An Update on Radish Breeding Strategies: An Overview

Abstract

In tropical, subtropical, and temperate climates, radish (Raphanus sativus L.) is a popular root vegetable. Radish diversity is intense from the eastern Mediterranean to the Caspian Sea. Many radish varieties have varied leaf morphology, root color, size, shape, flavor, vernalization requirements, and maturity times. Early radish variants were long and tapered rather than cylindrical, bulbous, elliptic, or spherical. For black Spanish radish, European-cultivated variety, and Asian-cultivated radish, three separate domestication processes occurred. The original radishes were black, followed by white in the 1500s then red and round in the 1700s. These are R. sativus L. var. radicula (sativus) or R. sativus L. var. niger radishes. Because of protogyny, self-incompatibility, open architecture, and biennial bolting, radish crosses readily. The fundamental methods for using heterotic breeding potential are SI, CMS, and doubled haploids (DH). This chapter discusses the various breeding strategies like inbred line development by the use of self-incompatibility, hybrid development by using male sterility system, population improvement, mutation breeding, haploid breeding, breeding strategies for biotic and abiotic stresses, QTL mapping, and genome wide and genomic tool in radish. Rapid developments in our understanding of advanced biotechnology technologies will increase our ability to identify cultivars and parental lines, check seed genetic purity, analyze phylogenetic links and genetic diversity, and add specific transgenic traits.

1. Introduction

Radish (Raphanus sativus L.) is an annual herbaceous plant with two sets of nine chromosomes [1]. It is a member of the Cruciferae family and consumed raw as a salad component, garnish, and shredded radish [2]. Furthermore, radish has been used in cuisines all throughout the globe. Radish is often used in eastern Asian cuisines [3]. Priority is given to the creation of superior radish cultivars suitable for tropical and subtropical climates [4]. Furthermore, breeding research on a variety of agronomic qualities, such as disease resistance and suitability to human use, has been done. High yield, early maturity, late bolting, pungency, cold-hardiness, drought resistance, heat tolerance, and soil adaptation are all important features for radish breeding [2]. There are correlations between the consistency of the radish and its sugar concentration, pungency, cell complexity, water content, and pore size [5]. To develop radish varieties, mass selection or pedigree techniques are being used, with an emphasis on the red globe, oval red, and white forms [6]. The most difficult problem has been adapting radish cultivation to many growth seasons [7]. For a successful radish breeding procedure, significant genetic data on chromosomes and inheritance information for numerous genes relevant for agronomic, biochemical, and biotic and abiotic stressors must be collected [8, 9]. It is required to undertake research utilizing novel methods, such as chromosomal or gene modification [10].

Physical attractiveness, including length, form, size, and skin tone, has a significant influence on consumer desire and marketing judgment [11]. The skin is generally white, but it may take on pink, red, purple, yellow, and green tones. Red radishes, on the other hand, are around 40 cm long and have a mild flavor (not as spicy) [5]. The anthocyanin pelargonidin is responsible for red colors, whereas a cyanidin derivative is responsible for purple hues. Even while quality-related characteristics are highly heritable, cultivation practices often have a significant impact on them. Radishes’ swelling taproots may take the shape of an oval, tapering, or cylindrical object [12]. Furthermore, cylindrical root variations are often collected mechanically [13]. Radish roots are high in antler velvet, generate beneficial phytochemicals, have cancer-preventive effects, and increase the flavor of Brassica products greatly [14]. Furthermore, radishes provide us with complex carbohydrates, dietary fiber, and organic nutrients and minerals [15].

Omics methodologies based on next-generation sequencing (NGS) techniques provide a significant amount of genomic research [16], which also allows for the distribution and acquisition of positional markers on the chromosomes as well as the identification of new genes and sequences [17]. Furthermore, genome-wide studies show the genetic foundation of some characteristics [18]. The ability to re-sequence genomes allows for genome-wide investigation of important markers and higher-throughput genotyping [19]. Less research has been generated that examines the most crucial historical events as well as current achievements in radish breeding. As a consequence, we have a wealth of information on all aspects of radish breeding and its countless accomplishments in this sector. We anticipate that vegetable breeders will benefit from this chapter in the future.

