Abstract
Grain legumes are prized for their high protein content and abundance of phytochemicals, which are essential in the human diet. Scientists have made significant advancements in discovering novel genetic features in legumes, including, but not limited to, productivity, tolerance/resistance to various environmental stresses, and improved nutritive value. The contemporary surge in genetic resources of grain legumes has facilitated the integration of advanced molecular breeding techniques such as transgenic methodologies, genome modification, and genomic selection, to augment the crop’s overall performance. This chapter discusses the application of CRISPR/Cas9-based genome editing tools for the improvement of grain legumes. Furthermore, it elaborates upon the latest developments in plant-specific genetic modification techniques, while also addressing the challenges and prospective benefits that come with enhancing grain legumes with significant agronomical attributes. Genome editing techniques have been proficiently employed in diverse legumes, encompassing model legumes such as Medicago, alfalfa, and lotus, alongside other widely cultivated legumes like soybean, cowpea, and chickpea. The advent of gene-editing methodologies in legume breeding has presented exciting opportunities for enhancing important agronomic characteristics.
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1 Introduction
Legumes, comprising over 19,500 species and 751 genera, are the third-largest angiosperm family [1]. They encompass a diverse range of food crops that are crucial sources of plant-based proteins and essential amino acids. Grain legumes play a crucial role in agricultural sustainability by promoting soil fertility through symbiotic nitrogen fixation (SNF) and the discharge of abundant organic matter into the ground. Notwithstanding their environmental benefits and health advantages, poor productivity has impacts on legume agronomy. The implementation of innovative approaches like genomics-assisted selection (also known as marker-assisted selection or genomic selection) and genome editing can help address the challenging problem of boosting productivity and reliability.
Over the course of the past 10Â years, significant advancements have been achieved in the sequencing of the genomes of leguminous crops. The draft genomes and transcriptome data of about 35 different legume species have been substantially assembled [2]. To select for intricate traits and generate better cultivars of grain legume crops, these genomic resources are proving invaluable [3]. The detailed assessment of the application of marker-assisted backcrossing (MAB), marker-assisted selection (MAS), and genomic selection (GS) techniques for improving the production of legume crops have been exhaustively investigated [3,4,5]. This chapter focuses on the latest developments in genome-editing technology and its efficacy as a precision breeding method for improving leguminous crops.
Genome editing involves making use of engineered nucleases and the inherent DNA repair mechanisms of cells to effectuate accurate and tailored modifications to an organism’s genetic makeup. The conception of gene-editing techniques commenced approximately thirty years ago, following the important revelation that targeted double-stranded breaks can be instigated in chromosomes using a meganuclease, namely I-SceI [6]. The effective use of meganucleases in genome editing was constrained by the limited number of target regions in the majority of genes. With the arrival of customizable zinc finger nucleases (ZFNs) [7] and transcription activator-like effector nucleases (TALENs) [8], the development of effective genome editing technologies witnessed a major acceleration. The remarkable progress in this state-of-the-art methodology was attained by successfully incorporating RNA-guided Cas9 nuclease, which originates from the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) adaptive defense mechanism, to facilitate genetic modification in eukaryotic organisms [9, 10].
2 Genome Editing Technology
The implementation of genome editing is a groundbreaking technological innovation that has the potential to significantly advance the field of plant biology and agricultural breeding [11]. This method relies on site-directed nucleases (SDNs) such as meganucleases, ZFNs, TALENs, and the CRISPR/Cas system [12, 13]. The CRISPR/Cas system’s growing popularity in genome modification tool development can be attributed to its simplistic nature and facile manipulability [14]. The fundamental CRISPR/Cas mechanism necessitates the presence of two integral constituents: a Cas endonuclease, such as Cas9 or Cpf1, and a guide RNA (gRNA) [15, 16]. The gRNA may be directed to bind to the desired DNA sequence and then use the Cas nuclease to create a double-strand break (DSB) at that specific location. The predominant mechanism for DSB repair in plants is the non-homologous end joining (NHEJ) pathway, which is known to be error-prone and often results in the insertion or deletion of bases, leading to genetic changes at the site of repair [16]. Thus far, a multitude of base and prime editor tools have been devised leveraging CRISPR/Cas technology to achieve greater precision in the editing process [17].
