Genetic Modification Of Plants Research Articles

Construction of peptide/plasmid DNA complexes for plant gene transfection via the basic leucine zipper domain

AbstractAn important method for plant genetic modification is using peptide/pDNA complexes to transfer genes into plant cells. With conventional carrier peptides, the peptide sequence must contain a high amount of cationic amino acids to condense and introduce pDNA. As a result, the dissociation of pDNA from the complex is inefficient, often causing problems. Herein, we designed a new peptide carrier that mimics the basic leucine zipper (bZIP) domain of DNA-binding proteins, in which (LU)4 is the leucine zipper motif and (KUA)3 is the basic DNA-binding and cell-penetrating motif (U = α-aminoisobutyric acid). After (KUA)3-(LU)4 peptide was mixed with pDNA, DNA molecules were condensed to form nanoparticles of approximately 130 nm. Furthermore, when complexes of (KUA)3-(LU)4 peptide and pDNA were introduced into the leaves of Arabidopsis thaliana (A. thaliana), expression of the reporter protein was detected in the plant cells. Thus, (KUA)3-(LU)4 peptide that mimics the bZIP domain is a novel and efficient carrier for pDNA with high dissociation efficiency.

Partial sequence analysis and relative expression of the HSP70 gene of Vasconcellea pubescens

Environmental factors affect nearly all land areas on the planet. Global warming is one of the most destructive of these factors because it has adverse effects on crop production systems. Plants are sessile organisms that have evolved complex mechanisms to cope with stress factors. Heat shock proteins (HSPs) are one of those mechanisms. In this study, we analyzed a partial gene sequence that encodes for HSP70 protein in Vasconcellea pubescens. We also measured the relative expression of the gene in plantlets of Vasconcellea pubescens and performed biochemical assays under heat stress. The plantlets were exposed to three temperatures 25° C (control), 45 °C and 55 °C (stress temperatures) for 4 hours. The bioinformatic analysis led to the first description of a partial sequence of the HSP70 gene and its evolutionary history in V. pubescens. We found significant differences for relative expression of theHSP70 gene, percentage of electrolyte leakage, and proline content between plants subjected to heat stress and those in the control group. Our results showed that V. pubescens displays thermotolerance even under extreme temperatures. V. pubescens is a poorly studied species that may contain genes of biotechnological interest (such as HSP70) that could be used for plant genetic modification.

Denovo induction of a DNA-histone H3K9 methylation loop on synthetic human repetitive DNA in cultured tobacco cells.

Genetic modifications in plants are crucial tools for fundamental and applied research. Transgene expression usually varies among independent lines or their progeny and is associated with the chromatin structure of the insertion site. Strategies based on understanding how to manipulate the epigenetic state of the inserted gene cassette would help to ensure transgene expression. Here, we report a strategy for chromatin manipulation by the artificial tethering of epigenetic effectors to a synthetic human centromeric repetitive DNA (alphoid DNA) platform in plant Bright-Yellow-2 (BY-2) culture cells. By tethering DNA-methyltransferase (Nicotiana tabacum DRM1), we effectively induced DNA methylation and histone methylation (H3K9me2) on the alphoid DNA platform. Tethering of the Arabidopsis SUVH9, which has been reported to lack histone methyltransferase activity, also induced a similar epigenetic state on the alphoid DNA in BY-2 cells, presumably by activating the RNA-dependent DNA methylation (RdDM) pathway. Our results emphasize that the interplay between DNA and histone methylation mechanisms is intrinsic to plant cells. We also found that once epigenetic modification states were induced by the tethering of either DRM1 or SUVH9, the modification was maintained even when the direct tethering of the effector was inhibited. Our system enables the analysis of more diverse epigenetic effectors and will help to elucidate the chromatin assembly mechanisms of plant cells.

