Methods for Plant Genetic Modification

CHAPTE R 15

Methods for Plant Genetic Modification Coralia Bleotu*,**, Lilia Matei*, Laura D. Dragu*, Liana Grigorescu†, Carmen C. Diaconu*, Gabriela Anton* *Stefan S. Nicolau Institute of Virology, Bucharest, Romania; **University of Bucharest, Bucharest, Romania; †Prof. Alexandru Obregia Clinical Hospital of Psychiatry, Bucharest, Romania

1 Introduction Genetic modifications (GM) are a group of methods that alter the genetic composition of a plant or animal to improve its nutritional content, shelf life, flavor, color, texture, agronomic properties, and processing characteristics. Plant genetic manipulation methods can be divided into those requiring in planta transformation or tissue culture (Darbani et al., 2008). In planta transformation is a direct transformation method that does not require tissue culture and regeneration steps, thus avoiding somaclonal variation. This method can produce large amounts of transgenic plants that accumulate high concentrations of soluble proteins in a short time. However, the mean frequency of transformed progeny is relatively low and very variable (Darbani et al., 2008). Two Agrobacterium-mediated in planta methods (floral dip and vacuum infiltration) are usually used for both mono and dicot plants. Floral dip consists of dipping the floral elements in Agrobacterium culture, but it has low transformation efficiency and randomly integrates the foreign gene into the host genome. Vacuum infiltration produces transgenic plants through Agrobacterium infection of flowering plants. Transgenic plants are selected on media using a screening marker (Shamloul et al., 2014). The floral dip and vacuum infiltration methods are frequently used in agronomic crops (Solahil et al., 2016). Only a limited number of species are transformed in the absence of a tissue culture-based regeneration system. The disadvantages of tissue culture-based transformation methods are that they are time consuming and produce somaclonal variation that can affect the qualitative and quantitative characteristics of the plant (Labra et al., 2004). The use of tissue culture-based methods can lead to unwanted genetic (e.g., point mutations and chromosomal aberrations) and epigenetic changes (e.g., methylation). Epigenetic changes are thought to underpin various well-known tissue culture phenomena (Smulders and de Klerk, 2011) and epigenetic silencing is considered for the silencing of transgenes. Epigenetic interference with the stability of transgene expression is reduced in plants whose plastid genome has been engineered for stable transgene expression (Moeller and Wang, 2008). Genetically Engineered Foods http://dx.doi.org/10.1016/B978-0-12-811519-0.00015-7

385

Copyright © 2018 Elsevier Inc. All rights reserved.

386  Chapter 15

2  Methods for Genetic Modification There are two main techniques for genetic manipulations: genetic and nongenetic engineering; however, there is not a dichotomy between genetic and nongenetic engineering as some mechanisms are common to both techniques.

Methods for genetic modification

Nongenetic engineering

Genetic ­engineering

• Simple selection (the selection of plants for continued propagation according to a desired phenotype) • Embryo rescue (the rescue of plants from hybrid embryos that cannot survive in vivo) • Crossing (obtaining a hybrid by mating sexually compatible individuals of different varieties) • Somatic hybridization (obtaining new hybrids through the in vitro fusion of protoplasts derived from different plants) • Somaclonal variation (genetic variability found among progeny of cells/tissue cultures in vitro) • Mutation breeding (the use of chemicals or physical mutagens to produce new plant varieties) • Cell selection (the isolation and cell-culture growth of cells selected for desired phenotypes that can be used to regenerate the whole plant) • Targeted manipulation of genetic material (e.g., cloning, viral vectors, microprojectile bombardment, electroporation, or microinjection) • Nontargeted manipulation of genetic material (random DNA mutations induced by chemicals or physical mutagens)

2.1  Nongenetic Engineering Methods The oldest method of engineering consisted of selecting (over several generations) individual seed populations that met the desirable features (e.g., special phenotype or physical characteristics). This technique allowed the production of genetically homogenous seeds from heterogeneous ones and the production of new varieties. The lengthy selection process resulted in the generation of food plant species that were significantly different from the wild variant from which they were derived (Moeller and Wang, 2008). The method is still used but is now based on molecular analysis that identifies certain selection markers (mainly those that address resistance to plant diseases) thus allowing a more rapid selection of species with important agronomic traits. Crossing allows researchers to obtain hybrids from two sexually compatible plants; however, this is a laborious method as recombination is random and requires a large number of hybrid intermediates to be produced before those with the desired features are obtained. Improved variants using this technique are currently in use. Interspecies crossing can be achieved either naturally (in particular conditions) or through human intervention and involves the integration

Methods for Plant Genetic Modification  387 of genes from one species into the genome of a closely related species. Hybrids produced using this method have the characteristics of both parents. Cytogenetic manipulations were coupled with interspecific hybridization to transfer disease- and insect-resistance genes into crops. (Jauhar, 2006). There are several patents that have used interspecies crossing to obtain new varieties of plants (i.e., apomictic soybean varieties having a determinate growth habit, patent number 9210847/2015; tomato plants that exhibit resistance to Botrytis cinerea and have commercially desirable characteristics, patent number 8829280/2010; lettuce (Lactuca sativa L.) plants with resistance against the lettuce aphid Nasonovia ribisnigri (Mosley); and hybrid pepper plants resistant to geminiviruses, tobamoviruses, and damage by Xanthomonas). Chromosomal translocations can be performed to transfer large chromosomal segments between related species. Although the method has proved valuable for obtaining plant varieties, the length of the chromosomal segments also allows the transfer of some unwanted genes. Recent advances in chromosome engineering have facilitated the transfer of small DNA fragments and produced better results in the nutrition and food industries (Singh, 2011), mostly for genes that confer resistance to herbicides and insects (Yu et al., 2016). There are cases where hybrid embryos produced from natural pollination between species (interspecies crossing) have not been capable of maturation. Embryo rescue technology (or embryo culture) is based on the insertion of the embryo in a special culture medium before seed abortion can occur and is used to breed parental lines with incompatible genomes (Mahgoub, 2015). The technology has been used to breed various crop species and has contributed to the production of useful species for the diet. The disadvantages of this method (i.e., the numerous selection processes, the inability to remove some undesirable traits, and the inability to use transgenics from unrelated species) required the implementation of modern technologies (Moeller and Wang, 2008). Somatic hybridization (cell fusion) is a nonsexual genetic method based on the fusion of protoplasts derived from different plants. The removal of cellular walls through enzymatic techniques (i.e., degradation of polysaccharides by cellulase, hemicellulase, and pectinase) and the pooling of protoplasts (in the presence of a chemical/electrical fusogen) results in the production of a heterokaryon (Mahgoub, 2015) which has genetic material from both plant sources. This method overcomes the obstacles of hybridization pollination but not those related to chromosomal incompatibilities and is not currently used (National Research Council Committee, 2004) because it requires tissue cultures which may lead to genetic instability and somaclonal variation. Somaclonal variation is an important source of phenotypic variation in plants and is caused by chromosomal rearrangements (that lead to spontaneous mutations) or epigenetic changes (induced by stress) during tissue culture (Mahgoub, 2015). Plant tissue culture refers to techniques that propagate plant cells/tissues/organs using a specific nutrient substrate. The occurrence of somaclonal variations had been attributed to numerous stress factors, including

