Biotechnological Approaches for Plant Protection and Growth Promotion

1. Introduction

Projections based on conservative estimates suggest that the human population will persist in its growth trajectory, with an expected increase of around 2 billion in the next 30 years. As a result, the population is projected to reach approximately 10 billion by 2050, up from the current 7.7 billion [1]. This presents significant challenges in feeding an exponentially expanding population. To tackle this issue, agriculture must assume a pivotal role in boosting food yields.

Throughout history, staple crops have been susceptible to both biotic and abiotic stresses, leading to food shortages and substantial economic losses. To address this issue, plant breeders have been developing resistant crop varieties for several years to minimize such losses. However, these stresses have eventually been able to overcome the resistance. To combat crop losses, numerous pesticides have been developed, resulting in heavy dependence on these compounds for plant disease control. Unfortunately, the use of pesticides has also incurred significant costs to both public health and the environment. The combined efforts of conventional breeding programs and plant biotechnology, involving different techniques such as gene transformation, omics approaches, and plant transformation, have the potential to make substantial contributions to sustainable agriculture. Agricultural biotechnology has seen intensive research in the area of crop protection, with a focus on genetic engineering strategies to safeguard crops from different stresses that can impair productivity, such as biotics stresses; fungi, bacteria, viruses, insects, nematodes, abiotic stresses; drought, heat, nutrient deficiencies. In this chapter, the focus will be on the utilization of advanced biotechnological tools, including gene transformation, omics techniques, genome editing, and other sophisticated methods. These techniques make use of transcriptomic, proteomic, metabolomic, phenomics, RNA interference, and epigenome modifications to enhance plant resilience.

3. Biotechnological approaches for crop improvement

Biotechnological approaches for crop improvement refer to the application of modern biotechnology techniques to enhance the characteristics and performance of crops. These methods offer numerous benefits, including increased yield, improved resistance to pests and diseases, enhanced nutritional content, and adaptation to various environmental conditions [2]. Some of the key biotechnological approaches used for crop improvement include: genetic engineering, Marker-Assisted Selection (MAS), genomic selection, RNA interference (RNAi), genome editing, tissue culture and cloning, metabolic engineering, synthetic biology.

3.1. Gene Transformation

Gene transformation involving cry genes, fusion proteins, lectins, alpha-amylase inhibitor, and chitinase is an advanced biotechnological approach used to enhance crop resistance against pests and diseases. Each of these components plays a specific role in providing the plant with defense mechanisms, leading to improved protection against various threats.

3.1.1. Cry genes

Gene transformation, particularly the introduction of cry genes, is a widely used biotechnological approach for crop improvement. The protein which is known as crystal, also known as the cry toxin (cry from crystal). Crystal inclusions are produced during sporulation by the soil bacterium Bt. The inclusions are hazardous proteins expressed by Cry genes that have been demonstrated to be poisonous to several types of protozoa, nematodes, and insects are among them [3]. The harmful proteins (Bt) don't harm people or unintended organisms. Numerous studies have shown that Bt produces Cry toxins, which discontinues the formation of disease causing insect larvae. Some strains of Bt, such as Bt israelensis, can produce a poisonous crystal known as cytolytic protein, or Cyt toxin, in addition to the Cry toxins. The name "Cyt toxin" (or protein) originates from its ability to lyse various invertebrate and vertebrate cells in-vitro, where in dipteran, this Cyt toxin enhances the effectiveness of Bt [4].

The risk of insect pests developing resistance to broad-spectrum chemical insecticides has led to the engineering of various crop plants with different cry genes, providing resistance against major insect pests [5,6,7]. However, this development of Bt crops has also resulted in insects developing resistance to Bt toxins. There are a number of example available now where, Cry gene transformation used for crop improvement. For instance, Cry1Ac and Cry2Ab genes have been widely incorporated into cotton plants to confer resistance against bollworms (Helicoverpa spp.) and other lepidopteran pests. Bt cotton has been commercially grown in several countries, and its cultivation has shown significant reduction in insecticide usage and increased yields [8]. To guard maize against specific pests like the European corn borer (Ostrinianubilalis) and corn rootworm (Diabrotica spp.), the Cry1Ab, Cry1F, and Cry3Bb1 genes have been inserted. Many nations, especially in North and South America, have widely embraced Bt maize [9]. Similarly, to increase resistance against lepidopteran pests including the soybean looper (Chrysodeixis includens) and velvet bean caterpillar (Anticarsia gemmatalis), the Cry1Ac and Cry1F genes have been put into soybean plants. The development and marketing of Bt soybeans are currently under consideration [10]. Furthermore, Cry1Ab, Cry1Ac, and Cry2A genes have been introduced into rice to protect against the rice stem borer (Chilo suppressalis) and other lepidopteran pests, without experiencing any decreased yields. It was discovered that the chimeric B. thuringiensis toxin Cry2AX1 produced in rice was efficient against a number of significant lepidopteran insect pests [11,12,13]. In addition, Cry3A gene has been incorporated into potatoes to confer resistance against Colorado potato beetle (Leptinotarsa decemlineata), a major pest affecting potato crops. Bt potato varieties have been developed and are being evaluated for commercial cultivation [14].