2. An overview of radish breeding

2.1 Origin and distribution

Radish is an annual vegetable in the Cruciferae family. From the Mediterranean to the Black Sea, the genus Raphanus was separated into the Raphanus DC. and Hesperidopsis Boiss sections. Each part has six species (R. sativus, Raphanus raphanistrum, R. microcarpus, R. rostratus, R. landra, and Rumex maritimus) and one species (Raphanus aucheri) [19]. Kitamura [19] also proposed that R. sativus may be grown in the Mediterranean region by natural or artificial hybridization of R. landra with R. maritimus. Banga [20] and Hida [21] proposed that four wild species (Raphanistrum raphanistrum, R. maritimus, R. landra, and R. rostratus) may have aided in the evolution of radish. Panetsos and Baker’s [22] study on wild R. sativus and R. raphanistrum confirmed the species differentiation. The wild R. sativus has a white or partially purple flower on a white background as well as a delicate, fairly thick pod made up of spongy parenchyma. R. raphanistrum, on the other hand, bears yellow blooms and slender, robust pods. The ripe pods disintegrate into bits. The aforementioned two species flourished in California in intermediate forms that may have arisen from natural hybridization. The F₁ plants produced by artificially crossing the two species demonstrated intermediate features, including chromosomal configurations of 1IV + 7II at initial metaphase (MI) of pollen mother cells (PMCs) and fertility of 50% in both pollen production and seed setting. Based on these findings, it was proposed that the F₁ plants had reciprocal translocations in one pair of chromosomes. When R. sativus wild type was spontaneously backcrossed with cultivated radish, some progeny lacked quadrivalent chromosomes and had a high seed setting rate. It was therefore suggested that gene flow (or introgression) from R. raphanistrum into radish cultivars be encouraged. Eber [23, 24] found comparable findings in their study of hybrids between the wild R. raphanistrum and the cultivated R. sativus in France. In F₁ hybrids of R. sativus and R. raphanistrum L. ssp. landra, Kato and Fukuyama [25] revealed correct chromosomal organization of 9II during meiosis and robust seed setting. Based on these findings, it was hypothesized that R. raphanistrum might undergo chromosomal reconstruction. Harberd [26] proposed that the genus Raphanus be classified as a cytodeme based on chromosomal number, chromosome layout at MI in PMCs, and fertility studies. This finding was corroborated by Prakash [27]. Tsunoda [28, 29] thought that all wild radish species belonged to R. raphanistrum and evolved around the Mediterranean-Black Sea coast. R. raphanistrum was common in Russia and the New World, but it was not found in China, Japan, or India [30, 31]. Raphanus was recently separated into two species, R. sativus and R. raphanistrum, the latter of which contains additional wild species as R. raphanistrum subspecies [31]. R. sativus var. hortensis f. raphanistroides Makino [19], also known as Hama-daikon or R. raphanistrum ssp. maritimus [31], grew wild along the East Asian shoreline. Another kind of wild radish, Nora-daikon or No-daikon, thrived in areas far from the sea. It was thought that these wild radishes were the result of cultivated radishes escaping [19, 32, 33, 34] or the migration of weedy radishes tainted with cereals such as wheat and oat. Numerous research, however, support the first point of view. Germplasm resources for understanding the origins of farmed radish and improving the radish crop include Hama-daikon and Nora-daikon. Molecular studies of DNA and genomes, in addition to morphology, ecology, and cytogenetics, may provide insight into the origin, differentiation, and domestication of radish.

2.2 Botany of radish

Radish (R. sativus L.), an entomophilous flower, is an allogamous plant [35]. When it appears as three florets at the tip of each branch of the panicle during normal flowering, each flower is capable of producing a pod up to 1 to 3 inches long and holding one to six seeds [36]. The radish blossom’s fresh corolla blooms in the morning and lasts till the following day [37]. The flower’s pollen receptivity is present only for a brief duration each day, according to Kremer. Its clawed petals, four erected sepals, six stamens, and 1.5 to 2 cm broad, pink to purplish with purple veins, blooms in a 3 to 4 cm long style [38, 39]. A siliqua, sometimes called a seedpod, is a radish seed capsule that is 1.5 cm wide and 3 to 7 cm long. It bears a long, conical, and seedless beak and 6–12 seeds per pod [5]. The inflorescence of the radish is a typical Cruciferae raceme that is long, erect, and rectangular [40, 41]. Radchenko [42] studied the pollination of radish. When Crane and Mather [43] investigated how to cross-pollinate radish, they found that the “Icicle” and “Scarlet Globe” cvs were self-incompatible and pollinated by bees [44]. The research found that the number of honeybees visiting the radish blooms significantly affected the quantity of seeds produced [37]. Honeybees pollinate radish blossoms at a rate of 77 to 99 percent on average, according to Radchenko [42], which increases crop yield by 22% and enhances seed quality. Consequently, it is thought that radish is almost entirely insect-pollinated [45]. While the fruit is developing, the color of the seeds is somewhat yellow, and they eventually become reddish-brown [46, 47]. The lyrate, pinnately distinct mature radish leaves feature a larger terminal lobe and smaller lateral lobes. They are arranged in a rosette condition, and alternate form [48]. Longer root types include winter radishes, daikon or mooli, and oriental radishes, which may grow up to 60 cm in length and with leaves as large as 45 cm by 60 cm in width [48].

