*8.2. Proteomics and Metabolomics*

Proteomics is a method used to profile all the proteins that are expressed in an organism. It is broken down into four separate categories: sequence, functional, structural, and expression proteomics [225,226]. Traditional proteomics includes size exclusion chromatography (SEC), exchange chromatography (IEC), and affinity chromatography. Western blotting and an enzyme-linked immune sorbent assay can be utilized to analyze specific proteins (ELISA). Additionally, more advanced methods for the separation of proteins have been developed and employed, including SDS-PAGE, 2-DE, and 2-D differential gel electrophoresis (2D-DIGE).

The numerous proteins involved in host–pathogen interaction and their function in protecting the host plant from pathogen attacks can be uncovered using a proteomics method [227,228]. Many proteins have been linked to significant host–pathogen interactions, including the establishment of the pathogen in a host plant that is vulnerable to it, as well as the host plant's defense against pathogen invasion [229–232]. These proteins range from syntaxins to subtilin-like proteases in different plant species in response to FW infection. They include chitinases, -1,3-glucanases, xylem proteinases, proteinase inhibitors, leucine-rich repeat proteins, proline-rich glycolproteins, pathogenesis-related (PR) proteins, cellulose synthases, ankyrin repeat-containing protein, and PR-5b [228,233–235].

The genotypes JG 62 (FW-susceptible) and Digvijay (FW-resistant) of chickpea were both found to contain a variety of defense-related proteins against FW infection [228]. Several ROS-activating enzymes, including glutaredoxin, glutathione peroxidase, ascorbate peroxidase, glutathione S-transferase, and peroxiredoxin, were identified in higher concentrations in Digvijay than in JG 62. This is similar to how Digvijay was able to reduce FW pathogen assault compared to the FW-sensitive cultivar JG 62 due to the genotype's excess of PR proteins [228]. Proteomics may therefore improve our understanding of the unknown proteins linked to numerous signal transduction pathways that cause host innate immunity in grain legumes to be triggered in response to FW attack.

Metabolomics is the complete study of metabolites that participate in many cellular processes in a biological system. The total collection of metabolites generated by metabolic pathways in the plant system is referred to as the "metabolome", instead [236,237]. The early metabolic system of an organism can be employed to predict its genome sequencing using metabolomics and NGS technology [238]. In one study, information was combined using the genome sequencing method (NGS) and metabolite measurement method (MS) to generate crop enhancement methods [239]. This can improve our understanding of how plants respond metabolically to stress via contact with pathogens or under stress.

Our understanding of many metabolites, hormonal interactions, and signaling components associated with plant defense systems against FW infection in agricultural plants, including grain legumes, may facilitate the development of resistant cultivars [228]. Hexokinase, trehalose, invertase, sucrose synthase, -amylase, and glucose-6-phosphate are examples of sugars that are generated in the reaction to FW [240]. These sugars act as an oxidative burst substrate, supplying energy, generating ROS, acting as a signaling molecule in coordinate on with various phytohormones, and enhancing lignification of the cell wall in order to activate plant innate immunity, and plays a crucial role in plant defense against pathogen attacks [241,242]. There are many different proteins that are involved in the TCA and glycolysis processes in Digvijay, as well as defense-related metabolites such as endo beta-1,3-glucanase, caffeic acid O-methyltransferase, chitinases, and caffeoylCoA O-methyltransferase; phytoalexins such as luteolin, genistein, and quinone; and phenolic compounds, including flavonoids [228]. A considerable decrease in specific amino acids and carbohydrates, like sucrose and fructose, in a vulnerable crop enables FW pathogens to enter and hasten the development of disease [228].

The function of PR proteins, chitinases, ROS activating enzymes, flavonoids, phenolic compounds, and phytoalexins in conferring wilt resistance is further supported by thorough analyses of plant transcriptomes, metabolomes, and proteomes in response to FW disease [243–245].

#### **9. Genomic Selection (GS)**

A promising method called genomic selection (GS) uses molecular genetic markers to create new breeding programs and new marker-based models for genetic valuation [246]. It offers chances to boost the genetic gain of complex traits per unit of effort and expense in plant breeding. For GS, weighing the pros and cons of working in crop plants is crucial. The most crucial elements for its successful and efficient application in crop species are the availability of genome-wide high-throughput, affordable, and flexible markers, and its low as certain bias, suitable for large population sizes, as well as for both model and non-model crop species with or without the reference genome sequence [247]. However, in order to achieve evaluable genetic gain from complex traits, these marker technologies may be paired with high-throughput phenotyping.

