Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY.

In vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both in vitro and ex vitro HRT were optimized in soybean using the RUBY reporter. Different parameters including A. rhizogenes strain, optical density of the bacterial cell culture (OD600), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the in vitro assay was more efficient than the ex vitro assay in terms of the percentage of induction of hairy roots and transformed roots (expressing RUBY). Nonetheless, the ex vitro technique was deemed faster and a less complicated approach. The highest transformation of RUBY was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in » B5 medium to OD600 (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of RUBY through two-step ex vitro hairy root transformation. Finally, using machine learning-based modeling, optimal protocols for both assays were further defined. This study establishes efficient and reliable hairy root transformation protocols applicable for functional studies in soybean.

Identification of Novel QTLs Associated with Frost Tolerance in Winter Wheat (Triticum aestivum L.).

Low temperature (cold) and freezing stress is a major problem during winter wheat growth. Low temperature tolerance (LT) is an important agronomic trait in winter wheat and determines the plants' ability to cope with below-freezing temperatures; thus, the development of cold-tolerant cultivars has become a major goal of breeding in various regions of the world. In this study, we sought to identify quantitative trait loci (QTL) using molecular markers related to freezing tolerance in winter. Thirty-four polymorphic markers among 425 SSR markers were obtained for the population, including 180 inbred lines of F12 generation wheat, derived from crosses (Norstar × Zagros) after testing with parents. LT50 is used as an effective selection criterion for identifying frost-tolerance genotypes. The progeny of individual F12 plants were used to evaluate LT50. Several QTLs related to wheat yield, including heading time period, 1000-seed weight, and number of surviving plants after overwintering, were identified. Single-marker analysis illustrated that four SSR markers with a total of 25% phenotypic variance determination were linked to LT50. Related QTLs were located on chromosomes 4A, 2B, and 3B. Common QTLs identified in two cropping seasons based on agronomical traits were two QTLs for heading time period, one QTL for 1000-seed weight, and six QTLs for number of surviving plants after overwintering. The four markers identified linked to LT50 significantly affected both LT50 and yield-related traits simultaneously. This is the first report to identify a major-effect QTL related to frost tolerance on chromosome 4A by the marker XGWM160. It is possible that some QTLs are closely related to pleiotropic effects that control two or more traits simultaneously, and this feature can be used as a factor to select frost-resistant lines in plant breeding programs.

Response of Maize Varieties (Zea mays L.) to the Application of Classic and Stabilized Nitrogen Fertilizers-Nitrogen as a Predictor of Generative Yield.

The study presents the results of a 3-year field trial aimed at assessing the yield and efficiency indicators of nitrogen application in the cultivation of three maize cultivars differing in agronomic and genetic profile. The advantages of the UltraGrain stabilo formulation (NBPT and NPPT) over ammonium nitrate and urea are apparent if a maize cultivar capable of efficient nutrient uptake in the pre-flowering period and effective utilization during the grain filling stage is selected. Therefore, the rational fertilization of maize with urea-based nitrogen fertilizer with a urease inhibitor requires the simultaneous selection of cultivars that are physiologically profiled for efficient nitrogen utilization from this form of fertilizer ("stay-green" cultivar). The interaction of a selective cultivar with a high genetically targeted potential for nitrogen uptake from soil, combined with a targeted selection of nitrogen fertilizer, is important not only in terms of production, but also environmental and economic purposes.

CRISPR/Cas9 in Planta Hairy Root Transformation: A Powerful Platform for Functional Analysis of Root Traits in Soybean.

Sequence and expression data obtained by next-generation sequencing (NGS)-based forward genetics methods often allow the identification of candidate causal genes. To provide true experimental evidence of a gene's function, reverse genetics techniques are highly valuable. Site-directed mutagenesis through transfer DNA (T-DNA) delivery is an efficient reverse screen method in plant functional analysis. Precise modification of targeted crop genome sequences is possible through the stable and/or transient delivery of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) reagents. Currently, CRISPR/Cas9 is the most powerful reverse genetics approach for fast and precise functional analysis of candidate genes/mutations of interest. Rapid and large-scale analyses of CRISPR/Cas-induced mutagenesis is achievable through Agrobacterium rhizogenes-mediated hairy root transformation. The combination of A. rhizogenes hairy root-CRISPR/Cas provides an extraordinary platform for rapid, precise, easy, and cost-effective "in root" functional analysis of genes of interest in legume plants, including soybean. Both hairy root transformation and CRISPR/Cas9 techniques have their own complexities and considerations. Here, we discuss recent advancements in soybean hairy root transformation and CRISPR/Cas9 techniques. We highlight the critical factors required to enhance mutation induction and hairy root transformation, including the new generation of reporter genes, methods of Agrobacterium infection, accurate gRNA design strategies, Cas9 variants, gene regulatory elements of gRNAs and Cas9 nuclease cassettes and their configuration in the final binary vector to study genes involved in root-related traits in soybean.