Many Cas9 variants have been synthesized that exhibit great fidelity, including those with paired nickase, point mutations, chimeric dCas9-FokI, etc. These modified Cas9 variants have cleavage activity with decreased off-target effects (refer to www.addgene.org). Thus choosing an appropriate Cas9 endonuclease variant is important yet difficult. With the use of these editing tools, breeders can manipulate target genes in the way they want to boost crop production and quality, endurance to biotic and abiotic stress, and herbicide resistance [11]. As a result, genome editing is seen as the breeding approach of the future.
The approval of the commercialization of genome-edited crops necessitates a framework of legislation and regulation [18]. Genome editing generates minor indels, base-pair changes, and targeted short sequence modifications by homology-directed repair (HDR), which are identical to those caused by natural mutations. As a result, in several countries, these sorts of mutants are not classified as genetically modified organisms (GMOs) and hence are excluded from GMO restrictions [19, 20].
3 Limitations in Genetic Modification of Legumes
The availability of a suitable genetic transformation protocol to introduce DNA into the plant is a prerequisite for achieving a fruitful plant transformation. Most of the grain legumes are grouped as recalcitrance to in vitro regeneration and transformation [21]. This phenomenon is further compounded by the intricate fact that only certain tissues (immature cotyledons, mature cotyledons with embryonic axis, embryonic axis or hypocotyl) within leguminous crops exhibit the ability to transform, while others possess the capacity for regeneration. It is noteworthy that these two distinct events do not invariably manifest within the same tissue. Achieving successful in vitro rooting can be a significant obstacle, particularly for legumes with large seeds. The impediments to large-scale transformation in legumes include method specificity and other factors. Traditional methods of genetic transformation are inadequate for achieving optimal results. Nevertheless, regeneration procedures for several legumes have not been successful, mostly because of poor in vitro roots during regeneration. It has been posited that conventional breeding methods are insufficient in tackling these obstacles [22, 23].
The development of efficient transformation techniques is crucial for validating the role of genes in targeted crops [24]. Various methodologies, including sonication-assisted Agrobacterium transformation, have garnered attention as a means to augment the genetic transformation procedure in leguminous plants. Improving the pace of genetic transformation in leguminous crops can be achieved through significant approaches such as optimizing explant, improving the affinity of host plant interaction, and refining culture media additives [25]. Additional research is necessary to explore the barriers to legume transformation and potential remedies. Advancements in the field of molecular science are expected to generate novel ideas and provide insights into the rapid enhancement of legume transformation rates.
The Agrobacterium-mediated transformation method is the primary means of genetic modification for the majority of grain legumes, with the biolistic method being utilized in a limited number of instances. Of the various grain legumes, genetic modification has been successfully achieved in soybean, and the CRISPR/Cas9 system has also been extensively utilized [26]. Nevertheless, the utilization of CRISPR/Cas9 technology has the potential to overcome these limitations and obstacles [27]. The development of reliable and repeatable regeneration techniques results in the commercially successful production of several species of genetically engineered grain legumes.
4 Application of CRISPR/Cas9 in Grain Legumes
The CRISPR-mediated gene-editing technique has been successfully applied to various legumes, such as soybean, chickpea, lentil, and Medicago truncatula, among others. Leguminous plants are cultivated extensively across the globe. The implementation of CRISPR/Cas9 technology has demonstrated enhancements in crop productivity, quality, and resilience against both biotic and abiotic stressors.
Legume species domestication has been extensively researched, and currently, the CRISPR/Cas9 mechanism is being employed in the process of domestication to introduce and enhance diverse characteristics. It is anticipated that the forthcoming process of crop domestication shall witness a significant acceleration, with the aid of CRISPR/Cas9, which shall effectively enhance numerous crop traits of commercial significance [28]. In recent times, there have been major advances in improving the nutritional value of legumes through the application of CRISPR/Cas9 technology.