Transfomasi Genetik : Peluang dan Tantangan

Plants have an important role in the development of human civilization. Human civilization progresses along with the development of the agricultural system and vice versa. Therefore humans have been trying to improve cultivated plants for thousands of years by trial and error. Traditionally, mankind have improved plants through repeated selection. However, the human population is increasing so rapidly that it necessitates a doubling of the increase in world food production. The program is constrained by the fact that we cannot increase arable land. So one solution is through biotechnology which is expected to produce more food without demanding an increase in agricultural land. At present, the application of biotechnology in agriculture can be done by genetic engineering such as genetic transformation and gene editing which are almost entirely aimed at forming transgenic plants. One of the developments in biotechnology that has been widely used is GMO (Genetically Modified Organism) or plants that have been genetically modified or are popularly known as transgenic plants. Genetic modification of plants has become the most promising approach for increasing crop yields, providing nutrients, exploiting stressed land, overcoming the energy crisis, and for producing cost-effective biopharmaceuticals.

Evaluation of the expression of induced genes in response to dehydration stress of Camelina (Camelina sativa) calli.

Plants are constantly exposed to various biological and non-biological stresses that endanger their lives. Drought stress is one of the abiotic stresses that have a great impact on the yield and life of plants and is one of the main causes of reduced crop yields. Reducing the effects of environmental stresses such as drought using methods such as irrigation, fertilizer application and appropriate planting methods is limited. Therefore, genetic modification of plants is an important effort to minimize the effect of environmental stresses. in this research, Twenty disinfected camelina seeds were cultured on the MS medium containing 3% sucrose, 0.8% agar and pH 5.8 under a laminar hood. After 14 days, the cotyledon explants (about 1 cm) were separated from the seedlings and placed on the callus induction medium. The MS callus induction medium containing 0.5 mg / l kinetin, 2 mg / l -2,4 D, 3% sucrose, 0.8% agar and pH 5.8. Samples were subcultured every two weeks to the same medium and calli were formed after 4 weeks. Then the calli were transferred to the medium containing a concentration of 30% PEG. To study gene expression, first callus samples were treated with liquid nitrogen and to study the effect of drought stress on gene expression, this sample was sent to Zagros Bioidea Company located in the Razi University Incubator. Gene expression was performed through microarray technology. The results showed that seven different genes whose expression increased by almost six times the control value can be mentioned, including Cold-acclimation protein (CAP160), NAC10, Abscisic acid (ABA), ABF4, CRK3, lysM domain receptor-like kinases (LYKs) and Basic/helix-loop-helix(bHLH130-like). Drought tolerance is not a genetically simple trait, but a quantitative and complex trait with various aspects that require the use of molecular methods to investigate the relevant mechanisms. This study aimed to investigate the expression of different genes of callus tissues of the Camelina plant under stress and non-stress conditions by microarray method.

Evolution and epidemiology of global populations of nursery-associated <i>Agrobacterium</i>

Agrobacterium tumefaciens is unique in that it can facilitate the interkingdom transfer of DNA and genetically modify its plant host. While Agrobacterium has been coopted for use in the genetic modification of plants, it is also a major pathogen, causing crown gall disease in the nursery, orchard, and vineyard industries. Pathogenicity in Agrobacterium is the result of two components. First is the Ti plasmid, which carries virulence genes and the transferred T-DNA region. The second component is the chromosome of Agrobacterium, which comprises diverse bacterial lineages and multiple species-level groups. The Ti plasmid can be transferred from strain to strain, diversifying the pathogen and complicating efforts to understand its epidemiology. This system provides an opportunity to study transmission of plasmids and their impact on disease persistence and spread. However, the movement of plasmids, and diversity of chromosomal lineages, means that conventional methods of using whole genome SNPs to track outbreaks are not sufficient, and new techniques must be developed. Additionally, Ti plasmids, like Agrobacterium, are genetically diverse and represent multiple plasmid types. Using a framework of 200 sequenced Agrobacterium genomes isolated from around the world, and a previously developed model of Ti plasmid types, we modelled their epidemiology. Key to this study was that we first separately analyzed plasmids and strain. Combining results revealed links between nurseries, potential horizontal transfer of the plasmid between strains within nurseries, global spread of plasmids, and long-term persistence of plasmids in the agricultural system. Agricultural practices have the potential to promote the diversification of pathogens and the emergence of new pathogen lineages.