388  Chapter 15 explant preparation (e.g., wounding and sterilization), imbalances of media components (i.e., sugar and plant growth regulators, such as auxin and cytokines), and the in vitro culture environment (e.g., lighting and humidity conditions) (Krishna et al., 2016; Sato et al., 2011). Point mutations or chromosome rearrangements induced in somaclonal variation segregate, especially in the first generation. This technique can be used to improve plant traits (mostly for crops) and to select complicated traits (i.e., yield, soluble solids, sweetness, texture, or shelf life). No in vitro methods have been developed to date (Krishna et al., 2016). Selection failure can occur due to negative modifications, positive changes effected in a negative way, or instability of the changes after selfing or crossing (Karp, 1992). However, somaclonal variation is cheaper than other methods of genetic manipulation. Tissue culture is one of the most important steps in plant transformation as novel variants have been reported among somaclones, thus highlighting the effect of tissue culture on locations of the genome that are not accessible through conventional/mutation breeding. The studies conducted so far have made tissue culture systems available for a greater number of plants than could be handled by transformation. There is no need to identify the genetic foundation of the trait using this method, nor is there a need to isolate and transduce the responsible gene. Using cell culture to create somaclones avoids the risks of receiving chimeric expression. Mutagenesis induced by chemicals and physical factors is used to induce random modifications in the genomes of plants. The randomness and nonreproducibility of these mutations makes the methods not useful for food. Ethyl methanesulfonate, calcium phosphate (Chowdhury et al., 2004), and lipofection (Kawata et al., 2003) are commonly used chemicals; electroporation, biolistics, vacuum infiltration, silicon carbide whiskers, ionizing radiation, and shock wave mediated transformation are commonly used physical factors (Rivera et al., 2014). Vacuum infiltration is used to facilitate the entry of some strains of Agrobacterium (under negative pressure) into the intercell spaces of certain cell types and has the advantage of low somaclonal variation levels as no tissue culturing is used (Rivera et al., 2014). The main application of this technique is the production of plant-derived vaccines for human clinical trials (Lai and Chen, 2012). However, induced mutagenesis can lead to deleterious mutations and has been used for more than 2000 varieties of crop in the United States.

2.2  Genetic Engineering Genetic engineering involves the targeted change of a gene sequence to produce a specific result. In this regard, genes encoding proteins of interest are identified, isolated, inserted into a delivery system, and transferred into an appropriate host through complex processes known as recombinant DNA technology. The methods of introducing DNA into plant cells can be divided into two major categories: indirect delivery through biological systems (via bacteria, usually Agrobacterium tumefaciens) and direct delivery using an intermediate host (usually biolistic and protoplast transformation).

Methods for Plant Genetic Modification  389 2.2.1  Biological-based transformation systems Biological-based transformation methods use plasmids, which are circular extrachromosomal double-stranded molecules of DNA found in bacteria, yeast, and some eukaryotic cells. These molecules are not essential for host survival but they can confer them some advantages in certain environmental conditions or over other organisms from the same ecological niche (Actis et al., 1999). Plasmids are frequently used as vectors to deliver genes into a host. The most common method for generating recombinant plasmid uses restriction enzymes and DNA ligases. In this regard, the donor DNA and the plasmid are cut by restriction enzymes at specific targeted sites (each type of restriction enzyme recognizing a specific DNA targeted sequence) and DNA molecules are combined by DNA ligases and a chimera DNA molecule containing both donor gene and plasmid DNA is formed (Griffiths et al., 2000; Lodish et al., 2003). The recombinant plasmid is subsequently introduced into bacterial cells where it replicates due to its autonomous replication capacity (Carattoli, 2013); plasmid vectors contain a replication origin that assures replication when initiated by host-cell enzymes. A selectable gene, usually for drug resistance, is also inserted in the plasmid vector structure to allow transformed bacterial cells to be selected (Griffiths et al., 2000; Lodish et al., 2003). Other method to generate plasmid vectors uses fusion polymerase chain reaction, in which primers that overlap different DNA fragments allow the combination of two different DNA molecules by their fusion during PCR. Vectors can also be obtained using different recombinatorial cloning systems that allow modification of the fungal genome (Schoberle et al., 2013). Biological-based transformation systems mainly use Agrobacterium-mediated transformation in plants [e.g., A. tumefaciens-mediated (T-)DNA transfer]. A. tumefaciens is a natural genetic engineering instrument. It is a motile, aerobic bacterium that causes crown gall disease, and has the capacity to transfer its genome into the plant cell by tumor-inducing (Ti) or root-inducing (Ri) plasmids. Moreover, they contain contain genes encoding hormones and opines and assure the formation of a niche for Agrobacterium growth and proliferation. They also contain a region encoding proteins responsible for DNA transfer from the bacterium to the plant (Montagu and Zambryski, 2017). To use T-DNA as a vector, the Ti region is removed and the donor DNA is inserted between the border regions. This natural system is used in plant transformation, being responsible for the majority of GM plants on the market (Christie and Gordon, 2014; Gelvin, 2003; Ziemienowicz, 2014). The mechanism of T-DNA integration is still not clear. T-DNA enters into the host as singlestranded DNA (Park et al., 2015; Rashid and Lateef, 2016). The integration system of T-DNA is activated by phenolic and sugar molecules that induce virulence proteins in the plasmid thus leadingT-DNA into the host cell by VirD2 protein through the Agrobacterium type IV secretion system formed by VirB1–11 and VirD4 proteins. VirD2 is an endonuclease that cut T-DNA from plasmids and remains attached at the 5’ end of the T-strand. The nuclear targeting of the VirD2/T-strand complex is mediated by importin α proteins which allows