4.1 Medicago truncatula (Alfalfa)
A forage crop, Medicago truncatula is used as a model crop due to its self-fertility, short life cycle, relatively simple transformation, diploidy, and smaller genome,. It is an excellent model for studying the molecular and physiological bases of leguminous crops. The CRISPR/Cas9 system exhibits a sub-optimal level of efficacy when applied to the polyploid alfalfa genome such as the tetraploid M. sativa and M. falcata. For effective gene editing in alfalfa, an improved CRISPR/Cas9 gene-editing system will be required. The MsSGR gene of M. truncatula has been effectively edited using CRISPR/Cas9. The outcomes indicated substantial differences in colour among the mutants. The presence of colour variation plays a crucial role in the attraction of insects and birds, thereby facilitating successful pollination. The experimental results indicated that mutations exhibited a greenish colour and suggested that the CRISPR/Cas9 method of knocking out alfalfa genes holds significant potential for future research [29]. Bottero and colleagues employed the CRISPR/Cas9 system to modify the NOD26 gene in alfalfa to enhance protein levels [30]. Phytoene desaturase (PDS) genes are a popular choice among researchers due to their easily observable phenotypic traits, which can be used to assess the efficacy of CRISPR/Cas9 gene-editing tools. Meng and Haji [31] devised a CRISPR/Cas9 mechanism to induce specific changes in the MtPDS gene of M. truncatula. Their findings revealed that out of 309 T0 transgenic plants, 32 displayed the albino phenotype. Sixteen of these 32 transgenic plants were arbitrarily chosen for sequencing, and the results confirmed that all the tested albino plants had alterations in the intended region of the MtPDS gene. The genome-wide association studies (GWAS) employing CRISPR/Cas9 were carried out to study the function of potential genes for nodulation in M. truncatula [32]. Additionally, Trujillo et al. [33] and Wang et al. [34] have conducted mutational studies on five different nodule-specific PLAT domain (NPD1–5) and nitrate peptide family (NPD) genes.
4.2 Glycine max (Soybean)
Soybean (G. max (L.) Merr.) holds significant value as a crop for its oil and protein content, making it a prime candidate for genetic enhancement through the CRISPR/Cas9 technology [35]. The soybean plant is a diploid species that has undergone an evolution from a palaeotetraploid ancestor. The soybean genome exhibits significant duplication, thereby presenting a significant barrier to conventional genetic methodologies aimed at elucidating gene functionality. One of the difficulties encountered in soybean transformation is the limited efficacy of Agrobacterium-mediated techniques, which is influenced by the type of tissue or cultivar used. Consequently, Agrobacterium rhizogenes-mediated hairy root transformation has acquired significant attention since it can be used to efficiently assess the effectiveness of gRNAs before the whole-plant transformation. This is due to the prompt way of obtaining transgenic hairy roots, which can be obtained in a matter of weeks [36]. In 2015, there were multiple reports of the effective utilization of the CRISPR/Cas9 technology for gene-editing purposes in soybean [37,38,39,40]. After these preliminary accomplishments, soybean researchers have attempted to improve gene-editing technology. Di et al. [41], for instance, used eleven different GmU6 promoters to determine which would be the most effective for driving gRNA expression in soybean hairy roots and found that the GmU6–8 and GmU6–10 promoters were the most active in improving editing efficiency (20.3% and 20.6%, respectively) than the rest of the nine GmU6 promoters (ranging from 2.8% to 17%). Additionally, the CRISPR/Cas9 technique has been effective in targeting three GmLox genes (GmLox1, GmLox2, and GmLox3) that encode three lipoxygenases (LOX1, LOX2, and LOX3) that produce a beany flavour that limits human intake [42]. They observed that the associated lipoxygenase activities had been eliminated in 60 T0-positive transgenic plants harboring various sgRNAs and mutations (two of them are triple mutants and one is a double mutant). Li et al. [36] employed the CRISPR/Cas9 methodology to modify the conglycinin (7S) and glycinin (11S) storage protein genes in soybean. The researchers observed gene-editing efficiencies of 5.8%, 3.8%, and 43.7% for Glyma.20 g148400, Glyma.03 g163500, and Glyma.19 g164900 genes, respectively. Furthermore, the manipulation of soybean plant architecture has been observed through the utilization of the CRISPR/Cas9 system.
