T1 Events Research Articles

Rearrangements during slow compression of a jammed two-dimensional emulsion.

As amorphous materials get jammed, both geometric and dynamic heterogeneity are observed. We investigate the correlation between the local geometric heterogeneity and local rearrangements in a slowly compressed bidisperse quasi-two-dimensional emulsion system. The compression is driven by evaporation of the continuous phase and causes the area packing fraction to increase from 0.88 to 0.99. We quantify the structural heterogeneity of the system using the radical Voronoi tessellation following the method of Rieser etal. [Phys. Rev. Lett. 116, 088001 (2016)]0031-900710.1103/PhysRevLett.116.088001. We define two structural quantities characterizing local structure, the first of which considers nearest neighbors and the second of which includes information from second-nearest neighbors. We find that droplets in heterogeneous local regions are more likely to have local rearrangements. These rearrangements are generally T1 events where two droplets converge toward a void, and two droplets move away from the void to make room for the converging droplets. Thus, the presence of the voids tends to orient the T1 events. The presence of a correlation between the structural quantities and the rearrangement dynamics remains qualitatively unchanged over the entire range of packing fractions observed.

Predominant types of regional cold waves in North China and their historical changes

Cold wave (CW) events in East Asia have a significant impact on the social economy, ecosystems, and human health. Based on daily CW records from October to March during 1980–2019 and K-means clustering, this study identified two types of regional CW (T1 and T2) in China. T1 regional CWs mainly struck Northeast China, whereas T2 regional CWs primarily affected the northern and eastern regions of China. Compared to T1, T2 regional CWs were more intense, lasted longer, and had a broader region of influence. During 1980–2019, there was a significant increase in the frequency of T1 regional CWs, while the frequency of T2 regional CWs showed no trend. Although amplification of the Siberian high was observed in both T1 and T2 regional CW events, there were differences in the wave train patterns in the middle troposphere between the two regional CWs. During T1 regional CW events, a wave train with negative–positive 500-hPa geopotential height anomalies over Siberia–Mongolia weakened the East Asian trough, causing an eastward movement of cold air from Siberia. The increasing trend of T1 regional CW frequency may be attributable to the linear trends of 500-hPa geopotential height caused by global warming. The wave train associated with T2 regional CW events formed ridge–trough–ridge circulation anomalies over the Ural Mountains, Mongolia, and North China. This led to a southeastward intrusion of the cold air. The conclusions of this paper indicate that the Siberian high amplification and mid-tropospheric wave train pattern should be jointly considered in forecasting regional CW events in China.摘要寒潮事件对东亚地区的社会经济, 生态系统和人体健康影响巨大. 根据1980−2019 年间10月至次年3月的每日寒潮记录和K-means聚类, 本文识别出了中国两种不同类型的区域性寒潮(T1 和 T2). T1区域性寒潮主要影响东北地区, T2区域性寒潮则主要影响华北和东部地区. 与T1区域性寒潮相比, T2区域性寒潮强度更强, 持续时间更长, 影响范围更广. 1980–2019年期间, T1区域性寒潮的频率显著增加, 而T2区域性寒潮的频率则没有趋势变化. T1和T2区域性寒潮事件均与西伯利亚高压增强有关, 然而与它们相关的对流层中层波列明显不同. 在T1区域性寒潮事件发生期间, 西伯利亚-蒙古上空出现负-正模态的500-hPa位势高度异常波列, 削弱了东亚大槽, 导致西伯利亚冷空气东移. T1区域寒潮频次增加趋势可能与全球变暖引起的500-hPa位势高度的线性趋势变化有关. 与T2区域寒潮事件相关的波列则在乌拉尔山脉, 蒙古和华北地区形成了脊-槽-脊环流异常, 导致冷空气向东南方向入侵. 本文得出结论, 由于两类区域寒潮影响不同, 西伯利亚高压增强和对流层中层波列模态在我国区域性寒潮事件的预报中应综合考虑.

Generating active T1 transitions through mechanochemical feedback.

Convergence-extension in embryos is controlled by chemical and mechanical signalling. A key cellular process is the exchange of neighbours via T1 transitions. We propose and analyse a model with positive feedback between recruitment of myosin motors and mechanical tension in cell junctions. The model produces active T1 events, which act to elongate the tissue perpendicular to the main direction of tissue stress. Using an idealised tissue patch comprising several active cells embedded in a matrix of passive hexagonal cells, we identified an optimal range of mechanical stresses to trigger an active T1 event. We show that directed stresses also generate tension chains in a realistic patch made entirely of active cells of random shapes and leads to convergence-extension over a range of parameters. Our findings show that active intercalations can generate stress that activates T1 events in neighbouring cells, resulting in tension-dependent tissue reorganisation, in qualitative agreement with experiments on gastrulation in chick embryos.