Heterologous expression of β-alanine betaine biosynthesis gene increases <i>Nicotiana tabacum</i> resistance to abiotic stresses

Plant genetic modification in order to increase their tolerance to various abiotic stresses has been of exceptional importance in recent years. Heterologous expression of glycine betaine (GB) biosynthetic genes leads to increased salt and drought tolerance in various plant species by maintaining the osmotic balance with the environment and stabilizing the quaternary structure of complex proteins. However, GB biosynthesis in transgenic plants is limited by choline availability. Members of the Plumbaginaceae family accumulate -alanine betaine (AB) instead [1]. The synthesis of AB is not limited by the availability of choline, as it follows the methylation pathway of the aproteinogenic amino acid -alanine.
 For the first time, we have generated Nicotiana tabacum plants expressing the -alanine N-methyltransferase (LlBANMT) gene of Limonium latifolium. Transgenic plants were much less affected by such abiotic stresses as increased salinity, excessive illumination, and low temperature. The experimental Nicotiana tabacum lines had lower rates of chlorophyll degradation under stress conditions compared to the control plants. LlBANMT expression also resulted in less biomass loss under stress conditions, which was associated with higher activities of reactive oxygen species detoxification systems and healthier cell membranes. The presented data demonstrate for the first time the protective properties of LlBANMT heterologous expression and shed light on the mechanisms of its action.

Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture

Of the more than 370 000 species of higher plants in nature, fewer than 0.1% can be genetically modified due to limitations of the current gene delivery systems. Even for those that can be genetically modified, the modification involves a tedious and costly tissue culture process. Here, we describe an extremely simple cut-dip-budding (CDB) delivery system, which uses Agrobacterium rhizogene to inoculate explants, generating transformed roots that produce transformed buds due to root suckering. We have successfully used CDB to achieve the heritable transformation of plant species in multiple plant families, including two herbaceous plants (Taraxacum kok-saghyz and Coronilla varia), a tuberous root plant (sweet potato), and three woody plant species (Ailanthus altissima, Aralia elata, and Clerodendrum chinense). These plants have previously been difficult or impossible to transform, but the CDB method enabled efficient transformation or gene editing in them using a very simple explant dipping protocol, under non-sterile conditions and without the need for tissue culture. Our work suggests that large numbers of plants could be amenable to genetic modifications using the CDB method.

The chloroplast genome of Farsetia hamiltonii Royle, phylogenetic analysis, and comparative study with other members of Clade C of Brassicaceae