2.3 Characteristics traits

It is a self-incompatible allogamous species. The considerable genetic variety of radish landraces and wild radish populations is paralleled in cultivar DNA polymorphism. Self-incompatibility may be overcome by bud pollination or high-concentration CO2 treatment, permitting the development of self-compatible progeny [49]. However, they exhibit inbreeding depression, making it difficult to get inbred lines. S-receptor kinase (SRK) is the recognition molecule of the stigma, similar to the self-incompatibility of Brassica species, whereas SP11, also known as SCR, is the recognition molecule of pollen [50]. These recognition molecule genes, SRK and SP11/SCR, have several alleles and are passed down through generations as the S haplotype. There are numerous S haplotypes in R. sativus [51], and the nucleotide sequences of some S haplotypes in R. sativus are similar to those of Brassica rapa, indicating that S haplotypes possessed by an ancient species were inherited by species in both Raphanus and Brassica without significant nucleotide sequence modification [50]. The majority of radish cultivars are root vegetables. The size, shape, and color of radish roots are all important. There has been evidence that quantitative trait loci (QTLs) influence root structure and color [52, 53]. Other factors, such as blossoming, influence root thickness. The QTL with the highest LOD score corresponded to a QTL for bolting time in our QTL analysis of root thickness using offspring obtained by crossing “Aokubi” with a white thick root and a rat’s tail radish cultivar. This might be natural since early blossoming is thought to reduce root thickness. The transcriptome of developing roots was studied, and genes involved in root thickening were found. The color of the radish root surface is caused by anthocyanins. Pelargonidin and cyanidin are the pigments responsible for the red and purple colors of radish varieties. The finding of purple roots in a hybrid of a red root line and a white root line shows that the red and white had knockout mutations in separate genes involved in cyanidin synthesis and that the functional alleles in the red and white functioned as complementing genes. In red root cultivars, alleles of the flavonoid 3′-hydroxylase (F3′H) gene exhibit Ty3/gypsy transposon or helitron insertions [54]. A dihydroflavonol reductase (RsDFR) and anthocyanidin synthase (RsANS) gene are expressed in the epidermal tissues of red-skinned cultivar roots but not in white-skinned cultivar roots [55]. The huge seed size of radish is a distinguishing feature among Brassicaceae species. Radish seeds weigh nearly five times as much as B. rapa seeds. Because of the large seed size, the cotyledons and hypocotyls of seedlings are larger than those of Brassica. The bigger seedling size allows for direct sowing in the field and produces sprouts that are larger than Brassica. The form of siliques is connected with the property of large seeds. Amphidiploid plants of intergeneric hybrids of R. sativus and B. rapa have intermediate siliques with a few seeds in both the beak and valvar regions. Although the amphidiploid plants are mostly sterile, a small number of seeds may be obtained. Furthermore, the seed size is about midway between Raphanus and Brassica. Isothiocyanates, which are responsible for the pungent flavor of radish, are produced when glucosinolates are digested. The flavor of grated fresh radish and radish salad is significantly influenced by glucosinolates. The major glucosinolate in radish roots is glucoraphasatin (also known as 4-methylthio-3-butenyl glucosinolate, dehydroerucin), and the glucosinolate composition changes slightly. The glucoraphasatin content in Japanese radish cultivars, on the other hand, varies greatly [56]. There have been reports of QTLs impacting glucosinolate concentration in the root [57], and the genes inferred are putatively involved. Ishida et al. (2015) identified a mutant with a high quantity of glucoerucin but no glucoraphasatin, and the gene responsible for this mutation was discovered [56]. Although most radishes are salt tolerant, R. sativus var. raphanistroides is especially so [58]. Although genetic research into the salt tolerance of R. sativus var. raphanistroides has not yet developed, radish salt tolerance genes will be significant in the evolution of Brassica crops. High-temperature stress has become a major concern in radish growing. High-temperature stress causes the center of a radish root to become reddish brown, resulting in unmarketable products. However, since sensitivity to high temperature stress varies, it should be possible to develop a resistant cultivar. When radishes bolt, their roots become fibrous and unsuitable for sale. As a result, the characteristic of late bolting is favored. On the other hand, cultivars for oil production or rat’s tail radish are needed to bloom even in tropical settings. Although vernalization is required for floral induction, rat’s tail radish may bloom without it. Radishes, like many other winter crops, have varying vernalization needs. A QTL with a significant LOD score in a region containing an FLC gene was found using progeny from a hybrid between “Aokubi” and rat’s tail radish. Although Plasmodiophora brassicae’s clubroot poses a serious danger to crop yield, there are techniques to control it [58].

Among Brassica crops, Japanese and South Korean radish cultivars are often resistant to clubroot. Radish may be used to breed resistance in Brassica plants [59]. A quantitative trait locus (QTL) promoting clubroot resistance has been found on LG1 [60], which correlates with Rs5 [61]. Fusarium yellow, caused by Fusarium oxysporum, is one of the most serious diseases in radish production. This disease causes wilting of the leaves and browning of the vascular tissue in the root. Certain cultivars and landraces have a high degree of resistance to Fusarium yellow. Because of its low ability for plant regeneration, radish is difficult to produce in tissue and cell cultures. There have been few reports of successful protoplast, anther, or isolated microspore culture [62], and protoplast, anther, and isolated microspore cultures are unusual [63, 64]. The function of isolated genes in radish cannot be shown due to the intricacy of plant transformation. The development of an efficient plant transformation technique is critical for scientific and practical radish research. Because plant regeneration capability must be genetically diverse, the first step in developing in vitro culture techniques would be to find cultivars or lines with high regeneration potential.

2.4 Breeding goals

The strong nutritional value and chemical components of the Brassicaceae family have significantly improved human health and well-being [65]. Root length is highly valued by consumers, who constantly rate radishes in terms of root length, diameter, and color. These factors are all highly important throughout the purchasing experience, and therefore, visual signals like the label’s color and location on the product are crucial. Too many gene-mapping studies have been devoted to the categorization of significant color-gene relationships in various plants [66]. The development of color genetics, which is essential to the success of radish crops, would be aided by the discovery and detection of key plant genes involved in radish color [67]. Numerous studies have been conducted thus far on the inheritance patterns of radish skin [68]. It was discovered that the most extensively studied varieties of radish, including red (30%), white (13%), and black (6%), had a total of 609 distinct chemical elements, distributed across 23 different groups [69]. The main plant sections from which the nutrients, anti-oxidants, and phytochemicals described in this study were derived were the roots, sprouts, and leaves [69]. Researchers have been interested in the natural red pigment that is abundant in red flesh radishes and is used widely in the food, wine, and cosmetics sectors [70]. Achieving uniformity in radish breeding in terms of color, size, and yield is becoming increasingly crucial [71]. Radishes’ esthetic appeal and health advantages are significantly influenced by color [72]. However, there is little study on the detection, characterization, and quantification of flavonoids in multicolor radish. Despite this, Zhang [73] discovered that the anthocyanin molecules that gave red and purple radishes their color pigment were relatively similar. These substances included pelargonin, callistephin, and red cyanidin [73]. Purple ZJL contains cyanidin o-syringic acid and cyanin, but dark red TXH has more callistephin and pelargonin. The metabolites that give colorful radishes their distinctive colors are more often associated with the secondary plant chemical biosynthesis pathway than SZB genes, in contrast. This approach could be useful for creating new, high-quality varieties of radish [73].