390  Chapter 15 the passage of the complex into the nucleoplasm through nuclear pores. Another protein implicated in the nuclear targeting of T-strands is VirE2, which also protects T-strands from nucleolytic degradation in the host cell (Gelvin, 2012; Rashid and Lateef, 2016; Tzfira and Citovsky, 2006; Ziemienowicz, 2014). T-strands are uncoated by proteolysis in the nucleus and converted into a double-stranded form by DNA repair machinery. The resultant intermediary form (ds T-DNA) is recognized as a broken DNA fragment and integrated into the host genome by an unknown pathway (Park et al., 2015; Rashid and Lateef, 2016; Tzfira and Citovsky, 2006). Advantages of this system are based on the possibility to transfer one or more genes through direct cloning of cassettes into T-DNA and plant transformation with a separate helper (vir) plasmid; however, the system’s have several disadvantages like specific targeting of cell nucleus and not organelles, limited transfer rate of DNA into monocots, and risk of transferring the insert to fungi. These disadvantages have led to alternative strategies based on P-DNA fragments (Darbani et al., 2008). Plant-derived (P-)DNA fragments replace Agrobacterium transfer T-DNA and have the potential to produce genetically engineered plants that contain only native DNA (Conner et al., 2007). Biological-based transformation systems also include viral-based transformations which uses plant viral vectors derived from geminivirus, tobacco mosaic virus, potato virus X, and cowpea mosaic virus (Gleba et al., 2014). Although these viral systems are highly expressed in plants (even at a systemic level) and have the potential for inducible expression, they are transiently expressed and show a lower infectivity for larger transgenes (Marillonnet et al., 2005). The most concerning aspect of viral vectors is their potential to induce posttranscriptional gene silencing, thus higher levels of transgene expression can be achieved via agroinoculation (Liu and Kearney, 2010). The development of new technologies for the transient and stable expression of recombinant proteins in plants using viruses and bacteria outside of the Agrobacterium host range is extremely valuable in plant science. These techniques allow precise genome modifications (Hartung and Schiemann, 2014). Sequence-specific nucleases accomplish targeted DNA modifications by creating double-strand breaks in the genes to be transformed (Voytas, 2013). Double-strand breaks represent DNA lesions that can lead to cell cycle arrest and, if not repaired, to cell death (Ambrosio et al., 2016). Cells have developed different DNA repair pathways to ensure the genome integrity, the two most important pathways being homologous recombination and nonhomologous DNA end joining. These repair pathways can be further controlled to reach the desired sequence modifications (Voytas, 2013). Zinc finger proteins (ZFP, ZFN) are a class of transcription factors, with Cys2-His2 zinc finger domain being one of the most common DNA-binding domains in the plant genome. ZFP are formed of a cleavaged domain derived from Fok (an IIS restriction enzyme) and a binding domain. The binding domain of a ZFP wraps around the DNA molecule in the presence of a zinc atom, each finger binding with 3–4 base pairs (bp). ZFP forms a compact ββα structure

Methods for Plant Genetic Modification  391 with the DNA molecule targeting a site of 9 bp (Maeder and Gersbach, 2016). To ensure double-strand DNA breaks, two binding sites of a ZFP located on the top and bottom strands of the DNA substrate in an inverted orientation, dimerize, and facilitate the nuclease domain activity. Since each ZFP from the dimerized complex requires two copies of 9 bp sequences in a tail-to-tail orientation, the specific recognition site must have 18 bp to induce cleavage of the DNA molecule (Durai et al., 2005). The ZFP binding domain has a modular structure. To generate new functional endonucleases with different sequence specificity and to use them in genetic engineering, the binding sites responsible for sequence-specific interactions should be changed while maintaining the backbone of the ZFP (Durai et al., 2005). With regard to this, the fingers can be treated as independent modules and assembled in accordance with target sequences or constructed based on developed protocols and strategies (Carroll, 2011; Maeder et al., 2009). Maeder et al. (2009) described a protocol of ZFN generation based on an “open source” combinatorial selection method called oligomerized pool engineering (OPEN). The ZFN engineering process can be divided into several stages in this process. First, the ZFN target sites within the gene of interest are selected using web-based software. The second step presumes the construction of B2H selection strains containing ZFN target half sites. Next, libraries encoding three-finger ZFN are created. The zinc finger arrays are constructed using OPEN, with each OPEN pool containing a collection of plasmids that encode three-finger proteins. Sequences encoding finger proteins are merged by overlapping PCR and cloned into an expression vector. The final step involves quantification binding activity of constructed ZFN in B2H reporter strains. Transcription activator-like effector nucleases (TALEN) are formed of a DNA-binding domain specific to TALE proteins (original from Xanthomonas) and a DNA-cleavage domain (the catalytic fragment of Fok1). The formation of dimers in essential for the induction of double-strand DNA breaks. The DNA-binding domain is formed of a tandem of 33–35 amino acids, followed by a single repeat of 20 amino acids. The amino acids in positions 12 and 13 of each repeat (repeat-variable diresidues) are responsible for recognizing and binding a single nucleotide base. There are currently several methods to engineer TALEN arrays. One limitation of TALEN is the presence of thymine in the 1 position directed by the constant N-terminal domain. The size and repetitive nature of TALENs challenges the stability of the delivery system (Cermak et al., 2011; Maeder and Gersbach, 2016). The generation of a TALEN construct involves the selection of target sites within the gene of interest and assembling the repeat modules into intermediary arrays of 1–10 repeats in plasmids with a selection gene. The correct arrays identified using restriction enzymes and electrophoresis are then joined into a backbone to make a final construct (with ampicillin resistance gene for selection). The structure of the final construct confirmed by DNA

