Technological Development and Application of Plant Genetic Transformation.

Genetic transformation is an important strategy for enhancing plant biomass or resistance in response to adverse environments and population growth by imparting desirable genetic characteristics. Research on plant genetic transformation technology can promote the functional analysis of plant genes, the utilization of excellent traits, and precise breeding. Various technologies of genetic transformation have been continuously discovered and developed for convenient manipulation and high efficiency, mainly involving the delivery of exogenous genes and regeneration of transformed plants. Here, currently developed genetic transformation technologies were expounded and compared. Agrobacterium-mediated gene delivery methods are commonly used as direct genetic transformation, as well as external force-mediated ways such as particle bombardment, electroporation, silicon carbide whiskers, and pollen tubes as indirect ones. The regeneration of transformed plants usually involves the de novo organogenesis or somatic embryogenesis pathway of the explants. Ectopic expression of morphogenetic transcription factors (Bbm, Wus2, and GRF-GIF) can significantly improve plant regeneration efficiency and enable the transformation of some hard-to-transform plant genotypes. Meanwhile, some limitations in these gene transfer methods were compared including genotype dependence, low transformation efficiency, and plant tissue damage, and recently developed flexible approaches for plant genotype transformation are discussed regarding how gene delivery and regeneration strategies can be optimized to overcome species and genotype dependence. This review summarizes the principles of various techniques for plant genetic transformation and discusses their application scope and limiting factors, which can provide a reference for plant transgenic breeding.

Technology Invention and Mechanism Analysis of Rapid Rooting of Taxus × media Rehder Branches Induced by Agrobacterium rhizogenes.

Taxus, a vital source of the anticancer drug paclitaxel, grapples with a pronounced supply-demand gap. Current efforts to alleviate the paclitaxel shortage involve expanding Taxus cultivation through cutting propagation. However, traditional cutting propagation of Taxus is difficult to root and time-consuming. Obtaining the roots with high paclitaxel content will cause tree death and resource destruction, which is not conducive to the development of the Taxus industry. To address this, establishing rapid and efficient stem rooting systems emerges as a key solution for Taxus propagation, facilitating direct and continuous root utilization. In this study, Agrobacterium rhizogenes were induced in the 1-3-year-old branches of Taxus × media Rehder, which has the highest paclitaxel content. The research delves into the rooting efficiency induced by different A. rhizogenes strains, with MSU440 and C58 exhibiting superior effects. Transcriptome and metabolome analyses revealed A. rhizogenes' impact on hormone signal transduction, amino acid metabolism, zeatin synthesis, and secondary metabolite synthesis pathways in roots. LC-MS-targeted quantitative detection showed no significant difference in paclitaxel and baccatin III content between naturally formed and induced roots. These findings underpin the theoretical framework for T. media rapid propagation, contributing to the sustainable advancement of the Taxus industry.

Agrobacterium spp. nosocomial outbreak assessment using rapid MALDI-TOF MS based typing, confirmed by whole genome sequencing.

Background: A number of episodes of nosocomial Agrobacterium spp. bacteremia (two cases per year) were observed at Bern University Hospital, Switzerland, from 2015 to 2017. This triggered an outbreak investigation. Methods: Cases of Agrobacterium spp. bacteremias that occurred between August 2011 and February 2017 were investigated employing line lists, environmental sampling, rapid protein- (MALDI-TOF MS), and genome-based typing (pulsed field gel electrophoresis and whole genome sequencing) of the clinical isolates. Results: We describe a total of eight bacteremia episodes due to A. radiobacter (n = 2), Agrobacterium genomovar G3 (n = 5) and A. pusense (n = 1). Two tight clusters were observed by WGS typing, representing the two A. radiobacter isolates (cluster I, isolated in 2015) and four of the Agrobacterium genomovar G3 isolates (cluster II, isolated in 2016 and 2017), suggesting two different point sources. The epidemiological investigations revealed two computer tomography (CT) rooms as common patient locations, which correlated with the two outbreak clusters. MALDI-TOF MS permitted faster evaluation of strain relatedness than DNA-based methods. High resolution WGS-based typing confirmed the MALDI-TOF MS clustering. Conclusions: We report clinical and epidemiological characteristics of two outbreak clusters with Agrobacterium. spp. bacteremia likely acquired during CT contrast medium injection and highlight the use of MALDI-TOF MS as a rapid tool to assess relatedness of rare gram-negative pathogens in an outbreak investigation.

Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants.

For non-model plants, functional characterization of genes is still hampered by lack of efficient stable transformation procedures. Here, we report a simple, fast and efficient transformation technique with Agrobacterium rhizogenes for generating stable transgenic roots in living plants to facilitate functional studies in vivo. We showed that injection of A. rhizogenes into stems of various plant species lead to stable transgenic root generation, which can sustain plant growth after the original, non-transgenic roots were cut off. A transformation system was established for pigeon pea, a major woody food crop, after optimizing the selection of A. rhizogenes strains, bacterium concentration, injection position and seedling age. RT-PCR and fluorescence observation indicated a transgenic root induction efficiency of about 39% in pigeon pea. Furthermore, induction of hairy roots was achieved in nine out of twelve tested economically important plants at an efficiency of 15-39%. As proof of concept, bimolecular fluorescence complementation (BiFC) assay was applied to test the interaction between CcCIPK14 and CcCBL1/2 in pigeon pea. Additionally, ectopic expression of the bZIP transcription factor MdHY5 from apple confirmed the utility of the transformation technique for engineering anthocyanin synthesis in roots. Taken together, we show that this method allows fast in vivo studies of gene function in a wide range of plant species.

Remediation of textile azo dye acid red 114 by hairy roots of Ipomoea carnea Jacq. and assessment of degraded dye toxicity with human keratinocyte cell line.

Bioremediation has proven to be the most desirable and cost effective method to counter textile dye pollution. Hairy roots (HRs) of Ipomoea carnea J. were tested for decolourization of 25 textile azo dyes, out of which >90% decolourization was observed in 15 dyes. A diazo dye, Acid Red 114 was decolourized to >98% and hence, was chosen as the model dye. A significant increase in the activities of oxidoreductive enzymes was observed during decolourization of AR114. The phytodegradation of AR114 was confirmed by HPLC, UV-vis and FTIR spectroscopy. The possible metabolites were identified by GCMS as 4- aminobenzene sulfonic acid 2-methylaniline and 4- aminophenyl 4-ethyl benzene sulfonate and a probable pathway for the biodegradation of AR114 has been proposed. The nontoxic nature of the metabolites and toxicity of AR114 was confirmed by cytotoxicity tests on human keratinocyte cell line (HaCaT). When HaCaT cells were treated separately with 150 µg mL(-1) of AR114 and metabolites, MTT assay showed 50% and ≈100% viability respectively. Furthermore, flow cytometry data showed that, as compared to control, the cells in G2-M and death phase increased by 2.4 and 3.6 folds respectively on treatment with AR114 but remained unaltered in cells treated with metabolites.

Assessment of ptxD gene as an alternative selectable marker for Agrobacterium-mediated maize transformation.

KEY MESSAGE: Bacterial phosphite oxidoreductase gene and chemical phosphite can be used as a selection system for Agrobacterium -mediated maize transformation. Application of phosphite (Phi) on plants can interfere the plant metabolic system leading to stunted growth and lethality. On the other hand, ectopic expression of the ptxD gene in tobacco and Arabidopsis allowed plants to grow in media with Phi as the sole phosphorous source. The phosphite oxidoreductase (PTXD) enzyme catalyzes the conversion of Phi into phosphate (Pi) that can then be metabolized by plants and utilized as their essential phosphorous source. Here we assess an alternative selectable marker based on a bacterial ptxD gene for Agrobacterium-mediated maize transformation. We compared the transformation frequencies of maize using either the ptxD/Phi selection system or a standard herbicide bar/bialaphos selection system. Two maize genotypes, a transformation amenable hybrid Hi II and an inbred B104, were tested. Transgene presence, insertion copy numbers, and ptxD transcript levels were analyzed and compared. This work demonstrates that the ptxD/Phi selection system can be used for Agrobacterium-mediated maize transformation of both type I and type II callus culture and achieve a comparable frequency as that of the herbicide bar/bialaphos selection system.

Assessment of the genetic and phenotypic diversity among rhizogenic Agrobacterium biovar 1 strains infecting solanaceous and cucurbit crops.