Most of the molecular markers, which have both large and small marker effects, are what determine the GS. Molecular markers are chosen based on their total genome coverage, and all QTLs should be in linkage disequilibrium with at least one marker [248]. The training population and the testing population are two separate sorts of populations that are employed in GS. The testing population, which is related to the breeding population, is used to estimate the genomic selection model parameter. A testing population is a population group in which genetic selection is employed.

One important issue with marker-assisted selection is that it can only target significant QTLs or genes. It is now commonly acknowledged that a multitude of genomic regions, each of which has just a tiny amount of genetic control, are involved in many complex traits, such as yield or broad-spectrum disease resistance. In many situations, it is highly advantageous to select for all or a few QTLs linked to the desired characteristic [249]. In this case, genomic selection, which has the capacity to capture several genes with minor additive effects, could prove beneficial for crop breeding. Genomic prediction, which relies heavily on the availability of high-throughput genotyping, along with accurate phenotyping data, is the key to success in GS breeding [90]. GS + de novo GWAS and haplotype-based GS + de novo GWAS approaches, together, have potential for developing capable chickpea genotype(s) [90].

#### **10. In Vitro Selection against Fusarium Wilt Disease Tolerance/Resistance in Chickpea**

Both biotic and abiotic stressors have a significant impact on legume crops. Therefore, it is essential to undertake efforts to cultivate plants that are tolerant to stress in order to increase agricultural yield. Growing stress-tolerant plants using tissue culture-based in vitro selection has become a practical and economical approach in recent years [250–252]. Applying selective agents to the culture media, such as pathogen culture filtrate [253], fusaric acid phytotoxin [254] or the pathogen itself (for disease resistance)—NaCl (for salt tolerance), and PEG [255] or mannitol for drought tolerance—may aid in the development of plant tolerance to both biotic and abiotic factors. Many efforts have been made in this respect for the screening and development of chickpea cultivars [256].

The optimal outcome depends on the availability of an appropriate selection agent. Fungal culture filtrate or a well-known toxin, such as oxalate acid or fusaric acid, are typically utilized as the selection agents [257]. In vitro pathogen resistance selection is possible by including a phytotoxin, such as fusaric acid, that is unique to the host. Fusaric acid (C10H13O2N), a metabolite generated by many strains of *Fusarium oxysporum*, is employed as a "selecting agent" for cell culture and callus culture to stop the germination of fungus. In comparison to plants derived from tissue culture without selection, several pathogenproduced non-specific phytotoxins, such as deoxynivalenol (DON), crude pathogen culture filtrate, or sometimes, the pathogen itself, have been shown to increase the frequency of resistant/tolerant plants [258]. Because there is a link between toxin tolerance and disease tolerance, toxin or filtrate can be used to make an agent decision based on reality. By exposing somatic embryos, shoots, embryogenic calli, or cell suspensions [259,260] to pathogen toxins, pathogen culture filtrate, or the pathogen itself, these selections can be made.

*Fusarium oxysporum* cultural filtrate affected the levels of total peroxidase, phenol, and beta 1, 3 glucanase in chickpea and reduced callus growth [261]. Resistance was apparent in chickpea plants that had grown back after being exposed to culture filtrate (*Fusarium oxysporum*) [262]. According to research conducted by Hamid and Strange [257] on the relationship between disease and the susceptibility of chickpea shoots to toxins (Solanapyrone A, B, and C) and the culture filtrate of *Fusarium oxysporum* (*Ascochyta rabiei*), the enzyme glutathione s-transferase may prove useful for boosting resistance.

