Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute(Corchorus capsularis)

Jute(Corchorus capsularis L.)is the second most important natural plant fiber source after cotton.However,developing an efficient gene editing system for jute remains a challenge.In this study,the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4,an elite jute variety widely cultivated in China.The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation(BiFC)assays.The CHLOROPLASTOS ALTERADOS 1(CcCLA1)gene,which is involved in the development of chloroplasts,was targeted for editing at two sites in Meifeng 4.Based on this hairy root transformation,the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and-GhU6.9 promoters,respectively,to assess the efficiency of gene editing.Results indicated the 50.0%(GhU6.7)and 38.5%(GhU6.9)editing events in the target 2 alleles(gRNA2),but no mutation was detected in the target 1 allele(gRNA1)in transgenic-positive hairy roots.CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation.Two CcCLA1 mutants were albinic,with a gene editing efficiency of 5.3%.These findings confirm that the CRISPR/Cas9 system,incorporating promoter GhU6.7,can be used as a gene editing tool for jute.

Comparison of Biolistic and Agrobacterium-mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium _mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG_CAM, into rice (Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium _mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium _mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two_cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium _mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium _ mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium _mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.

Agrobacterium-mediated Transformation of Kentucky Bluegrass

Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.

Establishment of the Agrobacterium-mediated Genetic Transformation System of Ginkgo biloba and the Construction of the Expression Vector of Gb-DXR

[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.

Agrobacterium-Mediated Transformation of Rice: Constraints and Possible Solutions

Genetic transformation of rice(Oryza sativa L.) by introducing beneficial traits is now a central research instrument in plant physiology and a practical tool for plant improvement. Many approaches are verified for stable introduction of foreign genes into the plant genome. The review examined the different constraints that limit the success of rice genetic transformation via Agrobacterium-mediated approach and suggested possible solutions. Explant identification, gene transfer technique and construct to tailor the integration, transgene expression without collateral to genetic damage and transformant selection are among the technical challenges affecting the rice transformation. Despite the contests, Agrobacteriummediated transformation system has been a better option for producing transgenic rice varieties because of its exact T-DNA processing and simple integration of low copy-number transgene. This information is necessary for improving the transformation system for recalcitrant rice varieties.

Agrobacterium-Mediated Transfer of Arabidopsis ICE1 Gene into Lemon (Citrus Limon (L.) Burm. F. cv. Eureka)

The Arabidopsis ICEI （inducer of CBF expression 1） gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon （Citrus Limon （L.） Burm. F. cv. Eureka） genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICEI gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome.

In planta Agrobacterium-Mediated Transformation of Rice

The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog(MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD_(600) = 0.8–1.0 and the co-cultivation at 25 ℃ for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gus A gene by PCR using the gus A specific primers in the T_0 lines, there were 4 transgenic lines from 286 T_0 lines(1.4% transformation efficiency). However, histochemical glucuronidase(GUS) assay demonstrated that only three of four transgenic lines exhibited gus A expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future.

Effect of Phytosulfokine-α on Agrobacterium-Mediated Transformation in Rice

Phytosulfokine- α （PSK- α ）, a biologically active peptide acting as a growth factor, plays a key role in cellular differentiation and proliferation. To test if PSK- α has some influence on agrobacterium-mediated transformation in rice, PSK-α at a series of concentrations was added into co-culture medium respectively. The results showed that PSK- α indeed affected the recovery of resistant calli and the transformation frequency of rice varieties Taipei 309 and Lijiangxintuanheigu, PSK- α at the concentration of 10 nmol/L could increase induction of resistant callus and efficiency of transformation, with a 11% and 4.9% top increase, respectively than the control. However, PSK- αat 200 nmol/L could inhibit the induction of the resistant calli. Further more, the effect of PSK-α on agrobacterium-mediated transformation is related with the concentration of 2, 4-D in selection medium. Higher induction rate of resistant calli was obtained from tissues treated with PSK- α plus 2 mg/L 2, 4-D.

Development of an Agrobacterium-mediated CRISPR/Cas9 system in pea(Pisum sativum L.)

