Keywords

10.1 Introduction

Practise of plant breeding started nearly 10,000 years ago that brought first grain crops under domestication and selective breeding (Hickey et al. 2019). Subsequent discovery of Mendel’s law, hybrid vigour and experimental designs not only improved the understanding of genetic elements underlying various plant traits, but also rendered plant breeding more systematic and efficient. Conventional plant breeding remains a key technology to facilitate crop improvement; however, it has limitations such as polyploidy, zygosity and longer generation time. Also, trait introgression from wild to cultivated varieties through hybridization and selection is extremely difficult (Zamir 2001; Warschefsky et al. 2014). Similarly, utilization of mutants generated through chemicals and/or irradiation is restricted either due to the mutational load or low mutation frequency in the targeted genomic region controlling trait(s) (Jung et al. 2018). Now molecular breeding approaches that integrate genomics and high-throughput phenomics and multipotent genetic material offer faster delivery of improved varieties (Varshney et al. 2009, 2018; Appels et al. 2013, 2015). Though biotechnological tools that could precisely engineer plant traits are available such as genetic transformation, these face challenges from regulators and policy makers. Furthermore, the cost of regulating GMOs is much higher than non-GMO crops and the entire process consumes considerable time even after developing improved products (Sprink et al. 2016). To address these challenges, precise modification of crop genes and/or regulatory elements has now become possible through genome editing. Recent years have seen genome editing gaining attention of researchers because it offers predictable allelic series to optimize both quantitative and qualitative traits (Kumar et al 2020; Scheben and Edwards 2018; Biswal et al. 2019).

Domestication and modern breeding practices favouring certain genomic regions have eroded genetic variation in current cultivated pools of different crop species. For instance, transition of domesticated rice from prostrate (wild rice) to erect growth (modern rice cultivars) resulted from the selection of an important single mutation prostrate growth 1 (PROG1) gene (Jin et al. 2008; Tan et al. 2008). Therefore, endeavouring precise modification of crop gene(s) by generating beneficial alleles with site-specific nucleases for desire phenotype will make huge impact on trait discovery and accelerate domestication of crop species (Scheben and Edwards 2018; Nogué et al. 2016). And with genome editing tools in place, it is possible to achieve this in much shorter duration (Scheben and Edwards 2018); however, their acceptance is still in obscurity. The onus is thus on the scientific community to provide ample evidences and generate awareness regarding technically different nature of genome editing products that lack foreign DNA, thus rendering this similar to the plants improved using conventional breeding tools. Researchers argue that the edited plants developed through genome editing should not be treated as GMOs (Araki and Ishii 2015). These technologies should be kept free from the hurdle of GMO legislation to allow their speedy adoption in routine genetic improvement programmes not only in developed countries but also in developing countries.

The present review aims to underscore the potential of modern genome editing tools for developing improved crop varieties for sustainable food production. This article evaluates genome editing with respect to environment and consumer risk. Also, the constraints that limit adoption of the crops improved with genome editing are briefly discussed.

10.2 Introducing Mutations Through Advanced Genome Editing Tools

10.2.1 Zinc Finger Nuclease (ZFN)

The term ZFN was initially used by Lusser et al. (2011) and successively by new technique working group (NTWG) in 2012. In this technique, a synthetic restriction endonuclease is customized to cut double-stranded deoxyribonucleic acid (DNA) at specific sequences (Wyman and Kanaar 2006). It comprises a zinc finger domain that allows recognition of a specific DNA sequence, enabling both site-specific mutation and integration of gene(s) into the plant genome (Bibikova et al. 2002; Wyman and Kanaar 2006). ZFN acts as a heterodimer, and therefore, ZFN transcribing genes are transported in a designed expression vector to plant cells (Söllü et al. 2010). The transfer of gene through ZFN technology involves electroporation (Wright et al. 2005), transfection (Szczepek et al. 2007), whiskers (Shukla et al. 2009), microparticle bombardment (Ainley et al. 2013), and Agrobacterium (De Pater et al. 2009). The viral vectors are also used for gene(s) transfer into the plant genome. ZFN causes double strand breaks at unambiguous site in the genome, which activate the repair mechanism of the host plant (Petolino 2015). Afterwards, both homologous recombination (HR) and DNA inclusion take place (Fig. 10.1a, e). This technique involves three artificial restriction enzymes, namely ZFN-1, ZFN-2 and ZFN-3 (Bibikova et al. 2001). (1) ZFN-1: Here ZFN is transported to the plant genome without taking repair template. Once it reaches the plant genome, it creates double-stranded breaks (DSB) to the host DNA that leads to non-homologous end-joining (NHEJ) of DNA (Puchta 2005), which either generates site-specific random mutations or small insertion or deletion. (2) ZFN-2: In contrast to ZFN1, a homology-directed repair (HDR) along with short repair template is delivered to plant genome along with ZFN enzyme (Lusser et al. 2011). The template DNA is homologous to target DNA, which binds to specific sequence causing a double-stranded break. The template starts repairing competing with endogenous repair machinery which led to site-specific point mutations through homologous recombination (HR). (3) ZFN-3: When ZFN transcribing gene is transported to the plant genome along with large repair template (for gene addition or replacement), it is called ZFN3 (Lusser et al. 2011; Araki et al. 2014). It binds to double-stranded DNA and causes site-specific double-stranded cleavage followed by HR. The end sequence flanking the double-stranded cleavage is the homologous results insertion of DNA stretch in a site-specific manner. ZFN-3 also helps in addition or replacement of the gene of interest, and for trait stacking in crops, such as herbicide resistance in plants (Townsend et al. 2009).

