Keywords

1 Introduction

Plant secondary metabolites (PSMs) are one of the most diverse group of compounds as they have tremendous diversity in their structure and functionality. Plants produce a plethora of low molecular weight organic compounds, being classified as primary and secondary on the basis of their direct involvement in growth and development of plants. Earlier PSMs are considered less important for the growth and development, being termed as “secondary.” Progressive research in this area revealed their role in interaction with the environment and in defense mechanism, so the term “specialized” will be more appropriate instead of “secondary” (Qari and Tarbiyyah 2021). Overall there is no boundary to distinguish the plant metabolite as primary or secondary because current understanding of plant metabolic engineering extends their area of functionalization (Sangwan et al. 2018; Erb and Kliebenstein 2020). These metabolites are of economically as well as ecological importance, therefore, special attention on their production is needed (Sangwan and Sangwan 2014; Sangwan et al. 2010). Moreover, there is no straight pathway to define their synthesis and degradation and it is often observed that these metabolites are restricted in their distribution and it may be limited up to plant family, genera, or species level and even at the tissue specificity (Fang et al. 2012; Jadaun et al. 2020; Kushwaha et al. 2013a). In some plants, they accumulate in special structures like trichome, vacuole, or specialized glands (Yadav et al. 2014; Yu et al. 2018; Maurya et al. 2019). These metabolites are not only crucial for plant life but also play significant role for humans. Pharmacological activities of these compounds made them attractive for industrial applications. Various biotechnological approaches are followed to enhance their production (Fierascu et al. 2020; Srivastava and Sangwan 2020; Narnoliya et al. 2021).

Secondary metabolites from plants are governed by several factors such as physiological, metabolic, environmental, and genomic (Sangwan et al. 2001a, 2017; Farooqi et al. 2000; Sangwan and Sangwan 2000; Singh et al. 2015; Srivastava et al. 2015; Shukla et al. 2003; Yadav et al. 2015). Conclusively, metabolism of SMs is the result of interaction between gene (G) and the environment (E) (Padilla-González et al. 2019). Generally, these metabolites are produced by plants in substantial amounts but variation in quantity and quality is noticed under fluctuation of biotic and abiotic factors (Sangwan et al. 2001a; Akula and Ravishankar 2011; Mishra et al. 2020). Another major factor responsible for accumulation pattern of SMs is the genotypic constitution (Maurya and Sangwan 2020). Polyploidy has gained the interest of numerous researchers due to various reasons: induction of polyploidy can be utilized to create genetic diversity in targeted crops, thus it has the potential to create new species. The proliferation of new species and genomes and the possible benefits of compatibility may have an impact on species evolution.

Genetic diversity of plants plays critical role in regulation of their metabolism either its primary or secondary. Quantitative trait locus (QTL) analysis is useful statistical method to employ the relationship between genotype and phenotypes (Miles, C. & Wayne). Metabolic quantitative trait loci (mQTLs) plays an important role to decode the involvement of gene in the production of a metabolite (Alseekh et al. 2015). There are different kinds of molecular markers which are available to record the genetic diversity in medicinal plants such as Simple Sequence Repeats (SSR), DNA polymorphism like Random Amplified Polymorphic DNA, and more specified ones such as Restriction Fragment Length Polymorphism, Amplified Fragment Length Polymorphism, and Single-Nucleotide Polymorphism (Sarwat et al. 2012) (Table 12.1). Now days Expressed Sequence Tag (EST) database is utilized to design the markers (Narnoliya et al. 2017).

Table 12.1 List of selected plants showing the assessment of genetic diversity by using molecular markers
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In this chapter, we will discuss various factors contributing towards the accumulation of secondary metabolites in plants and also the influence of the genetic variability with respect to their association with the secondary metabolite production.

2 Influence of Genetics on Secondary Metabolites

Secondary metabolites are classified into three categories: alkaloids, terpenoids, and phenyl propanoids. We will discuss these compounds in detail with special reference to the effect of genetics on the secondary metabolites using examples of leading and important medicinal and aromatic plants (Table 12.2).