Traditional in vitro strategies for sustainable production of bioactive compounds and manipulation of metabolomic profile in medicinal, aromatic and ornamental plants.

MAIN CONCLUSION: Precursor feeding, elicitation and culture medium parameters are traditional in vitro strategies to enhance bioactive compounds of medicinal, aromatic, and ornamental plants (MAOPs). Machine learning can help researchers find the best combination of these strategies to increase the secondary metabolites content of MAOPs. Many requirements for human life, from food, pharmaceuticals and cosmetics to clothes, fuel and building materials depend on plant-derived natural products. Essential oils, methanolic and ethanolic extracts of in vitro undifferentiated callus and organogenic cultures of medicinal, aromatic, and ornamental plants (MAOPs) contain bioactive compounds that have several applications for various industries, including food and pharmaceutical. In vitro culture systems provide opportunities to manipulate the metabolomic profile of MAOPs. Precursors feeding, elicitation and culture media optimization are the traditional strategies to enhance in vitro accumulation of favorable bioactive compounds. The stimulation of plant defense mechanisms through biotic and abiotic elicitors is a simple way to increase the production of secondary metabolites in different in vitro culture systems. Different elicitors have been applied to stimulate defense machinery and change the metabolomic profile of MAOPs in in vitro cultures. Plant growth regulators (PGRs), stress hormones, chitosan, microbial extracts and physical stresses are the most applied elicitors in this regard. Many other chemical tolerance-enhancer additives, such as melatonin and proline, have been applied along with stress response-inducing elicitors. The use of stress-inducing materials such as PEG and NaCl activates stress tolerance elicitors with the potential of increasing secondary metabolites content of MAOPs. The present study reviewed the state-of-the-art traditional in vitro strategies to manipulate bioactive compounds of MAOPs. The objective is to provide insights to researchers involved in in vitro production of plant-derived natural compounds. The present review provided a wide range of traditional strategies to increase the accumulation of valuable bioactive compounds of MAOPs in different in vitro systems. Traditional strategies are faster, simpler, and cost-effective than other biotechnology-based breeding methods such as genetic transformation, genome editing, metabolic pathways engineering, and synthetic biology. The integrate application of precursors and elicitors along with culture media optimization and the interpretation of their interactions through machine learning algorithms could provide an excellent opportunity for large-scale in vitro production of pharmaceutical bioactive compounds.

Perspectives on new opportunities for nano-enabled strategies for gene delivery to plants using nanoporous materials.

MAIN CONCLUSION: Engineered nanocarriers have great potential to deliver different genetic cargos to plant cells and increase the efficiency of plant genetic engineering. Genetic engineering has improved the quality and quantity of crops by introducing desired DNA sequences into the plant genome. Traditional transformation strategies face constraints such as low transformation efficiency, damage to plant tissues, and genotype dependency. Smart nanovehicle-based delivery is a newly emerged method for direct DNA delivery to plant genomes. The basis of this new approach of plant genetic transformation, nanomaterial-mediated gene delivery, is the appropriate protection of transferred DNA from the nucleases present in the cell cytoplasm through the nanocarriers. The conjugation of desired nucleic acids with engineered nanocarriers can solve the problem of genetic manipulation in some valuable recalcitrant plant genotypes. Combining nano-enabled genetic transformation with the new and powerful technique of targeted genome editing, CRISPR (clustered regularly interspaced short palindromic repeats), can create new protocols for efficient improvement of desired plants. Silica-based nanoporous materials, especially mesoporous silica nanoparticles (MSNs), are currently regarded as exciting nanoscale platforms for genetic engineering as they possess several useful properties including ordered and porous structure, biocompatibility, biodegradability, and surface chemistry. These specific features have made MSNs promising candidates for the design of smart, controlled, and targeted delivery systems in agricultural sciences. In the present review, we discuss the usability, challenges, and opportunities for possible application of nano-enabled biomolecule transformation as part of innovative approaches for target delivery of genes of interest into plants.