Fig. 10.1
figure 1

Structural representation of nucleases. (a) Structure of ZFN. ZFP represents zinc figure protein. The ZFN recognizes target site by the left and right ZFPs, and each engineered ZFP can recognize a target nucleotide. The ZFN monomer is contained a NLS (red) domain at N-terminal. The C-terminal comprises the Fok I endonuclease. The target sequence recognized by the left and right ZFPs which undergo for the dimerization of the Fok I endonuclease for activity. (b) TALEN contains an N-terminal domain comprising a nuclear localization signal (NLS); an essential domain typically formed of tandem TALE repeats to recognition a specific target DNA sequence; and a C-terminal domain with functional endonuclease Fok I. Each TALE repeat consists of 34-amino-acid with a variation at 12th and 13th amino acid position: NI (recognizes nucleotide A), NG (recognizes T), HD (recognizes C) or NN (recognizes G) (marked in black box). (c) Mode of action for TALEN. (d) Schematic representation of the CRISPR/Cas9 system structure and the principle for mutation induced through CRISPR. The synthetic guide RNA (sgRNA) complementary to the target DNA binding site and stem loops facilitates the binding of the Cas9 protein. The protospacer adjacent motif (PAM, NGG) is required for DSB which facilitate genome editing through error prone non-homologous end-joining (NHEJ) and homology-directed repair (HDR) repair pathway. (e) Mode of action for nucleases. The DNA double-stranded break DSB is repaired through HDR/NHEJ which causes base change or gene insertion or deletion in the target region (Adapted from Kumar et al. 2021)

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10.2.2 Transcription Activator-like Effector Nucleases (TALEN)

Transcription activator-like effector (TALE) proteins were discovered in the bacterial Xanthomonas sp. (Bonas et al. 1989). Bacterial system utilizes this to infect plants through injecting TALE protein in plant cell via the Type III secretion system, triggering effector specific genes in host (Römer et al. 2007). TALEs consist of effector proteins, which facilitate localization, activation and specific DNA binding (Miller et al. 2011). The DNA binding domain consists of TALE effector proteins that are highly conserved, and possesses tandem repeats of 5–30 (average of 17.5) amino acids which specifically recognizes target DNA sequences (Boch and Bonas 2010). The highly conserved domain shows variation at 12th and 13th position called as “repeat variable di-residues” (RVDs); it primarily determines the DNA specificity of TALE (Bogdanove and Voytas 2011). However, these tandem repeats end abruptly, leading to truncated repeated are termed as “half repeats” (Boch and Bonas 2010; Miller et al. 2011). The DNA binding efficacy of RVDs of TALEN to nucleotides (A, C, G and T) depends upon the amino acids Asn-Ile, His-Asp, Asn-Asn, Asn-Lys and Asn-Gly (Moscou and Bogdanove 2009). The deeper understanding of RVDs has allowed molecular biologist to modify naturally occurring TALEs for genome editing (Römer et al. 2007). The fusion of nikase Fok I to the C-terminus of TALEs results in development of specific TALEN for genome editing (Fig. 10.1b, c, e). The Fok I enzymes work in a dimeric state, hence pair of TALENs is required to facilitate DNA binding by Fok I heterodimer (Zu et al. 2013; Shin et al. 2014). Then the Fok I dimer cuts specific DNA region at the spacer site to create DSB. These DSB are repaired through NHEJ, which often yield indels within the target site of the genome. Further, the TALE protein can be fused with activator, repressor, nuclease or methylase to improve TALE based proteins for genome editing (Chen and Gao 2013). The application of TALENs was extended for—(a) introduction of exogenous sequences, e.g. fluorescent tags, etc.; (b) conditional gene expression and specific gene knockout; (c) controllable rearrangements of genomic DNA through deletions, inversions/reversions (Quétier 2016). Though widely used in animals, limited attempts have been reported so far in case of plant system due to the complex nature of TALEN construct (Araki and Ishii 2015).