392  Chapter 15 sequencing or digestion can be subsequently cloned into a vector of interest (Cermak et al., 2011). Clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR associated systems (CRISPR/Cas) represent an adaptive immune system of bacteria that is responsible for protection against plasmids and viruses. Short sequences of foreign nucleic acids are incorporated into CRISPR loci, transcribed, and processed into crRNA, which forms a complex with transactivating crRNA (tracrRNA) and CRISPR-associated proteins (Cas) and induce the cleavage of targeted foreign DNA by Cas nucleases (Maeder and Gersbach, 2016). There are three major types of CRISPR/Cas systems, type II being used for genetic engineering (Makarova et al., 2011). Type II CRISPR system components include a Cas9 protein, mature crRNA, and tracrRNA; for engineering purpose the last two components are fused into a single molecule of guide RNA (gRNA). The targeting specificity of CRISPR/ Cas9 system is determined by gRNA, specifically the crRNA sequence. The single limitation of this system is the need for a protospacer-adjacent motif (PAM), which must be positioned immediately downstream of the target site. The gRNA/Cas9 complex associates with PAM motifs and forms a DNA–RNA heteroduplex at the target site, inducing the cleavage of target DNA by Cas9 (Bortesi and Fischer, 2015; Kanchiswamy, 2016; Maeder and Gersbach, 2016). To generate a CRISPR/Cas system, a target-specific oligo is designed and annealed with a Cas9 specific scaffold oligo through the common complementary overlapping region. The resultant double-stranded DNA template is transcribed using the T7 promoter in the scaffold oligo structure. The functional gRNA is then purified from DNA (by treating the mixture with DNaseI) and from proteins, salts, and unincorporated nucleotides (using spin columns). The purified gRNA is introduced into a vector that also contains a Cas9 sequence and a selection gene and the structure of the final construct is confirmed by DNA sequencing (Ran et al., 2013). Generated constructs can be delivered into host cells using physical or chemical means or bacterial systems. 2.2.2  Nonbiological-based delivery methods Nonbiological-based methods of plant transformation include: (1) particle bombardment/ biolistics; (2) electroporation; (3) microinjection; (4) chemical transduction; (5) silicon carbide fibers; and (6) liposome-mediated transfection (lipofection). Microprojectile bombardment is also known as the biolistic method. Klein et al. (1992) were the first to report the delivery of DNA into plant cells using high-velocity microprojectiles. In this process, microprojectiles carrying donor DNA or RNA were accelerated and penetrated cell walls and membranes without killing them. This method allows gene delivery in many

Methods for Plant Genetic Modification  393 subcellular organelles, fungi, bacteria, plant cells and animal cells and does not require a vector (Rivera et al., 2012). Microparticles should be chemically inert, have a high DNA-binding affinity and be able to release DNA once inside the cell. They are usually made out of gold, tungsten or platinum and are 0.5–5 µm in diameter. DNA-coated microparticles are used as projectiles accelerate using a gene gun hitting a porous screen. Donor DNA is released after hitting the cells and it can integrate randomly into the host DNA (Brandenberg et al., 2011; Rivera et al., 2012). Different nanoparticle systems have been developed for donor DNA delivery, including mesoporous silica nanoparticles, calcium phosphate nanoparticles and polydendrimers. Calcium phosphate nanoparticles and polydendrimers can deliver donor DNA through coculture methods and do not need any assistance (Brandenberg et al., 2011; Rashid and Lateef, 2016). A series of techniques were elaborated to deliver donor DNA into protoplasts. These methods are based on the totipotent capacity of plant cells (Vasil and Vasil, 1972), which makes the regeneration possible from a protoplast. Protoplasts (plant cells without cell walls) are obtained by enzymatic removal of cell walls (Zhai et al., 2009), and donor DNA (e.g., naked DNA plasmids) can be introduced using electroporation and microinjection or chemical transduction. Electroporation induces the transient formation of micropores in the cellular membrane under an electrical field. Plant cells are transformed in protoplasts to ensure the access of donor DNA into the cell, which has been permeabilized by the application of an electrical impulse. Plant protoplasts can also take up other macromolecules (i.e., lipids and proteins) from the surrounding fluid. Electroporation is limited by the need of regeneration of the transformed cells and the limited transformation rate (Brandenberg et al., 2011; Rashid and Lateef, 2016; Rivera et al., 2012). Other techniques for protoplast transformation include microinjection, in which donor DNA is injected directly into the cell through a glass microcapillaryinjection pipette. The transformation of oil palm protoplasts (fixed in an alginate substrate) by microinjection of an exogenous DNA showed only 14% efficiency (Masani et al., 2014), although other research groups have reported efficiencies of 20%–53% in tobacco (Kost et al., 1995; Schnorf et al., 1991). However, this method remains laborious and also requires special equipment (Brandenberg et al., 2011; Rivera et al., 2012). Protoplasts can also be chemically transformed. The most commonly used technique is PEG treatment, which determines reversible cellular membrane permeabilization that allows donor DNA to enter into the host cell either as naked DNA or encapsulated into liposomes (Brandenberg et al., 2011). Poly-l-ornithine, poly-l-lysine, and dextran sulfate have also been used to increase the transfer efficiency while the use of spermidine protected DNA from shearing (Darbani et al., 2008). Other methods use plasmid DNA precipitation with calcium phosphate, the resulted complex invading through plasmatic membrane (Brandenberg

394  Chapter 15 et al., 2011). Although these methods are easy, plant regeneration from transfected protoplasts has a reduced efficiency thus limiting its utility. Several other methods have also been described; for example, Chen et al. (2007) used short arginine-rich intracellular delivery peptides to deliver plasmid DNA into the nucleus of living plant cells without protoplast formulation. Another method used for plant transformation is based on silicon carbide whiskers (silicon carbide fibers). Arshad group first describes this technique in 2008 and reported a transformation efficiency of 94% in cotton plants. The cotton transformants were stable and fertile and transgenes were transmitted in a Mendelian manner (Arshad et al., 2013; Asad et al., 2008). Silicon carbide fibers have high transformation efficiency in crops, such as maize and tobacco (Kaeppler et al., 1990) as they can introduce small holes into cellular walls without affecting viability. This method is easy and rapid and consists of mixing (under vortexing) silicon carbide fibers, a suspension of tissue (e.g., cell clusters, immature embryos, or callus) and plasmid DNA. The efficiency of this method depends on several parameters of the target cell, the fibers, and the vortex characteristics. The simplicity of transferring silicon carbide whiskers with DNA into the cells makes this method very attractive; however, the time required for embryogenic tissue culture to generate fertile plants is similar to other methods using transgenes. (Petolino and Arnold, 2009). Liposomes are spherical lipid bilayer vesicles of artificial origin, which can transfer the transgene into a target organism. Their transfer ability relies on the lipid bilayer fusing with the cell wall. Liposomes have several advantages that makes them appropriate vectors for gene delivery, such as their ability to protect transgenes against nucleases, their increased transfer efficiency, their ability to deliver to a variety of cell types, and their ability to deliver small organelles. (Gad et al., 1990).

