**6. Use of transgenic approaches to introduce foreign genes into maize**

The adoption of genetically modified (GM) traits has been rapidly embraced by farmers worldwide, making it one of the fastest innovations in agriculture. As per findings presented by Brookes and Barfoot, [91], the worldwide economic benefits derived from genetically modified crop varieties amounted to US\$ 225 billion during the period spanning from 1996 to 2018, with developing countries accounting for 52% of these gains. In 2019, transgenic crops were grown across 190.4 million hectares in 29 countries, intended for both food and feed purposes. This represents a substantial rise from 1.7 million hectares in 1996, marking a remarkable 112-fold increase.

The most widely adopted GM crops include soybean, maize, cotton, and canola. Of these crops, maize takes the lead. In 2019, 31% of the total global maize cultivation area, encompassing 60.9 million hectares across 14 countries, was allocated for growing genetically modified maize varieties [92].

In the last three decades, GM maize varieties have been effectively introduced to the market, providing farms with traits like resistance to herbicides and insects. The initial wave of genetically modified maize featured a single gene with a precise mechanism targeting a particular order of insects to confer insect resistance. In subsequent generations, the approach included crossbreeding herbicide and insect-resistant traits, as well as diverse insect-resistant traits, to establish multiple mechanisms of action against a range of insect orders. Farmers have found these stacked varieties to be remarkably effective, delivering evident and comprehensive phenotypic results [93]. Developing traits linked to quantitative characteristics such as tolerance to abiotic stress, efficient nutrient utilization, and increased yield presents a more intricate challenge. These characteristics are influenced by numerous genes and are sensitive to environmental factors, which adds complexity to the development process. To examine how individual genes affect complex traits, companies have established extensive biotechnology pipelines that involve evaluating genes in real field conditions on a large scale [94]. A standard biotechnology pipeline comprises various stages, which encompass discovery, demonstrating feasibility, initial development, advanced development, pre-launch, and the eventual release of commercial varieties. Certain stages might briefly coincide, particularly when a promising lead is identified early in the discovery phase, leading to the initiation of optimization efforts before validation is finished. The phase of gene discovery entails the quest for potential genes, which can be arduous, expensive, and uncertain, especially for traits such as drought tolerance and yield, which demand clearly defined phenotypic reactions. Extensive phenotypic screening of model plants like *Arabidopsis thaliana* and *Oryza sativa* is performed to assess hundreds of candidate genes [95, 96]. The proof-of-concept stage involves creating events for each candidate gene and conducting preliminary phenotypic evaluations in controlled settings as well as small-scale field experiments. During the early development phase, there is a focus on optimizing the lead to enhance stability and increase protein expression. Candidates demonstrating favorable agronomic performance, consistent trait expression, and heritability are chosen. These chosen candidates are subsequently subjected to molecular-level characterization and extensive field trials conducted across various locations and over multiple years [97].

In the advanced development stage, the validated leads are incorporated into commercial lines, often employing molecular markers to expedite the breeding process and guarantee the successful transfer of traits. In this phase, regulatory data related to the toxicity of gene products, allergenic potential, compositional analysis, as well as environmental and human safety aspects are collected. During the prelaunch stage, the production of seeds for the novel GM variety is expanded, quality control protocols are instituted to guarantee trait consistency and purity, a regulatory report is submitted, and arrangements are made for the commercial release of the new GM trait hybrid. The duration to finalize the pipeline, which varies according to the trait and available resources, generally averages around 11–13 years.

#### **6.1 Enhancing traits in genetically modified (GM) maize varieties**

Twenty-five years ago, the debut of the initial commercially accessible insect-resistant GM maize [98, 99] marked the beginning of a journey that led to the *Enhancing Maize (*Zea mays *L.) Crop through Advanced Techniques: A Comprehensive Approach DOI: http://dx.doi.org/10.5772/intechopen.114029*

approval of 148 GM maize events for global commercial utilization [93]. By 2019, worldwide GM maize cultivation had expanded to cover 61 million hectares, with the most substantial acreage located in the USA (33 million hectares), Brazil (15 million hectares), Argentina (6 million hectares), and South Africa (2 million hectares) [92]. Among crops, maize holds the record for the largest number of approved GM events, totaling 148 events across 35 countries. Most of these events integrate traits like insect resistance and herbicide tolerance. Furthermore, approved traits for maize encompass fertility restoration, male sterility, heightened drought tolerance, phytase production, modified amino acids and alpha-amylase expression, improved photosynthesis, and increased ear biomass. These authorized traits encompass a total of 39 individual genes, with the largest proportion associated with insect resistance (18 genes) and herbicide tolerance (11 genes). The forthcoming generation of GM maize varieties poised for market release incorporates events featuring novel insecticidal proteins, including Vpb4Da2, DvSnf7 RNA, and IPDO72Aa. These proteins are designed to manage insect populations that have developed resistance to Bt [100–102]. Additional prospective varieties seek to enhance grain yield by upregulating the zmm28 and ZM-BG1H1 genes [103, 104], and to bolster drought tolerance through the overexpression of ARGOS8 [105].