10.2.3 CRISPR: A Modern Editing Tool to Assist Plant Breeding

Bacterial and archaea genomes encode nucleases that trim invaders (bacteriophages) DNA. These small segments of foreign DNA are incorporated into the host genome as a long term permanent records of infectious genome (Barrangou and Doudna 2016). This yields direct repeats in bacterial genome intervened by short unique sequences (proto-spacers, 32 nucleotides), indeed representing a short sequence of foreign genome (Quétier 2016). The term CRISPR is an abbreviation for “clustered regularly interspaced short palindromic repeats”, whereas Cas represents the nuclease associated with CRISPRs assembly. Recent genome sequencing experiments have revealed occurrence of CRISPRs in almost 40% of bacteria and 90% archaea species (Horvath and Barrangou 2010). The bacterial genome encodes a range of Cas proteins, of which Cas9 represents Type II CRISPR/Cas system (Song et al. 2016). The CRISPR/Cas system was initially discovered in 1987 by Ishino and colleagues (Ishino et al. 1987). The principle that underlies CRISPR/Cas9 system was elucidated later in 2011 (Fig. 10.1d). The Cas9 associates with trans-activator crRNA (tracrRNA) and CRISPR RNA (crRNA, transcript of a protospacer) to create a double strand break in foreign DNA that matches the crRNA (Fig. 10.1d) (Deltcheva et al. 2011). Interestingly, these spacers are transcribed after each invasion and aligned with complementary nucleotides bases present in the foreign DNA, causing CRISPR/Cas mediated degradation of invaded DNA. The Cas9 protein consists of RuvC and HNH domains that create a blunt end DSB at the three base pairs upstream of protospacer at the 3´ end (Garneau et al. 2010). The DSB is repaired by NHEJ or HDR (Fig. 10.1e) mechanism which often results mutation such as indels (Xiong et al. 2015). Furthermore, the specificity of Cas9 also depends on its three-dimensional conformation. The nuclear DNA regulates the differential binding and residence time. For instance, extended binding time with target DNA sequences, whereas a shorter period for off-targets (Knight et al. 2015). To make this technique more robust, researcher fused the tracrRNA and crRNA to a single guide RNA molecule (sgRNA) (Jinek et al. 2012). The Cas9 nuclease specifically cleaves the RNA/DNA complex followed by DNA repair. With this modification CRIPSR/Cas9 genome engineering is achieved with much higher efficiency. A recent modification involves development of Cas9 variant using Fok I (from Streptococcus pyogenes), Cpf1 (Cas12; from Francisella novicida U112) and C2c2 (Cas13; from Alicyclobacillus acidoterrestris) nucleases (Tsai et al. 2015; Shmakov et al. 2015; Zetsche et al. 2015). Genome editing CRISPR technology has been extended beyond site-specific mutagenesis (Barrangou and Doudna 2016). Recent research has shown transcriptional regulation by deactivating the Cas9, and fusing the guide RNA with activator or repressor (Fig. 10.2) (Qi et al. 2013; Gilbert et al. 2014). Likewise, fusion of fluorophores enables Cas9 sequence-specific DNA visualization or chromatin imaging (Chen et al. 2012; Mao et al. 2016). Additionally, RNA manipulation has been reported using CRISPR/Cas13 in eukaryotes, including plants. RNA editing is a post-transcriptional mechanism, which converts adenosine to inosine (A to I) (Matsoukas 2018). Cox et al. (2017) reported that CRISPR/Cas13 in a programmable manner to alter the coding potential in mammalian cells. Further, Abudayyeh et al. (2017) and Aman et al. (2018) used Cas13 system to target mammalian and plant cells to knockdown of either endogenous or reporter transcripts and RNA virus, clearly indicating the potential applications in agricultural biotechnology (Ali et al. 2018). In recent years, CRISPR application has been extended to epigenetic modifications in genome to activate gene through promoters and enhancers by fusing to acetyltransferases to Cas9 (Hilton et al. 2015; Kearns et al. 2014). Unlike ZFNs and TALENs, CRISPR/Cas9 offers RNA guided genome editing in an cost-efficient and user-friendly manner (Nagamangala Kanchiswamy et al. 2015). These advancements have inspired increasing use of CRISPR/Cas9 technology in crop and animal breeding (Quétier 2016; Song et al. 2016).