BackgroundFarsetia hamiltonii Royle is a medicinally important annual plant from the Cholistan desert that belongs to the tribe Anastaticeae and clade C of the Brassicaceae family. We provide the entire chloroplast sequence of F.hamiltonii, obtained using the Illumina HiSeq2500 and paired-end sequencing. We compared F. hamiltonii to nine other clade C species, including Farsetia occidentalis, Lobularia libyca, Notoceras bicorne, Parolinia ornata, Morettia canescens, Cochlearia borzaeana, Megacarpaea polyandra, Biscutella laevigata, and Iberis amara. We conducted phylogenetic research on the 22 Brassicaceae species, which included members from 17 tribes and six clades.ResultsThe chloroplast genome sequence of F.hamiltonii of 154,802 bp sizes with 36.30% GC content and have a typical structure comprised of a Large Single Copy (LSC) of 83,906 bp, a Small Single Copy (SSC) of 17,988 bp, and two copies of Inverted Repeats (IRs) of 26,454 bp. The genomes of F. hamiltonii and F. occidentalis show shared amino acid frequencies and codon use, RNA editing sites, simple sequence repeats, and oligonucleotide repeats. The maximum likelihood tree revealed Farsetia as a monophyletic genus, closely linked to Morettia, with a bootstrap score of 100. The rate of transversion substitutions (Tv) was higher than the rate of transition substitutions (Ts), resulting in Ts/Tv less than one in all comparisons with F. hamiltonii, indicating that the species are closely related. The rate of synonymous substitutions (Ks) was greater than non-synonymous substitutions (Ka) in all comparisons with F. hamiltonii, with a Ka/Ks ratio smaller than one, indicating that genes underwent purifying selection. Low nucleotide diversity values range from 0.00085 to 0.08516, and IR regions comprise comparable genes on junctions with minimal change, supporting the conserved status of the selected chloroplast genomes of the clade C of the Brassicaceae family. We identified ten polymorphic regions, including rps8-rpl14, rps15-ycf1, ndhG-ndhI, psbK-psbI, ccsA-ndhD, rpl36-rps8, petA-psbJ, ndhF-rpl32, psaJ-rpl3, and ycf1 that might be exploited to construct genuine and inexpensive to solve taxonomic discrepancy and understand phylogenetic relationship amongst Brassicaceae species.ConclusionThe entire chloroplast sequencing of F. hamiltonii sheds light on the divergence of genic chloroplast sequences among members of the clade C. When other Farsetia species are sequenced in the future, the full F. hamiltonii chloroplast will be used as a source for comprehensive taxonomical investigations of the genus. The comparison of F. hamiltonii and other clade C species adds new information to the phylogenetic data and evolutionary processes of the clade. The results of this study will also provide further molecular uses of clade C chloroplasts for possible plant genetic modifications and will help recognise more Brassicaceae family species.

New Insights Into Tissue Culture Plant-Regeneration Mechanisms.

Plant regeneration occurs when plants repair or replace damaged structures based on the totipotency and pluripotency of their cells. Tissue culture is one of the most widely used regenerative technologies. Recently, a series of breakthroughs were made in the study of plant regeneration. This review summarizes two regenerative pathways in tissue culture: somatic embryogenesis and de novo organogenesis. Furthermore, we review the environmental factors influencing plant regeneration from explant sources, basal culture medium, plant growth regulators, and light/dark treatment. Additionally, we analyse the molecular mechanisms underlying two pathways. This knowledge will promote an understanding of the fundamental principles of plant regeneration from precursor cells and lay a solid foundation for applying plant micropropagation and genetic modification.

Evaluation of Antibiotic-Based Selection Methods for Camelina sativa Stable Transformants.

Camelina sativa (Camelina) is an oilseed crop that in recent years has gained importance due to its closeness to the plant model organism Arabidopsis thaliana (Arabidopsis), its low agronomical requirements, and the ability to grow under temperate conditions. To explore all the agronomical and biotechnological possibilities of this crop, it is important to evaluate the usability of the molecular procedures currently available for plants. One of the main tools for plant genetic modification and genetic studies is stable plant transformation. In the case of Arabidopsis, as well as Camelina, floral dipping is the easiest and most used method, which is followed by a selection for stable transformants. Commonly used selection methods for Camelina involve Discosoma sp. red protein (DsRed) fluorescence screening. However, many widely used plant transformation vector systems, for example those used in Arabidopsis and grasses, rely on antibiotic resistance selection. In this study, we evaluated the usability of different antibiotics including kanamycin (Kan), hygromycin (Hyg) and BASTA, and propose optimised protocols for selecting T1 and subsequent generation Camelina transformants, as well as crossing of Camelina lines expressing different transgenes. Finally, we also showed that overexpression of genes encoding enzymes from the seco-iridoid pathway of Catharanthus roseus using Hyg or BASTA-based expression constructs could be successfully achieved in Camelina, demonstrating the potential of these methods for metabolic engineering. Overall, in this study we show an efficient way to sterilize seeds, handle and perform selection of Camelina for use with transformation vectors designed for Arabidopsis thaliana. We also demonstrate a successful method to cross Camelina sativa and provide qRT-PCR results to prove its effectiveness.