Pea(Pisum sativum L.)is an annual cool-season legume crop.Owing to its role in sustainable agriculture as both a rotation and a cash crop,its global market is expanding and increased production is urgently needed.For both technical and regulatory reasons,neither conventional nor transgenic breeding techniques can keep pace with the demand for increased production.In answer to this challenge,CRISPR/Cas9 genome editing technology has been gaining traction in plant biology and crop breeding in recent years.However,there are currently no reports of the successful application of the CRISPR/Cas9 genome editing technology in pea.We developed a transient transformation system of hairy roots,mediated by Agrobacterium rhizogenes strain K599,to validate the efficiency of a CRISPR/Cas9 system.Further optimization resulted in an efficient vector,PsU6.3-tRNA-PsPDS3-en35S-PsCas9.We used this optimized CRISPR/Cas9 system to edit the pea phytoene desaturase(PsPDS)gene,causing albinism,by Agrobacterium-mediated genetic transformation.This is the first report of successful generation of gene-edited pea plants by this route.

Agrobacterium-Mediated Transformation of Embryogenic Calli of Anliucheng and Regeneration of Plants Containing the Chimaeric Ribonuclease Gene

Anliucheng (Citrus sinensis Osbeck), a very seedy and widely spread acidless sweet orange cul-tivar in south of China, was transformed by the strain of Agrobacterium Tumefaciens EHA105 carrying pTA29-barnase gene, which will induce pollen sterility in transgenic plants. The embryogenic calli of Anliucheng were co-cultivated with Agrobacterium tumefaciens for 3 days, and then transferred to selective medium containing 50 mg I/1 basta (a kind of herbicide) for 5 weeks. The resistant calli were recovered and regenerated 118 embryoids. A total of 13 entire plants were obtained after micro-grafted on trifoliate orange. These regenerated plants were verified by PCR amplification and confirmed by PCR-Southern blotting analysis.

A Review on Sources,Extractions and Analysis Methods of a Sustainable Biomaterial:Tannins

Condensed and hydrolysable tannins are non-toxic natural polyphenols that are a commercial commodity industrialized for tanning hides to obtain leather and for a growing number of other industrial applications mainly to substitute petroleum-based products.They are a definite class of sustainable materials of the forestry industry.They have been in operation for hundreds of years to manufacture leather and now for a growing number of applications in a variety of other industries,such as wood adhesives,metal coating,pharmaceutical/medical applications and several others.This review presents the main sources,either already or potentially commercial of this forestry by-materials,their industrial and laboratory extraction systems,their systems of analysis with their advantages and drawbacks,be these methods so simple to even appear primitive but nonetheless of proven effectiveness,or very modern and instrumental.It constitutes a basic but essential summary of what is necessary to know of these sustainable materials.In doing so,the review highlights some of the main challenges that remain to be addressed to deliver the quality and economics of tannin supply necessary to fulfill the industrial production requirements for some materials-based uses.

Drilling-based measuring method for the c-φ parameter of rock and its field application

The technology of drilling tests makes it possible to obtain the strength parameter of rock accurately in situ. In this paper, a new rock cutting analysis model that considers the influence of the rock crushing zone(RCZ) is built. The formula for an ultimate cutting force is established based on the limit equilibrium principle. The relationship between digital drilling parameters(DDP) and the c-φ parameter(DDP-cφ formula, where c refers to the cohesion and φ refers to the internal friction angle) is derived, and the response of drilling parameters and cutting ratio to the strength parameters is analyzed. The drillingbased measuring method for the c-φ parameter of rock is constructed. The laboratory verification test is then completed, and the difference in results between the drilling test and the compression test is less than 6%. On this basis, in-situ rock drilling tests in a traffic tunnel and a coal mine roadway are carried out, and the strength parameters of the surrounding rock are effectively tested. The average difference ratio of the results is less than 11%, which verifies the effectiveness of the proposed method for obtaining the strength parameters based on digital drilling. This study provides methodological support for field testing of rock strength parameters.