3  The Role of Epigenetics in Plant Transformation One of the issues raised by transgenic technologies is the epigenetic mechanism involved in the regulation of gene expression. Epigenetic mechanisms (e.g., DNA methylation, histone modification, and noncoding RNAs) modulate gene expression and play an important role in defending against pathogens. Studies focused on the unstable expression of transgenes showed that methylation mechanisms involving promoters correlate with chromatin alterations. Two types of transgene-silencing phenomena have been reported, which are determined by the position of the gene insertion in the host chromosome or the number of copies of the same or homologous sequence (Rajeevkumar et al., 2015). In homologydependent gene silencing interactions between homologous DNA sequences leads to transgene silencing. When interacting genes display homology in promoter regions, silencing occurs at transcriptional level and is determined through methylation. The first breakthrough in this regard came from Matzke et al. (1989) who noted that transgene silencing in

Methods for Plant Genetic Modification  395 Nicotiana tabacum was associated with DNA methylation in the transgene promoter (Matzke et al., 1989). Briefly, silencing was determined through the interaction between two promoter counterparts present in two T-DNA constructs, of which only one displayed methylation. DNA methylation of transgenes is also accompanied by other epigenetic changes, such as chromatin remodeling, which protects the transgene against host endonucleases. Moreover, subsequent studies in transgenic tobacco plants (T-DNA containing a cDNA of the potato spindle tuber viroid) demonstrated the contribution of RNA to de novo DNA methylation at homologous regions. Small RNA molecules are also involved in targeting homologous DNA sequences for methylation and for degrading through dsRNA formation. Small RNAs can not only contribute to silencing through target specificity (Meyer, 2013), but can also transmit the signal to other plant organs, which could lead to the silencing of undesired genes. Noncoding RNA-mediated gene silencing has been explored for the development of noncoding RNAbased GM plants (Ramesh, 2013).

4  Potential Risks Related to the Use of Genetically Modified Foods GM may induce unintended changes that are associated with unwanted health effects. For this reason, the entire modified feature induced by the genetic modification (intended or unintended) must be tested for adverse health effects caused by the intake of a new substances or an intolerable dose of the substance. Detailed food composition assessments are determined using current analytical methods and DNA/RNA/protein profiling. Differences in composition must be correlated with their biological significance. To prevent adverse health effects, the unintended health effects of genetically engineered foods must be compared with the unintended effects of conventional genetically modified foods. Several human health hazards are associated with GM plants, such as the accidental introduction of a new allergen or toxin or antibiotic resistance. Gene transfer from one cell type to another leads to the synthesis of proteins that were absent in the parental cells. The amino acid sequence of the novel protein structure is the main risk for developing food allergies to transgenic foods. The term allergy defines the pathological immune reaction to an antigen in a specific food component. Proteins are considered to be the main food allergens and their consumption may induce sequential skin reactions and alterations in the functioning of the respiratory and circulatory system that can lead to anaphylactic shock (Bernstein et al., 2003; Kramkowska et al., 2013; Ladics et al., 2011). Proteins obtained as a consequence of GM have allergenic potential if their sequence is homologous to an allergen sequence. At a global level approximately 2% of adults and 6% of children have food allergies. (Bernstein et al., 2003; D’Agnolo, 2005; Kramkowska et al., 2013). Special attention is given to allergies resulting from the consumption of transgenic foods. One example is the highly publicized Aventis case, which highlighted the adverse effects of genetically modified maize. The modified plant (StarLink) contained a

396  Chapter 15 transgene that produces resistance to pesticides. The genetic information transferred from Bacillus thuringiensis to the maize cell induced the expression of the highly allergenic protein, Cry9c. StarLink corn was approved to enter the trade market exclusively as animal feed; however, shortly after commercialization the transgenic corn was found in human food. The dissemination of information through the media was followed by numerous reports of consumers experiencing symptoms associated with food allergies (i.e., headache, diarrhea, nausea, and vomiting) (Batista and Oliveira, 2009; Bernstein et al., 2003; Domingo, 2007; Kramkowska et al., 2013). Another example of a GM food allergy involves the production of soy enriched in methionine, an amino acid obtained by synthesis of a gene that was isolated from Brazil nuts. Walnuts and walnut plants are known to cause allergic reactions; therefore, the consumption of genetically modified soy represents a danger in people sensitive to nuts (Key et al., 2008; Kramkowska et al., 2013). GM foods that cause allergies are most commonly linked with the induction of novel genes and genes derived from organisms that have allergenic potential. In the case of Lucerne, the introduction of genetic material from sunflower seeds triggered allergies similar to those induced by the consumption of Brazil nuts (Kramkowska et al., 2013). Another negative effect of genetically modified plants is represented by the cellular synthesis of toxic products, which can increase the activation of neoplastic processes. This can be exemplified when rapeseed oil derived from genetically modified plants was allowed to enter the market in Spain in 1983. The consumption of rapeseed oil led to the death of many consumers, although previous studies in rats had demonstrated the existence of adverse effects. Research into this phenomenon has involved several centers and the intoxication is thought to have been induced by toxic oil syndrome (oil contamination with aniline or aniline derivatives). Experiments by Suarez et al. (1985) showed no significant differences between rats fed for 2 months with oil contaminated with aniline and control rats fed with uncontaminated oil. Quero et al. (2011) also pointed out that genetic diversity is one of the factors involved in the toxic oil syndrome response (Kramkowska et al., 2013; Suarez et al., 1985). Negative effects of genetically modified foods were also reported in the United States in 1989. In this event transgenic tryptophan caused pain (mainly in muscles and joints) and even several deaths. Eosinophilia-myalgia syndrome was a reaction at ltryptophan consumption. l-tryptophan is a dietary supplement and is usually used to treat insomnia or depression. In this case the toxicity was primarily related to the technological processes and purification of the l-tryptophan containing compounds (Kramkowska et al., 2013). The health effects of transgenic foods are thoroughly investigated in animals. Different researchers have presented data on the harmful influences of MON810 maize (with pest resistance) on pancreas, intestine, liver, and kidneys in rodents (Kramkowska et al., 2013). Similar studies have shown that different varieties of GM maize (MON810 and MON863) that are resistant to insects, NK603, and the herbicide Roundup, can be toxic to the liver and

