Capsicum: Breeding Prospects and Perspectives for Higher Productivity

Abstract

Chipotle peppers were grown in America before being carried to Europe by Columbus. Capsicum breeding began with choosing wild species for desired characteristics, with additional development based on precision selection. To improve capsicum yields, traditional methods such as mass selection, pedigree, single-seed descent, backcrossing, and hybridization are being used. Capsicum has a high level of genetic diversity due to multiple new gene rearrangements. Capsicum fruits are high in nutrients that are beneficial to human health. As a result, the world market for and consumption of capsicum has lately grown. Capsicum breeding programmes aim to improve yield, biotic, abiotic resistance, and nutritional quality. Recent breakthroughs in capsicum breeding have included introgression, mutation breeding, polyploidy, haploidy, embryo rescue, and the use of genetic markers. Molecular technology has grown into an important tool that, when coupled with classic selection and hybridization procedures, has the potential to result in great success in an established capsicum genetic breeding programme.

1. Introduction

Capsicum is cultivated all over the world for a variety of applications needing different levels of quality and characteristics. Fresh or dried capsicums have been considered a foodstuff for many years due to the fact that they contain all of the required nutrients. Capsicum fruits have double the amount of vitamin C found in citrus fruits. In contrast, dried red chilies are high in vitamin A and β-carotene, making them a healthy snack option [1]. According to photochemical studies, it also has cancer-prevention due to the presence of higher antioxidant properties [2]. Capsaicin lotion is used therapeutically to treat arthritis and other painful chronic conditions [3]. Capsicum extracts are used in the production of cosmetics and pharmaceuticals. Cultivating capsicum in pots or gardens as an ornamental plant is becoming trendier these days [4]. Sweet pepper is the most important spice traded across the globe. It is possible to classify the capsicum market into five broad divisions depending on the fruit’s shape and intended purpose. In the fresh market, whole fruits are available in green or red; in the fresh-processing market, sauce, paste, canning, and pickles are available; in the dried spice market, whole fruits and pepper powder are available; and in the decorative market, ornamental varieties are available [5]. The ultimate objective of capsicum breeding projects will change according to the needs of farmers and consumers. Biotic and abiotic stresses are taken into account during the breeding of capsicum varieties and hybrids.

2. Breeding history

Capsicums are believed to have originated in the Western Hemisphere and have been used as a food source since 7500 BC. They are said to have originated in South America and spread to Central America. Capsicum was brought to Europe by Christopher Columbus, and it quickly spread across Africa and Asia. The genetic inheritance of important agro-horticultural traits, mutant forms, male sterility, disease, pest resistance, and quality characteristics were all required for the purpose of early capsicum study. It has been said in a number of places that these characteristics are driven by single genes with a dominant or recessive mode of action, and that some of these characteristics are governed by quantitative trait loci. Capsicum annuum is by far the most significant member of the Capsicum family, owing to the fact that it is the species of the genus Capsicum that is most often economically produced. When plant breeding first started, it was based mostly on the ability of individuals to be selected, which was a time-consuming and unstructured process. Bringing the notions of Mendelian genetics and inheritance into the field of vegetable breeding was a pivotal moment in the history of the field. Currently, plant breeders are using a variety of ways to select for desirable characteristics in their crops, with the methodology chosen depending on the objectives of the breeding programme [6, 7]. The primary goals of capsicum breeders are genetic enhancement in productivity, biotic and abiotic resistance and nutraceutical compounds.

2.1 Contemporary objectives of Capsicum breeding

Depending on the location, the breeding goals for capsicum, both hot and bell pepper, varies depending on the nation of culture, the purpose of cultivation, the growing conditions, the end user, and the preferences of the customers. Some countries prefer peppers that are fiery and pungent, while others prefer peppers that are sweet. The diseases that harm the crop also differ depending on the climate that prevails in the different countries. Biotic and abiotic resistance breeding, on the other hand, is one of the most important goals in the development of capsicum varieties [8]. A comprehensive depiction of the numerous pest and diseases that affect capsicum has been presented by Pohronezny [9]. The process of disease resistance breeding begins with the discovery of resistant sources, followed by a study of their genetics, and finally with the introduction of promising genotypes. It has been shown that in the case of capsicum, a significant amount of disease and pest resistance from wild species has been introduced into commercial cultivars in order to increase disease resistance. It is also necessary to assess the amount of crossability across species when developing an interspecific hybridization programme for resistance gene introgression. The utilization of wild materials for the insertion of biotic resistance genes into desirable cultivars has produced significant contributions to crop improvement, most notably in terms of increased yield and quality, as well as stability in capsicum production, among other applications. Introgression efforts to introduce disease resistance genes into superior cultivars have frequently failed when disease resistance traits are under polygenic control and linked with undesirable horticultural and economic characteristics. To counteract the ongoing growth and emergence of new disease races and strains against presently existing resistant genotypes, it is vital to seek for and deploy new resistant sources on a regular basis.