A comparison study on structure-function relationship of polysaccharides obtained from sea buckthorn berries using different methods:antioxidant and bile acid-binding capacity

In this study,the structural characters,antioxidant activities and bile acid-binding ability of sea buckthorn polysaccharides(HRPs)obtained by the commonly used hot water(HRP-W),pressurized hot water(HRP-H),ultrasonic(HRP-U),acid(HRP-C)and alkali(HRP-A)assisted extraction methods were investigated.The results demonstrated that extraction methods had significant effects on extraction yield,monosaccharide composition,molecular weight,particle size,triple-helical structure,and surface morphology of HRPs except for the major linkage bands.Thermogravimetric analysis showed that HRP-U with filamentous reticular microstructure exhibited better thermal stability.The HRP-A with the lowest molecular weight and highest arabinose content possessed the best antioxidant activities.Moreover,the rheological analysis indicated that HRPs with higher galacturonic acid content and molecular weight showed higher viscosity and stronger crosslinking network(HRP-C,HRP-W and HRP-U),which exhibited stronger bile acid binding capacity.The present findings provide scientific evidence in the preparation technology of sea buckthorn polysaccharides with good antioxidant and bile acid binding capacity which are related to the structure affected by the extraction methods.

Efficient Regeneration and Agrobacterium-mediated Transformation of Brassica napus Cultivar Qingza No.5

Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB ＋ 5 mg/L thidiazuron （TDZ） ＋7.5 mg/L AgNO3 ＋ 0.1 mg/L NAA ＋ 2 mg/L proline （L-pro） ＋ 250 mg/L casein acid hydrolysate （CH） ＋ 3% sucrose; the appropriate growth medium was 1/2 MSB ＋ 1 mg/L IBA ＋ 2 mg/L L-pro ＋ 250 mg/L CH ＋ 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB ＋ 0.2 mg/L IAA ＋ 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.

Material point method simulation of hydro-mechanical behaviour in twophase porous geomaterials: A state-of-the-art review

The material point method(MPM)has been gaining increasing popularity as an appropriate approach to the solution of coupled hydro-mechanical problems involving large deformation.In this paper,we survey the current state-of-the-art in the MPM simulation of hydro-mechanical behaviour in two-phase porous geomaterials.The review covers the recent advances and developments in the MPM and their extensions to capture the coupled hydro-mechanical problems involving large deformations.The focus of this review is aiming at providing a clear picture of what has or has not been developed or implemented for simulating two-phase coupled large deformation problems,which will provide some direct reference for both practitioners and researchers.

Deciphering gastric inflammation-induced tumorigenesis through multi-omics data and AI methods

Gastric cancer(GC), the fifth most common cancer globally, remains the leading cause of cancer deaths worldwide. Inflammation-induced tumorigenesis is the predominant process in GC development;therefore, systematic research in this area should improve understanding of the biological mechanisms that initiate GC development and promote cancer hallmarks. Here, we summarize biological knowledge regarding gastric inflammation-induced tumorigenesis, and characterize the multi-omics data and systems biology methods for investigating GC development. Of note, we highlight pioneering studies in multi-omics data and state-of-the-art network-based algorithms used for dissecting the features of gastric inflammation-induced tumorigenesis, and we propose translational applications in early GC warning biomarkers and precise treatment strategies. This review offers integrative insights for GC research, with the goal of paving the way to novel paradigms for GC precision oncology and prevention.

Volumetric lattice Boltzmann method for pore-scale mass diffusionadvection process in geopolymer porous structures

Porous materials present significant advantages for absorbing radioactive isotopes in nuclear waste streams.To improve absorption efficiency in nuclear waste treatment,a thorough understanding of the diffusion-advection process within porous structures is essential for material design.In this study,we present advancements in the volumetric lattice Boltzmann method(VLBM)for modeling and simulating pore-scale diffusion-advection of radioactive isotopes within geopolymer porous structures.These structures are created using the phase field method(PFM)to precisely control pore architectures.In our VLBM approach,we introduce a concentration field of an isotope seamlessly coupled with the velocity field and solve it by the time evolution of its particle population function.To address the computational intensity inherent in the coupled lattice Boltzmann equations for velocity and concentration fields,we implement graphics processing unit(GPU)parallelization.Validation of the developed model involves examining the flow and diffusion fields in porous structures.Remarkably,good agreement is observed for both the velocity field from VLBM and multiphysics object-oriented simulation environment(MOOSE),and the concentration field from VLBM and the finite difference method(FDM).Furthermore,we investigate the effects of background flow,species diffusivity,and porosity on the diffusion-advection behavior by varying the background flow velocity,diffusion coefficient,and pore volume fraction,respectively.Notably,all three parameters exert an influence on the diffusion-advection process.Increased background flow and diffusivity markedly accelerate the process due to increased advection intensity and enhanced diffusion capability,respectively.Conversely,increasing the porosity has a less significant effect,causing a slight slowdown of the diffusion-advection process due to the expanded pore volume.This comprehensive parametric study provides valuable insights into the kinetics of isotope uptake in porous structures,facilitating the development of porous materials for nuclear waste treatment applications.