Methods for Plant Genetic Modification  397 kidneys. This was confirmed in experiments where rats were fed for 90 days with 11 and 33% transgenic corn components; however, the study results were contested due to a small number of rats in the test group (80) compared to rats in the control group (4 times higher). In addition, toxic effects should have been evaluated over a longer period (i.e., 2 years) (de Vendomois et al., 2009, 2010). One study that was extended over a period of 2 years demonstrated that animals fed with maize NK603 and especially animals exposed to Roundup presented palpable tumors (Seralini et al., 2014). Female rats also showed a higher sensitivity to foods containing toxic compounds. One possible explanation could be the low concentration of flavonoid antioxidants, such as ferulic acid (reduced by 16%–30%) and caffeic acid (reduced by 21%–53%), caused by overexpression of 5-enolpyruvylshikimate-3-phosphate synthase from A. tumefaciens, which provide plant resistance to Roundup (Seralini et al., 2012). Modulating effects of isoflavonoids on the estrogen receptor have been reported in previous studies (Duke et al., 2003; Seralini et al., 2012). Agglutinin and lectin synthesized in GM potatoes are toxic to mammalian growth and development. A diet rich in genetically engineered potatoes, which contains lectin gene expression from Galanthus nivalis (Domingo, 2007; Key et al., 2008) causes deregulated cell division within gastric mucosal cells; however, it should be noted that the effects are linked not only to the presence of the transgene, but also to other genetic factors. It should also be added that the described experiments involved a small number of animals and a short study duration (10 days) (Domingo, 2007). Another aspect that must be mentioned is the risk of developing antibiotic resistance due to the transference of antibiotics resistance genes from GM organisms. Early biotechnological studies commonly used bacteria as markers to enable the identification of transformed cells. Regular use of therapeutic agents as transformation agents can lead to the transfer of resistance genes from physiological and pathogenic bacteria to digestive tract microbiota. Consequently, disease-causing pathogens can develop resistance to specific antibiotics. To avoid adverse health effects, it is recommended to eliminate antibiotic as selection markers (D’Agnolo, 2005; Kramkowska et al., 2013).

5 Conclusions There are many methods for obtaining GM plants and each has its advantages and disadvantages in terms of productivity in plants, but most importantly, in terms of their effects on human health. Increasing the concentration of substances with beneficial health effects can be accompanied by adverse health effects (e.g., due to the production of antinutritive compounds, toxins, or allergens). Therefore it is necessary that plants with new characteristics be tested for extended periods of time using successive generations of animals to eliminate possible risks to human health.

398  Chapter 15

References Actis, L.A., Tolmasky, M.E., Crosa, J.H., 1999. Bacterial plasmids: replication of extrachromosomal genetic elements encoding resistance to antimicrobial compounds. Front. Biosci. 4, D43–62. Ambrosio, S., di Palo, G., Napolitano, G., Amente, S., Dellino, G.I., Faretta, M., Pelicci, P.G., Lania, L., Majello, B., 2016. Cell cycle-dependent resolution of DNA double-strand breaks. Oncotarget 7, 4949–4960. Arshad, M., Zafar, Y., Asad, S., 2013. Silicon carbide whisker-mediated transformation of cotton (Gossypium hirsutum L.). Methods Mol. Biol. 958, 79–92. Asad, S., Mukhtar, Z., Nazir, F., Hashmi, J.A., Mansoor, S., Zafar, Y., Arshad, M., 2008. Silicon carbide whiskermediated embryogenic callus transformation of cotton (Gossypium hirsutum L.) and regeneration of salt tolerant plants. Mol. Biotechnol. 40, 161–169. Batista, R., Oliveira, M.M., 2009. Facts and fiction of genetically engineered food. Trends Biotechnol. 27, 277–286. Bernstein, J.A., Bernstein, I.L., Bucchini, L., Goldman, L.R., Hamilton, R.G., Lehrer, S., Rubin, C., Sampson, H.A., 2003. Clinical and laboratory investigation of allergy to genetically modified foods. Environ. Health Perspect. 111, 1114–1121. Bortesi, L., Fischer, R., 2015. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol. Adv. 33, 41–52. Brandenberg, O., Dhlamini, Z., Sensi, A., Ghosh, K., Sonnino, A., 2011. Introduction to molecular biology and genetic engineering. Food and Agriculture Organization of the United Nations, Rome, Italy. Carattoli, A., 2013. Plasmids and the spread of resistance. Int. J. Med. Microbiol. 303, 298–304. Carroll, D., 2011. Genome engineering with zinc-finger nucleases. Genetics 188, 773–782. Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A., Somia, N.V., Bogdanove, A.J., Voytas, D.F., 2011. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic. Acids Res. 39, e82. Chen, C.P., Chou, J.C., Liu, B.R., Chang, M., Lee, H.J., 2007. Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation. FEBS Lett. 581, 1891–1897. Chowdhury, E.H., Kunou, M., Nagaoka, M., Kundu, A.K., Hoshiba, T., Akaike, T., 2004. High-efficiency gene delivery for expression in mammalian cells by nanoprecipitates of Ca–Mg phosphate. Gene 341, 77–82. Christie, P.J., Gordon, J.E., 2014. The Agrobacterium Ti plasmids. Microbiol. Spectr. 2, 1–18. Conner, A.J., Barrell, P.J., Baldwin, S.J., Lokerse, A.S., Cooper, P.A., Erasmuson, A.K., Nap, J.-P., Jacobs, J.M.E., 2007. Intragenic vectors for gene transfer without foreign DNA. Euphytica 154, 341–353. D’Agnolo, G., 2005. GMO: human health risk assessment. Vet. Res. Commun. 29 (Suppl. 2), 7–11. Darbani, B., Farajnia, S., Toorchi, M., Zakerbostanabad, S., Noeparvar, S., Neal Stewart, J.C., 2008. DNA-delivery methods to produce transgenic plants. Biotechnology 7, 385–402. de Vendomois, J.S., Roullier, F., Cellier, D., Seralini, G.E., 2009. A comparison of the effects of three GM corn varieties on mammalian health. Int. J. Biol. Sci. 5, 706–726. de Vendomois, J.S., Cellier, D., Velot, C., Clair, E., Mesnage, R., Seralini, G.E., 2010. Debate on GMOs health risks after statistical findings in regulatory tests. Int. J. Biol. Sci. 6, 590–598. Domingo, J.L., 2007. Toxicity studies of genetically modified plants: a review of the published literature. Crit. Rev. Food Sci. Nutr. 47, 721–733. Duke, S.O., Rimando, A.M., Pace, P.F., Reddy, K.N., Smeda, R.J., 2003. Isoflavone, glyphosate, and aminomethylphosphonic acid levels in seeds of glyphosate-treated, glyphosate-resistant soybean. J. Agric. Food Chem. 51, 340–344. Durai, S., Mani, M., Kandavelou, K., Wu, J., Porteus, M.H., Chandrasegaran, S., 2005. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res. 33, 5978–5990. Gad, A.E., Rosenberg, N., Altman, A., 1990. Liposome-mediated gene delivery into plant cells. Physiol. Plant. 79, 177–183.