3.1.2. Fusion proteins

A fusion protein is created by combining two or more functional protein domains into a single polypeptide chain. This approach allows for the introduction of multiple traits or functionalities into a crop plant through a single transformation event. Generation of fluorescent fusion proteins have found widespread application as powerful tools for directly visualizing protein localization and dynamics within cells. The novel method facilitates robust co-expression of chimeric fluorescent fusion proteins in plants, compatible with current fluorescent protein-based bio-imaging. It addresses conventional limitations by employing a single expression vector containing multiple semi-independent expressing cassettes with individual promoters, fluorescent tags, target proteins, and terminators [15]. The use of a single expression vector to produce chimeric fluorescent fusion proteins is completely compatible with various technologies such as CRISPR-Cas9, RNAi, and protein over-expression. These technologies have been employed to investigate the functions and interactions of multiple genes in plants, as indicated by studies [16,17,18]. Lectin has been utilized as a carrier protein to create several effective fusion proteins. The lectin domain has been identified as the factor responsible for the observed rise in insect mortality caused by fusion proteins, as it promotes the binding process and facilitates the entry of the toxin into the insect more effectively [19]. Here are recent examples of studies related to fusion proteins in crop gene transformations, for instance, disease resistance; fusion proteins have also been investigated for disease resistance in crops. The recent study focused on a fusion protein combining an antimicrobial peptide with a plant defensin. This fusion protein enhanced resistance against fungal pathogens in tomato plants [20]. Nutritional enhancement; the fusion protein combining a high-lysine protein with a storage protein. This fusion protein resulted in increased lysine, content in rice grains, addressing a nutritional deficiency in this staple crop.

3.1.3. Lectins

Lectins are proteins that occur naturally in various organisms, such as animals, plants, and microorganisms. They have the ability to bind specifically to carbohydrates, such as sugars and glycoproteins, and play various roles in biological processes. There are four primary categories of plant lectins that may be identified based on the overall domain architecture: merolectins, hololectins, chimerolectins, and superlectins. According to Peumans and Damme [21], lectins' main roles include acting as storage proteins, cell surface adhesion, and recognition molecules in the immune system, and defensive mechanisms for plants against infections and pests that invade their environments. In the field of genetic engineering and crop improvement, lectins have been explored for their potential applications in several ways, few of them are discussed in this chapter. For instance, antifungal activity; plants have a predominance of lectins that bind to chitin, a complex polymer found in the cell walls of fungi. CBL lectins attach to chitin and prevent fungal development. Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Colletotrichum lindemuthianum, and Aspergillus niger were all susceptible to the antifungal effects of a galctose binding lectin that was isolated from seeds of Bauhinia ungulata L. (Caesalpinoideae) [22]. Similarly, by introducing lectin genes into plants, they aim to create crops that can produce lectins toxic to specific insect, reducing the need for chemical insecticides. Extensive documentation exists regarding the insecticidal properties of plant lectins against a variety of hemipteran insects [23]. Lepidoptera, Coleoptera, and Homoptera, three key insect orders, have all been discovered to be noxious to lectins from a variety of sources. Larval weight loss, mortality, feeding inhibition, delays in total developmental time, adult emergence, and fecundity in the first and second generations is some of the negative impacts of lectins on biological parameters of insects [24].

3.1.4. Alpha-amylase inhibitors (AAIs)

It has been investigated as a tool in gene transformations for crop improvement. AAIs are proteins that inhibit the activity of alpha-amylase, an enzyme involved in the breakdown of starch. By introducing AAI genes into crops, researchers aim to enhance resistance against pests and improve various agronomic traits. Mehrabadi et al. [25] reported that numerous phytophagous insects, including those found in stored products, rely on alpha-amylases for their survival, as they consume diets that are rich in polysaccharides. The insects convert starch to maltose and subsequently to glucose using alpha-amylases. It has been observed that only alpha-amylases capable of breaking down starch or glycogen have been identified in insects. When alpha-amylases are inhibited by AAIs, it can lead to compromised nourishment and a lack of energy. This has led to the exploration of AAIs as a potential means of conferring pest resistance to crops. Insects that feed on crops require alpha-amylase to digest starch, and the inhibition of this enzyme can disrupt their digestion and reduce their fitness. To enhance resistance against pests that rely on starch as a primary energy source, such as certain beetles, weevils, or caterpillars, researchers have incorporated AAI genes into crops. According to research [26], the presence of wheat alpha-amylase inhibitor (WAAI) in transgenic tobacco plants has been shown to elevate the mortality rate of lepidopteran larvae by 30-40%. Similarly, transgenic pea and azuki plants expressing alpha-amylase inhibitor derived from common bean have been observed to exhibit absolute resistance to bruchid beetle and weevil infestations.