Cross over to collective rearrangements near the dry-wet transition in two-dimensional foams

Liquid foams respond plastically to external perturbations over some critical magnitude. This rearrangement process is directly related to the mechanical properties of the foams, playing a significant role in determining foam lifetime, deformability, elasticity, and fluidity. In this paper, we experimentally investigate the rearrangement dynamics of foams near a dry-wet transition. When a foam transforms from a dry state to a wet state, it is found that considering collective events, separated T1 events propagate in dry foams, while T1 events occur simultaneously in wet foams. This cross over to collective rearrangements is closely related to the change in local bubble arrangements and mobility. Furthermore, it is also found that a probability of collective rearrangement events occurring follows a Poisson distribution, suggesting that there is little correlation between discrete collective rearrangement events. These results constitute progress in understanding the dynamical properties of soft jammed systems, relevant for biological and material sciences as well as food science.

Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac.

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

Predicting and following T1 events in dry foams from geometric features

Machine learning techniques have been recently applied in predicting deformation in amorphous materials. In this study, we extract structural features around liquid film vertices from images of flowing 2D foam and apply a multilayer perceptron to predict local yielding. We evaluate their importance in the description of the T1 events and show that a high level of predictability may be achieved using well-chosen combinations of features as the prediction data. The most relevant features are extracted by performing the predictions separately for isolated sets of features, and these findings are verified using principal component analysis. Using this approach, we determine which properties of the images are most important with regard to the physics of the processes. Our findings indicate that film lengths and angles between the liquid films joining at the vertex are the most important features that predict the local yield events. These two features describe 83% of the yield events. As an application, we extract the statistics of event waiting times from the experiment.

Expression interference of a number of Heterodera avenae conserved genes perturbs nematode parasitic success in Triticum aestivum

The cereal cyst nematode, Heterodera avenae is distributed worldwide and causes substantial damage in bread wheat, Triticum aestivum. This nematode is extremely difficult to manage because of its prolonged persistence as unhatched eggs encased in cysts. Due to its sustainable and target-specific nature, RNA interference (RNAi)-based strategy has gained unprecedented importance for pest control. To date, RNAi strategy has not been exploited to manage H. avenae in wheat. In the present study, 40 H. avenae target genes with different molecular function were rationally selected for in vitro soaking analysis in order to assess their susceptibility to RNAi. In contrast to target-specific downregulation of 18 genes, 7 genes were upregulated and 15 genes showed unaltered expression (although combinatorial soaking showed some of these genes are RNAi susceptible), suggesting that a few of the target genes were refractory or recalcitrant to RNAi. However, RNAi of 37 of these genes negatively altered nematode behavior in terms of reduced penetration, development and reproduction in wheat. Subsequently, wheat plants were transformed with seven H. avenae target genes (that showed greatest abrogation of nematode parasitic success) for host-induced gene silencing (HIGS) analysis. Transformed plants were molecularly characterized by PCR, RT-qPCR and Southern hybridization. Production of target gene-specific double- and single-stranded RNA (dsRNA/siRNA) was detected in transformed plants. Transgenic expression of galectin, cathepsin L, vap1, serpin, flp12, RanBPM and chitinase genes conferred 33.24–72.4 % reduction in H. avenae multiplication in T1 events with single copy ones exhibiting greatest reduction. A similar degree of resistance observed in T2 plants indicated the consistent HIGS effect in the subsequent generations. Intriguingly, cysts isolated from RNAi plants were of smaller size with translucent cuticle compared to normal size, dark brown control cysts, suggesting H. avenae developmental retardation due to HIGS. Our study reinforces the potential of HIGS to manage nematode problems in crop plant.