Table 12.2 Effect of polyploidy on secondary metabolites production
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2.1 Withania Species (Solanaceae)

The genus Withania belongs to Solanaceae family and it is used in traditional medicines since ancient times. It has immense importance as an ingredient in different medicinal systems like Ayurveda, Unani, and Siddha medicinal practices. It shows health beneficial effects by controlling the aging process through its rejuvenating properties. Genus Withania comprises approximately 23 species, but only few species get attention for their usage and popularity. W. somnifera and W. coagulans are two of most studied species (Kushwaha et al. 2013b; Sangwan et al. 2014). Research is carried out significantly on W. somnifera followed by Withania coagulans (Mishra et al. 2013; Mishra et al. 2016; Jadaun et al. 2017a, 2017b, 2020). Although both species are very well studied for their ethnopharmacology and a number of phytomolecules have been isolated and identified from the species. Most of the pharmacologically active compounds belong to the terpenoid category of secondary metabolites and collectively this group of compound is termed withanolides.

Withania somnifera, Ashwagandha, the popular Indian ginseng or gooseberry, is a perennial, evergreen small shrub that is distributed globally in wide geographical regions. Although the whole plant possesses medicinal properties, the roots are preferably used for extraction of “Rasayana” (Sangwan et al. 2007). Many pharmacological activities have been attributed due to this Rasayana like adaptogenic, anti-sedative, anticonvulsion, anti-inflammatory, immunomodulatory activities (Mukherjee et al. 2021). Interestingly, it is effective against several brain and nervous system-related disorders like Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease (Dar and Ahmad 2020). The neuroprotective activity of W. somnifera is mediated through anti- oxidative stress mechanism. Withanolide A, withanolide D, β-sitosterol and stigmasterol are the major metabolites against Alzheimer’s diseases (Hannan et al. 2020). There is transcriptomic data of W. somnifera available, which also reflects the tissue-specific accumulation of different withanolides (Gupta et al. 2013; Sangwan et al. 2014). There is a repository of transcription factors which are involved in regulation of secondary metabolites metabolism (Tripathi et al. 2017). There is variation in ploidy level of W. somnifera. Plants may have diploid (2n = 24), tetraploid (2n = 48), and hexaploid (2n = 72) levels, although the predominating ploidy is diploid (2n = 24). W. somnifera shows significant variation in morphogenetic characters. By using the EST-SSR markers, genetic diversity in 36 genotypes of W. somnifera is accessed (Parita et al. 2018). Application of ISSR marker showed genetic diversity in W. somnifera species collected from diverse geographical locations (Hiremath et al. 2021). Sangwan and his coworkers have carried out several experiments to explore more and more about the genetic and chemotypic diversity of W. somnifera plants collected from different locations in India (Sangwan et al. 2004). Different types of chemotypes (NMITLI-101, 110, 133, 104, 128, 141, 108, 109, 118, 135, and 144) have been identified (Tuli and Sangwan, 2009). The phytochemical analysis includes polypeptide polymorphism, isoenzymes, along with withanolide profiles. RAPD analysis reveals that they cluster together on the basis of genetic similarity. In another study, thirty cultivars are selected from eight different states and cultivated for approaching their stability and adaptability. Out of thirty, cultivars W 20, W 1 (cv. Pratap), W 2, W 3 (cv. Chetak), W 4 and W 6 were found to be highly stable for the root yield (Lal 2015). Five different varieties of W. somnifera (Jawahar, Nimithli, Chetak, Pratap, Poshita and) were examined for the withanolide content. There is a significant variation in all the varieties, with the highest content of alkaloids (withaferin A and withanolide A) found in Poshita, followed by Jawahar-20 (Singh et al. 2018; Mishra et al. 2020). In another study performed in Sri Lanka, withaferin A content was estimated in two varieties of W. somnifera, LC1 and FR1, these varieties were grown under the same climatic conditions. Different tissues (leaf, bark stem and root) of both varieties are screened for alkaloid content and results showed highest content of withaferin A in leaves of LC1, with minimum content found in the root of FR1 (Siriwardane et al. 2013). Srivastava et al. (2018) reported genetic variability in 53 genotypes of W. somnifera collected from different geographical locations. According to pathway coefficient analysis, there is positive correlation in fresh root weight, total alkaloid (%) in leaf and deoxywithastramonolide in the root (%). Effect of genotype-environment interaction on withanolide content was studied on 16 genotypes of W. somnifera. A total of 12 characters were considered along with alkaloid content and a variation was observed in characters of all the genotypes grown under different locations (Kumar et al. 2020). Mirjalili et al. (2009) reported the genetic (by using the RAPD anlysis) and withaferin A diversity in Iranian natural populations of W. somnifera and W. coagulans.