Fig. 10.2
figure 2

Schematic illustration of crop improvement. (a) Loss of genetic diversity prior to domestication resulting in domesticated crop with desired trait(s), (b) conventional breeding approach for crop improvement which involves up to 6–15 yrs., (c) genome editing assisted breeding for faster improvement of existing elite cultivars (Adapted from Kumar et al. 2021)

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10.3 Showcasing of the Candidate Genes Through Genome Editing of Crop Plants

Site-specific nucleases have allowed the introduction of targeted sequence-specific changes in both plant and animal system. Initially adopted in animal systems for targeted genome modification, ZFN and TALEN protein-guided recognition tools were later extended to create mutations or indels in the target gene of various plant species (Table 10.1) (Gaj et al. 2013; Lor et al. 2014; Sawai et al. 2014). In model plant Arabidopsis ZFN technique was employed to generate several mutants (Qi et al. 2013). For instance, Arabidopsis loss of function mutant for endogenous gene aba-insensitive-4 (ABI4) was generated for ABA and glucose insensitivity (Osakabe et al. 2010), deletion mutants for alcohol dehydrogenase-1(ADH1) and transparent testa-4 (TT4) which have shown heritable behaviour (Zhang et al. 2010). In maize, ZFN technique conferred herbicide tolerance through disruption of target gene IPK1, which alters inositol phosphate profile (Shukla et al. 2009). A similar approach in tobacco demonstrated disruption of an endogenous endochitinase gene CHN50 through a ZFN construct that consisted of a herbicide resistance PAT gene flanked by short stretches of endochitinase (Cai et al. 2009). Similarly, mutations in acetolactate synthase genes—SuRA and SuRB of tobacco improved tolerance against herbicides (Townsend et al. 2009). The heritable nature of the genetic modifications caused by gene editing was confirmed in soybean for target 10 genes: a transgene “GFP transgene” and nine endogenous genes (DCL1a, DCL1b, DCL2a, DCL2b, DCL4a, DCL4b, RDR6a, RDR6b and HEN1a) (Curtin et al. 2011). Recent examples for ZFN mediated modifications in plants include apple and fig (Peer et al. 2015), populus (Lu et al. 2016), tomato (Hilioti et al. 2016) and tobacco (Schneider et al. 2016).

Table 10.1 List of genomes edited crops developed through genome editing—ZFNs, TALENs and CRISPR/Cas9
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Like ZFNs, TALENs have also been implemented for the improvement of crop species (Gaj et al. 2013). In monocot species, nearly 12 genes were targeted to generate desirable knockout mutants through TALENs technique (Zhang et al. 2013). In rice, Os11N3 (OsSWEET14, member of SWEET sucrose-efflux transporter family) gene is responsible for bacterial blight susceptibility (Antony et al. 2010; Chen et al. 2012). This gene in rice was mutated through TALEN and thus transgenic plants gained desired resistance to bacterial blight disease (Li et al. 2012). In barley, the promoter of HvPAPhy_a (from phytase gene family) was targeted as it accounts for the maximum of the phytase activity during seed development (Wendt et al. 2013). The mildew-resistance locus (MLO) gene was targeted which encodes for a protein that suppresses defence against powdery mildew disease (Wang et al. 2014). With TALEN technology three homoeoalleles of MLO were disrupted in bread wheat to confer heritable resistance against powdery mildew (Wang et al. 2014). In tomato DELLA protein is encoded by procera (PRO) gene (Carrera et al. 2012), and it negatively regulates the GA signalling pathway (Zentella et al. 2007). Tomato pro mutant possesses enhanced levels of GA, but it partially retained some GA response, suggesting a leaky phenotype of the mutant protein (Van Tuinen et al. 1999). In order to completely block the DELLA protein function, PRO gene mutants of tomato were raised through TALEN, which displayed a similar phenotype as pro mutant (Lor et al. 2014). TALENs have been implicated for improving postharvest quality of potato. The cold storage of potato induces formation of reducing sugars, which react with free amino acids at high temperature to form acrylamide (Kim et al. 2015). Recently, Clasen et al. (2016) obtained TALEN based knockout of the vascular invertase gene, whose tuber produces negligible level of reducing sugars and its processed chips consisted undetectable amount of acrylamide.

A growing body of literature indicate successful application of the CRISPR/Cas9 method in model and crop plants (Shan et al. 2014; Belhaj et al. 2015; Liu et al. 2017; Collonnier et al. 2017). This technique was effectively used to generate mutants in both monocots (rice and sorghum), and dicots (Arabidopsis and tobacco) (Jiang et al. 2013). In wheat, CRISPR/Cas9 system is successfully applied for mutating inositol oxygenase and phytoene desaturase (Upadhyay et al. 2013), and MLO gene (Shan et al. 2013; Wang et al. 2014). Recently, IPK gene function was neutralized in maize by using two sgRNA in the CRISPR/Cas9 (Liang et al. 2014). Mutated chlorophyll a oxygenase 1 (CAO1) and LAZY1 gene in rice caused loss of Chlorophyll b in the mutant leaf and noticeable tiller-spreading during the tillering stage, respectively (Miao et al. 2013). Similar alterations in the promoter regions of OsSWEET14 and OsSWEET11 genes in rice yielded resistance against bacterial blight (Jiang et al. 2013).