Strategies for crop nutraceutical content enhancement, a review

AbstractThe increase in cardiovascular and degenerative diseases around the world, in addition to other metabolic alterations derived from an inadequate diet, have highlighted the need to consume more nutritious and healthy foods, that can also provide added value in the prevention of diseases. In this sense, nutraceutical‐rich functional foods have become a sustained growing market derived from consumer demands. Different strategies have been developed to enhance the content of nutraceuticals compounds in crops and end products, as well as to increase the bioavailability of them. These strategies have been grouped in biofortification techniques (e.g., foliar, and soil application of nutrients, germination, and soaking of seeds, genetic modification of plants and crops) and techniques for nutraceutical enrichment (e.g., fermentation of food byproducts, nanoparticles, and induced post‐harvest stress). The utilization of these techniques thrives a new approach for novel biofortified, and nutraceutical enriched foods that meet the needs of the most demanding and health‐conscious consumers.

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences.

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.

All Plant Breeding Technologies Are Equal, but Some Are More Equal Than Others: The Case of GM and Mutagenesis.

A pervasive opposition to genetically modified (GM) foods has developed from the notion that they pose a risk to human and environmental health. Other techniques for the genetic modification of plants, such as sexual crossing and mutagenesis breeding, have mostly remained unchallenged. This research aims to investigate public perception of plant breeding technologies. Specifically, sexual crossing, mutagenesis, transgenics (GM) and gene editing. It was expected that attitudes and intentions would be most positive and the perception of risk lowest for plant genetic modification through sexual crosses. Scores on these variables were expected to be similar between mutagenesis, GM and gene editing. It was also expected that attitudes, intentions and risk perception would change (becoming more positive) once participants learned about foods developed through these technologies. Participants reported their attitudes, intentions and risk perception at two points in time. At Time 2, they were presented with pictures of food items developed through sexual crossing, GM and mutagenesis. The results showed that mutagenesis stood out as the most negatively perceived technology, whereas genetic development via sexual crosses was generally perceived as positive. The results highlight the importance of messaging, framing in consumer attitudes.

Genetic Modification of Plants: An Emerging Technology

Genetically modified (GM) plants can be created by adding specific DNA sequences obtained from the same plant species or different species. Which aims to achieve higher yields, disease and pest resistance, herbicide tolerance, production of antibodies, and other pharmaceutical molecules by manipulating gene expression to alter the protein properties. Insect resistant plants reduce the damage caused by pests and diseases. Herbicide glyphosate or glufosinate-tolerant GM plants gave promising results in combating weed. The properties of plants such as metal uptake, transport, accumulation, and detoxification of organic pollutants can be enhanced by genetically manipulating the fast-grown and introducing the responsible gene from the hyper accumulative species. GM plants can be used to produce cost-efficient and manageable drugs, vaccine, and biopharmaceuticals, if certain limitations are to be considered such as quality of final products, techniques for extraction and processing of biopharmaceuticals, and biosafety. Despite all these benefits, its adverse effects on the environment and human health have always been a matter of concern. The main limitation includes a horizontal transfer of the transgene to other species which may code for the specific antibiotic and herbicide resistance. It might be the possible transmission of resistance from the food products to the whole human population via intestinal bacteria. To address this several methods, need to be adopted to either keep away or eliminate marker genes from the transformed plants before growing in the field. Many scientists have come up with strategies to generate marker-free transgenic plants to give us safe and reliable GM technology.