Rhizogenic Agrobacterium biovar 1 strains have been found to cause extensive root proliferation on hydroponically grown Cucurbitaceae and Solanaceae crops, resulting in substantial economic losses. As these agrobacteria live under similar ecological conditions, infecting a limited number of crops, it may be hypothesized that genetic and phenotypic variation among such strains is relatively low. In this study we assessed the phenotypic diversity as well as the phylogenetic and evolutionary relationships of several rhizogenic Agrobacterium biovar 1 strains from cucurbit and solanaceous crops. A collection of 41 isolates was subjected to a number of phenotypic assays and characterized by MLSA targeting four housekeeping genes (16S rRNA gene, recA, rpoB and trpE) and two loci from the root-inducing Ri-plasmid (part of rolB and virD2). Besides phenotypic variation, remarkable genotypic diversity was observed, especially for some chromosomal loci such as trpE. In contrast, genetic diversity was lower for the plasmid-borne loci, indicating that the studied chromosomal housekeeping genes and Ri-plasmid-borne loci might not exhibit the same evolutionary history. Furthermore, phylogenetic and network analyses and several recombination tests suggested that recombination could be contributing in some extent to the evolutionary dynamics of rhizogenic Agrobacterium populations. Finally, a genomospecies-level identification analysis revealed that at least four genomospecies may occur on cucurbit and tomato crops (G1, G3, G8 and G9). Together, this study gives a first glimpse at the genetic and phenotypic diversity within this economically important plant pathogenic bacterium.

A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

Immunogenic assessment of plant-produced human papillomavirus type 16 L1/L2 chimaeras.

Cervical cancer is caused by infection with human papillomaviruses (HPV) and is a global concern, particularly in developing countries, which have ~80% of the burden. HPV L1 virus-like particle (VLP) type-restricted vaccines prevent new infections and associated disease. However, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. Thus, there is an urgent need for cheap second-generation HPV vaccines that protect against multiple types. The objective of this study was to express novel HPV-16 L1-based chimaeras, containing cross-protective epitopes from the L2 minor capsid protein, in tobacco plants. These L1/L2 chimaeras contained epitope sequences derived from HPV-16 L2 amino acid 108-120, 56-81 or 17-36 substituted into the C-terminal helix 4 (h4) region of L1 from amino acid 414. All chimaeras were expressed in Nicotiana benthamiana via an Agrobacterium-mediated transient system and targeted to chloroplasts. The chimaeras were highly expressed with yields of ~1.2 g/kg plant tissue; however, they assembled differently, indicating that the length and nature of the L2 epitope affect VLP assembly. The chimaera containing L2 amino acids 108-120 was the most successful candidate vaccine. It assembled into small VLPs and elicited anti-L1 and anti-L2 responses in mice, and antisera neutralized homologous HPV-16 and heterologous HPV-52 pseudovirions. The other chimaeras predominantly assembled into capsomeres and other aggregates and elicited weaker humoral immune responses, demonstrating the importance of VLP assembly for the immunogenicity of candidate vaccines.

Assessment of factors influencing the Agrobacterium-mediated in planta seed transformation of brinjal (Solanum melongena L.).

An efficient and reproducible in planta transformation method was developed for brinjal using seed as an explant. The brinjal seeds were infected with Agrobacterium tumefaciens EHA 105 harbouring pCAMBIA 1301-bar plasmid, and the transformants were selected against BASTA®. Several parameters influencing the in planta seed transformation such as pre-culture duration, acetosyringone concentration, surfactants, duration of sonication, vacuum pressure and vacuum duration have been evaluated. The putatively transformed (T 0) brinjal plants were screened by GUS histochemical analysis. Among the different combinations and concentrations tested, when the 18-h pre-cultured brinjal seeds were sonicated for 20 min and vacuum infiltered for 3 min at 500 mm of Hg in Agrobacterium suspension containing 100 µM acetosyringone, 0.2 % Silwett L-77 favoured the Agrobacterium infection and showed maximum transformation efficiency. Among the five brinjal varieties evaluated, Arka Samhitha showed maximum transformation efficiency at 45.66 %. The transgene was successfully transmitted to progeny plants (T 1) which was evidenced by GUS histochemical analysis, polymerase chain reaction and Southern hybridisation. The in planta protocol developed in the present study would be beneficial to transfer the economically and nutritionally important genes into different varieties of brinjal, and the transgenic brinjal plants can be produced in less time (approximately 27 days).