The ARGONAUTE7 (AGO7) gene in tomato regulates biogenesis of a group of sgRNAs which control the expression of auxin response factor gene (Husbands et al. 2009). Induction of mutations in tomato AGO7 through CRISPR/Cas9 system resulted in leaf deformities and affected pollen viability (Brooks et al. 2014). Recent work exploring CRISPR/Cas9 system in tomato involved mutagenesis genes such as PDS and phytochrome interacting factor PIF4 (Pan et al. 2016), downy mildew resistance 6 (de Thomazella et al. 2016) and ripening inhibitor (Ito et al. 2015). This technique generates desired mutations at the specific site of interest that are inheritable. Mutagenesis of multiple genes by CRISPR/Cas9 through expressing more than one sgRNAs suggests its immense implications for improving quantitative traits. For example, 30% yield advantage was achieved in rice following CRISPR/Cas9-driven manipulation of 13 genes associated with abscisic acid biosynthesis (Miao et al. 2018). CRISPR/Cas9 approach has been applied in the model plant Arabidopsis (Upadhyay et al. 2013) and tomato (Brooks et al. 2014), and monocot plants like rice (Zhang et al. 2014). Interestingly, a deletion of 10–1000 nucleotides can be created through multiplexing the sgRNA (Belhaj et al. 2013), thus can also lead to deletion of gene clusters due to chromosomal deletion (Zhou et al. 2014). Other examples of CRISPR/CAS9 based modification in plants include targeting multiple loci in Arabidopsis to enhance yield and resistance (Mao et al. 2016; Osakabe et al. 2016; Peterson et al. 2016), gemini virus resistance in tobacco (Zaidi et al. 2016), disease resistance in tomato (de Thomazella et al. 2016), starch modification and herbicide resistance in rice (Baysal et al. 2016; Sun et al. 2016, Wang et al. 2017), improvised fatty acid accumulation in Camelina (Jiang et al. 2016), resistance against Phytophthora sojae in soybean (Fang and Tyler 2016), canker resistance in citrus (Peng et al. 2017), starch modification in wheat (Liang et al. 2017), gibberellins metabolism in rice (Lu et al. 2016), etc.

Change in the expression level and/or organization of the genes resulting from mutations in cis-regulatory regions is known to create quantitative and qualitative variation of the traits (Wittkopp and Kalay 2012). Gene expression is fine-tuned by cis-regulatory elements (CREs) present in the promoter region. Recently, Rodriguez-Leal et al. (2017) used the CRISPR/Cas9 to modify the CREs in the promoters of tomato WUS (SlWUS) and CLV3 (SlCLV3) genes, that control fruit size, and inflorescence architecture. The induced novel cis-regulatory mutant alleles increased the tomato fruit size and locule number, similar to the natural QTL variants. Base-editors (BEs) are another CRISPR/CAS9 technique, which enables direct, irreversible conversion of one base to another at a target locus. Given the majority of the agronomic traits are controlled by point mutations (Huang et al. 2010), recent findings indicated that by fusing a nuclease-deactivated Cas9 (dCAS9) to a cytidine deaminase or adenosine deaminase induces C.G and A.T base pairs (bps) to T.A and G.C (Brooks and Gaj 2018). Though BEs approach was initially applied in mammalian systems, and the same was successfully employed in rice, wheat, maize, and tomato (Zong et al. 2017; Shimatani et al. 2017; Lu et al. 2016).

10.4 Challenges for Genome Editing

One of the major challenges in successfully achieving genome editing is plant genetic transformation and regeneration, which are the bottlenecks in agriculture biotechnology. Various technologies are available to improve plant transformation. For example, floral dip transformation is an attractive solution, it eliminates the plant tissue culture step, but the limitation is that not compatible to several crop plants, except in Arabidopsis and Camelina sativa (Lu and Kang 2008) and its transformation efficiency is very low. Further, to improve plant transformation and regeneration methods, high throughput, efficiency and novel transformation technologies are required. Development of novel methodologies could dramatically enhance plant genomics knowledge to feed the world (Altpeter et al. 2016).