Selecting Aragonez Genotypes Able to Outplay Climate Change–Driven Abiotic Stress

High temperatures and extreme drought are increasingly more frequent in Portugal, which represents a strong threat to viticulture in certain regions of the country. These multifactorial abiotic stresses are threatening viticultural areas worldwide, and the problem can hardly be overcome only by changing cultural practices. This scenario has raised a major challenge for plant scientists to find ways to adapt existing varieties to the new conditions without loss of their characteristic flavors, yield, and associated varietal character of wines. Aragonez (syn. Tempranillo) is one such variety, widely cultivated in Portugal and Spain, with specific characteristics associated with terroir. In this context, insight into intravarietal variability to enable its exploitation for selection becomes an important tool to mitigate the effect of multifactorial stresses driven by climate changes. The present work describes an innovative selection approach: selection for abiotic stress tolerance, measured by the leaf temperature of clones under environmental conditions of drought and extreme heat. This evaluation was complemented with values of yield and quality characteristics of the must (pH, acidity, °Brix, and anthocyanins). The application of this methodology was done in an experimental population of 255 clones of Tempranillo for 3 years. The genotypes were then ranked according to their level of tolerance to abiotic stress without loss of yield/quality. To understand the differences at the transcription level that could account for such variability, several of the most tolerant and most sensitive genotypes were analyzed for key genes using reverse transcriptase–quantitative polymerase chain reaction. The results enabled the selection of a group of genotypes with increased tolerance to stress, in relation to the average of the variety, which maintained the typical must quality of Aragonez. In parallel, several transcripts previously acknowledged as markers for abiotic stress tolerance were identified in several clones and are possible targets for plant breeding and genetic modification and/or to develop screening procedures to select genotypes better adapted to the abiotic stress driven by climate change.

Improving the Efficiency of Adventitious Shoot Induction and Somatic Embryogenesis via Modification of WUSCHEL and LEAFY COTYLEDON 1.

The induction of adventitious organs, such as calli, shoots, and somatic embryos, in tissue culture is a useful technique for plant propagation and genetic modification. In recent years, several genes have been reported to be adventitious organ inducers and proposed to be useful for industrial applications. Even though the Arabidopsis (Arabidopsis thaliana) WUSCHEL (WUS) and LEAFY COTYLEDON 1 (LEC1) genes can induce adventitious organ formation in Arabidopsis without phytohormone treatment, further improvement is desired. Here, we show that modifying the transcriptional repression/activation activities of WUS and LEC1 improves the efficiency of adventitious organ formation in Arabidopsis. Because WUS functions as a transcriptional repressor during the induction of adventitious organs, we fused it to an artificial strong repression domain, SUPERMAN REPRESSION DOMAIN X (SRDX). Conversely, we fused the strong transcriptional activation domain VP16 from herpes simplex virus to LEC1. Upon overexpression of the corresponding transgenes, we succeeded in improving the efficiency of adventitious organ induction. Our results show that the modification of transcriptional repression/activation activity offers an effective method to improve the efficiency of adventitious organ formation in plants.

PHYSIOLOGICAL RESPONSE OF CONVENTIONAL AND TRANSGENIC MAIZE TO WATER STRESS

Genetic modifications of plants can affect their ability to use environmental resources. It is known that the anthropic action has caused global climate changes, such as increases in temperature and drought. In view of this, the objective of this study was to evaluate if the addition of the Bt gene influences the physiological performance of isogenic maize hybrids grown under water stress and optimal conditions. Three Bt and non-Bt maize hybrids (Agromen 30A95, Dow 2B707 and BG 7049Y) were cultivated in soil with moisture content at 60% (water stress) and 100% of the field capacity (FC). Among the hybrids, the 30A95 presented greater variation between Bt and non-Bt hybrids in regard to physiological variables. The addition of the Bt gene practically had no impact on the photosynthetic performance of the 2B707 and BG7049 hybrids. The presence of the Bt gene increased the sensitivity to water stress in the hybrids, with reduction of maximal fluorescence and chlorophyll content in the plants. Regardless of the genetic factor (Bt or non-Bt), water stress reduced transpiration, stomatal conductance, photosynthetic rate, and maximum quantum yield of the PSII in maize plants. It was concluded that the presence of the Bt gene in maize indicates a reduction in the photosynthetic potential of the plant grown under water stress.