2.2 Artemisia (Asteraceae)

Artemisia is a rich genus of aromatic plants with around 500 species including both herbs and shrubs (Valles and Arthur 2001). Several Artemisia species are perennial. However, few Artemisia species are annual or biennial (Vallès et al. 2003) because of its greater population and morphological complexities, the taxonomy of genus Artemisia is not clear (Hayat et al. 2009). Based on recent molecular studies, the genus Artemisia has been divided into five deferent subgenera. These subgenera are Absinthium, Artemisia, Dracunculus, Tridentata, and Pacifca (Vallès et al. 2011). The diversity of Artemisia is mainly found in Asia, Europe, and North America. China (around 150 accession) and Europe (174 accession) are main regions of Artemisia diversity. Japan (50 species), Pakistan (38 species), and Iran (35 species) also add diversity to the genus (Hayat et al. 2009). Only few Artemisia species (Artemisia annua, Artemisia Absinthium, and Artemisia vulgaris) are commonly utilized as traditional medicine in Asia and Europe from ancient time.

Artemisia annua (sweet wormwood or qinghao in China) is the most popular among Artemisia species due to its antimalarial properties from ancient times (Hong et al. 2015). Artemisia annua is native to Asian, North American, and European countries. The antimalarial properties of Artemisia annua are due to an important compound, artemisinin. The acceptance of artemisinin as antimalarial drug was gained due to the emergence of chloroquine resistance in the 1950s (Ma et al. 2020). Artemisinin, the sesquiterpenoid-based therapy has so far proved efficient in the treatment of millions of people in developing countries (Ma et al. 2020). The Chinese scientist Youyou Tu was honored with the 2015 Nobel Prize in Physiology or Medicine for her discovery of artemisinin, which became a promising drug (Liu and Liu 2016). Recently, artemisinin-derived compounds such as artesunate and artemether are basically used in medicine preparation for malarial drugs (Tu et al. 1981; Wright 2002).

The main chemical constituents of Artemisia species are polyphenols and essential oils, but also coumarins, acetylenes, sesquiterpene-lactones, alcohols, flavonoids, monoterpenes, and sesquiterpene derivatives. The chemical diversity of constituents attributes Artemisia species with different pharmacological bioactivities including antibacterial, antimalarial, anticancer etc. (Padalia et al. 2016; Nigam et al. 2019; Sangwan et al. 2010). Vast research is ongoing with A. annua for the development of drugs to treat COVID-19 (Bisht et al. 2021).

Artemisinin, the endoperoxide sesquiterpene-lactone synthesized in the eight-celled trichome glands of A. annua, is an active ingredient in the artemisinin combination therapy-based treatment of malaria. The content of artemisinin varies among the varieties of A. annua from different origins. Even Indian population of A. annua plant exhibited very high level of genetic variation in RAPD analysis (Sangwan et al. 1999). Advances in genetics and metabolic engineering approaches widen the path to in planta artemisinin production. Microbial-based metabolic engineering of artemisinic acid (the precursor of artemisinin) had demonstrated some potential, but the further chemical synthesis of artemisinin are costly (Ro et al. 2006). Thus, the agricultural production of artemisinin is the only viable source for ACT. A. annua is an undeveloped crop and plant-based production of artemisinin is a challenging task. Development of improved varieties for artemisinin production purposes is the realistic target. Different approaches have been applied to improve in planta artemisinin yield.