#### **6.2 Concerns and regulations related to GM crops**

It's crucial to emphasize that scientific evidence confirms that GM crops do not present any heightened risks to both humans and the environment in comparison to conventional crops (National Academies of Sciences, Engineering, and Medicine, 2016). Nevertheless, public apprehensions and restrictions related to GM technology, notably within the European Union, continue to hold substantial importance [106]. To tackle these concerns and potentially shift public opinion, there is an ongoing exploration of new plant breeding technologies (NPBTs), including cisgenesis, intragenesis, and genome editing. Proficient communication of these technologies to the public holds the potential to impact public approval [107, 108]. Following 7 years of GM crop cultivation with no observable health impacts, apprehensions about potential environmental hazards, particularly gene transfer to other species, have gained more prominence than concerns regarding food safety. Pollen and seeds released into the environment may convey genetic characteristics to neighboring crops or wild relatives. Self-pollinating crops like wheat, barley, and potatoes have minimal chances of gene transfer, whereas cross-pollinating crops like sugar beets and corn are of greater concern in this regard. Although numerous cultivated crops lack wild counterparts in their present cultivation regions, the places of origin for these crop species are notably vulnerable to the infiltration of transgenic traits into native varieties or landraces. There is apprehension that transgenic varieties possessing a competitive edge might progressively supplant valuable genetic diversity. Consequently, Mexico, a nation harboring over 100 distinct corn varieties, has enforced a ban on the cultivation of transgenic corn.

### **7. Omics technologies in maize improvement**

Advancements in the fields of biotechnology and computational sciences have paved the way for the generation of omics data on a large scale for various plant sets, including different varieties and species [109]. The application of diverse omics

**Figure 2.** *Representation of different omics approaches used for maize crop improvement.*

techniques has facilitated the discovery of genes, their respective functions, the specific types of RNA or proteins involved, their structural attributes, and the pathways influencing the development of final morphological traits. These identified genes can be subject to manipulation or transfer to create novel varieties or hybrids possessing advantageous traits. The multi-omics approach has proven successful in enhancing crop yields and developing resistance to stresses in agriculture (**Figure 2**). Molecular biology methods encompassing various omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, have played instrumental roles in advancing these research endeavors [110, 111].

Genomics is dedicated to the sequencing, characterization, and comprehensive exploration of a plant's genetic makeup, encompassing its composition, structure, functions, and intricate networks within the genome [112]. Novel approaches to plant breeding have been made possible by developments in plant genomics, which effectively enhance and expedite various aspects of the breeding process. These innovations include techniques such as marker-assisted selection, gene pyramiding, association mapping, breeding by design, genomic selection, and more [113–119]. In a study conducted by Vinayan and colleagues [120], genomic regions linked with fodder traits were pinpointed, and a prediction study on genomic regions was carried out using 1026 DH lines and 276 elite lines as prediction sets from bi-parental crosses.

In order to determine the expression profiles of both coding and non-coding RNA in response to different stresses, high-throughput sequencing platforms were used by transcriptomics to generate transcript data. It also incorporates RNA sequencing, microarray and serial analysis of gene expression (SAGE) [121]. The genetic makeup of the transcripts that have differential gene expression in particular cells has been revealed by a number of studies. These transcripts can affect phenotypic variations in maize, such as growth, yield components, disease tolerance, environmental response and quality traits. For instance, transcriptome correlation and comparisons signaling network analysis were used in a study by Liu and Zhang [122] to identify six genes essential to the control of the MAP Kinase cascade and HY5 module in the presence of blue light. In maize, these genes are essential for regulating stomata formation and dispersion. qRT-PCR and transcriptome analysis studies in maize roots, that are