2.5 Breeding methods

The form of the leaves, the color of the petiole and leaf blades, and the quantity of surface hair are the important factors in radish breeding. Roots that are beyond their prime, on the other hand, are picked based on their internal solidity and outer morphology. Watts [74] formulated an immersion method for the solidity test in which the roots are immersed in water; acceptable solid roots sink and are selected for planting, but unsuitable pithy roots float and are discarded. In the nineteenth and twentieth centuries, mass and repeating selections were utilized to boost productivity and uniformity. However, hybrid breeding began in the 1950s to examine the prospects of heterotic vigor. Twenty-first-century biochemical, molecular, and biotechnological tools facilitated and diversified breeding tactics for quality and stress tolerance. Population improvement techniques for radish, like those used for other crops, include mutation breeding, backcross breeding, hybrid breeding (synthetics, heterotic F₁ hybrids), molecular and transgenic methods, gamete selection, family selection, line breeding, recurrent selection, and mass-pedigree breeding [75, 76].

2.6 Radish breeding by using self-incompatibility system

Self-incompatibility (SI) is a mechanism that promotes stigmatization of self-pollen, prohibits self-fertilization and inbreeding, and demands outcrossing. Selfing may be prevented via embryo abortion; however, SI is pre-zygotic and precludes embryo development. The sporophytic type of incompatibility system causes pollen grains to fail to germinate and form pollen tubes on the surface of stigma epidermal cells (papilla) as well as the deposition of callose inside the papillae. Stout [77] discovered sporophytic SI in radish, and Bateman was the first to demonstrate how it was inherited. SI is caused by the pollen tube’s inability to penetrate the papillae as well as a lack of adhesion, hydration, and pollen grain germination. Dickinson [78] reported that homomorphic SI is typically controlled by a single S-locus containing two multiallelic genes encoding the S-locus glycoprotein (SLG), S-locus receptor kinase (SRK), and S-locus cysteine rich protein/S-locus protein 11 (SCR/SP11), all of which are expressed on the stigma. So far, massive amounts of S-alleles have been discovered [79, 80]. A significant number of S-haplotypes in Brassica oleracea, B. rapa, and R. sativus have been discovered using a variety of techniques, including pollination tests, electrophoretic analysis of stigmatic proteins, DNA polymorphism in SLGs or SRKs, and determination of SLG, SRK, and SCR sequences [51, 81, 82, 83]. The SI technique has the benefit of enabling two parental lines to be homozygous for independent S alleles, allowing F₁ hybrid seed to be produced. Unlike cole crops, most radish genotypes have brittle and unstable SI systems. The majority of Indian radish genotypes tested at the IIVR in Varanasi, Uttar Pradesh, India, are self-compatible to mildly self-incompatible, with only a few genotypes, particularly red radish, exhibiting moderate self-incompatibility and a red genotype VRRAD-130 exhibiting severe self-incompatibility. Raphanus has been related to genetic variances in SI levels [84, 85]. However, it is less reliable since hybrid seeds always carry the danger of generating an unwanted number of siblings and because reproducing SI lines by bud-pollination (BP) is difficult. The SI system in Brassica, including radish, must be broken down by BP, CO₂ treatment, and NaCl treatment in order to maintain and propagate self-incompatible lines. In contrast to cole crops, most radish genotypes have rather weak and unstable SI systems; as a consequence, hybrid seeds including sib-seeds are always possible.

Because radish is a self-incompatibility crop with significant heterosis, the generation of F₁ hybrids based on self-incompatibility is desired to remove the time-consuming manual emasculation [86]. The basic purpose of a plant breeder is to identify S haplotype breeding lines. The plant breeder can keep the parental lines from crossing [87]. The S haplotypes of parental lines must be determined in order to achieve F₁ hybrid breeding because each parental line’s S haplotype must indicate compatibility between parental lines [88, 89]. The abundance of S haplotype establishes a specific S haplotype using traditional procedures such as the test cross method, pollination, isoelectric focusing, immunoblot analysis, and pollen tube fluorescence analysis [85, 90]. The S alleles of the S haplotype are highly diverse [89]. In addition, Nikura and Matsuura found 37 alleles in Radish [91]. Raphinus sativus contains many S haplotypes, which are labeled S-1, S-2, S-3, and so on based on polymorphism in the SLG, SRK, and SCR/SP11 sequences [91]. Despite the fact that radish is not a part of the Brassica genus, Brassica SP11, SRK, and SLG alleles are interleaved in the evolutionary trees of these genes, indicating that the diversification of these alleles predates the speciation of these taxa [87]. Some S haplotypes in radish feature SP11, SRK, and SLG alleles that are very similar to some S haplotypes in Brassica, and one S haplotype in radish has been shown to have the same recognition specificity as one S haplotype in B. rapa [87]. Comparisons of the nucleotide sequences of the SP11 and SRK alleles, as well as recognition specificities across related S haplotypes of radish and Brassica, may aid in understanding the molecular structures of SP11 and SRK proteins. However, different studies number S haplotypes in radish; therefore, the nucleotide sequence data on S haplotypes are uncertain (Nishio and Sakamoto 2017). S haplotype in Raphanus and Brassica is also identified using the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) approach, which examines SLG and SRK [47, 50, 86, 92]. However, PCR-RFLP has two inherent limitations: First, it is difficult to design a universal primer that can amplify SLG and SRK alleles, and second, the presence of several homologous genes in Brassicaceae plants makes PCR amplification of specific SLG or SRK alleles more difficult [87]. To help in radish hybridization, the Ogura CMS approach created further advanced radish cultivars (cultivars with improved yield and quality) [13]. Because variety displays such as bulk selection, mixed mass pedigree selection, or bud pollination might take eight to twelve years to develop a new variety, new varieties must be created by different genetic processes [93].