Several plant breeding-based techniques are used to develop improved variety of A. annua in terms of higher artemisinin yield. Conventional breeding approach includes identification of superior parental lines with desired traits and crossing them to develop hybrid lines with improved artemisinin yield (Delabays et al. 2001; Cockram et al. 2012; Townsend et al. 2013). Artemisinin biosynthesis in A. annua is controlled by genetic factors (Ferreira et al. 1995b; Delabays et al. 2001, 2002). The vegetative phase of Artemisia plant can be maintained under long photoperiods, while flowering can be induced under short days. Flowering time of different varieties varies under field conditions, but can be induced in greenhouse. Delabays et al. (1992) had crossed artemisinin (1.1%, w/w) rich, late-flowering Chinese clone with European plants and developed new variety with 0.64% to 0.95% artemisinin content and dry leaf yields between 14 to 21 t/ha (Delabays et al. 1992). Banyai et al. (2010) have developed the tetraploid cultivar of A. annua by doubling the chromosomes number with higher level of artemisinin content. Graham et al. (2010) studied genomics of A. annua where all the genes of the pathway are shown in genetic linkage with marker traits. Artemis (1.4% artemisinin) is commercially important variety of A. annua developed by cross between two genetically different heterozygous parental genotypes C4 and C1 (Delabays et al. 2001). Graham et al. (2010) have developed genetic linkage and QTL maps for Artemis and validated positive QTL for artemisinin yield. 34,419 SNPs with mean SNP frequency of 1 in 104 base pairs were identified from the five EST databases of Artemis F1 hybrid material (Graham et al. 2010). Further, polymorphism confirmation with 19 AFLP primer combinations revealed 322 polymorphic markers. 49 SSR markers which are segregated in the Artemis F1 population were also identified through in silico approach (Graham et al. 2010). The mapping population of the Artemis F1 exhibited variation in plant phenotype, artemisinin content (0.93 to 20.65 mg/mg dry weight), glandular trichome density (4.89 to 19.11 mm2), leaf area (508.76 to 4696.08 mm2) and plant fresh weight (160 to 4440 g). Graham et al. (2010) found strong segregation distortion of the advantageous alleles QTL on C4LG1 in favor of artemisinin yield trait (a product of artemisinin concentration and plant fresh weight). Although, artemisinin is present in A. annua plant leaf and stem tissues, but artemisinin yield depends on plant genetic potential (Ferreira et al. 1995a). Heterozygous nature of A. annua is also a challenge for plant breeders because developed plant exhibit varying degrees of artemisinin content (Delabays et al. 2001; Graham et al. 2010; Larson et al. 2013).

Besides A. annua, several other Artemisia species such as A. absinthium, A. aff. Tangutica, A. apiacea, A. bushriences, A. campestris, A. cina, A. diffusa, A. dracunculus, A. dubia, A. indica, A. japonica, A. lancea, A. moorcroftiana, A. parviflora, A. roxburghiana, A. scoparia, A. sieberi, A. sieversiana, A. spicigeria, and A. vulgaris are reported to have artemisinin (0.05–0.034%) in their aerial parts (Mannan et al. 2010; Singh and Sarin 2010; Rashmi et al. 2014). Nganthoi and Sanatombi (2019) had studied artemisinin contents in four Artemisia species (viz., A. parviflora, A. vulgaris L., A. myriantha, and A. nilagarica) of Manipur region in India and also established the genetic relationship among the four Artemisia species by using RAPD (15), ISSR (11) as well as IRAP (3) markers. Out of total 267, 203 and 58 reproducible fragments, 240, 187 and 51 were polymorphic with high average polymorphism (89.88% for RAPD, 92.5% for ISSR and 87.93% for IRAP) (Nganthoi and Sanatombi 2019). The highest similarity index in RAPD (0.5), ISSR (0.5) as well as IRAP (0.79) markers was found between A. nilagarica and A. myriantha (both belongs to Sub-genus Artemisia) with similar artemisinin content per gram DW in leaf (0.031%–0.044%), young flower (0.042%–0.052%), mature flower (0.031%–0.047%) and stem (0.008%–0.01%) (Nganthoi and Sanatombi 2019). While A. parviflora (belongs to the Sub-genus Dracunculus) depicted least similarity index with rest three Artemisia species and high level of artemisinin content in leaf (0.078%–0.087%). A. nilagarica and A. myriantha had depicted low genetic variability and have a closer evolutionary relationship, while A. parviflora had depicted higher genetic variability.