Creating a tobacco line with a weaker antifeedant property against colorado potato beetle

Background . Genetic modification of plants is one of the promising strategies to increase their resistance to insect pests. The development of metabolic or RNA interference systems for plant protection requires appropriate models of host-insect interactions. Nicotiana tabacum L. is a classical model plant used in molecular and metabolic engineering. We consider tobacco as a model for developing protective strategies against Colorado potato beetle ( Leptinotarsa decemlineata Say, CPB). Normally, tobacco is toxic for CPB due to high content of nicotine and related alkaloids in leaves. Modification of the tobacco genome could provide tobacco genotypes with altered metabolism suitable for CPB feeding. It is known that different mutations in Berberine Bridge-Like (BBL) genes cause different alterations in tobacco leaf alkaloid levels. In the current study, the Cas9/gRNA system targeting members of the BBL gene family of tobacco was used to create a line which can serve as a diet for CPB. Results . In order to obtain tobacco with modified alkaloid content, two gRNAs matching target sequences in six BBL genes were selected. Each gRNA was cloned into a gRNA/Cas9 generic vector. The created constructs were mixed and used for biolistic transformation of tobacco leaf explants together with the pBI121 plasmid harboring the kanamycin resistance gene nptII and the reporter E.coli betaglucuronidase (GUS) gene. Regenerants were selected on 100 mg/l of kanamycin and checked for transgene presence by histochemical GUS-assay. Unexpectedly, the regenerated plants displayed a variety of adverse phenotypic effects including different degree of growth and rooting inhibition, early flowering, increased number of internodes, changes in leaf shape, fusion of flowers, longostyly, and partial sterility. Only one from seven obtained calli produced a population of regenerated plants without severe phenotypic abnormalities. The NtaBBL5-14 line of clonally propagated plants was selected from this population and used for a CPB feeding experiment. It was shown that CPB larvae consume the leaves of NtaBBL5-14 line ten times more efficiently than the leaves of control plants (97±0.5% vs. 9±3% in 24 h respectively). Conclusion . The NtaBBL5-14 tobacco line is suitable for CPB feeding and can be further used as a model for studies in plant-pest interaction. The modification of other genes regulating nicotine metabolism can be a promising strategy to obtain tobacco plants edible for CPB with less pleiotropic effects.

Challenges and opportunities of transgenic agricultural products in Iran: convergence of perspectives using Delphi technique

BackgroundWidespread production and use of transgenic products across the world in recent decade has led to different reactions and point of views. Iran is also one of the consumers of these products. However, there has been an ever-increasing controversy between supporters and opponents of the use of these products in Iran and no constructive agreement has been attained on this issue. Accordingly, the main goal of present study was to determine the challenges and opportunities of transgenic agricultural products in biotechnology and plant breeding specialists’ point of view.MethodologyDelphi technique was used to achieve this goal. The statistical population was Iranian specialists in plant breeding, biotechnology, and genetic modification. The snowball sampling approach was employed to select the samples.ResultsIn general, 16 concepts in the dimension of opportunities and 25 concepts in the dimension of the challenges were identified. Analyzing the agreement rates showed that the panel of experts agree most on 14 concepts out of 16 concepts identified for opportunities. In the dimension of challenges for transgenic products, only 17 concepts received acceptable agreement scores (over 80%).ConclusionIn general, categorizing the concepts of transgenic products’ opportunities led to identification of two main aspects namely “food security” and “biosafety”. Challenges were also categorized into four strata of structural, educational, cognitive, and informational challenges. In addition, it should be mentioned that the lack of familiarity with methods, goals, and results of genetic engineering and gene transferring methods in transgenic plants is the most important aspect of people’s concern. Although it is not possible to get full knowledge about genetic engineering and genetic manipulation methods for all those who are the consumers of transgenic products, their awareness and recognition about the goals and positive/negative effects of the use of these products can be increased using specific strategies. For instance, one of the strategies is to focus and invest on mass media that which provides high availability, vast coverage, and low-cost. Accordingly, mass media (such as radio and television) can act as the best educational and informational tools for development and application of transgenic plants.