Plumular meristem transformation system for chickpea: an efficient method to overcome recalcitrant tissue culture responses

Major biotic and abiotic stresses have led to the reduction of chickpea productivity, creating a strong barrier for its utilization as major food legume. Genetic transformation has revolutionized the chickpea improvement programs for biotic stress tolerance. Recent knowledge on genomic resources offers a range of approaches for genetic improvement of chickpea with a greater resolution, but relies on the transgenic establishment which is the major bottleneck in this process. Poor tissue culture responses along with insensitivity to in vitro rooting hindered the transgenic chickpea development through tissue culture based transformation methods. In the present report, Agrobacterium-mediated plumular meristem transformation was adapted in chickpea to bypass the recalcitrancy of regenerable tissue. Embryonic axis of immature seedlings of chickpea cultivar was decapitated followed by pricking of plumular meristem present in shoot apex and cotyledonary nodes. Pricking is followed by infection by Agrobacterium tumefaciens carrying binary vector pBI121. Three days co-cultivation was performed with infected explants in 6-benzylaminopurine and α-naphthaleneacetic acid supplemented modified Murashige and Skoog medium. Transient GUS expression was observed in co-cultivated explants and PCR based screening strategy allowed the establishment of primary transgenic events with a frequency of 60%. Kanamycin mediated stringent selection of T1 events helped to eliminate the chimeric plants and 44% of T1 progenies were confirmed through PCR. Further, Southern hybridization was performed to identify transgene integration in T1 events. Additionally, prominent GUS activity in T1 events confirmed the expression of transgene. Plumular transformation method, based on culture dependent Agrobacterium-infection and culture independent plant selection as well as establishment, was reported for the first time in chickpea. This transformation method will boost the recovery of novel genotypes with improved agronomic traits and mutant development for functional genomics in chickpea. Agrobacterium-mediated transformation of plumular meristem in chickpea is established, following in vitro co-cultivation of explants and culture independent selection of transgenic plants, at higher frequency.

Local description of foam flow, deformation and pressure drop in narrow constricted channels

We study in extensive detail the flow of dry and wet gas-liquid foams through a narrow two-dimensional channel comprising a complex constriction. The effects of constriction profile and size of aperture, foam flowrate, liquid holdup and liquid viscosity are experimentally investigated and a sophisticated image analysis procedure is developed to extract local fields of foam flow and deformation. Foam dynamics are scrutinised and interpreted in terms of local velocity distribution, elastic strain (texture tensor), plastic deformation (T1 events) and pressure drop. Important foam transformations occur in the vicinity of the constriction. For the first time, the relationship between pressure drop, flowrate and size of constriction aperture is investigated.

Stress banding in compressed quasi-two-dimensional aqueous foams

We present results from a study of the compression of a quasi-two-dimensional aqueous foam in a Hele-Shaw cell. Our results show that during compression, the spatially averaged normal-stress difference localizes in bands with a wavelength of the order of the mean diameter of a bubble. Stress field colormaps are constructed by image analysis and used to visualize the bands. We investigate the phenomenon by varying the mean bubble diameter and polydispersity. We quantify the extent of (global) compression by measuring the average of an anisotropy ratio for the eigenvalues of the statistical strain tensor and find the probability distribution of the angle between corresponding eigenvectors of the statistical stress and strain tensors. We show that these stress bands are ruptured in regions in which avalanches of neighbor-switching T1 events occur, which suggests local stress relaxation in these regions.

Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera

Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80–100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25–80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.

Host Delivered RNAi of Two Cuticle Collagen Genes, Mi-col-1 and Lemmi-5 Hampers Structure and Fecundity in Meloidogyne incognita.

Root-knot nematodes have emerged as devastating parasites causing substantial losses to agricultural economy worldwide. Tomato is the most favored host for major species of root-knot nematodes. Control strategies like use of nematicides have proved to be harmful to the environment. Other control methods like development of resistant cultivars and crop rotation have serious limitations. This study deals with the application of host generated RNA interference toward development of resistance against root-knot nematode Meloidogyne incognita in tomato. Two cuticle collagen genes viz. Mi-col-1 and Lemmi-5 involved in the synthesis and maintenance of the cuticle in M. incognita were targeted through host generated RNA interference. Expression of both Mi-col-1 and Lemmi-5 was found to be higher in adult females followed by egg masses and J2s. Tomato var. Pusa Ruby was transformed with the RNAi constructs of these genes to develop transgenic lines expressing the target dsRNAs. 30.80–35.00% reduction in the number of adult females, 50.06–65.73% reduction in the number of egg mass per plant and 76.47–82.59% reduction in the number of eggs per egg mass were observed for the T1 events expressing Mi-col-1 dsRNA. Similarly, 34.14–38.54% reduction in the number of adult females, 62.34–66.71% reduction in number of egg mass per plant and 67.13–79.76% reduction in the number of eggs per egg mass were observed for the T1 generation expressing Lemmi-5 dsRNA. The multiplication factor of M. incognita reduced significantly in both the cases and the structure of adult females isolated from transgenic plants were heavily distorted. This study demonstrates the role of the cuticle collagen genes Mi-col-1 and Lemmi-5 in the structure and development of M. incognita cuticle inside the host and reinforces the potential of host generated RNA interference for management of plant parasitic nematodes (PPNs).