The study carried out by Bao et al. [35] involved the selective targeting of squamosal promoter-binding protein-like genes, namely GmSPL9a, GmSPL9b, GmSPL9c, and GmSPL9. The results of the study indicated that the T2 double homozygous mutant spl9a/spl9b exhibited a reduced plastochron length. Also, it has been observed that T4 mutant plants exhibit an increment in the number of nodes on the main stem as well as an increase in branch numbers. Zheng et al. [43] described simple binary vector systems using Cas9 and egg cell-specific promoters (ECp). The two genes, GmAGO7a and GmAGO7b, which encode ARGONAUTE7 (AGO7), were specifically targeted due to their significant role in regulating leaf patterns in soybean. Their findings indicate that the promoters can generate mutations and that it is possible to obtain several distinct mutations independently. Virdi et al. [44] conducted a study wherein they employed CRISPR/Cas9 mutagenesis to generate several knockout alleles including one in-frame allele for the β-ketoacyl synthase 1 (KASI) gene, which is involved in the conversion of sucrose to oil. The findings of the study revealed that the function of the genes was lost.
Because of the importance of Soybean, a substantially higher number of CRISPR studies have been conducted on it compared to other legumes. These studies primarily focus on modifying its nutritional value and plant architecture, specifically leaf patterns and nodule numbers. Nonetheless, the establishment of stable soybean genetic transformation remains elusive due to the recalcitrant nature of this crop towards transformation. The enhancement of transformation efficiency has the potential to propel CRISPR research in soybean toward subsequent genetic research, owing to its efficiency, multiplex editing, and high-throughput mutagenesis capability [35].
4.3 Cicer arietinum (Chickpea)
Chickpea is a crop of significant commercial importance on a global scale. The application of genome-editing techniques presents a viable solution for addressing the challenges encountered during its cultivation. Badhan et al. [45] conducted a study with the objective of editing genes associated with drought tolerance, namely 4-coumarate ligase (4CL) and Reveille 7 (RVE7), using CRISPR/Cas9 technology in chickpea protoplasts. The results suggested that the knock-out of the RVE7 gene displayed a high level of editing efficiency in vivo. The findings of this study indicate that the utilization of protoplasts enables the application of the CRISPR/Cas9 DNA-free gene editing technique for enhancing drought tolerance in chickpea genes. This study represents the initial and singular instance in which CRISPR/Cas9 gene editing has been employed in chickpea research.
4.4 Arachis hypogea (Peanuts or Groundnut)
Groundnut is an important leguminous crop that exhibits a notable concentration of oleic acid. One of the primary breeding goals for peanuts is to increase their oil content. The oil possesses significant industrial utility and benefits, such as prolonged shelf life and antioxidant properties. The CRISPR/Cas9 gene-editing system was leveraged in a study that edited the ahFAD28 gene, which is responsible for the conversion of oleic acid to linoleic acid in fatty acids. The CRISPR/Cas9 system has effectively modified the gene to introduce desirable traits. The targeted mutation of this gene was achieved using groundnut protoplast and culture [46]. In a separate experiment, the utilization of the CRISPR/Cas9 tool was explored to edit the allergen gene (Ara h 2) in peanuts. The altered version of this gene resulted in enhanced nutritional qualities of peanuts for individuals with peanut allergies. Shu et al. [47] performed a study wherein CRISPR/Cas9 was employed to investigate the functions of Nod factor receptors (NFRs) in peanut nodule formation, particularly in the initiation of a symbiotic relationship with rhizobia. The mutants that underwent editing to contain two AhNFR5 genes exhibited a Nod-phenotype. Conversely, the mutants that were picked for containing two AhNFR1 genes were still capable of forming nodules following inoculation.