The second goal for which capsicum breeders across the globe are trying is to increase yield, which will ultimately result in increased total production. In this regard, the heterosis breeding programme is becoming more important. It is preferred that more emphasis be placed on the development of F₁ hybrids based on available male sterility systems, since this reduces the amount of time and work necessary for hybrid seed production. In order to make hybrids, both genetic (GMS) and cytoplasmic male sterility (CMS) systems have been used, with the cytoplasmic male sterility system being the most extensively used. The discovery of new CMS sources, the identification of their maintainers, and the diversity of CMS systems all become key goals as a result of this process. The discovery of restorers with excellent general and specific combining capacity, as well as the insertion of resistance genes into these CMS lines and restorers, should also be a priority for the generation of hybrids.

Capsicum breeding aims are also influenced by market demand and end-use usability, among other factors. This involves breeding for horticultural economic and nutritional quality traits, among other things. Fresh market breeders are looking for characteristics such as fruit color at the unripe stage, which is often green (light, medium, or dark), fruit size-length, width, and pericarp thickness. Furthermore, the amount of pungency is an important and distinct feature of capsicum breeding that should not be overlooked. Understanding consumer preferences for pungency in a given location is an extremely important aspects of the research process. Pungency, which is a major economic characteristic of capsicum, is attributable to the presence of a chemical complex of alkaloids known as capsaicinoids in the plant [10]. Capsaicin and dihydrocapsaicin are the two major capsaicinoids in capsicum, accounting for around 90% of the total capsaicinoids in most pungent varieties.

One of the most important quality attributes that capsicum breeders consider when developing commercial varieties is the amount of capsaicin present in the plant [11]. Capsanthin concentration in capsicum is estimated using the high-performance liquid chromatography (HPLC) analytical method. The capsanthin-capsorubin synthase (CCS) enzyme is found only in the Capsicum genus, and it is responsible for the production of two red pigments, capsanthin and capsorubin [12]. According to the USDA, red capsicum is being bred for higher capsanthin content in order to be used as a dried spice and for industrial extracts viz, paprika oleoresin, capsaicinoids, and carotenoids. The red color of chilli peppers is indicative of the presence of capsorubin and capsanthin, whereas the yellow color of chilli peppers is due to the presence of β-carotene and violaxanthin [13]. In general, the higher the ASTA color rating, the deeper the genotype’s ripening red color. It has a capsanthin content of between 70 and 100 ASTA units (low), 71–100 ASTA units (medium), and 101–150 ASTA units (high). The ASTA color affects the brightness of a product, while the surface color affects the hue. As a consequence, another major aim of capsicum breeding is the development of paprika varieties that meet the great demand for nonpungent pods with a high color value for industrial uses. Dry matter content is a critical trait to breed for in dry capsicum, since it is used to make dry powder and whole dried fruits. Additionally, these are the most often requested characters for export reasons. While a high dry-matter content in red chilli fruit is useful commercially, there is no correlation between the dry-matter content and the fruit’s capsaicin concentration [14]. It is critical to have a thin pericarp for dry capsicum in order to facilitate drying. The surface of fruits with a thick pericarp gets wrinkled and their appearance becomes bland as a consequence of drying. In response to increasing industrialization, the risk of crop failure associated with climate change, and consumer demand (both domestic and international) for more nutritious and safer foods, increased emphasis is being placed on breeding genotypes with increased tolerance to high temperatures, drought, and wider adaptability.

2.2 Major goals of Capsicum breeding

A variety of colors, including medium or dark green at the unripe stage and red, yellow, or orange at the mature stage, are among the key targets of genetic enhancement of sweet peppers. Research in this initiative aims to find and develop new varieties of capsicum that are rich sources of antioxidants as well as vitamins. Flavonoids and carotenoids (red, yellow, and orange carotenoids), which contain vitamin A precursors such as alpha and beta carotene, as well as β-cryptoxanthin are also included in this category [15, 16]. Breeding efforts are also focused at increasing fruit set and yield in varying climatic situations, including open and protected. Low temperatures, drought, and salt stress are all being studied as part of breeding efforts to combat abiotic threats. Breeding for long-term storage stability of carotenoid extract and resistance to Phytophthora fruit rot are also on the research agenda. Powdery mildew, anthracnose, Phytophthora fruit rot, bacterial wilt and viruses, are some of the most common diseases that affect sweet peppers in open-field as well as in the green house production. Another important objective is to develop sweet pepper genotypes that are more tolerant to tropicalization, since this is a cool-season crop. Produce will be accessible throughout the year for a longer amount of time in places that are not usual [17]. The primary breeding goals in protected culture are to develop sweet pepper lines with an indeterminate growth habit, tolerant to training and pruning systems, blocky fruit, resistance to biotic stress, and resistance to root-knot nematode [18].