2.3 Ocimum (Lamiaceae)

Ocimum genus belongs to the Lamiaceae family and holds immense importance in the plant word due to its medicinal and aromatic properties. The plant is well adapted to survive under tropical warm and temperate conditions and is an inhabitant of tropical Africa, tropical Asia, and tropical America (Paton et al. 2004). The genus contains approximately 150 species out of which 66 species have been reported online (Chowdhury et al. 2017; Gurav et al. 2021). Several Ocimum species are studied in great details for their pharmaceutical constituents being the major ones O. tenuiflorum, O. basilicum, O. Americanum, O. africanum, O. gratissimum, O. kilimandscharicum, and O. citriodorum (Gupta et al. 2018; Gurav et al. 2021; Maurya et al. 2019). In India, nine species, including three exotic species namely O. adscendens, O. basilicum, O. canum, O. gratissimum, O. kilimandscharicum, O. tenuiflorum, O. americanum L., O. minimum L., and O. africanum Lour have been reported (Chowdhury et al. 2017). Ocimum is rich in chemical diversity which makes it extremely useful in culinary, fragrance, therapeutic, and the cosmetic industry (Pandey et al. 2016). Ocimum is a repertoire of compounds like terpenoids, alkaloids, phenols, tannins, anthocyanins, lignins, quinones, saponins, flavonoids etc. Essential oils present in Ocimum contain linalool, chavicol, methyl chavicol, eugenol, camphor, 1,8-cineole, α-terpineol 6.5%, β-pinene, camphene, sabinene, cis-ocimene, trans-ocimene, ß-caryophyllene, geraniol, limonene, 10-Heptadecen-8-ynoic acid; Cyclopentane, thymol, etc. (Maurya et al. 2019; Enegide and Charles 2021). These compounds impart hepatoprotective, anti-diabetic, antioxidant, neuroprotective, anti-inflammatory, antiseptic, antidiarrheal, antispasmodic, and antimicrobial properties. They also play a role in the treatment of skin, respiratory tract infections, stomach ache, kidney malfunctions etc. (Prakash and Gupta 2005; Dhama et al. 2021; Enegide and Charles 2021). Basil seeds are a rich source of proteins (11.4–22.5 g/100 g), carbohydrates (40–63.8 g/100 g), dietry fiber (7.1–22.6 g/100 g) along with calcium, magnesium, and phenolic compounds (orientine, vicentine, and rosmarinic acid) (Calderón Bravo et al. 2021). Microencapsulation of essential oil from O. basilicum was conducted to develop a packaging material which could control the bacterial growth in packaged food and also increase the shelf life of food by inhibiting change in pH (Amor et al. 2021). Similarly, seed mucilage from O. basilicum was used with montmorillonite (MMT) as nanofiller to synthesize bionanocomposite films to be used as packaging material (Rohini et al. 2020). Its use as a seasoning, sauces, salads is well accounted for. These traits of Ocimum makes its essential oil very important in the national and international market and thereby an 8% compound annual growth rate is expected from 2019 to 2023 (Gurav et al. 2021).

A number of different approaches have been used to increase secondary metabolites in terms of concentration and components. With the aim of incorporating desired traits and increase the genetic stock; plant breeding has been practiced for many decades (Ahmar et al. 2020). In Ocimum basilicum var. glabratum Benth chemotypes which were morphologically similar but contained different chemical compounds, i.e., eugenol, methylchavicol, and camphor were established. It was assumed that the variation in accumulation of compounds with different origins (eugenol, methylchavicol are phenylpropanoids while camphor is terpenoid) was due to the presence of a multi-allelic gene which expresses differentially (Gupta et al. 2005). Similarly, a eugenol rich and methyl chavicol rich line had also been developed by breeding (Gang et al. 2001). In another study, a cross between basil varieties “Perrie” (rich in eugenol) and “Cardinal” (rich in methyl chavicol) on segregation generated eugenol chemotype (23%–25%), methyl chavicol chemotype (23%–25%, and an intermediate mixture of the two compounds (~50%). The segregation followed 1:2:1 pattern, thereby suggesting involvement of a single bi-allelic gene with incomplete dominance (Dudai et al. 2018). Other than these a large number of chemotypes have been developed by conventional breeding such as CIM ayu; eugenol rich variety of O. sanctum, CIM Angana; high yielding variety of O. sanctum (Shyam tulsi); CIM Saumya; methyl chavicol and linalool rich short duration variety of O. basilicum (Lai 2003; Lal et al. 2004, 2008). Higher ploidy level is associated with higher essential oil content (Lavania 2005). Attempts were made to induce autotetraploidy by colchicine treatment at different plant stages and at different concentrations. Polyploidy resulted in large stomata, pollen grains and increased chloroplast number (Omidbaigi et al. 2010).