2.7 Radish breeding by use of male sterility system

Male sterility (MS) is a condition in which plants are unable to produce viable pollen, which is required for efficient hybrid seed commercial production. It often manifests itself in floral development as an incompatibility of nuclear-mitochondrial interaction in alloplasmic lines created by spontaneous mutation. It may also occur in wide crosses (intraspecific, interspecific, and intergeneric). Staminal MS systems are common in radish. Male sterility systems that contain nuclear and/or mitochondrial genomes include genic male sterility (GMS), cytoplasmic male sterility (CMS), and cytoplasmic-genic male sterility (CGMS). Transgenic male sterility (TMS), a new kind of male sterility, was developed using biotechnological technologies. Based on the process of male sterility induction and fertility restoration, all TMS systems developed to date may be classified into five kinds [94].

Plants with cytoplasmic male sterility, which is inherited from their mothers, are unable to generate effective pollen. The CGMS hermaphrodite state is restored by a collection of nuclear genes known as restorers of fertility (Rf), which inhibit the CMS genes’ activity. The Ogura CMS system has been applied in a number of contexts and is now commercially available. Ogura [95] found the CMS in a Japanese radish cultivar, giving rise to the term Ogura CMS. Ogura CMS is regulated by a recessive nuclear gene (msms) in association with sterile cytoplasm (S-cytoplasm).

The genotype of male sterile plants is Smsms, whereas the genotype of the maintenance line is Nmsms. Several CMS systems have also been identified in the Brassicaseae family. Polima [96, 97], napus [98, 99], Ogura [95], and Anand [100] are well-characterized CMS systems from the Brassica genus; however, the following systems, such as Ogura CMS [101] Raphanus and Brassica species, lack Rf genes, but all fertile The Rf genes are widely distributed in the Japanese wild radish, regardless of cytoplasm type (R. raphanistrum). The bulk of cultivated radishes in Japan and India lacks restorative genes in their populations, although European and Chinese variations do [102]. However, the Rf gene is essential for crops that require pollination and fertilization for economic growth, such as chili, tomato, eggplant, and melon. CMS lacking the Rf gene benefits from simple transfer in diverse backgrounds and is utilized in a variety of vegetables where the vegetative element is economically valuable, such as root crops, cole crops, tuber crops, and leafy vegetables.

Similar to CMS, the genetic emasculation approach in radish allows for the harnessing of heterotic vigor for yield, uniformity, adoption, and earliest maturity as well as the production of high-quality seeds. Despite being one of India’s most important salad crops, the first CMS-based radish hybrids and Public Sector CMS lines were reported in 2018 from ICAR-IIVR, Varanasi, UP, (2018) by Singh and colleagues.

2.8 Population improvement

Cross-pollinated crops and Brassica vegetables have benefited from population improvement. In India, mass selection has been routinely utilized to enhance the genetics of radish. This approach is useful for genetic gain of simply inherited monogenic characteristics, but it is inefficient for qualities regulated by polygenes. Significant genetic variety for variables of relevance, as found by several studies in radish [103, 104, 105, 106, 107, 108], is a precondition for crop development. Many cultivars have been produced across the globe with various goals in mind. Based on progeny appraisal, changes such as mass pedigree and family selection are superior to basic mass selection. The decision between these strategies is determined by the population’s homogeneity. Recurrent selection is a preferable choice for improving quantitative traits, particularly those controlled by additive gene activity. This approach is helpful for improving leaf morphology, root form, size, color, yield, and other economic features. Kashi Lohit, a red radish cultivar, was developed at ICAR-IIVR in Varanasi, Uttar Pradesh, using a simple recurrent selection to target red color root, tapering root form, and yield. Kashi Lohit has around 30–125% more nutrients such as ascorbic acid, total phenolics, anthocyanins, and antioxidants than white-rooted commercial radish cultivars [109]. Inbred lines may be produced by breaking down SI barriers using either BP or chemical induction procedures (NaCl or CO2). Many cultivars have been released in India by public sector organizations including Kashi Hans, Kashi Sweta, Kashi Mooli-40, Kashi Lohit, and Kashi Aardra as some of the characters. VRRAD-150 (ICAR-IIVR, Varanasi); Pusa Desi, Pusa Reshmi, Pusa Chetki, and others. Pusa Himani, Pusa Mridula, Pusa Jamuni, and Pusa Gulabi [ICAR-Indian Agricultural Research Institute (IARI), New Delhi]; Hisar Sel-1 [Hisar Agricultural Research Institute]. Hisar University (HAU)]; Kalyanpur-1 [Chandra Shekhar Azad University of Technology] Kanpur’s College of Science, Agriculture, and Technology (CSAUAT)]; Chaudhary, Palam Hriday Sarwan Kumar Himachal Pradesh Krishi Vishvavidyalaya, Palampur]; Punjab Safed and Punjab Pasand [Punjab Agricultural University (PAU)]; as well as Arka Nishant [ICAR-Indian Institute of Horticulture Research] (IIHR), Bengaluru].

2.9 Mutation breeding

Mutation breeding refers to the technique of developing and using genetic variation via induced mutagenesis. It is an effective method for improving complex traits, especially in crops with limited genetic bases, vegetative reproduction, and self-pollination. More than 3000 mutant cultivars of various crops have been published in more than 60 countries; of these, 776 mutants have been generated for various nutritional quality traits, including minerals [110]. When compared with wild-type radish, mutants displayed a 30% higher net photosynthetic rate and a 36% higher total chlorophyll content.