3. Breeding strategies

3.1 Conventional breeding approaches

Capsicum crop improvement has been achieved by the applying of conventional breeding procedures such as mass selection, pureline selection, pedigree breeding, single-seed descent method, backcross breeding, and heterosis breeding. Different types of breeding strategies, including mutation breeding and polyploidy breeding, have also been used in an effort to generate variety in capsicum, which may then be used in improvement initiatives. At the time of the beginning of systematic plant breeding, many tactics for capsicum improvement were used: mass selection, pureline selection, pedigree breeding, single-seed descent method, and backcross breeding were among those employed. Mass selection is one of the most straightforward strategies that has been utilized to increase the quality of capsicum. Improvement for many qualities with simple inheritance may be done at the same time without having to worry about the pedigree of the individuals involved. Initially, it was employed to enhance landraces or open-pollinated cultivars of capsicum, which were previously unproductive. Characters with high heritabilities may be readily repaired using this technique, but an acceptable amount of variability is also retained. Traditional landraces and local cultivars were the primary targets of pure line selection since farmers were cultivating them. This strategy involves selecting better plants, harvesting them individually, then evaluating their progenies the following year in order to determine plant performance. Progenies that exhibit better performance and are free of genetic variability are collected in large quantities and assessed further in repetitive experiments against check cultivar(s). So, this approach has been widely utilized to develop various types of capsicum for commercial cultivation, and it is still being used today.

In a pedigree selection method, selection is conducted between and within families of individuals, and selected individuals are issued a pedigree number, allowing any offspring in any generation to be traced back to its original crop that was picked in the F₂ generation. This has historically been one of the most often utilized methods for developing capsicum cultivars. Selecting superior parental cultivars is critical for the development of this approach. It is often employed in conjunction with backcrossing to effectively introduce essential genes into advanced inbreds. Using the single-seed descent (SSD) procedure, one seed from a single fruit is collected from each plant in a segregating generation. The segregating generation is produced in greenhouse conditions to increase the number of generations each year. Additionally, this enables the formation of a large number of pure inbred lines for use in test crossings for the development of hybrids, as well as the generation of recombinant inbred line populations for mapping research. In the capsicum breeding programme, the backcross method is the most often used way for disease resistance development. This method is most often used to transfer a single gene or a limited number of genes from primitive cultivars or wild forms to leading cultivars. In exceptional circumstances, even BC₂ families may be routed using the pedigree strategy (modified backcross) rather than the usual backcrossing process, which involves 5–6 backcrosses with the recurrent parent. While open-pollinated varieties of hot peppers and bell peppers are still commonly available, heterosis breeding has been found to increase hot pepper and bell pepper production. Numerous hybrids have been developed in the capsicum plant; nevertheless, the hybrid research effort should be continued to ensure that seeds are affordable to farmers. Capsicum F₁ hybrids are gaining popularity as a consequence of a large number of private sector seed companies investing in vegetable industry research and seed manufacturing. Male sterility is frequently used in the generation of hybrid seeds in the chilli plant to increase the cost-effectiveness of seed production. The discovery of various male-sterile mutants, which eliminate the need for more laborious emasculation techniques, together with the identification of several marker genes, has improved the detection of undesirable types at the seedling stage even further. GMS and cytoplasmic-genetic male sterility (CGMS) are two types of genetic male sterility that are now being economically exploited in chilli for hybridization. GMS has been suggested above CGMS for hybrid seed production because GMS exhibits male sterility and male fertility segregation, but CGMS does not.