3.1.5. Chitinase

Chitin, a crucial component of pathogenic fungal cell walls, can be targeted by chitinases, which can deactivate the fungi without causing harm to the plants. This can increase not only the plant's defense mechanisms against fungal infections but also stimulate plant growth and yield. The use of chitinases and recombinant technologies can be an effective approach to enhance plant resistance to fungal diseases. Chitinases have a high affinity for polymer chitin, which allows them to break it down into N-acetylglucosamine and low-molecular-weight COS (chitooligosaccharides). It has the potential to be a top contender in the near future for managing plant diseases due to its simplicity of use [27]. Here are some important examples of chitinases uses in gene transformations in different crops. For instance, a vector was designed to generate synthetic microRNA (amiR-24) that targets the chitinase gene of H. armigera, which is one of the most destructive polyphagous pests [28]. Through the use of host-induced RNA interference, tobacco and tomato plants having resistant against Helicoverpa armigera were developed [29,30]. The introduction of a chitinase gene from Spodoptera littoralis resulted in the production of transgenic maize plants that are resistant to insects [29]. In addition, chitinases have also been studied for their role in combating fungal diseases in crops. For instance, a gene for maize (Zea mays) chitinase “chitinase 2” effectively works against pathogen rot F. graminearium [31]. Nicotiana sp.'s osmotin gene (ap24) and Oryza sativa's chitinase gene (ch11) have been found to mitigate sheath blight disease caused by R. solani [32]. The chitinase I gene from the Hordeum vulgare cultivar Haider-93 inhibits the growth of the phytopathogenic fungi A. solani, R. solani, and V. dahliae. Additionally, Hordeum vulgare's class 11 endochitinase gene hinders A. solani from developing [33,34]. By expressing chitinase genes in crops, researchers aim to enhance the plants' defense against fungal infections and improve disease resistance.

3.1.6. Protease Inhibitors

Protease inhibitors (PIs) are predominantly protein molecules that obstruct the activity of proteases, which are enzymes that facilitate the disintegration of proteins, produced by pathogens. While the function of some individual PIs and their target enzymes has been extensively studied, it is uncertain whether this defensive mechanism occurs naturally in plants. In addition, several plants produce multiple types of PIs, and it was previously unclear whether these proteins work collaboratively to provide protection or if they serve additional purposes.

The categorization of PIs into families based on the specific reactive site present in their sequences was proposed by Laskowski and Kato [35]. The adoption of this nomenclature simplified the categorization of PIs into four major families, namely: (I) cysteine protease inhibitors, (II) metalloprotease inhibitors, (III) aspartic protease inhibitors, and (IV) serine protease inhibitors. Plant PIs are classified according to their functional and biochemical properties, such as cysteine protease inhibitors, cereal trypsin/α-amylase inhibitors, mustard trypsin inhibitors, metallo carboxypeptidase inhibitors, potato-type II protease inhibitors, potato type I inhibitors, serpins, soybean trypsin (Kunitz) inhibitors, Bowman-Birk serine protease inhibitors and squash inhibitors. Rawlings and Barrett [36] presented an updated classification system for PIs, in which they are grouped into families and clans, akin to the classification system for peptidases/proteases suggested by Laskowski and Kato [35]. Nevertheless, this system aims to mirror the evolutionary relationships among PIs and is structured hierarchically, comprising three main levels: inhibitors, families, and clans, with the clan being the highest level of evolutionary divergence [36].

Here we cite some successful examples to reveal the potential of these PIs (Table 1). Four distinct PIs that were effective against spider mite, insect, herbivore, and fungal attacks were identified in Arabidopsis thaliana. These PIs were AtkTI4, AtkTI5, AtSerpin1, AtWSCP, and UPI [37,38,39,40,41]. In Solanum tuberosum, six different PIs were also identified [42,43,44,45,46,47,48,49,50]. Plant-parasitic nematodes (PPNs) are a serious global problem to cereal production [51,52,53,54,55]. Different types of PIs, such as cowpea trypsin inhibitor (CpTI), cystatins and serine proteinase inhibitors [56], have been reported to act following attacks by PPNs. These proteinase inhibitors commence production and become effective against all classes of proteinases from nematodes [57]. Various genes have been employed to confer resistance to different PPNs. Examples include PIN2 from potato (Solanum tuberosum L.) introduced into durum wheat (Triticum durum Desf.) to protect against H. avenae, Mi gene from tomato to confer resistance against M. incognita, Hs1pro-1 from sugar beet (Beta vulgaris) to defend against H. schachtii, Gpa-2 from potato to provide protection against Globodera pallida, and Hero A from tomato to impart resistance against G. rostochiensis [56,58,59]. Likewise, the "cysteine proteinases," which are the primary digestive enzymes in many nematodes, have been targeted for the production of plants resistant to nematodes. Hartl et al. [60] also discovered four serine PIs from Solanum nigrum that provided protection against various natural herbivorous insects in both field and greenhouse experiments. To summarize, PIs are a potential approach to attain control over plant pests, as they have been shown to safeguard specific tissues, serve as storage proteins, and regulate the activity and release of proteases.

3.2. Omics approaches

The regulation of molecular factors determines plant responses to various biotic and abiotic stressors. Therefore, an integrated omics approach can be employed to comprehend the biological interactions and molecular mechanisms that plants invoke in response to these stressors. Genome sequencing of plants has identified a range of biotic and abiotic stress-responsive genes and broadened the genomic resources available for investigating stress tolerance within their gene pool. There are multiple “omics” studies have been reported i.e., genomics, transcriptomics, proteomics, epigenomics and metabolomics.