2.10 Breeding for biotic and abiotic stresses

Heat, rain, Alternaria blight, Fusarium wilt, white rust, aphids, and beetles are all major abiotic and biotic elements affecting radish cultivation. As a consequence, improving radish stress tolerance is an important breeding target. Radish takes a lot of breeding during the off-season. In India, breeding lines, cultivars, and hybrids resistant to heat stress (38–43°C), such as cvs, have been developed. Despite the North Indian plains’ subtropical environment which has three seasons—summer, rainy, and winter—Pusa Chetki, Kashi Mooli-40, VRRAD-200, Chetki group, VRRADH-41, and VRRADH-42; and tolerance to high humidity make it feasible to produce radish commercially practically year-round. There are several sources of resistance to Fusarium wilt, according to Ashizawa [111], Peterson and Pound [112], and Soh [113]. Furthermore, after screening 260 accessions from 9 Asian and European countries, Jeon [114] discovered 54 radish accessions that were resistant to Fusarium wilt. Ghimire [115] also tested radish for Alternaria leaf spot.

2.11 Molecular markers to QTL breeding

Radish has been demonstrated to have a variety of economically important characteristics, such as yield, insect resistance, and disease resistance [2]. Yield is a complex trait governed by polygenic characteristics; it is difficult to discover these traits through standard breeding since they depend on phenotypic expression and interact with the environment and genotype. These challenges are addressed by the novel molecular breeding approach, which uses DNA markers for quantitative trait identification and linkage mapping [116]. Several DNA markers are utilized in breeding programs, including restriction fragment length polymorphism (RFLPs), random amplified polymorphism DNA (RAPD), simple sequence repeats (SSRs), and single-nucleotide polymorphism (SNPs) [117]. Raphanus hortensis var. sativus and var. niger were demonstrated to have distinct origins and to have descended from distinct progenitors owing to the application of molecular markers like as RAPD [118]. Several Asian varieties feature darker skin and flesh as well as variations in root size, length, and weight. It is therefore hardly unexpected that var. hortensis has genetic heterogeneity. Furthermore, Lee [119] performed phenotypic studies after genome-wide association analysis (GWAS) using genotyping-by-sequencing (GBS) to find FW resistance loci [119]. The GWAS study revealed 20 possible candidate genes and 44 single nucleotide polymorphisms (SNPs) that were significantly associated with FW resistance. Four QTLs were discovered in an F₂ population derived from an FW resistant line and a susceptible line, one of which was co-located with the SNPs on chromosome 7. These markers are newly accessible tools for molecular breeding efforts and marker-assisted selection to generate resistant R. sativus cultivars Lee [119]. Furthermore, Yu [120] produced a genetic linkage map on the F₂ population to detect the disease Fusarium wilt, and they identified a total of 8 loci conferring FW resistance that were spread across 4LGs, namely 2, 3, 6, and 7 of the Raphanus genome. Synteny analysis using the linked markers QTL found similarities to A. thaliana chromosome 3, which contains clusters of disease-resistance genes, showing that resistance genes are conserved between both. The sites of important QTLs discovered in the radish are Crr3, Crs1, and Crs2 [121]. Researchers uncovered a novel QTL named qRCD9 that modulates root CD by using markers SRAP, RAPD, SSR, ISSR, RAMP, and RGA to generate a genetic map of an F₂ population [116]. Resistance to cyst nematode (Heterodera schachtii) was discovered using RAPD, dpRAPD, AFLP, and SSR markers [122]. They identified 8 and 10 quantitative characteristics in radish for morphological aspects such as ovule number per silique, seed number per silique, plant shape, pubescence, whole plant weight (g), upper part weight (g), whole root weight (g), and main root weight using recombinant inbred lines (g) [53]. In the locations where QTLs were discovered, nine SNP markers were recently developed. The expression and nucleotide sequences of these genes suggested a possible function in the production of 4MTB-GSL in radish roots [57]. Fan [123] discovered that the R2R3-MYB transcription factor, which is responsible for creating the anthocyanin pigment 2, is located on chromosome 2 (PAP2). The RsPAP2 gene, which encodes the amino acid sequence that gives radish its red skin color, was readily differentiated from previously identified RsMYB genes.

Biochemical- and DNA-based markers enable the identification and description of cultivars and parental lines of hybrids, assessing the genetic purity of seed, diversity in agricultural cultivars and their wild variants, phylogenetic analysis, and pinpointing the origin of the germplasm. The identification of isoenzymes was the first tool for genetic analysis [124]. This method was used by Tai-Young [125] to identify cultivars and validate the purity of radish seeds. DNA is now frequently examined by directly using PCR-based molecular approaches such as amplified fragment-length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and inter-simple sequence repeats (ISSR). Wang [126] classified 65 cultivated radish accessions from 21 European, Asian, and North African countries into four groups based on their origin (Europe, Middle East, South Asia, and East Asia) in a neighbor-joining tree. Along with RAPD and ISSR marker information, reliable descriptors based on isoenzymes were established for the different stages of radish development [127]. Furthermore, Nakatsuji [128] generated 417 radish SSR markers utilizing cDNA data and SSR-enriched genomic libraries, which may be used for genetic research in radish and related species. The genetic diversity of the collection was studied using 144 radish cultivars. An SSR-enriched collection was used to generate genomic SSR markers [129]. Furthermore, the genetic diversity of 126 radish F1 cultivars was assessed using 60 SSRs and 29 agronomic parameters [130].

