**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 selfpollen, 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

#### *An Update on Radish Breeding Strategies: An Overview DOI: http://dx.doi.org/10.5772/intechopen.108725*

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, 81–83]. 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 selfincompatible, 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 nuclearmitochondrial 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.