Modeling Agrobacterium-Mediated Gene Transformation of Tobacco (Nicotiana tabacum)-A Model Plant for Gene Transformation Studies.

The multilayer perceptron (MLP) topology of an artificial neural network (ANN) was applied to create two predictor models in Agrobacterium-mediated gene transformation of tobacco. Agrobacterium-mediated transformation parameters, including Agrobacterium strain, Agrobacterium cell density, acetosyringone concentration, and inoculation duration, were assigned as inputs for ANN-MLP, and their effects on the percentage of putative and PCR-verified transgenic plants were investigated. The best ANN models for predicting the percentage of putative and PCR-verified transgenic plants were selected based on basic network quality statistics. Ex-post error calculations of the relative approximation error (RAE), the mean absolute error (MAE), the root mean square error (RMS), and the mean absolute percentage error (MAPE) demonstrated the prediction quality of the developed models when compared to stepwise multiple regression. Moreover, significant correlations between the ANN-predicted and the actual values of the percentage of putative transgenes (R 2 = 0.956) and the percentage of PCR-verified transgenic plants (R 2 = 0.671) indicate the superiority of the established ANN models over the classical stepwise multiple regression in predicting the percentage of putative (R 2 = 0.313) and PCR-verified (R 2= 0.213) transgenic plants. The best combination of the multiple inputs analyzed in this investigation, to achieve maximum actual and predicted transgenic plants, was at OD 600 = 0.8 for the LB4404 strain of Agrobacterium × 300 µmol/L acetosyringone × 20 min immersion time. According to the sensitivity analysis of ANN models, the Agrobacterium strain was the most important influential parameter in Agrobacterium-mediated transformation of tobacco. The prediction efficiency of the developed model was confirmed by the data series of Agrobacterium-mediated transformation of an important medicinal plant with low transformation efficiency. The results of this study are pivotal to model and predict the transformation of other important Agrobacterium-recalcitrant plant genotypes and to increase the transformation efficiency by identifying critical parameters. This approach can substantially reduce the time and cost required to optimize multi-factorial Agrobacterium-mediated transformation strategies.

Methods for Chromosome Doubling.

The completely homozygous genetic background of doubled haploids (DHs) has many applications in breeding programs and research studies. Haploid induction and chromosome doubling of induced haploids are the two main steps of doubled haploid creation. Both steps have their own complexities. Chromosome doubling of induced haploids may happen spontaneously, although usually at a low rate. Therefore, artificial/induced chromosome doubling of haploid cells/plantlets is necessary to produce DHs at an acceptable level. The most common method is using some mitotic spindle poisons that target the organization of the microtubule system. Colchicine is a well-known and widely used antimitotic. However, there are substances alternative to colchicine in terms of efficiency, toxicity, safety, and genetic stability, which can be applied in in vitro and in vivo pathways. Both pathways have their own advantages and disadvantages. However, in vitro-induced chromosome doubling has been much preferred in recent years, maybe because of the dual effect of antimitotic agents (haploid induction and chromosome doubling) in just one step, and the reduced generation of chimeras. Plant genotype, the developmental stage of initial haploids, and type-concentration-duration of application of antimitotic agents, are top influential parameters on chromosome doubling efficiency. In this review, we highlight different aspects related to antimitotic agents and to plant parameters for successful chromosome doubling and high DH yield.

Betaine Aldehyde Dehydrogenase (BADH) vs. Flavodoxin (Fld): Two Important Genes for Enhancing Plants Stress Tolerance and Productivity.

