**7. Genomics**

Genomics is now at the core of crop improvement, and the radish crop has been exploited to study the underlying differences in genotypes. The rapid development of genomic data boosted the discoveries regarding the genetic basis of plant traits, such as increased yield, flowering or disease resistance [74]. Various studies of the radish have investigated their genomes' arrangement and the reorganisation of chromosomes during polyploidy events [75], of which draft genomic sequences have been assembled. Another study reported an Asian radish cultivar, WK10039 which was sequenced entirely by combining 454, Illumina and PacBio sequencing systems and bacterial artificial chromosome clones obtained through end sequencing was fully sequenced by using the end sequencing method and sequencing equipment from the ABI firm [76]; over the last decade, a variety of genomic studies on the cultivated radish have been performed [77]. Moreover, a chromosome-scale genome assembly (rs1.0) of WK10039, an Asian radish cultivar, was constructed compared with assemblies documented previously [78]. It revealed more details than those previously recorded (having greater coverage of the genome, a greater number of contigs and chromosome anchoring) [79]. However, Radish Base is a genomic and genetic database containing radish mitochondrial genome sequences [80]. This database presently 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 [81]. The previous study published the bioinformatics analysis in radish and identified 20 *COL* transcription factors in the radish genome among 54,357 coding genes [82]. Every *COL* gene in the 'Aokubi daikon' cultivar matched the *COL* gene in the 'kazusa' cultivar. A total of 20 radish *COL* genes were also searched in the cultivar 'WK10039' [82]. Besides, in the radish genome, 35 unique RsOFPs and five RsOFP-likes (with no/partial OVATE domain) were identified by BLASTP, and analysis of exon-intron organisation revealed that most genes were intron-less containing maximum coding sequences in the genome [82].

Based on 17-mer analysis, the estimated size of the genome came out to be 530 Mb. A 387.73 Mb was assembled into 44,820 high-quality scaffolds using SOAP denovo [83, 84] and SSPACE [85]. The assembly in this study showed excellent results with fosmid clones (98.86% covered). The assembly showed a much higher quality than the draft genome of *Raphanus raphanistrum* (254 Mb contigs) [86] and two assemblies (116.0 and 179.8 Mb) of *R. sativus* 'Aokubi' [87] which was released previously. After de novo assembly of the 'Okute-Sakurajima' genome, an estimated haploid genome size of 498.5 Mb was found. The de novo assembly showed a substantially heterozygous genome [88]. Subsequent long-read sequencing produced 36.0 Gb data (60.7 coverage of the estimated genome size) in 2.3 million reads with an N50 length of 29.1 kb. After two rounds of data polishing, the long-read assembly consisting of 504.5 Mb primary contigs (including 1437 sequences with an N50 length of 1.2 Mb) and 263.5 Mb alternative contigs consists of the other haplotypes with different alleles, also known as haploid sequences (including 2373 sequences with an N50 length of 154.6 kb) [88].

A study performed on the radish genome after polyploidy has shown fundamental information about the radish genome production and evolution, which provides valuable insights into radish genetics and breeding. The detailed data and genomic methods obtained through these investigations support a greater understanding of the radish triplicated genome composition. Additionally, these methods help radish breeding by promoting marker-assisted collection, comparative genomic studies and the transmission of knowledge from the reference data to other radish accessions [89]. Consequently, a portal that is home to considerable quantities of genomic information and various links to specific genome analysis methods is precious in radish research and breeding.

### **8. Genetic engineering**

Genetic engineering has pivotal role in agriculture by improving the characteristics in the crops and satisfying the need of poor nourished countries. The developments in gene technology and metabolic engineering systems accelerated the production of valuable germplasms [90]. Progress is being achieved in plant methods by improving the traits; researchers have successfully produced transgenic radishes with various agronomic characteristics [91–93]. Gene transmission is done with the help of pathogen, known as agrobacterium, which is extensively used for plant hairy root lines, which appear to yield better than other forms of root systems [94]. Herbaceous hairy roots have advantageous due to their longevity, pace of growth

and capacity to assist plants in growing from the root up [95]. The hairy roots are produced in nutrient solution with the help of increasing agrobacterium that contains unusual properties, including biochemically and bio-transforming different metabolites. It is best to use Agrobacterium to produce secondary metabolites since they help to enhance growth regulators [96]. Working on the hairy roots, new sources of natural compounds [97]. In addition, chromosomal disruption or amplification may affect the fertility of cultivated plants. Antibiotics, herbicides, metabolic analogues and non-toxic agents all facilitate transformed cells for survival. Kanamycin and hygromycin B hamper radish regeneration [98].