4.5 Vigna radiata (Mungbean)
Given the current accessibility of complete genome sequencing and the extensive collection of 1481 mung bean entries that have undergone comprehensive evaluation for different agronomical characteristics [48], there exists a significant opportunity for harnessing CRISPR/Cas9 gene editing in mung bean breeding initiatives. The successful implementation of CRISPR/Cas9-mediated gene editing in cowpea (V. unguiculata) has been reported, wherein a symbiosis receptor-like kinase gene was targeted to disrupt symbiotic nitrogen fixation [49]. The efficacy of CRISPR/Cas9 in a Vigna system implies the potential applicability of genome editing in additional species, such as mung bean. The initial objectives for gene editing in mung bean would encompass resistance to diseases and other desirable traits. The development of mung bean cultivars that exhibit resilience to fluctuating climate conditions would facilitate the global expansion of mung bean agriculture.
4.6 Vigna ungiculata (Cowpea)
Cowpea is a leguminous crop that is well-suited for cultivation in warm and arid regions [50]. It is considered as an orphan grain legume. Globally, the production of cowpeas increased from 9.7 MT in 2009 to 14.4 MT in 2019. Cowpeas exhibit a protein level of 25% by dry mass and exhibit a high-lysine content, thereby enabling them to serve as complementary dietary ingredients in cereal crop-based diets.
The recalcitrance of cowpea towards transformation has been observed to impede the regular application of CRISPR/Cas9 techniques. Che et al. [51] have reported enhanced transformation efficiencies ranging from 4.5% to 37% across nine cowpea varieties. Che and associates employed a CRISPR/Cas9 framework that drives the expression of Cas9 under the control of soybean elongation factor (GmEF1A2) promoter and a gRNA under the promoter of VuU6 to generate a total of 35 T1 plants. Che and colleagues specifically targeted the VuSPO11-1 gene to facilitate the production of asexual plants that are suitable for hybridization. Juranić et al. [52] designed a transient approach to evaluate CRISPR/Cas9 constructs within a 48-h timeframe using Agrobacterium infiltration of detached leaflets, as a means of surmounting the challenge of testing such constructs in stable cowpea transformants.
5 Conclusion
Legumes have been a staple in human and livestock diets since ancient times. The uniqueness of these plants lies in their possession of multiple nutritional benefits and their ability to withstand various diseases. Population growth has exerted a major supply burden for these crops on the food supply network. Researchers are introducing new variations for various crop traits [53, 54]. The application of the CRISPR/Cas9 tool has become increasingly significant in contemporary genome editing, with potential implications for achieving global food security. This widely used tool demonstrates crop modification without the use of transgenes. Legume crop genome editing using the CRISPR/Cas system has not yet reached its full potential [55] and it is anticipated that CRISPR/Cas9 in combination with other molecular approaches would significantly improve legumes and increase their yield.
CRISPR/Cas9 technology has undergone significant advancements, which have expanded the range of possibilities for accurately and effectively manipulating genes through genetic material addition or deletion. The utilization of CRISPR/Cas9 technology presents innovative opportunities for the exploration of functional genomics and the enhancement of diverse characteristics in grain legume crops. The efficacy of genome editing in enhancing legume quality depends on the presence of proficient procedures for plant transformation and complete plant regeneration, as well as a favorable regulatory framework and substantiation of societal endorsement of gene-edited crops.
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Das, D., Acharjee, S. (2024). Application of CRISPR/Cas-Mediated Genome Editing Techniques in Leguminous Crops. In: Ricroch, A., Eriksson, D., Miladinović, D., Sweet, J., Van Laere, K., Woźniak-Gientka, E. (eds) A Roadmap for Plant Genome Editing . Springer, Cham. https://doi.org/10.1007/978-3-031-46150-7_15
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