CGMS was discovered in capsicum for the first time by Peterson [19] and was designated as USDA accession PI164835. There have been no reports of any additional CMS sources so far. “orf507” and “tp6-2” are two aberrant mitochondrial genes found in the capsicum CMS system that have been linked to male sterility [20]. Because the genes are present in the mitochondria, they are passed down via the maternal line. It is also necessary that a nuclear gene for the restoration of fertility be absent in order for male sterility to be expressed. A restorer line is required for effective hybrid seed development when the restoration of fertility is driven by a single dominant gene, as is the case in most cases. In order to preserve male sterility, a maintainer line must include both fertile cytoplasm and the lack of a nuclear gene that would allow for fertility restoration. Due to the fact that the CGMS system of hybrid seed production necessitates the use of three lines, namely, the CMS line, the keeper of male-sterile line, and a restorer of fertility in hybrids, the system is referred to as the three-line system of hybrid seed production in the capsicum plant. The GMS technique has also been employed to develop capsicum, but to a lesser degree. In the GMS system, the expression of male sterility is regulated by homozygous recessive genes (ms/ms), while male fertility is controlled by homozygous dominant or heterozygous genes (Ms/MS or Ms/ms). Ms/Ms and Ms/ms are isogenic lines that vary solely at the Ms locus, and they are essential for the maintenance of male sterility in the GMS population. Intercrossing between these two lines results in offspring that are a combination of both male fertile (Mf/ms) and male sterile (ms/ms) sperm in equal quantities. Visual identification identifies male fertile plants in the field and rejects them, while male-sterile lines are utilized for hybrid seed production [21]. To begin with, the production of successful capsicum cultivars was largely dependent on the breeder’s expertise, perception, and good fortune in selecting promising genotypes. Cultivar development still relies on the breeder’s knowledge and perception, even in the age of cutting-edge breeding procedures. A blend of both science and art, plant breeding continues to be such. There are several more approaches for improving capsicum, including mutation breeding, polyploid and haploid development, transgenics, and marker aided breeding, all of which have had some success.

3.1.1 Colchiploidy breeding

After the newly produced polyploid has grown in strength, it will next adapt to its new environmental surroundings. It has been suggested that the advantage of polyploids over diploids might be due to the phenomena of transgressive segregation, which is the production of extreme phenotypes, as described by Van de Peer and co-workers [22]. It has been suggested by Malhova [23] that capsicum may react to variations in ploidy in the same manner as Solanum does. Capsicum ploidy levels may be intentionally increased or decreased in a very straightforward manner. Using colchicine to repair injured leaf axils, it is possible to achieve somatic doubling in plants. On the other side, synthetic autotetraploids do not seem to have any economic or breeding benefits over diploids. Polyploid capsicum is characterized by slowed growth and the presence of larger, thicker, and dark green leaves [24]. The presence of more chloroplasts and larger chloroplasts in polyploid leaves has been attributed to the polyploid leaves’ rich green tint [25]. When compared to diploid capsicum, the tetraploid capsicum has increased leaf, stem, and root dry weight, as well as increased leaf area and thickness. Tetraploids have been shown to have an increased capacity for water, NO₃–N, and K absorption, which correlates with an increase in photosynthesizing potential; they also generate small but more unified fruits irrespective of fruit loading; and they produce tinier but more uniform-sized fruits regardless of fruit loading [26].

It has been discovered that the tetraploid capsicum flowers about one month later than the diploids. The total number of flowers produced was reduced, with this reduction owing mostly to the non-branching character of the polyploidy [24]; nonetheless, the total number of flowers produced was increased. Raghuvanshi and Sheila [25] discovered that the colchiploids of Capsicum frutescens had delayed and protracted blooming, as well as a bigger and more diversified number of floral components than the diploids. Polyploids have larger blooms as well as larger pollen grains, which are also typical of polyploids [27].

Treatment of seeds with colchicine resulted in the generation of tetraploid plants of the C. annuum variety “Chigusa,” thus according to Ishikawa et al. [28]. Following a flow cytometric study of the seeds treated with colchicine, it was observed that 20% of the seeds were tetraploid. Tetraploid flowers had seven petals and filaments, 20 of ovaries, and 25% larger pollen grains than diploid flowers, which typically had six petals and anthers [29]. Tetraploid blooms were also 20% bigger than diploid blooms in diameter. Polyploids have also been shown to be sterile, which might be owing to abnormalities seen during the meiotic phase [30]. Following treatment with colchicine, researchers discovered that a plant of the chilli pepper cv. CO-2 had chromosomal counts ranging from 2n = 38 to 96. It possessed 4.95% pollen fertility but did not produce any seeds, and its development was inhibited as a result [31]. Although colchicines have been used to double the number of homozygotes produced by anther culture, these homozygotes have not yet been exploited to produce commercial F₁ hybrids capable of exhibiting heterosis. Instead, they have been used to investigate the genetic mechanisms of resistance to pests [32] and diseases [33].

Malhova [23] successfully established an interspecific hybrid of Capsicum pubescens and Capsicum annuum by fertilizing C. pubescens with autotetraploid Capsicum annuum pollen. The use of induced auto-tetraploidy to overcome post-fertilization hurdles may benefit future interspecific crosses of the capsicum genus, according to this research. Pochard [34, 35] revealed previously unknown trisomies in C. annuum. Pochard [34] demonstrated that trisomies may be used to identify genes on specific chromosomes, either via skewed segregation ratios seen in the offspring of trisomic F₁ hybrids or through dosage effects shown when trisomics are compared to conventional diploid humans [36]. These trisomics confirmed the presence of gene “C” (which determines pungency) on acrocentric chromosome number “XI” [34] and its location on the long arm [37], since the trait pungency segregated independently of the markers on the acrocentric chromosomes’ short arms [34].