Abiotic stresses, mainly salinity and drought, are the most important environmental threats that constrain worldwide food security by hampering plant growth and productivity. Plants cope with the adverse effects of these stresses by implementing a series of morpho-physio-biochemical adaptation mechanisms. Accumulating effective osmo-protectants, such as proline and glycine betaine (GB), is one of the important plant stress tolerance strategies. These osmolytes can trigger plant stress tolerance mechanisms, which include stress signal transduction, activating resistance genes, increasing levels of enzymatic and non-enzymatic antioxidants, protecting cell osmotic pressure, enhancing cell membrane integrity, as well as protecting their photosynthetic apparatus, especially the photosystem II (PSII) complex. Genetic engineering, as one of the most important plant biotechnology methods, helps to expedite the development of stress-tolerant plants by introducing the key tolerance genes involved in the biosynthetic pathways of osmolytes into plants. Betaine aldehyde dehydrogenase (BADH) is one of the important genes involved in the biosynthetic pathway of GB, and its introduction has led to an increased tolerance to a variety of abiotic stresses in different plant species. Replacing down-regulated ferredoxin at the acceptor side of photosystem I (PSI) with its isofunctional counterpart electron carrier (flavodoxin) is another applicable strategy to strengthen the photosynthetic apparatus of plants under stressful conditions. Heterologous expression of microbially-sourced flavodoxin (Fld) in higher plants compensates for the deficiency of ferredoxin expression and enhances their stress tolerance. BADH and Fld are multifunctional transgenes that increase the stress tolerance of different plant species and maintain their production under stressful situations by protecting and enhancing their photosynthetic apparatus. In addition to increasing stress tolerance, both BADH and Fld genes can improve the productivity, symbiotic performance, and longevity of plants. Because of the multigenic and complex nature of abiotic stresses, the concomitant delivery of BADH and Fld transgenes can lead to more satisfying results in desired plants, as these two genes enhance plant stress tolerance through different mechanisms, and their cumulative effect can be much more beneficial than their individual ones. The importance of BADH and Fld genes in enhancing plant productivity under stress conditions has been discussed in detail in the present review.

Modeling callus induction and regeneration in an anther culture of tomato (Lycopersicon esculentum L.) using image processing and artificial neural network method.

Doubled haploids, subsequent to haploid induction, have wide range of applications in basic and applied plant studies. Various parameters can affect the efficiency of haploid induction through an anther culture of tomato. The hybrid system of image processing-artificial neural network (ANN) was used to better understand callus induction and regeneration in an anther culture of tomato. The effect of parameters such as plant genotype, the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) plant growth regulators, the concentration of gum arabic (GA) additive, the cold pretreatment duration, and flower length on callus induction percentage and number of regenerated calli in an anther culture of tomato were studied using multiple linear regression (MLR) and ANN models. The precise flower bud length was measured using an image processing technique. The 4',6-diamidino-2-phenylindole (DAPI) analysis showed that the flowers with 5-6.9 mm length had the highest percentage of the mid- to late-uninucleate microspore stage. The best ANN model for both callus induction percentage and number of regenerated calli was a model with one hidden layer, 12-15 neurons in the first hidden layer, Levenberg-Marquardt learning algorithm, and Tan-Sigmoid transfer function in hidden layer, based on the root mean square error (RMSE), mean absolute error (MAE), and coefficient of determination (R2) statistics. The scatter plot of measured values versus the predicted values showed the superiority of the ANN to MLR model to predict the callus induction percentage in an anther culture of tomato. The sensitivity analysis of MLR and ANN models revealed the plant genotype and 2,4-D concentration as the most important factors affecting both callus induction percentage and number of regenerated calli. Since tomato is a recalcitrant plant to androgenesis-based pathway of haploid induction, therefore the results of the present study can be helpful to develop an efficient haploid induction protocol in tomato through an anther culture pathway.

Application of genetics and biotechnology for improving medicinal plants.

MAIN CONCLUSION: Plant tissue culture has been used for conservation, micropropagation, and in planta overproduction of some pharma molecules of medicinal plants. New biotechnology-based breeding methods such as targeted genome editing methods are able to create custom-designed medicinal plants with different secondary metabolite profiles. For a long time, humans have used medicinal plants for therapeutic purposes and in food and other industries. Classical biotechnology techniques have been exploited in breeding medicinal plants. Now, it is time to apply faster biotechnology-based breeding methods (BBBMs) to these valuable plants. Assessment of the genetic diversity, conservation, proliferation, and overproduction are the main ways by which genetics and biotechnology can help to improve medicinal plants faster. Plant tissue culture (PTC) plays an important role as a platform to apply other BBBMs in medicinal plants. Agrobacterium-mediated gene transformation and artificial polyploidy induction are the main BBBMs that are directly dependent on PTC. Manageable regulation of endogens and/or transferred genes via engineered zinc-finger proteins or transcription activator-like effectors can help targeted manipulation of secondary metabolite pathways in medicinal plants. The next-generation sequencing techniques have great potential to study the genetic diversity of medicinal plants through restriction-site-associated DNA sequencing (RAD-seq) technique and also to identify the genes and enzymes that are involved in the biosynthetic pathway of secondary metabolites through precise transcriptome profiling (RNA-seq). The sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) are the genome editing methods that can produce user-designed medicinal plants. These current targeted genome editing methods are able to manage plant synthetic biology and open new gates to medicinal plants to be introduced into appropriate industries.