Designing of ZFNs and TALENs is one of the most pain stacking jobs. The commercially available ZFNs are more efficient, but very expensive, than the publicly designed (Ramirez et al. 2008). In contrast, TALENs designing has become easy and efficient by using Golden Gate cloning—a DNA assembly technique (Engler et al. 2008). On the basis of available literature, some of the most important problems associated with genome editing are their low efficacy, regulatory vagueness and social acceptance (Shukla et al. 2009). Yet, the accurate estimation of efficiency is very difficult because efficiency depends on various factors such as the crop plant selection, methodology used, target gene and marker genes. For example, the efficiency of ZFN induced mutation in Arabidopsis is reported to be around 2% (de Pater et al. 2009) whereas, in the case of tobacco, it is 40% (Townsend et al. 2009). Over and above, one of the problems associated with ZFN and TALENs technologies is its non-specific binding which leads to create non-specific mutations (Pattanayak et al. 2011). These off target effects are also associated with CRISPR/Cas9 technology (Song et al. 2016). An improper concentration between Cas9 and sgRNA, or promiscuous PAM sites, or poor codon optimization of Cas9 during translation results off target/undesired cleavage of DNA sequence. It has been reported that high off-targets were found in humans, but low in mice, zebrafish and plants (Fu et al. 2013; Pan et al. 2016). Depending upon the species/cultivar the efficiency of the technology shows discrepancy. At the same time, T-DNA (foreign DNA) will be removed before proceeding for commercialization (Schaart et al. 2010). However, off target effects are expected with any genome editing tools as these are driven by several factors including sequence similarities. Nevertheless, researcher always selected the best phenotypic variants from genetically engineered lines, ruling out off target effect.

The regulatory cost of new plant breeding techniques is very high and the regulatory process alone takes 5–7 years, hence the acceptance of these techniques is low. When products become GMOs, it costs even higher and more time consuming compared to non-regulated classical breeding techniques (Kalaitzandonakes et al. 2007). Therefore, usage of these new techniques is limited. In particular, small companies are using these techniques only for limited traits of high value crops (Miller et al. 2011). Hence, it will be hard for plant breeders to invest in ventures where regulatory cost has a direct impact on the economic potential of the crops such as orphan crop and GM approaches-based product.

Once the plant is classified as GMOs, it has to be a method for identification and actual quantification on the newly introduced gene/s and has to be mentioned before going to the market (Kuzma and Kokotovich 2011). All contemporary available standard methods for GMO detection basically depends on the quality of DNA and efficiency of the techniques (PCR, qPCR, ELISA, etc.,). In order to evaluate the changes brought about by these genomes editing, those are mostly monitored by an expert committee, which is considered to be an important element of risk management (Glandorf et al. 2011). The prior knowledge of DNA sequence has an imperative role in the detection and identification of GMOs. The plant produced through ZFN1, ZFN2 and ZFN3 techniques can be detected by DNA based approaches only when there is prior information of flanking sequences of introduced modifications.

Genome editing has been mostly implemented in plant breeding to generate disease resistance and yield advantage for crops. Its application has to widen such as abiotic stress tolerance, nutritional quality enhancement and allergenicity elimination from various crops. There has been some report on RNAi based reduction of allergens from apple (Gilissen et al. 2005). Similarly, peanut allergens and gluten gene from various crops such as wheat, rye and barley can be reduced or abolished using these techniques (Gilissen et al. 2014; Smulders et al. 2015). Now breeders are opting for developing superior varieties through grafting. In case of grafting, plants are produced by joining of scions and rootstocks. When a non-GM scion is attached to the GM rootstocks, detection of scion derived products becomes impossible, but rootstock can be identified using usual genomics tools used for GM crops.

10.5 Genome Edited Crop: Social Acceptance and Regulatory Framework

Since the domestication of first agricultural plants nearly 10,000 years ago, plant breeding techniques have tremendously improved crop yields that can feed more than 70 million peoples (Palmgren et al. 2015). However, new breeding techniques and agronomic practices are required for a sustainable food future of 10 billion people by 2050. The unfavourable conditions like biotic and abiotic stress are conspicuous factors which have increased the losses of crop productivity over the years. Thus, the pressing demand for resilient crop species has invigorated researchers to discover the possibility through reverse genetic or genome editing. Although recent genetic engineering approaches in crop species have achieved considerable progress in crop improvement, its social acceptance is negligible due to lack of strong global policies (Araki and Ishii 2015). Argument for the accepting GMOs or genome edited plants occur not with the public; but surprisingly, it had also created a debate between researchers (Tanaka 2012; Freedman 2013; Lucht 2015). It is projected that acceptance of these genome edited plants will adversely affect the native crop germplasm resources and also human health. In order to broaden the public acceptance, constant discussion with the society is a prerequisite (Palmgren et al. 2015), excellent reviews have been published on regulatory vagueness and social acceptance (Jones 2015; Araki and Ishii 2015). In 2007, the European Union commission and member states decided to set up an expert committee on NPBTs to evaluate these new techniques with respect to GMO legislation (Schaart et al. 2016), and the commission highlighted an array of legal and social issues associated with GMOs (Lusser et al. 2012). According to committee view, these techniques may or may not involve genome alteration of the target plant species given their heterogeneous nature. The EU declaration defines GMO as “any organism having altered genetic material which does not occur by natural mating or by natural recombination” (Directive 2001/18/EC 2001).