3.1.2 Embryo rescue

Embryo rescue has been the most often used technique to overcome post-zygotic hybridization difficulties in interspecific crosses. The hybridization of capsicum species from separate gene pools has been observed, but incompatibility has also been reported within the same gene pool, such as between C. annuum and Capsicum chinensis or between C. annuum and Capsicum frutescens. Many fruits with shriveled seeds are created as a result of incorrect endosperm and/or embryo formation in numerous interspecific crosses in the Capsicum spp. As a result, many fruits with shriveled seeds are produced, which are unable to germinate properly. In the scientific literature, it has been reported that a hybrid embryo originating from interspecific crosses in the Capsicum genus has been successfully recovered. Fari et al. [38] performed the first successful attempt at embryo rescue in the capsicum genus, acquiring an embryo from a hybrid between C. annuum and C. baccatum. This was the first successful attempt at embryo rescue in the Capsicum genus. It is yet another example of extensive hybridization in which immature interspecific embryos or embryos from different species were/were rescued prior to abortion, as was the case with the hybridization of C. annuum and C. baccatum [39].

Technically, the procedures of embryo removal and in vitro embryo culture are both highly challenging to perform. The stage at which embryo abortion occurs during hybridization may also be determined by the genotypes of the individuals engaged in the cross, according to some researchers. Yoon et al. [40] reports that some researchers have been successful in saving interspecific embryos at the most advanced stages of development in the Capsicum genus, while other researchers have had to save them at the very beginning of development [40, 41]. Rescue of embryos at an earlier stage, on the other hand, is more difficult, and the possibility of recovering interspecific hybrids is lower at this time [42]. Anthracnose resistance exhibited in C. baccatum lines has been shown to have been transferred into C. annuum lines by the rescue of embryos obtained from interspecific crossings between the two species and subsequent culture of embryos resulting from those crosses [40].

Alternate methods of overcoming the aforementioned issue may be used, such as the construction of a genetic bridge based on the usage of species that are phylogenetically closer to the two species that are affected by crossability barriers. A bridge species must be used in conjunction with this method because it must be capable of crossing with both the target species and the target species’ predator. Initially, it is essential to cross the bridge species with one of the target species, and then to cross the resultant hybrid with the other target species [39]. After doing this research, it was shown that C. chinensis is an acceptable bridge species for performing widespread hybridization between the species C. annuum and C. baccatum [43].

3.2 Modern breeding approaches

3.2.1 Development of Capsicum Haploids

Capsicum annuum and Capsicum frutescens anther cultures were used to produce the first haploids in the genus Capsicum [44], which were then used to produce the first haploids in the genus Capsicum [45]. Because of the poor recovery of haploid plants from androgenic cultures seen in previous studies, researchers decided to construct experiments with the goal of identifying the elements that influence the induction of androgenesis. Based on the various experiments conducted on haploid induction, the androgenic response was determined to depend on growing conditions, age, the genotype of the donor plant [46], and developmental stage of microspores in the anther.

The development of doubled haploids is one of the most effective means of establishing full homozygosity in any crop species; nevertheless, because of the plant’s recalcitrance, its application in capsicum enhancement is still restricted [47]. Capsicum breeding requires a genetically stable and homozygous plant population in order to better understand genetics, as well as mapping and identification of genes for various morphological traits and biotic and abiotic stress-related morphotypes. Despite the limited frequency of findings, a number of researches on the practical side of haploid breeding in several capsicum species are now underway [48]. It has previously been reported that parental lines created utilizing doubled haploid (DH) technology may be used to create varieties and F₁ hybrids [49]. DH capsicum lines also had higher production attributes and dry matter content in their fruits [50]. Superior DH lines with great variation in plant and fruit features, as well as androgenic capsicum lines with favorable qualities, have been isolated [51]. In addition to enhanced production, it has been feasible to develop Capsicum DHs with improved quality characteristics such as fruit shape, flavor, fruit hardness, dry matter content, total soluble content, phenolic content, and antioxidant activity, such as CUPRAC and FRAP [52, 53].

