**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

### *Capsicum: Breeding Prospects and Perspectives for Higher Productivity DOI: http://dx.doi.org/10.5772/intechopen.104739*

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

### *Capsicum: Breeding Prospects and Perspectives for Higher Productivity DOI: http://dx.doi.org/10.5772/intechopen.104739*

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,

### *Capsicum: Breeding Prospects and Perspectives for Higher Productivity DOI: http://dx.doi.org/10.5772/intechopen.104739*

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 markerassisted 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

### *Capsicum: Breeding Prospects and Perspectives for Higher Productivity DOI: http://dx.doi.org/10.5772/intechopen.104739*

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, nonirradiated 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.
