**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 *Raphanu*s 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 intersimple 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 *RsOFP*s 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 cosuppressed, 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 Agrobacteriummediated 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].