Effective Screening of Transgenic Pigeonpea in Presence of Negative Selection Agents

Selection of putative transformants is an essential step in transgenic plant development program of important pulse crop pigeonpea. The multiple shoot induction of pigeonpea was studied under the influence of three negative selection agents kanamycin, hygromycin and glufosinate ammonium. Minimum inhibitory concentration of these selection agents on shoot regeneration was determined. These observations were then applied to genetic transformation studies where 100 mg/l kanamycin and 2.0 mg/l glufosinate ammonium were used to select the neomycin phosphotransferase II (nptII) and bar expressing T0 pigeonpea transformants, respectively. Hygromycin was found to be detrimental for shoot regeneration in all applied concentrations. T1 seeds obtained from nptII positive events were screened by incubating 5 h in 100 mg/l kanamycin solution prior to germination. T1 events containing bar gene were selected by painting 2.0 mg/l solution of glufosinate ammonium on adaxial surfaces of the leaves. Positive transformants survived in both the selection processes, were further confirmed by polymerase chain reaction analysis. Knowledge generated through this study will facilitate the incorporation of agronomically important traits in transgenic pigeonpea for its betterment.

Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.

Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T0 transformants were selected on the basis of PCR and protein expression profile. T1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T0, T1, and T2). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.

Experimental observation of local rearrangements in dense quasi-two-dimensional emulsion flow.

We experimentally study rearranging regions in slow athermal flow by observing the flow of a concentrated oil-in-water emulsion in a thin chamber with a constricting hopper shape. The gap of the chamber is smaller than the droplet diameters, so that the droplets are compressed into quasi-two-dimensional pancakes. We focus on localized rearrangements known as "T1 events" where four droplets exchange neighbors. Flowing droplets are deformed due to forces from neighboring droplets, and these deformations are decreased by nearby T1 events, with a spatial dependence related to the local structure. We see a tendency of the T1 events to occur in small clusters.

Fluctuations of particle motion in granular avalanches - from the microscopic to the macroscopic scales.

In this study, we have investigated the fluctuations of particle motion, i.e. the non-affine motion, during the avalanche process, discovering a rich dynamic from the microscopic to the macroscopic scales. We find that there is a strong correlation between the magnitude of the velocity fluctuation and the velocity magnitude in the spatial and temporal domains. The possible connection between this finding and the theory of the shear transformation zones is discussed based on the direct measurement of the T1 events. In addition, the velocity magnitude of the system and the stress fluctuations of the system are strongly temporally correlated. Our finding will pose challenges to the development of more rigorous theories to describe avalanche dynamics based on the microscopic approach. Moreover, our finding presents a plausible mechanism of particle entrainment in a simple system.

Scaling of critical velocity for bubble raft fracture under tension

The behavior of materials under tension is a rich area of both fluid and solid mechanics. For simple fluids, the breakup of a liquid as it is pulled apart generally exhibits an instability driven, pinch-off type behavior. In contrast, solid materials typically exhibit various forms of fracture under tension. The interaction of these two distinct failure modes is of particular interest for complex fluids, such as foams, pastes, slurries, etc. The rheological properties of complex fluids are well-known to combine features of solid and fluid behaviors, and it is unclear how this translates to their failure under tension. In this paper, we present experimental results for a model complex fluid, a bubble raft. As expected, the system exhibits both pinch-off and fracture when subjected to elongation under constant velocity. We report on the critical velocity vc below which pinch-off occurs and above which fracture occurs as a function of initial system width W, length L, bubble size R, and fluid viscosity for both monodisperse and polydisperse systems. Though both exhibit a transition from pinch-off to fracture, the behavior as a function of L/W is qualitatively different for the two systems. For the polydisperse systems, the results for the critical velocity are consistent with a simple scaling law vcτ/R∼L/W, where the fluid viscosity sets the typical time for bubble rearrangements τ. We show that this scaling can be understood in terms of the dynamics of local bubble rearrangements (T1 events). For the monodisperse systems, we observe a critical value for L/W below which the system only exhibits fracture.