3.2.1. Role of transcriptomics in plant protection

The transcriptome is the complete set of transcripts in a cell, during a particular stage of physiological developmental. The transcriptome issued to understand the functional and molecular constituents of the plants [78]. The aim of transcriptome study is to categorize all types of transcripts such as mRNA, large & small RNA and non-coding RNA [79]. From a few past decades, transcriptome (RNA-sequencing) has made robust advances for the development of different stress resistance plant. The RNA-Seq technique provides valuable knowledge about gene expression in various environments and aids in uncovering previously undiscovered genes [80,81,82,83,84,85,86,87,88,89,90,91,92,93,94]. This, in turn, contributes to a deeper understanding of metabolic and cellular processes. The key advantage of RNA-Seq is its capability to analyse and compare gene expression patterns across multiple samples. Before the rise of deep sequencing technology, microarray analysis served as the primary method for quantifying gene expression levels. Although hybridization techniques have been extensively employed, their limited sensitivity presents challenges in detecting low-abundance targets and subtle changes in the expression levels of the target gene. Consequently, RNA-Seq has been shown to be more accurate than microarray analysis. RNA-Seq can quantify the absolute amount of each molecule in a cell population and directly compare the results across experiments. Furthermore, RNA-Seq can assist in discovering novel genes since existing transcript annotations in databases may not be exhaustive. Additionally, RNA-Seq results can be self-assembled, bypassing the requirement for known genome annotations and enabling the identification of new genes [95].

3.2.2. Role of metabolomics in plant protection

Metabolomics, an emerging and captivating tool in the field of omics, has been widely employed in crop improvement. It plays a critical role in assessing the tolerance of crops to biotic and abiotic stress and in metabolic-assisted breeding. Thus far, noteworthy progress has been made in the creation of cutting-edge metabolomics tools aimed at improving crop yields [96]. There are two types of metabolites in the plant metabolome, namely primary and secondary metabolites. Analysing both primary and secondary metabolites provides a comprehensive understanding of the biochemical mechanisms underlying plant metabolism [97]. Complex metabolic pathways are closely interconnected with various primary and secondary metabolites present in plants. Metabolites such as terpenes, alkaloids, and phenolics are chemical adaptations that enable plants to cope with environmental stressors or provide defense against microorganisms, insects, predators, and even other plants (allelochemics) [98]. The deployment of advanced metabolomics techniques, such as liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and non-destructive nuclear magnetic resonance (NMR) spectroscopy, has enabled the efficient detection, identification, evaluation, and assessment of these metabolites [99]. Metabolomics has been extensively used in various studies aimed at enhancing the biotic and abiotic resistance of crop plants. For instance, Yadav et al. [100] utilized metabolic profiling based on GC-MS to explore the mechanism of drought tolerance in eight different wheat cultivars. They noted a rise in the levels of glutamine, serine, methionine, lysine, and asparagine. Similarly, Yang et al. [101] also performed metabolic profiling on maize exposed to drought stress, using RP/UPLC-MS. Their findings revealed an increase in lipid and carbohydrate metabolism, as well as an acceleration of the glutathione cycle. Nam et al. [102] used LC-MS and GC-MS-based metabolic profiling to confirm the variation in metabolite accumulation in soybean under drought stress. Similarly, for biotic stress such as Zymoseptoria tritici in wheat, Seybold et al. [103] utilized FT-ICR-MS to analyze the metabolome and uncover stress-responsive mechanisms. Cuperlovic-Culf et al. [104] performed metabolomic profiling on wheat exposed to Fusarium graminearum using NMR spectroscopy, and detected the presence of multiple disease-resistant biomarkers such as 3-hydroxybutyrate, trehalose, phenylalanine, myoinositol, asparagine and L-alanine.

3.2.3. Role of epigenomics in plant protection

According to Wu and Morris [105], the term "epigenetics" was introduced by Conrad Waddington in the 1940s. Waddington defined epigenetics as the study of inheritable modifications in gene function that do not entail changes in DNA sequence, and can be passed on during cell division or sexual reproduction. The genetic code, or DNA sequence, is only partially responsible for determining a trait, as the epigenetic code, or chromatin state, also plays a significant role. According to Grant-Downton and Dickinson [106,107], the nucleosome is the fundamental building block of chromatin, consisting of eight histone proteins (two of each H2A, H2B, H3, and H4) around which approximately 150 base pairs of DNA are coiled. Typically, DNA that is loosely packed around histones represents euchromatin, which is actively transcribed, while DNA that is tightly packed around histones represents heterochromatin, which is transcriptionally inactive, as discussed by Donà and Mittelsten Scheid [108], and Probst and Scheid [109]. The stability of a particular chromatin state is not guaranteed, as it can be altered by various processes. DNA undergoes methylation or demethylation, while histones can be subject to various post-translational modifications, including methylation, acetylation, ubiquitination, phosphorylation, sumoylation and biotinylation [110,111,112]. Of all the histone modifications, lysine residues' methylation and acetylation have received the most extensive research. Lysine acetylation introduces a negative charge to the histone, leading to the repulsion of the negatively charged DNA. This results in the relaxation or opening up of the chromatin structure (euchromatin), enabling the process of transcription. In contrast to acetylation, methylation of lysine residues does not change the charge of the histone. Each site of modification can experience mono-, di-, or tri-methylation of lysine residues, resulting in added functional diversity at these sites. According to Feng and Jacobsen [113], euchromatin is typically characterized by H3K4me3 and H3ac/H4ac, while heterochromatin is associated with H3K27me3, H3K9me2, and general histone deacetylation. The intricate modifications to chromatin ensure its flexibility in responding to diverse developmental and environmental stimuli. Several studies have reported changes in cytosine DNA methylation in response to drought stress in wheat seedlings, roots, and leaves, with tissue and genotype-specific modifications observed in two different wheat cultivars with varying drought tolerance [114]. Additionally, genome-wide analyses of histone acetyltransferases and deacetylases have provided evidence of the involvement of histone acetyl transferases in the response to drought stress [115]. Similar research has been conducted on the epigenetic changes resulting from PPN infection in various crops, such as tomatoes, soybean, and rice [116]. These studies have revealed the causal impact of hypomethylation on immunity, as demonstrated by a significant reduction in plant susceptibility upon treatment with the DNA methylation inhibitor 5-azacytidine.