#### **11. Speed Breeding in Chickpea Improvement**

Crop varieties that are resistant to disease can be developed using plant breeding techniques [258,259]. In order to protect global food security, it is urgently necessary to increase the existing pace of genetic gain in key food crops [260,261]. This may be helpful in the fast transfer of desired genes [262]. Lengthy breeding cycles/generations are mostly to blame for the poor advances in crop improvement [263]. Traditional/conventional breeding methods may not be sufficient to meet the demands of future generations. Speed-breeding approaches are increasingly applied at large/small scales to obtain rapid genetic gain in several crop species in order to overcome the limitations associated with traditional methods and to ensure food security [264]. Crop varieties can be developed more quickly through speed breeding. This involves a synthetic habitat that has longer daylight hours to extend the growing season and aid in the manipulation of photo insensitive crop life cycles [265].

The rapid generation cycling methods of double haploids [266], the in vitro culturing of immature embryos [267], the embryo rescue technique [268], and other methods have not been successful in the chickpea. Three generations per year in short-season conditions were supported in the first report on chickpea rapid generation development [269]. It may be advantageous to increase production and reduce life cycles using the recently established "speed breeding" technique in chickpea, which could let researchers conduct more generations per year [270,271]. In the pigeon pea plant, a rapid generation advancement approach, which showed 100% germination from immature seeds taken from 35-day-old plants, opened new possibilities for developing three to four generations in a year [272].

The induction of early blooming and the germination of immature seeds in a more recent study on chickpeas resulted in a shorter seed-to-seed cycle period [273]. A system for growing chickpeas in greenhouses with artificial light but no growth regulator has been developed. In this technique, the photoperiod must be extended to 22 h using a temperature-controlled greenhouse with working high-pressure lamps. This greenhouse provides for precise control of temperature, humidity, and lighting. Immature seeds were germinated at 20–23 days after flowering (DAF) to further shorten the generation cycle, and the photoperiod was increased to encourage early flowering. There were six accessions used, with two each from the early, medium, and late maturity groups. Six or seven generations occurred annually. This method may also be adopted for the screening of wilt-resistant plants, as it may save time.

According to Fikre and Tulu [274], a unique field-based rapid generation cycle strategy may increase breeding effectiveness and hasten the release of improved chickpea varieties for the farming community. Compared to other rapid generation progress technologies that require expensive infrastructure, the strategy is easy to use, effective, and requires little investment. Importantly, the field-based rapid cycle technique for chickpeas is best suited for breeding operations located in tropical and subtropical areas, where the climate allows for chickpea development all year round. However, because this strategy includes propagating plant generations outside, it is crucial to implement risk management procedures to safe guard priceless breeding resources from severe weather conditions and wildlife. Speed breeding strategies may also be applied to the development of Fusarium wilt-resistant chickpea varieties.

#### **12. Conclusions**

Biotic stressors significantly decreased the yield of the leguminous crop. After yield improvement, resistance to FW one of the most important breeding goals of crop improvement programs for chickpea. The development of efficient, innovative, conventional, and molecular breeding technologies can be used to strategically control breeding for FW resistance. This review has covered the many approaches that may be utilized to locate and incorporate novel wilt resistance gene in chickpea. The capacity to apply a QTL mapping strategy for the genetic study of stressors in chickpeas was made possible by recent advancements in the utilization of molecular marker technologies and the availability of high-density genetic maps. Draft chickpea genome sequences have since been made public. Due to the significantly increased chickpea genomic repertoire, we have a fantastic opportunity to examine the unique genetic determinants/haplotypes governing this stress across the full genome level using genome-wide association studies (GWAS). Several marker-assisted breeding methods, including MABC and MARS, are now being applied in chickpea breeding programs. To understand functional analyses, the molecular mechanisms of genes, and gene networks, these omics approaches—genomics, transcriptomics, proteomics, metabolomics, ionomics, and phenomics—have been employed. There is an urgent need for the identification of transcription factors that play an important role in limiting the pathogen activity of *Fusarium oxysporum* in the soil, as well as in chickpea. This review outlines approaches that can be used to manage the effect of FW on chickpea

production and suggests recommendations for improving chickpea wilt-resistant breeding programs. The adoption of these approaches is anticipated to be given more prominence in future breeding programs. This review includes information on the biotic limitations of chickpea production and future directions.

**Author Contributions:** Conceptualization, M.K.T., R.K.Y., and S.T.; writing—original draft preparation, R.K.Y., R.A., and V.P.; writing—review and editing, M.K.T., N.T., R.S.S., and D.K.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


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