Recent advancements in plant biotechnology indicate that radish could be genetically modified via a process called 'floral-dipping'. This technique involves co-suppression of the photoperiodic gene GIGANTEA in radish and contributes to the plant's ability to delay bolting and blooming. It can be used to boost a crop's medicinal value [98]. The prospects for improving transformation efficiency and selecting new traits for generating late-flowering radish are published [68]. In 2001, it was demonstrated that plants derived from plants dipped into an Agrobacterium suspension containing both the beta-glucuronidase (gusA) gene and the herbicide resistance gene (bar) between the flanking T-DNA border sequences could be used to generate transgenic radish (*R. sativus* L. longipinnatus Bailey) [91]. In the end, Southern blotting results revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits [91]. A study revealed that The *RHA2b* gene from radish encodes a transcription factor involved in abscisic acid (ABA) signal transduction and is responsible for seed dormancy and pre-harvest sprouting [99]. The study performed the experimentation in which The *RsRHA2b* gene was cloned and transferred into Zhengmai 9023 via Agrobacterium-mediated stem apex transformation [99]. The agrobacterium-mediated transformation became a more appropriate method for genetic transformation [100]. Using adventitious shoot growth on hypocotyl explants for Agrobacterium-mediated radish genetic transformation was investigated using transgenic radish (*Raphanus sativa* L., cv. Jin Ju Dae Pyong) grown on Murashige and Skoog medium [101]. Besides, northern blot results revealed that the GUS gene transcript was detected in a few regenerated plants, confirming genetic transformation. In addition, the techniques available for introducing pharmaceutical proteins into radish for on-site delivery of edible proteins into it are discussed by Curtis in his study [98]. The concerns of releasing transgenic radish to the field in pollen-mediated gene transfer have also been explored. Risks that might exist and the introduction of transgenic radish to the field are sometimes brought up in discussions about transgenic crops [91, 102, 103].

### **9. Conclusion and future directions**

For successful production of radish yield, inter- and intra-specific hybridizations are vital to genetic research and crop improvement because they enable the introduction of desirable agronomic traits into the population. The production of yields, early maturity and late bolting, pungency, cold-hardiness, drought resistance, heat tolerance and soil adaptability are just a few of the essential radish breeding traits. The radish genome contains self-incompatibility alleles, allowing for the generation of F1 hybrids without the labour-intensive and hand emasculation required in radish. When generating F1 combinations, it is critical to determine the S haplotypes of the parental lines to avoid hand emasculation. Collecting complete genetic data on

chromosomes and information on inheritance is critical. To better understand and forecast resistance, yield characteristics and fruit quality, researchers must understand the regulatory factors synchronising at various developmental stages for each attribute discussed. It remains necessary to develop a robust and long-lasting strategy for plant disease resistance, which is currently under consideration. This is because diseases are capable of evading resistance by generating novel bacterial strains.

Speed breeding is one such strategy; as genome sequencing costs continue to decline, RAD-sequencing and DNA microarrays will become more common, enabling faster genome mapping and tagging of new quantitative trait loci. These quantitative trait loci (QTLs) may incorporate resistance into high-yielding radish genotypes and combine them with significant resistance genes to increase the number of resistant radish genotypes. Additionally, GWAS (genome-wide association studies) can map characteristics to specific candidate genes on a genome-wide scale to improve crop production and quality in radish. The discovery of significant genetic and metabolic diversity paves the way to develop controlled harvest variations in agriculture and genetic enhancement via breeding.