3.2.4. Role of phenomics in plant protection

Phenomics involves analysing phenotypes on a large scale. Through advanced phenotyping techniques, it has become possible to predict genotypes that are susceptible to abiotic stress [117]. The implementation of an automated greenhouse system has demonstrated success as a high-throughput approach for plant phenotyping. The system enables non-destructive screening of plants using image acquisition techniques over a specific period. Capturing and analysing multiple plant images with advanced algorithms allows the prediction of specific phenotypes [117]. Plants displaying tolerant phenotypes have proven to be valuable sources of genomic resources and are frequently selected for diverse molecular techniques, such as high-throughput sequencing, to identify relevant alleles of interest. Nevertheless, phenomics has certain limitations as it may not always yield precise correlations between values obtained from pot culture and those from field experiments.

3.3. Genome editing technologies

The rapid growth of the global population necessitates the development of novel crop improvement techniques to satisfy the escalating need for food and nutritional security. Traditional approaches are inadequate and labour-intensive. The precise, efficient, and targeted alteration of genomic loci is made possible through a set of sophisticated molecular biology techniques collectively known as genome editing, as described in studies [118,119]. RNAi and CRISPR/Cas9 are innovative RNA-based methods, with RNAi being particularly useful in identifying stress-responsive genes. RNAi is a versatile tool for crop improvement, providing benefits such as increased yield, stress resilience, and improved nutrient content. However, updated regulatory frameworks, risk identification, and user-friendly approaches are necessary to fully leverage these benefits [120].

The four primary mechanisms of site-specific genome editing, namely mega nucleases (MegNs), zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENs), and CRISPR/Cas-9 (CRISPR/Cas-associated protein 9), have created new possibilities for breakthroughs in both medicine and agriculture.

3.3.1. Zinc finger nuclease (ZFNs)

The creation of zinc finger nucleases (ZFNs), which are artificial restriction enzymes designed for site-specific editing, marked the beginning of genome editing technology. ZFNs comprise multiple zinc finger domains and a Fok-1 endonuclease domain, creating hybrid heterodimeric proteins. The construction of ZFNs involves the utilization of zinc fingers, which are transcription factors that recognize 3-4 base pairs each. According to research [121], the zinc finger domain in eukaryotic transcription factors identifies DNA, and the nuclease domain, often derived from the bacterial restriction enzyme FokI, creates double-strand breaks. By combining the DNA-binding and DNA-cleaving domains, it is possible to create a highly precise set of "genomic scissors". The targeting of any gene in any organism is feasible using appropriately designed pairs of ZFNs. According to studies, zinc finger recognition depends on DNA sequence matching, and DNA repair mechanisms, including HR and NHEJ, are common in almost all species. Incorporating more zinc fingers (4, 5, and 6 finger pairs) in ZFNs has been shown to enhance specificity and efficiency. Additionally, the modular assembly of pre-characterized zinc fingers using standard recombinant DNA technology can improve targeting. As an illustration, the insertion of PAT gene cassettes resulted in herbicide tolerance and alterations in inositol phosphate profiles in developing maize seeds by disrupting the endogenous maize gene ZmIPK1, according to a study [122]. In maize, this technique has also been employed to stack multiple advantageous traits, allowing for greater potential in crop improvement [123,124].

3.3.2. Mega nucleases (MegNs)

Endo deoxyribonucleases known as Mega nucleases (MegNs) are naturally present in microbial life, eukaryotic mitochondria, and chloroplasts. These nucleases are typically encoded by genes found within self-splicing elements. MegNs are enzymes that exhibit high specificity, as they cleave ds-DNA selectively at recognition sites that comprise 14-40 base pairs. These nucleases hold great potential for various applications, such as the creation of therapies for inherited diseases caused by frameshift mutations or nonsense codons. Natural MegNs are not without limitations, as their use necessitates the introduction of a recognized cleavage site into the targeted region of interest. Additionally, separating the DNA-binding and DNA-cleavage domains presents challenges. Designing sequence-specific enzymes for all possible sequences is time-consuming, costly, and locating an enzyme that targets a specific locus is difficult. Consequently, routine utilization of MegNs in genome editing faces technical constraints and limitations [125].