Methods for Plant Genetic Modification  399 Gelvin, S.B., 2003. Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev. 67, 16–37. Gelvin, S.B., 2012. Traversing the cell: Agrobacterium T-DNA’s journey to the host genome. Front. Plant Sci. 3, 52. Gleba, Y.Y., Tuse, D., Giritch, A., 2014. Plant viral vectors for delivery by Agrobacterium. Curr. Top. Microbiol. Immunol. 375, 155–192. Griffiths, A.J.F., Miller, J.H., Suzuki, D.T.E.A., 2000. Making Recombinant DNA. W.H. Freeman, New York. Hartung, F., Schiemann, J., 2014. Precise plant breeding using new genome editing techniques: opportunities, safety and regulation in the EU. Plant J. 78, 742–752. Jauhar, P.P., 2006. Modern biotechnology as an integral supplement to conventional plant breeding: the prospects and challenges. Crop Sci. 46, 1841–1859. Kaeppler, H.F., Gu, W., Somers, D.A., Rines, H.W., Cockburn, A.F., 1990. Silicon carbide fiber-mediated DNA delivery into plant cells. Plant Cell Rep. 9, 415–418. Kanchiswamy, C.N., 2016. DNA-free genome editing methods for targeted crop improvement. Plant Cell Rep. 35, 1469–1474. Karp, A., 1992. The role of growth regulators in somaclonal variation. Br. Soc. Plant Growth Regul. Annu. Bull. 2, 1–9. Kawata, Y., Yano, S., Kojima, H., 2003. Escherichia coli can be transformed by a liposome-mediated lipofection method. Biosci. Biotechnol. Biochem. 67, 1179–1181. Key, S., Ma, J.K., Drake, P.M., 2008. Genetically modified plants and human health. J. R. Soc. Med. 101, 290–298. Klein, R.M., Wolf, E.D., Wu, R., Sanford, J.C., 1992. High-velocity microprojectiles for delivering nucleic acids into living cells. 1987. Biotechnology 24, 384–386. Kost, B., Galli, A., Potrykus, I., Neuhaus, G., 1995. High efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts. J. Exp. Bot. 46, 1157–1167. Kramkowska, M., Grzelak, T., Czyzewska, K., 2013. Benefits and risks associated with genetically modified food products. Ann. Agric. Environ. Med. 20, 413–419. Krishna, H., Alizadeh, M., Singh, D., Singh, U., Chauhan, N., Eftekhari, M., Sadh, R.K., 2016. Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech 6, 54. Labra, M., Vannini, C., Grassi, F., Bracale, M., Balsemin, M., Basso, B., Sala, F., 2004. Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip. Theor. Appl. Genet. 109, 1512–1518. Ladics, G.S., Cressman, R.F., Herouet-Guicheney, C., Herman, R.A., Privalle, L., Song, P., Ward, J.M., Mcclain, S., 2011. Bioinformatics and the allergy assessment of agricultural biotechnology products: industry practices and recommendations. Regul. Toxicol. Pharmacol. 60, 46–53. Lai, H., Chen, Q., 2012. Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations. Plant Cell Rep 31, 573–584. Liu, Z., Kearney, C.M., 2010. A tobamovirus expression vector for agroinfection of legumes and Nicotiana. J. Biotechnol. 147, 151–159. Lodish, H., Berk, A., Matsudaira, P., Kaiser, C.A., Krieger, M., Scott, M.P., Zipursky, S.L., Darnell, J., 2003. DNA cloning by recombinant DNA methods. In: Freeman, W.H. (Ed.), Molecular Cell Biology. fifth ed. Freeman, W. H. & Co, New York, pp. 361–371. Maeder, M.L., Gersbach, C.A., 2016. Genome-editing technologies for gene and cell therapy. Mol. Ther. 24, 430–446. Maeder, M.L., Thibodeau-Beganny, S., Sander, J.D., Voytas, D.F., Joung, J.K., 2009. Oligomerized pool engineering (OPEN): an “open-source” protocol for making customized zinc-finger arrays. Nat. Protoc. 4, 1471–1501. Mahgoub, S.E.O., 2015. Genetically Modified Foods: Basics, Applications, and Controversy. CRC Press, Boca Raton, USA, (e-Book PDF). Makarova, K.S., Haft, D.H., Barrangou, R., Brouns, S.J., Charpentier, E., Horvath, P., Moineau, S., Mojica, F.J., Wolf, Y.I., Yakunin, A.F., Van Der Oost, J., Koonin, E.V., 2011. Evolution and classification of the CRISPRCas systems. Nat. Rev. Microbiol. 9, 467–477.