Anthropogenic activity has dramatically changed agricultural strategies such as large-scale cultivation of new varieties in combination with affecting natural habitats of ancestral wild species of crop plants (von Wettberg et al. 2018). Notably, the important alleles and genetic variations present in the plant wild species allow sustainable growth in extreme environmental conditions and distant geographical regions (Hajjar and Hodgkin 2007; Lu 2013; Brozynska et al. 2016). Recent success in the field of genetic engineering has also overlooked the huge potential of wild species and their use in prebreeding for certain extent, as these modern techniques have potential to improve elite crops or domestication of crop wild relatives in shorter duration (Palmgren et al. 2015; Li et al. 2018). Numerous researchers believe that worldwide acceptance of these genome edited plants can severely affect the diversity of plant wild species, and even it could lead to extinction of some rare species (Stewart et al. 2003; Castañeda-Álvarez et al. 2016). Questions remain on the legal acceptance for the applied strategies, and also on the social, economic and ethical acceptance of them (Tanaka 2012). Hence, the major concern with the release of living modified organisms (LMOs) is their impact on the environment, biodiversity conservation and a human health risk due to consistent consumption of GMOs (Lucht 2015).

According to the Cartagena Protocol, plants raised using genome editing can be out boxed from GMO regulation as they do not possess any transgene (http://bch.cbd.int/protocol/text/). It is quite interesting that, the stringency of the regulation of GMO or LMOs considerably varies within countries. For example, in New Zealand and Europe, food obtained from the plants derived from the precision mutagenesis techniques should be compared similar to the food derived from the traditional mutagenic techniques (Lusser et al. 2011; Palmgren et al. 2015). However, the foremost challenge for GMOs is acceptance of them in public domain, which greatly relies on the mindset of citizen, farmers and decision makers (Araki and Ishii 2015). The controversies related to transgenic have led to their widespread public rejections, and limited commercialization. For instance, the expert committee of new plant biotechnology declares that ZFN-1 and 2 both create GMO and therefore, both fall under the directive of 2001/18/EC or Directive 2009 41/EC 2009 (Schiemann et al. 2009; Sprink et al. 2016). Also, the plant produced using ZFN-3 technology is transgenic and therefore, comes under the directive of 2001/18/EC (Schiemann et al. 2009; Araki et al. 2014). Similarly, several CRISPR/Cas9 mediated products including rice, maize, soybean, etc., are already developed and waiting for the approval of government regulatory bodies. Very recently, the Court of Justice of the European Union (ECJ) subjected CRISPR edited plants under tough GM laws by subjecting these plants to a 2001 directive, previously developed to control GM crops for food (Callaway 2018). However, researchers and plant breeders argue that CRISPR/Cas9 edited plants should be treated same as irradiation mutagenesis because it causes changes in DNA and does not involve the insertion of foreign genes, thus they can be exempt from the directive. Currently, the adoption of CRISPR system in agronomy has been remarkably increasing (Ricroch et al. 2017). As a result, several countries like USA, Canada, China, etc., have showed positive response towards CRISPR/Cas edited crop products (Lassoued et al. 2019); however, the developed edible food products from edited crops are of major concern. In fact, globally, the impact of genetically modified crops has been realized, especially due to the recent economic analysis obtained for the modified global crops such as maize and cotton (Brookes and Gaj 2018). For instance, in Spain and Portugal, over the 21-year period (1998 and 2018) the insect-resistant (IR) maize (aka corn) has increased farmers income by €285.4 million (US$322.9 million) by saving money on insecticides and producing more crop yields (Brookes and Gaj 2018). Additionally, use of IR maize maintained the required production by using lesser arable land because for the same production with conventional breeding material the farmers would have required an additional 15,240 hectares in the two countries. In 2014, the genetically modified soybean, cotton and canola saved 19, 9 and 1.5 million hectares of land globally (https://www.pgeconomics.co.uk/pdf/2017globalimpactstudy.pdf).

Recent reports have suggested that the genome editing tools have faded the boundaries between edited crops and regulatory bodies for social acceptance (Ishii and Araki 2016). Many products delivered through TALEN approach has been accepted (see Table 10.2). For instance, a TALEN mediated SU (sulfonylurea) Canola launched by Cibus (https://www.cibus.com/products.php) was commercially approved by the Canadian and United States governments in 2015 (Table 10.3). In the next 5 years other products from TALEN mediated genome edited products from Cibus such as glyphosate tolerant flax, soybean and maize breeds are under evaluation in the United States (Li et al. 2016) (Table 10.3). No wonder, more crop TALEN mediated genome edited products would be pushed to the market, as TALEN has proved its potential and critical role in genome editing breeding. It is obvious that the growing demand of food supply coupled with agronomic losses due to increased prevalence of diseases and abiotic stress needs supports of genome edited crops, which can provide elite varieties in very short duration. We speculate, in next two decades CRISPR/Cas9 mediated crop will have more products directly developed from the domestication of crop wild relatives (Li et al. 2018), which known to have several important features including higher nutritional quality and disease resistance, these products should be globally accepted to fulfil hunger need.