Uncertainties of landslide susceptibility prediction: Influences of random errors in landslide conditioning factors and errors reduction by low pass filter method

In the existing landslide susceptibility prediction(LSP)models,the influences of random errors in landslide conditioning factors on LSP are not considered,instead the original conditioning factors are directly taken as the model inputs,which brings uncertainties to LSP results.This study aims to reveal the influence rules of the different proportional random errors in conditioning factors on the LSP un-certainties,and further explore a method which can effectively reduce the random errors in conditioning factors.The original conditioning factors are firstly used to construct original factors-based LSP models,and then different random errors of 5%,10%,15% and 20%are added to these original factors for con-structing relevant errors-based LSP models.Secondly,low-pass filter-based LSP models are constructed by eliminating the random errors using low-pass filter method.Thirdly,the Ruijin County of China with 370 landslides and 16 conditioning factors are used as study case.Three typical machine learning models,i.e.multilayer perceptron(MLP),support vector machine(SVM)and random forest(RF),are selected as LSP models.Finally,the LSP uncertainties are discussed and results show that:(1)The low-pass filter can effectively reduce the random errors in conditioning factors to decrease the LSP uncertainties.(2)With the proportions of random errors increasing from 5%to 20%,the LSP uncertainty increases continuously.(3)The original factors-based models are feasible for LSP in the absence of more accurate conditioning factors.(4)The influence degrees of two uncertainty issues,machine learning models and different proportions of random errors,on the LSP modeling are large and basically the same.(5)The Shapley values effectively explain the internal mechanism of machine learning model predicting landslide sus-ceptibility.In conclusion,greater proportion of random errors in conditioning factors results in higher LSP uncertainty,and low-pass filter can effectively reduce these random errors.

Sparse Modal Decomposition Method Addressing Underdetermined Vortex-Induced Vibration Reconstruction Problem for Marine Risers

When investigating the vortex-induced vibration(VIV)of marine risers,extrapolating the dynamic response on the entire length based on limited sensor measurements is a crucial step in both laboratory experiments and fatigue monitoring of real risers.The problem is conventionally solved using the modal decomposition method,based on the principle that the response can be approximated by a weighted sum of limited vibration modes.However,the method is not valid when the problem is underdetermined,i.e.,the number of unknown mode weights is more than the number of known measurements.This study proposed a sparse modal decomposition method based on the compressed sensing theory and the Compressive Sampling Matching Pursuit(Co Sa MP)algorithm,exploiting the sparsity of VIV in the modal space.In the validation study based on high-order VIV experiment data,the proposed method successfully reconstructed the response using only seven acceleration measurements when the conventional methods failed.A primary advantage of the proposed method is that it offers a completely data-driven approach for the underdetermined VIV reconstruction problem,which is more favorable than existing model-dependent solutions for many practical applications such as riser structural health monitoring.

Numerical manifold method for thermo-mechanical coupling simulation of fractured rock mass

As a calculation method based on the Galerkin variation,the numerical manifold method(NMM)adopts a double covering system,which can easily deal with discontinuous deformation problems and has a high calculation accuracy.Aiming at the thermo-mechanical(TM)coupling problem of fractured rock masses,this study uses the NMM to simulate the processes of crack initiation and propagation in a rock mass under the influence of temperature field,deduces related system equations,and proposes a penalty function method to deal with boundary conditions.Numerical examples are employed to confirm the effectiveness and high accuracy of this method.By the thermal stress analysis of a thick-walled cylinder(TWC),the simulation of cracking in the TWC under heating and cooling conditions,and the simulation of thermal cracking of the SwedishÄspöPillar Stability Experiment(APSE)rock column,the thermal stress,and TM coupling are obtained.The numerical simulation results are in good agreement with the test data and other numerical results,thus verifying the effectiveness of the NMM in dealing with thermal stress and crack propagation problems of fractured rock masses.