The response of the statistics of the cumulative features on grains in nanomaterials to different grain growth phenomena

Each nanomaterial grain has some number of features, such as faces or triple junctions, on it. The sum of all the features on all grains in nanomaterials, herein called cumulative feature, can be obtained. During grain growth both the number of features per grain and the cumulative features on all grain in nanomaterials evolve randomly with time. Different mechanisms are responsible for grain growth in nanomaterials. This includes Grain Boundary Migration, Grain Rotation-Coalescence, T1 and T2 events. Evolution models for number of features per grain are known already, and not model for evolution of cumulative features. The present paper uses the tools of stochastic theory given by Random Marked Point Field to propose models for the temporal and thermal evolutions of the statistics of the random cumulative features on grains in nanomaterials under different grain growth mechanisms. The resulting differential equations are solved simultaneously using data from nanocrystalline aluminium. It is observed that the mean number of features per grain increases and density of grains in nanomaterials decreases during grain growth. It is revealed that grain growth results in decrease in moments of the cumulative features. It is shown that an increase in annealing temperature results in relatively higher increase in mean number of features per grain, further decrease in grain density, relative increase in mean cumulative features on grain and variable dispersions of cumulative features. It is also observed that the evolution of the statistics of the cumulative features depends on the nature of Galzier-diffusion term, the form of the critical number of faces per grain and the type of grain growth mechanisms. For some choices of the Glazier diffusion term, the dispersion of the cumulative feature evolves in a manner similar to that of the nanomaterials mechanical properties given by the Hall–Petch to Reversed-Hall–Petch Relationship. The variables results are explained to be consequences of different grain growth mechanisms, temperature and the diffusion termed. Thus, it can be concluded that processing route, processing conditions and the nature of evolution of the constituents of nanomaterials are simultaneously vital when designing or characterising nanomaterials.

Topological rearrangements and stress fluctuations in quasi-two-dimensional hopper flow of emulsions

We experimentally study the shear flow of oil-in-water emulsion droplets in a thin sample chamber with a hopper shape. In this thin chamber, the droplets are quasi-2D in shape. The sample is at an area fraction above jamming and forced to flow with a constant flux rate. Stresses applied to a droplet from its neighbors deform the droplet outline, and this deformation is quantified to provide an ad hoc measure of the stress. As the sample flows through the hopper we see large fluctuations of the stress, similar in character to what has been seen in other flows of complex fluids. Periods of time with large decreases in stress are correlated with bursts of elementary rearrangement events ("T1 events" where four droplets rearrange). More specifically, we see a local relationship between these observations: a T1 event decreases the inter-droplet forces up to 3 droplet diameters away from the event. This directly connects microscopic structural changes to macroscopic fluctuations, and confirms theoretical pictures of local rearrangements influencing nearby regions. These local rearrangements are an important means of reducing and redistributing stresses within a flowing material.

Agrobacterium-Mediated In Planta Transformation of Field Bean (Lablab purpureus L.) and Recovery of Stable Transgenic Plants Expressing the cry1AcF Gene

The amenability and reproducibility of a tissue culture-independent Agrobacterium tumefaciens-mediated transformation strategy was analyzed in field bean and the stability of the transgenes was examined. The protocol involves in planta inoculation of embryo axes of germinating seeds and allowing them to grow into seedlings ex vitro. Transformants were raised using a chimeric Bt gene, cry1AcF, and putative transformants were analyzed by PCR for both cry1AcF as well as the nptII genes. Bioassays against Helicoverpa armigera, the major pod borer, showed that several T1 plants performed well with 17% of T1 plants harboring the transgene. Further, enzyme-linked immunosorbent assay (ELISA) and quick dip strip test confirmed the expression of the chimeric Bt toxin. The stability of the transgenes was checked in three generations for integration, expression, and efficacy against the two insects, H. armigera and Spodoptera litura. Southern blot analysis of 10 high expressing plants confirmed the integration of the transgene, whereas single copy integration of the T-DNA in 5 events was also evident. Transcript accumulation of the cry1AcF gene by Northern analysis supported the expression analysis by ELISA. Likewise, Western blot analysis for the NPTII protein further confirmed the transgenic nature of the plants. At the end of the analysis in the T3 generation, five plants from five T1 events were selected as promising. Therefore, the study proved not only the amenability of the field bean to the transformation protocol but also the stability of the introduced genes through three generations.