concept and thinking of IT resources. The characteristics of flexibility, innovation, diversity and scale of cloud computing stimulate the innovation vitality of enterprises and promote the development of enterprises. At the same time, the enterprises in the process of digital transformation are faced with various difficulties, therefore, how to choose high quality and efficient enterprise digital transformation method has become an important task for the current enterprise development. This paper takes India’s digital service provider Infosys as an example and analyzes the future development direction of traditional IT outsourcing companies in the current digital environment.

Transgenic Peanut (<i>Arachis hypogaea</i> L.) Plants Conferring Enhanced Protection Against Fungal Pathogens by Expressing <i>Tc chitinase-I</i> Gene

In this study, an efficient Agrobacterium-mediated genetic transformation method followed by Tc chitiI gene for enhanced expression in peanut (Arachis hypogaea L.) cv ICG 7827 is reported. A. tumefaciens strain LBA 4404 harboring the plant transformation plasmid pBinAR contains the Tc chitI gene and nptII gene as a selectable marker. The precultured leaflet (LL) explants were infected with A. tumefaciens containing Tc chitiI gene and cocultivated on SIM (MMS+10 mg/L BAP+1 mg/L NAA+2 mg/L AgNO3) for four days. After co-cultivation, these explants were transferred to selection medium (SIM+75 mg/L Kan+250 mg/L Cefotaxime). In T0 generation, 70 % of transformation efficiency was recorded. T1 generation derived from the primary transgenic (T0) events revealed a Mendelian inheritance pattern (3:1) for the Tc chit I transgene. T1 transgenic peanut plants were tested for resistance against C. arachidicola, C. personatum, and P. arachidis by infection with the microspores using detached leaf assay. The Tc chitI gene expressed in T1 transgenic plants have shown longer incubation, longer latent period, and lower infection frequencies than non-transformed (WT) plants. A significant negative correlation was recorded between chitinase activity and the frequency of infection to the C. arachidicola, C. personatum and P. arachidis pathogens.

Facile Preparation of Aryl Nitriles from Aryl Aldehydes in PEG-DME 250

: A facie and efficient method for one-pot transformation of aryl aldehydes to aryl nitriles using hydroxylamine hydrochloride and phosphorus tribromide in PEG-DME 250 is reported. The conversion of various aryl aldehydes to the corresponding aryl nitriles occurred smoothly in high yields under the present reaction conditions.

An Efficient Agrobacterium-Mediated Transformation Method for Hybrid Poplar 84K (Populus alba × P. glandulosa) Using Calli as Explants.

A highly efficient Agrobacterium-mediated transformation method is needed for the molecular study of model tree species such as hybrid poplar 84K (Populus alba × P. glandulosa cv. ‘84K’). In this study, we report a callus-based transformation method that exhibits high efficiency and reproducibility. The optimized callus induction medium (CIM1) induced the development of calli from leaves with high efficiency, and multiple shoots were induced from calli growing on the optimized shoot induction medium (SIM1). Factors affecting the transformation frequency of calli were optimized as follows: Agrobacterium concentration sets at an OD600 of 0.6, Agrobacterium infective suspension with an acetosyringone (AS) concentration of 100 µM, infection time of 15 min, cocultivation duration of 2 days and precultivation duration of 6 days. Using this method, transgenic plants are obtained within approximately 2 months with a transformation frequency greater than 50%. Polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR) and β-galactosidase (GUS) histochemical staining analyses confirmed the successful generation of stable transformants. Additionally, the calli from leaves were subcultured and used to obtain new explants; the high transformation efficiency was still maintained in subcultured calli after 6 cycles. This method provides a reference for developing effective transformation protocols for other poplar species.