2.4 Mentha Species

The Mentha species (Lamiaceae) plants evolved through natural hybridization and selection, consisting of about 25 to 30 species (Brickell and Zuk 1997). Mentha essential oil is important for pharmaceutical, cosmetics, food, confectionery, liquor, and pesticide industries (Sangwan et al. 2000). Mentha is a rich source of plant secondary metabolites such as terpenes (Sangwan et al. 2000). Phylogenetic relationships and genetic diversity among Mentha species have been widely studied mainly by using molecular markers such as RAPD and amplified fragment length polymorphism (AFLP) primers-based genetic analyses (Gobert et al. 2002; Shinwari et al. 2011; Rodrigues et al. 2013; Virginia et al. 2021). Rodrigues et al. (2013) had studied the morphological, phytochemical, and genetic variation in 12 populations of Mentha cervina L. (Portuguese endangered medicinal plant) to access the level of diversity through ISSR markers. One hundred and twenty-one individuals of ISSR amplification gave a total of 175 bands (average: 82.4 fragments per individual) with 97.7% (171 band) of them being polymorphic. Out of twelve, four populations clustered together in morphological parameters as well as molecular studies, but the secondary metabolites, i.e., essential oils basis created different clustering (Rodrigues et al. 2013). Rodrigues et al. (2013) interpreted the observation as essential oils evolve more rapidly than the morphological traits, so the morphological traits were found more correlated with the genetic variation. Shinwari et al. (2011) studied the RAPD profile based similarity and diversity of two mint species (Mentha royleana and Mentha spicata) including 15 accessions of each. The polymorphism percentage observed in all the 15 accessions of M. royleana was 27.44% while in M. spicata, it was 47.2%. A high level of genetic polymorphism was noticeable among population, thereby indicating genetic richness and heterozygosity (Shinwari et al. 2011). RAPD profiles of different mint species/accessions Mentha arvensis L., two of Mentha spicata L., one each of Mentha spicata L. cv. viridis, Mentha × piperita L., Mentha × piperita L. cv. citrata and Mentha × gracilis Sole cv. RAPD markers could clearly identify various Mentha genotypes, accessions as well as hybrids. Fenwick and Ward (2001) identified 17 accessions of Mentha (three species) belonging to different geographical origins from the USA by using RAPD-based profiling. Momeni et al. (2006) had studied the genetic variation and taxonomic relationship between 17 accessions out of four Mentha species viz., M. spicata, M. piperita, M. suaveolens, and M. longifolia by RAPD fingerprinting. In another study, Virginia et al. (2021) had established the genetic variations between ten varieties of Mentha (Mentha longifolia, Mentha aquatica, Mentha x piperita, Mentha arvensis subsp. arvensis and Mentha spicata var. crispa and five commercial varieties) by using eight arbitrary RAPD primer based profiling and recorded 100% polymorphism. Hundred percent polymorphism was interpreted due to out-breeding and the wide dispersal of seed and pollen grains (Virginia et al. 2021).

2.5 Geranium sp.

Geranium (family geraniaceae) is a very popular aromatic crop of commercial importance. Essential oil of geranium is used in a variety of formulations of commercial, pharmaceutical, and agricultural importance (Gallardo et al. 2012; Narnoliya et al. 2019). Transcriptome information of P. graveolens proposed the pathways involved in primary and secondary metabolite biosynthesis (Narnoliya et al. 2017, 2018). Geranium essential oil has several terpenes as constituents majorly geraniol, citronellal, linalool, geranyl acetate, limonene, pulegone, citronellyl format, citronellylacetate, menthone isomenthone, 10-epi-γ-eudesmol, etc. (Rana et al. 2002; Jadaun et al. 2017a, 2017b). A large number of species diversity (~ 200 speices) is reported in the Geranium genera in reference to morphological features and essential oil composition (Tyagi et al. 2003; Yi et al. 2018). Commercially, four species: graveolens: odoratissimum: radens, and P. capitatum were grown for harvesting the essential oil. On the basis of variation in oil yield and oil constituents, different chemotypes of Pelargonium capitatum are reported (Demarne et al. 1993). Production of somaclonal variations may be a good source of induction of the genetic variability in plants. Kulkarni et al. (1997) reported about the intra clonal variations in Pelargonium sp. as they differ significantly in essential oil composition (isomenthone is major constituent instead of geraniol and linalool). Somaclonal variations were studied in an Indian cultivar, “Bourbon” of rose-scented geranium (Pelargonium graveolens), they differ in the phenotype (height, leaf shape, leaf size, leaf dentation) and phytochemical composition (Ravindra et al. 2004). Glass house grown somaclones, highly dentated leaves (HDL) and less dentated leaves (LDL) show variability in leaf dentation pattern, herb yield as well as variability in the oil yield also (Saxena et al. 2008; Tyagi et al. 2003).