The expression of the orf138 gene causes Ogura CMS in Brassicaceae [131, 132]. The orf138 gene contains at least nine known nucleotide sequence variants, including one by Yamagishi and Terachi [133] with a 39-nucleotide deletion (Kosena type). Additionally, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ area of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2-kbp fragment that was unique to the NWB CMS type of male sterility and absent from other CMS kinds of radish. Through de novo transcriptome analysis, Nie [134] found crucial genes involved for bolting and blooming in radish.

2.12 Haploid breeding

In traditional breeding, the inbreds that are selfed and chosen during 6–8 generations of selfing constitute a major component of the commercial seed production of F₁ hybrids, which is detrimental to inbred vigor and inbreeding-depressive effects in Brassica crops, including radish. The microspore-produced doubled haploid (DH), which creates inbred lines with 100% homozygosity in only one generation, is an attractive tool because using DHs as parental lines may speed up breeding operations, create novel hybrids and varieties, conduct fundamental genetic research, and save time [135, 136]. Male gametophytic cells are cultured in vitro to form haploid plants, which are then treated with chromosome doubling procedures to produce haploid cells at the microspore or immature pollen developing stage. Since the initial report of effective isolation and culture of microspores in Brassica napus, microspore culture technology is being used in Brassica breeding [137]. Chun [138], Sugimoto [139], Takahata [62], and Tuncer [140] all successfully used microspore cultivation in radish.

2.13 Genomics and genomics tools

Today, genomics drive crop production, and the radish crop has been employed to investigate the underlying genotypic differences. The rapid rise of genomic data has spurred research into the genetic basis of plant characteristics such as better production, flowering, and disease resistance [141]. Several comprehensive studies on radish genome structure and chromosomal rearrangement during polyploidy events have been conducted [142], from which several genomic sequences have been generated [61]. Another research demonstrated that by combining the 454, Illumina, and PacBio sequencing technologies with bacterial artificial chromosome clones created by end sequencing, the whole genome of the Asian radish cultivar WK10039 was sequenced [143]. Numerous genetic investigations on the cultivated radish have been undertaken during the last 10 years [144]. A chromosome-scale genome assembly (rs1.0) of the Asian radish cultivar WK10039 was also generated, and the findings were compared to prior assemblies [145]. It provided more information than previously known due to increased genome coverage, contigs, and chromosomal anchoring [146]. However, radish mitochondrial genome sequences are now available in Radish Base, a genomic and genetic resource [147]. This resource now includes the mitochondrial genomes of two newly sequenced radish species, one from the normal cytoplasm and the other from the male-sterile cytoplasm of Ogura [148]. A recent study’s bioinformatics analysis of the radish genome discovered 54,357 coding genes and 20 COL transcription factors [149]. Each COL gene in the “Aok I daikon” cultivar had a match with its corresponding COL gene in the “kaz sa” cultivar. Furthermore, the cultivar “WK10039” was screened for a total of 20 radish COL genes [149]. Furthermore, BLASTP analysis of the radish genome revealed 35 different RsOFPs and five RsOFP-like genes (with no/partial OVATE domains), with the majority of genes being intron-less and containing the bulk of the genome’s coding sequences [149]. The HiSeq2000 technology was also used to generate whole-genome shotgun sequences on the R. sativus inbred line XYB36–2, a 119.75 GB dataset [150]. Based on 17-mer analysis, the estimated genome size was 530 MB. A 387.73 MB was assembled into 44,820 high-quality scaffolds using SOAP denovo and SSPACE [151, 152]. This study’s assembly produced outstanding results using fosmid clones (98.86% coverage). The assembly was much greater in quality than the previously released entire genome of R. raphanistrum (254 Mb contigs) and two assemblies of R. sativus ‘Aok I (116.0 and 179.8 Mb). The “Okute-Sakurajima” genome was reconstructed from scratch, yielding an estimated haploid genome size of 498.5 MB. The de novo assembly showed a largely heterozygous genome [153]. Further, long-read sequencing produced 36.0 GB of data from 2.3 million reads with an N50 length of 29.1 kB (60.7 coverage of the predicted genome size). The long-read assembly of 504.5 MB primary contigs, including 1437 sequences with an N50 length of 1.2 MB, and 263.5 Mb alternative contigs, including 2373 sequences with an N50 length of 154.6 kB, contains the other haplotypes with different alleles, also known as haploid sequences, after two rounds of data polishing [153]. Following polyploidy, research on the radish genome has revealed vital insights about the radish genome’s origin and evolution, providing deep knowledge on radish genetics and breeding [66]. The detailed information and genomic approaches obtained as a result of these studies help to a better understand the radish triplicated genome structure. Furthermore, these strategies improve radish breeding by increasing the use of marker-assisted collection, comparative genomic study, and the distribution of knowledge from reference data to new radish accessions [154]. As a consequence, a gateway with a large volume of genomic data and many linkages to specific genome analysis methodologies is very useful for radish research and breeding.