3.3.3. Transcription activator-like effector nucleases (TALENs)

TALENs offer a cost-effective, efficient, and safer alternative to other genome editing tools. They possess the capability to target particular regions of the genome with precision. Similar to ZFNs and MegNs, TALENs require re-engineering for each targeted DNA sequence. Both TALENs and ZFNs are modular and possess natural DNA-binding specificities. TALENs are hybrid proteins formed by combining a non-specific Fok I restriction endonuclease domain with a DNA-binding domain that can recognize any arbitrary base sequence. The DNA-binding domain contains well-conserved repeats from transcription activator-like effectors (TALEs). The TALE protein comprises three domains: an amino-terminal domain with a transport signal, a DNA-binding domain with repeating 34 amino acid sequences arranged in tandem, and a carboxyl-terminal domain with a nuclear localization signal and a transcription activation domain. TALENs have been effective in editing plant genomes and hold promise for generating genetically modified laboratory animals to study human diseases. However, the TALEN technique may prove challenging for those with limited familiarity with molecular biology, and the size of TALENs is greater than that of ZFNs. In comparison to ZFNs, TALENs possess an advantage because each domain recognizes a single nucleotide. This feature simplifies the design of TALENs and results in less complex interactions between the DNA binding domains derived from TALENs and their targeted nucleotides [126,127]. Examples of the use of TALENs in plant genome editing include the creation of powdery mildew-resistant wheat, maize mutants exhibiting the glossy phenotype, and the improvement of cell wall composition and saccharification efficiency in sugarcane. In addition, TALENs can alter the nutritional profile of crops, for example, producing soybeans with high oleic acid and low linoleic acid contents, which can improve the shelf life and heat stability of soybean oil. Knocking out the vacuolar invertase (VInv) gene in potato tubers led to the production of tubers with reducing sugar levels that were undetectable. Moreover, TALENs have the potential to accelerate plant breeding by generating haploid plants that inherit chromosomes from only one parent, as evidenced in maize and Brassica oleracea. These instances highlight the diverse applications of TALENs in plant genome editing [128,129,130,131,132,133,134,135].

3.3.4. Clustered regularly inter spaced short palindromic repeats (CRISPR)

The CRISPR-associated (Cas) system is a genome-editing tool that is widely used in plants and other organisms to enhance our comprehension of gene function, diagnose diseases, and improve crop quality. Although Cas9 is frequently employed for DNA modifications, its use for RNA manipulation at the post-transcriptional level is limited. The discovery of CRISPR dates back to 1987 when Japanese researchers were investigating a crucial gene in Escherichia coli [136]. Later, they were characterized by Francisco Mojica from the University of Alicante, Spain in 1993 and found to be involved in regulating gene expression in various bacteria and archaea. Over a decade later, they were identified as a bacterial adaptive defense system that comprises of CRISPR, DNA-targeting spacers, and Cas operon [137,138,139,140]. The identification of the CRISPR/Cas system in prokaryotes has resulted in its utilization as a sophisticated technique for editing DNA and RNA in different organisms. CRISPR/Cas systems are categorized into two primary classes, depending on whether a multi-protein effector complex or a single protein is present to modify the target. Further divisions into types and sub-types are based on differences in Cas proteins within these two classes. Type VI CRISPR/Cas systems, particularly Cas13, enable accurate RNA manipulation without causing permanent alterations to the genome. As a result, Cas13 is effective in studying RNA-related phenomena such as viral interference, RNA knockdown, and RNA detection in diverse organisms. Cas systems belonging to Class 2, particularly types II and V, are predominantly utilized as tools for genome editing. Cas9, which was one of the first proteins to be extensively examined and employed for DNA editing in animals, plants, and bacteria, is among the most commonly used [141,142,143,144,145,146,147].

CRISPR/Cas9 technology has been employed in tomato crops to improve the yield potential and quality of the crops by modifying the cis-regulatory control of quantitative trait loci. By employing CRISPR/Cas9, researchers have induced mutations in the SlCLV3 promoters in tomato to systematically investigate the correlation between phenotypic traits and cis-regulatory regions. This approach may prove beneficial in the advancement of tomato breeding [148]. In addition, endogenous plant upstream open reading frames (uORFs) have been edited using CRISPR/Cas9 to regulate mRNA translation, leading to the development of a mutant lettuce variety with enhanced tolerance to oxidative stress and an increased ascorbate content [149]. The utilization of CRISPR/Cas9 in targeting genes associated with tomato morphology, flower and fruit production, and ascorbic acid synthesis has enabled the introduction of favourable traits into wild tomato accessions, thereby expediting the domestication of crops [145]. The two-line system is crucial to hybrid O. sativa breeding, with thermo-sensitive genetic male sterility (TGMS) being extensively employed to enhance the crop's yield potential. CRISPR/Cas system has been implemented in the development of new thermo-sensitive genetic male sterility (TGMS) lines that are free of transgenes. This approach has resulted in the production of 11 new TGMS cultivars within a year, underscoring the technology's potential to enhance the efficiency of hybrid O. sativa breeding [150]. The CRISPR/Cas system has been employed to induce mutations in genes (DEP1, Gna1, IPA1, and GS3) in O. sativa, leading to the development of T2 generation mutants that exhibit traits such as increased grain number, larger grain size, and denser erect panicles [151]. Moreover, it is utilized to impart resistance against viruses such as Tomato yellow leaf curl virus (TYLCV), beet severe curly top virus (BSCTV) and Potato virus Y (PVY) in plants like N. benthamiana, cucumber and potatoes, respectively [152,153]. It has also been employed to generate herbicide-resistant crops, including soybean, rice, potatoes, maize, and flax, as well as to confer drought-resistant characteristics in maize (refer to Table 2 for details) [154,155,156,157,158,159,160,161].