Table 10.2 Examples of genome edited crops approved through regulatory agency or are in pipeline
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Table 10.3 List of crop and traits targeted by commercial biotech company Cibus
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10.6 Intellectual Property Rights (IPRs) Protection and Freedom to Operate (FTO)

Biotechnology research has increased the availability of crop plants that are high-yielding, nutritious, stress tolerant, etc. (Díaz de la Garza et al. 2004; Vinocur and Altman 2005; Storozhenko et al. 2007; Nunes et al. 2009; Tamás et al. 2009; Varshney et al. 2011). However, the success of these genetically engineered plants is greatly dependent on the inventor incentives because of IPRs. IPRs include a set of laws to provide a legal protection to inventor or innovators for a fixed period of time against direct exploitation of their product or method (Malik and Zafar 2005). IPRs protect the biotechnology material through two major systems: patents and rights in plant varieties, but for a limited period of time. Patents provide a wide range of legal rights to retain, use, transfer it by sale or as a gift, and restrict others from similar rights for a duration of 17–20 yrs. (Gold et al. 2002; Graff et al. 2003). According to International Union for the Protection of New Varieties of plants (UPOV), a plant variety can be protected only if it is unique, stable, uniform and fulfil the novelty requirements (Jördens 2005). This grant provides an exclusive right to the owner to sell the plant materials such as reproductive organ or whole plant, which can be up to a period of 20–30 years. In the field of agro-biotechnology there is an exponential increase of the counts for filed application patents in USA, Germany and Japan (Graff et al. 2003). This surge was motivated by the royalties which can be obtained from invention (Barrows et al. 2014). The seed companies protect their genetically engineered seeds by IPRs (Oczek 2000; Frison et al. 2010), and sell their seed to farmers at monopolistic prices. It has also affected the conventional plant breeding, which is slow due to their need on the availability of the desired traits from the ancestral or closely related species. However, the rapid increase of the advancement in agricultural plant biotechnologies has increased the monitoring responsibility for the crop biosecurity which protects from the bioterrorism, biopiracy, genetic erosion, etc., (Evenson 1999; Chen and Puttitanun 2005). Furthermore, the surge of proprietary protection in crop species has intricate the exchange of germplasm which are required to develop new cultivars against destructive disease and environmental stress (Graff et al. 2003; Luby and Goldman 2016). This has led to an inability of researchers and breeders to obtain seed without acquiring permission through entering into an agreement such as material transfer certificate, license, etc. (Chi-Ham et al. 2012; Luby et al. 2015), hence, deprive researchers and breeders from genetic gain. These circumstances restrict the reach of quantity breed from a breeder and the researcher, and limit FTO for the purpose of crop breeding (Binenbaum et al. 2003; Le Buanec 2005). FTO allows researcher/people to determine whether a commercialization or testing can be done without violating any valid IPRs of others (Luby et al. 2015; Bjørnstad 2016; Zanga et al. 2016). To increase the accessibility of germplasm to breeders and researcher several open resource centre available such as an open source seed initiative (OSSI), Chinese crop germplasm information system (CCGIS), national small grains collection (NSGC), USDA soybean germplasm collection, tomato genetics resource centre (tgrc.ucdavis.edu/), etc., (Sachs 2009). Recently, efforts have been made to enhance the FTO in corn and carrot breeding through development of open source populations (Luby and Goldman 2016; Zanga et al. 2016). Similar attempts are required for improving the crop breeding because today crop genetics resources are intensely secure with IPRs.

10.7 Conclusion

The expectations of genome editing have risen that the technology would expedite progress towards sustainable crop productivity. Some products are at the final stage of development. The commercial use of the technology is relatively new in crop improvement. Genome editing allows generation of superior plants rapidly with higher efficiency and in an environmentally safe manner. Majority of the commercial crop varieties developed over the last 20 years by transgenesis, conventional and molecular breeding are now being explored through genome editing. Considering wider applications, CRISPR/Cas9 has gained more attention from researchers and breeders as compared to ZFNs, TALENs, grafting, reverse breeding, etc. As a result, breeders are now encouraging CRISPR edited plants crops to combat climate change and associated yield loss. The genome editing is accepted by the commercial sector because of its impending financial gain over alternative traditional techniques. However, a wider adoption of products derived from these techniques depends on several factors, including regulatory jurisdiction, the efficiency of the techniques and political expediency. Genome edited crop products are now available in a few countries such as the U.S. and Canadian government has approved genome edited canola and mushroom. We anticipate that CRISPR/Cas9 technology is likely to bridge the gap between GMO and society. The genome editing will be instrumental in meeting the challenge of feeding 12 billion people by the end of the twenty-first century.