2.14 Genetic engineering

Genetic engineering is significant in agriculture since it improves agricultural characteristics and meets the needs of undernourished nations. The improvement of metabolic engineering methods and gene technology has sped up the development of usable germplasms [155]. Plant approaches advance by enhancing attributes; scientists have successfully developed transgenic radishes with a variety of agronomic qualities [156]. According to Tzfira and Citovsky [157] and Lacroix and Citovsky [158], some radish types contain beneficial features, such as better yield, that are passed on to the host plant. Gene transfer is carried out with the assistance of the pathogen Agrobacterium, which is widely employed as a strategy for plant hairy root lines, which seem to develop better than other types of root systems [159]. Herbaceous hairy roots are sought after because of their robustness, quick development, and ability to promote root-up growth in plants [160]. Agrobacterium, which grows in nutrient solution and has unique capabilities such as biochemically and biotransforming different metabolites, produces hairy roots. Agrobacterium is the greatest choice for producing secondary metabolites since it aids growth regulators [161]. New sources of natural chemicals may be identified by focusing on the hairy roots [162]. In a cultivated situation, chromosome disruption or amplification may potentially affect a plant’s fertility. Herbicides, antibiotics, metabolic mimics, and non-toxic substances all aid in the survival of changed cells. Radish regeneration is inhibited by kanamycin and hygromycin B. Floral dipping is a process that might be used to genetically edit radish, according to current plant biotechnology breakthroughs. In this strategy, the photoperiodic gene GIGANTEA in radish is co-suppressed, which helps the plant postpone bolting and flowering. It might be used to increase the medicinal potential of a crop [163]. It is addressed how to improve transformation efficiency and choose new characteristics to produce late-flowering radish [118]. Transgenic radish (R. sativus L. longipinnatus Bailey) was created in 2001 using plants that had been dipped into an Agrobacterium solution containing both the beta-glucuronidase (GUSA) gene and the herbicide resistance gene (bar) between the flanking T-DNA border sequences [156]. Finally, Southern blotting data demonstrated that the GUSA and bar genes had been incorporated into altered plant genomes and were segregating as dominant Mendelian features [156]. The radish RHA2b gene encodes a transcription factor implicated in the abscisic acid (ABA) signal transduction process as well as preharvest sprouting and seed dormancy, according to one research [164]. The RsRHA2b gene was cloned and introduced into Zhengmai 9023 by Agrobacterium-mediated stem apex transformation, according to the researchers [164]. Agrobacterium-mediated transformation was found to be a superior method for genetic modification [165]. Transgenic radish (Raphanus sativa L., cv. Jin Ju Dae Pyong) grown on Murashige and Skoog medium was used to study the use of adventitious shoot development on hypocotyl explants for Agrobacterium-mediated radish genetic transformation [64]. Furthermore, Northern blot findings revealed that the GUS gene transcript was found in a few regenerated plants, indicating genetic alteration [64]. In his study, Curtis also investigates strategies for delivering therapeutic proteins into radish for on-site administration of consumable proteins [163]. Concerns have been raised about pollen-mediated gene transfer after the introduction of transgenic radish into the wild. Potential risks and the field planting of transgenic radish are sometimes raised in talks concerning transgenic crops [156, 166, 167]. Plant regeneration from hypocotyl explants and somatic embryogenesis from hypocotyls was used to produce branches in radish. By adding aminoethoxyvinylglycine (AVG), an inhibitor of ethylene synthesis, and AgNO₃, an inhibitor of ethylene action, to the regeneration medium, cultured radish hypocotyl explants were able to regenerate shoots at a rate of 40% [168].

3. Conclusions and future perspectives

A few of the crucial radish breeding features are higher yield, early maturity, late bolting, pungency, cold hardiness, drought resistance, heat tolerance, and soil adaptation. Self-incompatibility alleles found in the radish genome make it possible to produce F₁ hybrids without the time-consuming and labor-intensive manual emasculation necessary for radish. To prevent hand emasculation, it is essential to know the S haplotypes of the parental lines when creating F₁ combinations. Inter- and intra-specific hybridizations are essential for the effective generation of radish yield because they allow the introduction of favorable agronomic features into the population. It is crucial to get comprehensive genetic data on chromosomes as well as the knowledge of inheritance. Researchers must comprehend the regulatory variables that synchronize at different developmental stages for each of the above-mentioned features in order to better understand and predict resistance, yield characteristics, and fruit quality. It is still essential to create a reliable and long-lasting plan for plant disease resistance, which is now being thought about. This is due to the ability of diseases to produce new bacterial strains, which may evade resistance. Scientists will now have accurate knowledge on disease resistance genes for a range of diseases as well as genes encoding essential biochemical features of the plant thanks to the completion of large-scale sequencing of the radish genomes earlier this year. One such method is speed breeding; as the cost of genome sequencing decreases, RAD-sequencing and DNA microarrays will be used more often, allowing for quicker genome mapping and tagging of novel quantitative trait loci. In order to enhance the number of resistant radish genotypes, these quantitative trait loci (QTLs) may introduce resistance into high-yielding radish genotypes and combine them with important resistance genes. Additionally, to increase radish crop output and quality, GWAS (genome-wide association studies) may map traits to particular candidate genes on a genome-wide scale. Using trait-specific genetic resources, heterotic potential, hundreds of molecular markers, highly saturated genetic maps, and effective contemporary technologies will all contribute to the development of prospective radish varieties and hybrids with improved quality and stress tolerance. Significant genetic and metabolic variety has been found, opening the door to breeding for genetic improvement and controlled harvest variability in agriculture. Finally, the use of trait-specific genetic resources, as well as the availability of thousands of molecular markers, highly saturated genetic maps, and efficient modern tools, will undoubtedly aid in the development of potential radish varieties and hybrids with improved quality and stress tolerance. Future radish breeding strategies that are crucial for boosting output and productivity as well as the effective use of input resources include targeted breeding strategies to create model crop ideotypes, improve nutritional quality, increase sustainability of production, increase adaptability to various climatic conditions, and increase tolerance to insects and diseases and efforts to pyramid two- or multi-tiered breeding approaches to widen the genetic base.