Nowaczyk et al. [54] used DH technology to improve the shelf life of soft-flesh Capsicum spp. recombinants. DH lines derived from in vitro capsicum anther culture showed varying degrees of resistance to Xanthomonas campestris pv. vesicatoria [55] and Phytophthora capsici [55] and Phytophthora capsici [56]. These disease-resistant DH lines might be exploited to develop novel genotypes that are resistant to many diseases. Using anther culture, it was also possible to get PVY-resistant lines as well as lines with important qualitative and quantitative traits [57]. Todorova et al. [58] revealed their findings after using haploid culture to generate capsicum lines with high production, enhanced fruit attributes, and decreased sensitivity to Verticillium wilt. Microspore embryogenesis has been used to create genotypes with higher productivity, resistance to Verticillium dahliae Kleb [48] and resistance to tobacco mosaic virus [59].

3.2.2 Techniques of genetic modifications

Genetic transformation has been proposed as an alternative method for the enhancement of capsicum. Transgenic technology, in the case of capsicum, has many key benefits, one of which is that it allows for the transfer of valuable genes or the acquisition of distinctive features across interspecific and intergeneric barriers. A pioneering research on the transformation of capsicum was initially published in 1990 [60]. The lack of repeatability in the pepper plant, on the other hand, is a significant stumbling hurdle for capsicum transformation studies. There has been a great deal of work done on capsicum transformation for disease resistance, particularly against viruses such as tobacco mosaic virus (TMV), pepper mild mottle virus (PMMV) [61], tomato mosaic virus (ToMV) [62], cucumber mosaic virus (CMV) [63]. A transgenic virus resistance strategy that makes use of viral coat protein regions and satellite RNA is referred to as RNA silencing in the scientific community [64]. The use of transformation and overexpression of TsiI, a tobacco pathogenesis-related (PR) gene in capsicum, allowed us to demonstrate broad spectrum resistance against a variety of pathogens, including PMMV and CMV, as well as the bacteria Xanthomonas campestris pv. vesicatoria and the fungal pathogen Porphyromonas capsici [62]. A limit is placed on the number of transformation experiments in capsicum that are carried out on parameters other than disease resistance.

Harpster and colleagues discovered that the enzyme ripening-related endo-1.4-b-glucanase is inhibited in transgenic capsicum [65]. Transformation research in capsicum that includes the introduction of foreign genes from other plants or species are quite usual. It was possible to develop a dwarf transgenic capsicum after transformation with the OsMADS1 gene from rice [66, 67]. Ketoacyl-ACP reductase (CaKR1) was discovered by using RNA silencing to identify a unique gene in the capsicum plant that produces non-pungent fruits, which was named by the researchers [68]. A reporter gene for capsicum transformation studies has been most often used, and it is the GUS gene (β-glucuronidase) [61]. It has been most common to create capsicum transgenics using Agrobacterium-mediated transformation, with cotyledons and/or hypocotyls being used as explants in the vast majority of experiments [61]. Capsicum transgenic C. frutescens has recently been exposed to direct transformation by the gene gun, resulting in the development of a new variety of capsicum [69].

3.2.3 Marker-assisted breeding

In the field of capsicum enhancement, marker-assisted breeding (MAB), also called molecular-assist breeding, has gained favor. Capsicum has been studied using isozyme markers, amplified fragment length polymorphism (AFLPs), random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLPs), simple sequence repeat (SSR), single nucleotide polymorphism (SNP), and COS II markers. These markers have been widely used to study the transmission of important features, as well as to identify horticultural and disease resistance genes, as well as quantitative trait loci (QTLs).

3.2.3.1 Necessity of molecular breeding

However, environmental factors make it difficult to select for quantitatively passed-down complex characteristics, making it difficult to use direct selection on genotype or phenotypic values. As a result, the indirect selection is seen to be a preferable method of selection. Conventional breeding has had little success because of the polygenic regulation of resistance characteristics, the large variety of pathogen strains found in various habitats, the complexity of the host-pathogen relationship, and the great diversity in pathogenicity. There are several reasons for this. It’s possible to use indirect selection by looking at other, more readily measurable features that are closely connected to the desired traits, but which are more difficult to measure or which are impacted by the environment.

Due to other features, indirect selection for yield is constrained. The inability to select for certain genes is typically due to a lack of available tools, facilities, and resources. As a result of the development of molecular (DNA) markers, plant breeders now have an effective tool for doing gene selection. Marker-assisted gene selection is not a true form of gene selection, but it is the best method available for indirectly selecting target genes in DNA. A reliable and successful method is the marker-assisted selection (MAS). Both Collard and Mackill [70] and Kole and Gupta [71] have shown the benefits of MAS over traditional phenotypic selection. Compared to phenotypic breeding, selection utilizing molecular markers is easier.