Detoxification activity and energy cost is attenuated in whiteflies feeding on tomato yellow leaf curl China virus-infected tobacco plants.

The begomovirus Tomato yellow leaf curl China virus (TYLCCNV) can benefit its vector, the whitefly Bemisia tabaci, through suppressing the defences of their shared host plants. However, the mechanisms of this vector-virus mutualism remain largely unknown on the insect side of the interaction. Here, we compared the transcriptional profiles of female adult whiteflies of B. tabaci Middle East-Asia Minor 1 feeding on TYLCCNV-free and TYLCCNV-infected tobacco plants using the next-generation sequencing technique and quantitative real-time PCR. Interestingly, the genes involved in the oxidative phosphorylation (OXPHOS) pathway and detoxification enzyme were down-regulated in whiteflies feeding on virus-infected plants. Decreased detoxification activity costs less energy, which may reduce OXPHOS activity. Moreover, the genes involved in redox activity were also down-regulated, which may indicate that the reduced OXPHOS activity decreased reactive oxygen species production. Reduced detoxification activity is likely to attenuate energy costs, thereby enhancing the performance of whiteflies on virus-infected plants. These results provide further insight into the mechanisms of the plant-mediated whitefly-virus mutualism. Moreover, our study suggests that investigating the transcriptional profiles on the insect side of the interaction can advance our understanding of the tripartite interactions.

Efficiency of neural network-based combinatorial model predicting optimal culture conditions for maximum biomass yields in hairy root cultures.

KEY MESSAGE : ANN-based combinatorial model is proposed and its efficiency is assessed for the prediction of optimal culture conditions to achieve maximum productivity in a bioprocess in terms of high biomass. A neural network approach is utilized in combination with Hidden Markov concept to assess the optimal values of different environmental factors that result in maximum biomass productivity of cultured tissues after definite culture duration. Five hidden Markov models (HMMs) were derived for five test culture conditions, i.e. pH of liquid growth medium, volume of medium per culture vessel, sucrose concentration (%w/v) in growth medium, nitrate concentration (g/l) in the medium and finally the density of initial inoculum (g fresh weight) per culture vessel and their corresponding fresh weight biomass. The artificial neural network (ANN) model was represented as the function of these five Markov models, and the overall simulation of fresh weight biomass was done with this combinatorial ANN-HMM. The empirical results of Rauwolfia serpentina hairy roots were taken as model and compared with simulated results obtained from pure ANN and ANN-HMMs. The stochastic testing and Cronbach's α-value of pure and combinatorial model revealed more internal consistency and skewed character (0.4635) in histogram of ANN-HMM compared to pure ANN (0.3804). The simulated results for optimal conditions of maximum fresh weight production obtained from ANN-HMM and ANN model closely resemble the experimentally optimized culture conditions based on which highest fresh weight was obtained. However, only 2.99 % deviation from the experimental values could be observed in the values obtained from combinatorial model when compared to the pure ANN model (5.44 %). This comparison showed 45 % better potential of combinatorial model for the prediction of optimal culture conditions for the best growth of hairy root cultures.

Results of a 90-day safety assessment study in mice fed a glucan produced by Agrobacterium sp. ZX09.

Salecan is a novel water-soluble glucan produced by Agrobacterium sp. ZX09. It has potential application as a food additive with a unique chemical composition and excellent physicochemical properties. The objective of this study was to investigate the acute and subchronic toxicity of Salecan. The oral LD50 of Salecan in ICR mice was greater than 3000 mg/kg body weight. In the subchronic study, ICR mice (10/sex/group) were fed diets containing 0%, 1.0%, 2.5% and 5.0% of Salecan (weight/weight) for 13 weeks. Based on the results from the subchronic study, the overall health, body weight gain, food consumption and clinical pathology parameters were comparable between the groups feed Salecan and the control. No dose-related effects were observed in the treated animals. The only exception was the observation that blood glucose in female mice fed Salecan was lower than in the control group. In addition, the fecal matter from Salecan fed mice exhibited increased water content versus the control animals. The no observed adverse effect level (NOAEL) of 14478 mg/kg body weight/day was determined. The results from this study support the conclusion that Salecan is non-toxic at the levels tested and does not pose a risk to human health when used in food.