400  Chapter 15 Marillonnet, S., Thoeringer, C., Kandzia, R., Klimyuk, V., Gleba, Y., 2005. Systemic Agrobacterium tumefaciensmediated transfection of viral replicons for efficient transient expression in plants. Nat. Biotechnol. 23, 718–723. Masani, M.Y., Noll, G.A., Parveez, G.K., Sambanthamurthi, R., Prufer, D., 2014. Efficient transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. PLoS One 9, e96831. Matzke, M.A., Primig, M., Trnovsky, J., Matzke, A.J., 1989. Reversible methylation and inactivation of marker genes in sequentially transformed tobacco plants. EMBO J. 8, 643–649. Meyer, P., 2013. Transgenes and their contributions to epigenetic research. Int. J. Dev. Biol. 57, 509–515. Moeller, L., Wang, K., 2008. Engineering with precision: tools for the new generation of transgenic crops. Bioscience 58, 391–401. Montagu, M.V., Zambryski, P., 2017. Agrobacterium and Ti Plasmids. Reference Module in Life Sciences, Elsevier Inc. Available from: https://doi.org/10.1016/B978-0-12-809633-8.06024-6. National Research Council Committee, 2004. National Research Council Committee on Identifying and Assessing Unintended Effects of Genetically Engineered Foods on Human Health. National Academies Press, Washington. Park, S.Y., Vaghchhipawala, Z., Vasudevan, B., Lee, L.Y., Shen, Y., Singer, K., Waterworth, W.M., Zhang, Z.J., West, C.E., Mysore, K.S., Gelvin, S.B., 2015. Agrobacterium T-DNA integration into the plant genome can occur without the activity of key non-homologous end-joining proteins. Plant J. 81, 934–946. Petolino, J.F., Arnold, N.L., 2009. Whiskers-mediated maize transformation. Methods Mol. Biol. 526, 59–67. Quero, C., Colome, N., Rodriguez, C., Eichhorn, P., Posada de la Paz, M., Gelpi, E., Abian, J., 2011. Proteomics of toxic oil syndrome in humans: phenotype distribution in a population of patients. Chem. Biol. Interact. 192, 129–135. Rajeevkumar, S., Anunanthini, P., Sathishkumar, R., 2015. Epigenetic silencing in transgenic plants. Front Plant Sci. 6, 693. Ramesh, S.V., 2013. Non-coding RNAs in crop genetic modification: considerations and predictable environmental risk assessments (ERA). Mol. Biotechnol. 55, 87–100. Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., Zhang, F., 2013. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308. Rashid, A., Lateef, D., 2016. Novel techniques for gene delivery into plants and its applications for disease resistance in crops. Am. J. Plant Sci. 7, 181–193. Rivera, A.L., Gomez-Lim, M., Fernandez, F., Loske, A.M., 2012. Physical methods for genetic plant transformation. Phys. Life Rev. 9, 308–345. Rivera, A.L., Gómez-Lim, M., Fernández, F., Loske, A.M., 2014. Genetic transformation of cells using physical methods. J. Genet. Syndr. Gene Ther., 5. Sato, M., Hosokawa, M., Doi, M., 2011. Somaclonal variation is induced de novo via the tissue culture process: a study quantifying mutated cells in Saintpaulia. PLoS One 6, e23541. Schnorf, M., Neuhaus-Url, G., Galli, A., Iida, S., Potrykus, I., Neuhaus, G., 1991. An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis. Transgenic Res. 1, 23–30. Schoberle, T.J., Nguyen-Coleman, C.K., May, G.S., 2013. Plasmids for increased efficiency of vector construction and genetic engineering in filamentous fungi. Fungal Genet. Biol. 58-59, 1–9. Seralini, G.E., Clair, E., Mesnage, R., Gress, S., Defarge, N., Malatesta, M., Hennequin, D., de Vendomois, J.S., 2012. Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Food Chem. Toxicol. 50, 4221–4231. Seralini, G.E., Clair, E., Mesnage, R., Gress, S., Defarge, N., Malatesta, M., Hennequin, D., de Vendomois, J.S., 2014. Republished study: long-term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Environ. Sci. Eur. 26, 14. Shamloul, M., Trusa, J., Mett, V., Yusibov, V., 2014. Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana. J. Vis. Exp. (86), 1–13, e51204. Singh, R.J., 2011. Medicinal Plants. Genetic Resources, Chromosome Engineering and Crop ImprovementCRC Press, Boca Raton, USA. Smulders, M.J.M., de Klerk, G.J., 2011. Epigenetics in plant tissue culture. Plant Growth Regul. 63, 137–146.

Methods for Plant Genetic Modification  401 Solahil, A.J., Zabta, K.S., Sabir, H.S., Armghan, S., Muhammad, A.Z., Nazir, A., 2016. In-planta transformation: recent advances. Rom Biotechnol. Lett. 21, 11085–11109. Suarez, A., Viloria, M.D., Garcia-Barreno, P., Municio, A.M., 1985. Toxic oil syndrome, Spain: effect of oleoylanilide on the release of polysaturated fatty acids and lipid peroxidation in rats. Arch. Environ. Contam. Toxicol. 14, 131–136. Tzfira, T., Citovsky, V., 2006. Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr. Opin. Biotechnol. 17, 147–154. Vasil, I.K., Vasil, V., 1972. Totipotency and embryogenesis in plant cell and tissue cultures. In Vitro 8, 117–127. Voytas, D.F., 2013. Plant genome engineering with sequence-specific nucleases. Annu. Rev. Plant Biol. 64, 327–350. Yu, W., Yau, Y.Y., Birchler, J.A., 2016. Plant artificial chromosome technology and its potential application in genetic engineering. Plant Biotechnol. J. 14, 1175–1182. Zhai, Z., Jung, H.I., Vatamaniuk, O.K., 2009. Isolation of protoplasts from tissues of 14-day-old seedlings of Arabidopsis thaliana. J. Vis. Exp. (30), 1–3, e1149. Ziemienowicz, A., 2014. Agrobacterium-mediated plant transformation: factors, applications and recent advances. Biocatal. Agric. Biotechnol. 3, 95–102.