Selecting a single plant with high dependability may be done at any step of the plant’s life cycle, in addition to this. Gene localization and the generation of novel genotype combinations with high yield and stress-resistant genes have both been made possible by the advent of molecular markers. This speeds up the breeding process considerably. They’ve helped researchers learn more about how certain genes work. Gene placement and selection aren’t the only things that molecular markers help with; they may also be used to analyze genetic diversity, monitor quality, and aid in breeding. The use of molecular markers is critical to expediting the speed of improvement programmes in order to fulfill the rising demand for increased capsicum yield and disease-resistant genotypes. Capsicum molecular markers [72] have been used for DNA fingerprinting, genetic diversity analysis, QTL analysis of important biotic stresses, and MAS [73].

Capsicum genotypes may be reliably differentiated by estimating their genetic diversity. Different kinds of marker systems such as isozymes, RAPD [74], AFLP [75], and SSR [76] have been used for genetic diversity study and varietal identification in capsicum. The use of molecular markers to determine genetic diversity is helpful for a variety of reasons, including choosing different parent combinations for hybrid production, understanding the evolutionary link of various Capsicum species, and accurately identifying varietals. In order to protect and make use of plant genetic resources, it is necessary to do molecular characterization on germplasm. Conventional plant breeding has certain drawbacks, which MAS attempts to alleviate using molecular selection. Numerous genetic markers in capsicum, including mapped microsatellites and single nucleotide polymorphisms (SNPs), have been utilized successfully in genomics [77].

SSRs and SNPs have been used to clone and define genes in capsicum that influence stress tolerance, quality traits, and other aspects of plant growth. These genes are valuable assets for molecular-assisted breeding. Capsicum researchers now employ SSRs as the most common markers, in part because of their widespread availability in the public domain, as well as their ease of use and efficacy [78]. Genomes/QTLs for a wide range of important traits in capsicum, such as pungency, fertility restoration, soft flesh and deciduous fruits [79], capsanthin content, fruit size and shape [80], male sterility [81], parthenocarpy [82], resistance to CMV [83], potyvirus, have been identified in the genomes of various species of capsicum.

3.2.4 TILLING and Eco-TILLING approaches

Genetic differences are created via mutations, which are the most common cause of genetic diversity. It is currently considered to be a cornerstone of contemporary plant breeding. In the case of capsicum, it has been discovered that mutation breeding is a successful and efficient breeding strategy. Daskalov [84] has provided an in-depth analysis of this topic matter. The seeds of capsicum are the most appealing portions to be treated with mutagenic agents. It is advisable to utilize seeds of uniform size and germinability (96–100%), as well as seeds with a low moisture content (approximately 13%), in order to achieve high repeatability of findings. Ionizing radiation utilized as a mutagen should result in a 40–60% chance of survival [85], but chemical mutagens should result in a 70–80% chance of survival [86]. Bell peppers, as opposed to spicy peppers, are often more radiosensitive in general. Pollen grains have also been treated with gamma or X-rays and utilized for the pollination of emasculated, non-irradiated flowers immediately after irradiation. In order to prevent cross-pollination, the M₁ generation (first generation after mutagen treatment) plants must be cultivated on separate plots (at least 700 m away from other capsicum plants) followed by bagging of the M₁ flowers to prevent out-crossing. Each experiment requires the cultivation of at least 3000–5000 M₁ plants. Plants are produced in the following generation at a rate of 20–25 M₂ plants per M₁ plant or 10–15 M₂ plants per M₁ fruit (with 2–3 fruits per M₁ plant) in the first generation. According to the M₂ field population size estimates, the population size ranges between 70,000 and 100,000 plants, however this varies depending on the kind of selection to be done and the number of observations to be made. The M₂ generation is the one in which the majority of the work is done in terms of mutant selection. All identified mutants must be selfed, which is commonly accomplished by bagging the flowers, to enable offspring testing.

In capsicum, the method of mutation breeding has been employed extensively for functional gene annotation as well as for the creation of new variability that can be exploited in breeding [87] used the sweet pepper cultivar “Maor” to develop a mutation population that was later used for the isolation and characterization of genes controlling plant architecture and flowering [87]. Similar mutant populations have been established in chilli peppers using the cultivar “Yuwol-cho,” which is a cross between two different cultivars [88]. TILLING (targeted induced local lesions in genome) technique was used in the same cultivar “Yuwol-cho” by Jeong et al. [89], and they were successful in isolating a line that was resistant to the tobacco etch virus (TEV).