#### *Enhancing Maize (*Zea mays *L.) Crop through Advanced Techniques: A Comprehensive Approach DOI: http://dx.doi.org/10.5772/intechopen.114029*

infected by *Holotrichia parallela* larvae, were the focus of another study project conducted in 2020 by Pan and colleagues [123, 124]. This showed the expression of twelve differently expressed genes linked to the pathways of benzoxazinoid production and Jasmonic acid-mediated signaling, which are in charge of maize roots' defense mechanisms against invaders. Zhou and colleagues [125] used bulked sergeant transcriptome analysis (BSTA) to study the mechanism behind maize's resistance to drought stress. On chromosome 2, four highly expressed candidate genes that confer *Gibberella* ear rot disease resistance in maize were found by transcriptome profiling of several inbred lines of maize [126]. Together, these results highlight the significance of transcriptomics in maize research, as it facilitates the discovery of essential regulatory components for enduring abiotic and biotic challenges, as well as the annotation of gene functions and the identification of candidate genes. Breeders will be able to solve present and future economic, ecological, and environmental concerns and ensure food security by using this information to gain the insights they need to create improved varieties of maize [69, 127, 128].

Proteomics involves a comprehensive investigation of proteins within a biological system, encompassing plants and animals, at a specific moment in time [129]. The analysis of proteomics serves the purpose of quantifying the abundance of various proteins, discerning alterations resulting from diverse post-translational modifications, and elucidating their functions and localization [130]. It offers a snapshot of diverse metabolic processes, their ensuing interactions, and their impacts on other regulatory pathways. Consequently, proteomic studies are indispensable for deciphering the diverse reactions within pathways under various stress conditions and timeframes [131]. The Proteomics field has attracted considerable interest from scientists seeking to examine physiological differences at the proteomic level under varying stress conditions. For example, Zhang et al. [132] performed a proteomic analysis of maize leaves in an attempt to evaluate proteome-level changes in corn when infected by the *Ostrinia furnacalis* (Asian corn borer). A total of 62 defense-responsive proteins were found, with a special focus on thioredoxin M-type and pathogenesisrelated protein 1 (PR1), a chloroplastic precursor that significantly impacted the development of corn borer larvae and pupae. Comparative proteome profiling was done on resistant and susceptible lines exposed to *Puccinia polysora* (southern corn rust) in a study by Wang et al. [133]. This study demonstrated that resistance in the resistant lines was inhibited by a particular remorin protein (ZmREM 1.3). A comparative proteome profiling of drought-tolerant and susceptible maize lines was carried out by Dong et al. [134]. Plants use the development of defense-associated proteins (DAPs) in conjunction with the down-regulation of redundant proteins as a stress-reduction and energy-saving strategy.

Metabolomics is a cutting-edge biotechnique that seeks to identify functionally active metabolites, clarify their functions, and provide insight into the various biochemical processes that occur in plant genotypes and the phenotypic expressions that follow [135]. All metabolites, primary and secondary, with a molecular weight of less than 1500 Da, as well as their precursors and intermediates within the corresponding metabolic processes, are included in the metabolomes. Based on their particular goals, metabolic investigations can be divided into two categories: targeted and untargeted. The goal of targeted metabolomics is to precisely quantify one or a small number of metabolites from a predetermined list of recognized compounds. This method helps identify metabolites linked to particular features because of its high sensitivity and quantitative nature. Untargeted metabolomics, on the other hand, increases the possibility of identifying unintentional impacts by measuring the mass

spectrometric properties of metabolites with unknown identities [136]. The application of metabolomics has proven invaluable in understanding how maize plants respond to various stress conditions i.e., heat, salinity and drought. For example, a metabolomic investigation involving salt-tolerant and salt-sensitive maize genotypes revealed differences in metabolite accumulation in both roots and seedlings under salt stress. In seedlings, salt stress induced glucose and acid metabolism. Thirty common chemicals, including metabolites linked to basic metabolism such as deoxyadenosine, adenine, L-pyroglutamic acid, cis-9-palmitoleic acid, and galactinol compounds, were found in the roots of both salt-sensitive and tolerant cultivars [137]. Heat stress effects on pollen male sterility in maize, especially at the most vulnerable tetrad stage, has also been clarified by metabolic pathway study. A reduction in pyruvate levels and an enhancement in sucrose levels were found in this research. In the meantime, other genes linked to signaling, unfolded protein stress, and auxin synthesis did not alter [138]. More importantly, a study by Ganie et al. [139] revealed metabolic pathways impacted by phosphorus stress situations, offering insights into strategies for improving phosphorus efficiency. The analysis, conducted using gas chromatography-mass spectroscopy, the investigation revealed a drop in fatty acids like cholesterol and stigmasterol, which are critical for membrane fluidity, and an increase in sugar alcohols like glucitol and mannitol under P-limitation, which are essential for membrane fluidity. In cases of severe phosphorus starvation, plants will scavenge phosphorus from these fatty acids, disrupting membrane fluidity. Additionally, elevated levels of serine and glycine indicated an increase in photorespiration rates.