3.4. RNA interference

RNA interference (RNAi) is a group of molecular processes that utilize a small RNA fragment to selectively target specific nucleic acid sequences and control gene expression [143]. Noncoding RNA-based gene regulation is an ancient process that may have existed prior to the emergence of cellular life [143,162,163]. It was first proposed by Jacob and Monod [164], but it took several decades before it could be experimentally verified. Since then, the topic has gained significant attention and has been thoroughly investigated, particularly during the 1980s [165].

RNAi employs small RNA molecules to regulate gene expression at the transcriptional and post-transcriptional levels. Insights into the molecular architecture and functional modules of RNAi have been gleaned from recent structural studies, providing valuable knowledge. Structural studies have examined proteins and nucleic acid complexes involved in RNA biogenesis, including Argonaut, RNase III, Dicer, PIWI, Drosha, and DGCR8. These studies have elucidated the mechanisms underlying nucleic acid recognition and cleavage [166]. Despite its intricacy, the RNAi phenomenon is mediated by only three principal proteins: Ago-Piwi, Dicer-like protein (which typically comprises RNAseIII and helicase domains), and RNA-dependent RNA polymerase (RdRP), along with several auxiliary proteins [167]. At least some members of four out of the five eukaryotic supergroups possess these three components. However, excavates either lack homologs of Dicer or possess Dicer-like proteins that lack either the helicase component or the tandem RNase III portion. As a result, the existence of RNAi in most excavates is uncertain [168]. However, it is believed that a functional RNAi system containing these three proteins may have been present before the most recent common ancestor of existing eukaryotes, based on the "starphylogeny" of the five supergroups [169,170]. In addition, the dispersed distribution of the ancient paralogous pair Ago-Piwi across four eukaryotic supergroups suggests that this duplication occurred prior to the divergence of the supergroups [167,171]. RNAi is a mechanism that suppresses gene expression at the transcription or translation stage of specific genes and is composed of two primary pathways: small interfering (si) RNA-mediated gene silencing and miRNA-based pathways. These pathways defend against viruses and transposable elements and regulate gene expression in eukaryotes, respectively [172].

3.5. Marker-assisted selection

Marker-assisted selection (MAS) is a technique that entails identifying genes associated with particular traits in crops, such as quality traits, disease resistance, and tolerance to different stresses, for the purpose of selection [173,174,175,176]. In addition to identifying cultivars, assessing genetic diversity and purity, selecting parents, and studying heterosis, MAS is also a valuable tool for enhancing crop yield and nutritional quality. This could aid in narrowing the gap between the demand and supply of food needed to sustain the ever-increasing population [177]. The selection of plants carrying genomic regions that control the expression of desirable traits through the use of molecular markers is a key aspect of the MAS process [178]. MAS involves examining markers to identify DNA segments that are genetically associated with and impart resistance to specific diseases. Using DNA markers, it has proven to be a successful approach in developing disease-resistant cultivars, as demonstrated by several studies. An example of the successful integration of genes/QTLs, including Pi2, Pi9, GM1, and GM4, through marker-assisted selection to confer resistance against blast and gall midge in a rice variety (CRMAS2621-7-1) was reported by Das and Rao [179]. Jamaloddin et al. [180] employed gene-specific markers (xa13prom (xa13), pTA248 (Xa21), RM224 (Pi1), and Pi54MAS (Pi54)) to create two rice lines, TH-625-491, and TH-625-159 which harbour four genes and exhibit a strong resistance to bacterial blight and blast diseases. Hanson et al. [181] also used MAS to create F7 multiple disease-resistant tomato lines that exhibited resistance to yellow late blight, leaf curl disease, grey leaf spot, bacterial wilt, and tobacco mosaic virus. Similarly, Calayugan et al. [182] utilized marker-assisted selection to develop a nutritionally valuable rice variety.

3.6. Anther culture

Plant tissue culture is a laboratory technique that allows for the cultivation of plant cells, tissues in a sterile and controlled environment. In the field of plant breeding, haploid culture is a tissue culture method that employs plant reproductive organs as explants. By using haploid culture, recessive genes associated with tolerance to environmental stressors like drought, low temperature, and nutrient deficiency in self-pollinating plants can be identified. Afterwards, these haploid plants can be duplicated to generate double haploid (DH) lines, leading to the production of homozygous plants that possess the desired traits [183].