Capsicum mutant populations were generated by Daskalov [90, 91] by the use of X-rays and gamma irradiation, and these populations were studied further. Novel male-sterile lines were isolated from these populations and then described to determine their suitability for use in breeding programmes. These populations were also used to generate capsicum cultivars that have desirable features like resistance to the cucumber mosaic virus (CMV), superior taste, greater yield, and compact plant height, among others [90, 91]. Japanese researchers Honda and colleagues [92] generated mutants using heavy ion beams (12C and 20Ne), however the majority of the screening was done in the M₁ generation, which is the first generation of mutants. Capsicum has been subjected to ultraviolet irradiation in order to produce mutants with higher levels of vitamin C and E [93]. Three male-sterile lines were obtained from a capsicum mutant population produced by gamma irradiation and used in hybrid development by Daskalov and Mihailov [93].

Tomlekova and colleagues [15, 16] have recovered mutants with altered shoot architecture in hot pepper [86], some induced mutants in sweet pepper [92], and capsicum with enhanced β-carotene and orange color on maturity [15, 16]. Capsicum annuum L. dry seeds were gamma irradiated, and numerous intriguing mutants were developed, the most interesting of which were induced male-sterile mutations, which were acquired after gamma irradiation of the seeds. Male sterility is controlled by a small number of recessive genes, which are designated as ms-3, ms-4, ms-6, ms-7, and ms-8. Using the male-sterile lines Pazardjishka kapia ms-3 and Zlaten medal ms-8 that were recovered following mutagen treatment, the researchers were able to evaluate their combining capacity against the original male-sterile line that was utilized for hybridization. The results obtained suggest that there is no statistically significant difference in the combining ability for early and total yields, according to the findings. Several male-sterile lines were crossed with a huge number of other lines in order to produce hybrid combinations that might be used for a variety of reasons. When it came to early yield, the majority of the hybrid combinations outperformed the check. There was also a rise in overall yield in several hybrids, which was observed. Two-hybrid combinations, designated “Krichimski run” and “Lyulin” were released as cultivars based on the male-sterile lines retrieved from the mutant population and used in the development of the hybrid combinations.

4. Limitations of capsicum breeding

Crop improvement via conventional plant breeding relies on manipulating plant genomes inside the core gene pool of a genus. Hybridization and selection are used in conjunction with backcross breeding, mutagenesis, and somatic hybridization to develop novel combinations of genomes from various species. Segregating progeny phenotypic evaluations are used to identify economically significant novel characteristics. Beyond a certain point, traditional plant breeding’s relevance in improving quality and output becomes exceedingly challenging. Modern breeding distinguishes itself from traditional breeding by separating phenotypes from genotypes. The phenotype is a manifestation of a person’s inherited genes in a particular environment. Genotype selection and screening are focused on phenotypic expression rather than genetic variation. As a consequence, new cultivars include qualities that breeders want, but they also have undesirable characteristics that were not taken into account during the selection process, and this transfer of undesirable traits from existing to new varieties is almost always unavoidable via traditional breeding. Breeders face a second problem when they attempt to make use of the genetic variation that is present in groups that are incompatible with each other. Wide-scale hybridization and intensive backcrossing of created hybrids with recipient parents provide novel features into cultivated types. Nevertheless, the targeted features of interest don’t appear on their own; they’re accompanied by larger portions of wild chromosomes and so are linked to linkage drag, which may include unwanted genes. Traditional breeding cannot regulate the expression of target genes in a new genetic background, which is the third constraint. Using current breeding methods like marker-assisted selection helps speed up the introgression process while also reducing linkage drag. Plant breeding will continue to use conventional techniques to create new and better varieties, in other words. In contrast to traditional phenotypic selection, molecular breeding plays an important role since it is more accurate, quick, and cost-effective than conventional phenotypic selection.

5. Future perspectives

The success of Capsicum breeding demonstrates the program’s potential for future growth. Capsicum has a lot of opportunities for development and expansion. The genetic diversity of capsicum has been discovered using a whole-genome sequence and a genotyping by sequencing technique to locate single nucleotide polymorphisms (SNPs). Because of this genomic information, we may now believe that the genetic makeup of capsicum can be changed to a far greater extent than previously anticipated. The number of studies that relate genetic variation to observable phenotypic variation, on the other hand, is still fairly low. Finding unique linkages between the generated genetic resources and crucial capsicum characteristics such as fruit size, production, pungency, resilience to abiotic stress, nutritional content, and disease resistance is a major research subject. Furthermore, the utilization of transgenic technology in capsicum is slow because of the difficulty of changing and regenerating capsicum. Now that the capsicum genome sequence is accessible, researchers may look at the most recent genome-editing technologies and their potential use in genetic upgrading of capsicum. The capacity of organisms to profit from gene/genome editing is greatly limited by a lack of well-characterized target gene information. Combining cutting-edge genetic breeding technologies with tried-and-true processes like as traditional selections and crosses will be important in capsicum breeding in the future.

Conflict of interest

The authors declare no conflict of interest.