The anther is a male reproductive structure found in flowers, typically consisting of two lobes and a sac-like shape. Within the anther, microsporangia produce pollen grains through meiotic cell division. Androgenesis is a procedure that entails aseptically isolating the anther from the bud of flower and cultivating it on nutrient media to generate haploid plantlets [184]. The effectiveness and replicability of DH production are closely tied to the management of crucial elements in the process. An example of the importance of optimizing the composition of culture media can be seen in androgenesis, where it plays a crucial role in determining the fate of microspores [185]. Top of FormSome microspores follow a direct embryogenesis pathway, while others undergo an indirect organogenesis pathway. In the direct embryogenesis method, the anther behaves like a zygote and forms embryoids. These embryoids are then transferred to a favourable growth medium that promotes the development of the radical and plumule, leading to the eventual formation of haploid plantlets. In the indirect organogenesis method, the anther undergoes repeated cell divisions, resulting in the formation of callus tissue. After hormonal treatment, the callus tissue differentiates into shoot and root tissues, eventually giving rise to haploid plantlets. Through anther culture, various rice varieties like PSBRc50 ‘Bicol’ [186]; Risabell, Jankas, Abel [187]; CR Dhan 801 (CRAC2224-1041, IET18720); hybrid ‘CRHR 32’ [188] were released. Among wheat varieties, AC Andrew [189], Huapei 8 [190], Kharoba [191] and GK Déva [192] were developed through anther culture.

3.7. Embryo culture

It refers to the in-vitro cultivation of isolated immature or mature embryos, which include extraction of mature embryo before drying from completely formed seeds and put in a culture medium for in-vitro growth. Culturing embryos as explants offer several advantages, as outlined in Table 3.

Although zygotic or seed embryos are often utilized as explants to initiate callus cultures and development is facilitated by nourishing endosperm tissue, when two distantly related species are crossed, the endosperm tissue degenerates, impeding embryo development and preventing the formation of a viable plant. In such cases, embryo rescue techniques are employed to recover the hybrid embryo. Embryo rescue, performed through in vitro culturing of the embryo by various approaches such as embryo culture, ovule culture, and ovary culture [193].

3.8. Protoplast fusion

The term protoplast refers to living plant cells that have lost their cell walls due to enzymatic digestion. Their totipotent nature emphasizes their potential as a potent biotechnological tool for in vitro manipulation and crop improvement, bypassing sexual reproduction [194]. To isolate protoplasts, there are two commonly used methods: mechanical isolation (physical disruption) and enzymatic isolation (cellulose and pectinase enzymes). Protoplasts are delicate and have fully exposed outer plasma membranes, necessitating the removal of the cell wall without causing damage.

Somatic hybridization, achieved through protoplast fusion, is a valuable technique for producing hybrids between different species or genera. It involves merging protoplasts from two distinct genomes, identifying somatic hybrid cells, and subsequently regenerating hybrid plants [195]. It was first successfully demonstrated 50 years ago using Nicotiana glauca Graham and Nicotiana langsdorffii Weinm, enables gene transfer between species and facilitates the integration of parental nuclear and cytoplasmic genomes. Table 4 illustrates several examples of somatic hybridization in plants involving interspecific, intergeneric, and intertribal crosses. Protoplast fusion techniques can be categorized into chemical fusogen and electric fusion methods. The optimal chemical fusion of protoplasts is achieved by combining PEG with calcium [196]. To perform electric fusion, protoplasts are inserted into a culture vessel with electrodes and a potential difference is applied. This method is more effective and less harmful to the protoplasts, but it demands costly equipment [197].

Protoplast fusion can lead to the formation of cybrids in variable frequencies. Generating cybrids at high frequencies can be achieved through two methods: irradiating one parent protoplast before fusion or another approach is to generate enucleate protoplasts of one species and then fusing them with normal protoplasts of another species [198]. Protoplasts can also be used as an alternative to Agrobacterium for direct gene transfer. Agrobacterium's capacity to manipulate cells stems from its plasmids, and the first successful recovery of a transgenic plant was achieved through Agrobacterium-mediated transformation of tobacco protoplasts. Protoplast-mediated gene transfer has provided a breakthrough in genetic engineering [199].

3.9. Somaclonal variation

Somaclonal variation is the spectrum of differences observed in plants regenerated through somatic cell culture in vitro. It is a dynamic and innovative approach that enhances genetic diversity, expands the genetic foundation, and thereby enriches the genetic pool for significant and progressive advancements in crop improvement [200]. Somaclonal variation encompasses genetic or epigenetic changes in the DNA that result in discernible phenotypic differences compared to the original parent plant. These variations can arise from factors such as gene mutations, chromosomal abnormalities, genetic rearrangements, methylation changes, the presence of transposable elements, availability of phytohormones, extended periods and composition of in vitro culture and other mechanical aspects during the culturing process [201]. Moreover, the applicability of somaclonal variation is reduced by genotype dependency and genetic fidelity issues. Culture-induced genetic variations are heritable, whereas epigenetic variations are typically unstable, non-heritable, and tend to disappear in sexually reproducing plants. Molecular markers and reversed-phase HPLC (RP-HPLC) are useful tools for evaluating these genetic and epigenetic variations. Somaclonal variation can be categorized as androclonal, protoclonal, gynoclonal and calliclonal, and gametoclonal based on the tissue cultures used, including gametic, anther, ovary, protoplast, and callus. In vitro techniques, such as plant tissue culture, have been employed to induce various types of variation to expand and enhance the genetic diversity (Table 5).