For identification of genetic diversity in Geranium species different types of DNA markers are also used. RAPD and ISSR analysis in cutting (diploid) and polyploidy seedlings revealed geraniol, geranyl formate, and linalool concentration was quite higher in polyploid plants (Yi et al. 2018). RAPD analysis was performed to trace the phylogenetic relationship among the following species of Geranium: G. dissectum L. (sec. Dissecta); G. persicum Schönb.-Tem., G. tuberosum L., G. kotschyi Boiss., G. platypetalum Fisch. & C. A. Mey., G. gracile Ledeb. Ex Nordm., G. ibericum Cav. (sec. Tuberosa subsect. Mediterranea R. Knuth). G. stepporum P.H.Davis (sec. Tuberosa subsect. Tuberosa (Boiss.) Yeo); G. columbinum L., G. rotundifolium L., G. sylvaticum L., G. collinum Stephan ex Willd, G. pratense (Yin et al. 2021).

2.5.1 Molecular Markers in Association to Secondary Metabolism

Molecular markers have been used extensively in characterization of accessions, germplasm as well as mutant lines. Along with the progress that has markedly taken place during past two to three decades in molecular techniques in developing markers such as RAPD, RFLP, ISSR, microsatellites etc., the markers have been associated with various traits of interest. The plants with secondary metabolites have also been studied, and the co-relation with various markers to assist the analysis related to association between secondary metabolites and markers. RAPD markers were quite popular owing to ease in establishment and also not requiring genomic information (Sangwan et al. 1999, Shukla et al. 2005). Medicinally important plant punarnava (Boerhavia diffusa) which is used in herbal and medicinal puoposes. The plant grows in wild and majorly two varieties, white flowered and red flowered are in the trade (Shukla et al. 2005). The attempts to develop better varieties through conventional breeding approaches and also mutant generation through mutagens of physical and chemical nature were attempted. The genetic differences were well differentiated by the RAPD marker as well as by isozymic pattern analysis. The performance of RAPD markers in associating the trait was much better as compared to isozymes. Also the mutant which had unique characters, including flower color, exhibited a unique RAPD pattern.

Similarly, in Cymbopogon species, the fragrant members of Poaceae family were examined with RAPD, isozyme and protein polymorphic markers basis. Cymbopogons are aromatic species and differ in their ploidy status as well. Cymbopogon martinii is one of the most prominent species preferred by the farmers for cultivation owing to its rose like aroma. The essential oil contains geraniol and geranyl acetate in elite varieties of sofia and motia. The genetic score, gene diversity, heterozygosity, etc., were computed. Such genetic marker analysis could distinguish elite genotypes of Cymbopogon species (Sangwan et al. 2001a, 2007; Sangwan Neelam et al. 2003). Furthermore, information related to the genetic basis of wild and collected germplasm as well as requirement for trait introgression can be envisaged from the diverse growing plant types.

Similarly in another study, important trade type of Cymbopogon species were studied using RAPD marker to distinguish diversity of trade type varieties and species differing substantially in their oil types (Sangwan et al. 2001a, 2003). The study correlated essential oil metabolic diversity with the genetic diversity parameters. Markers also clustered together Cymbopogon flexuosus, C. pendulus and C. khasianus in one cluster, owing to the similarity in their essential oil constituent profile. Diagnostic markers and stand-alone markers were identified and associated with constituents (Sangwan et al. 2001a, 2003). In another study, genetic variation was created through somaclonal variation in Cymbopogon hybrid Jamrosa. Large numbers of growth parameters were impacted including essential oil content (Nayak et al. 2003). Gross genetic variations in RAPD pattern of parent and somaclones of Jamrosa indicated the extent of variation in several physiological parameters as well secondary metabolites (Nayak et al. 2003). Superior clones with respect to higher terpinol geraniol content were evaluated in field trials for consistency in performance and characterization through RAPD molecular marker (Nayak et al. 2003).

Withania somnifera is one of the traditional resources of medicinal herb. Molecular markers including RAPDs, and SSRs have been used in studying the genetic diversity and characterizing the genotypes in metabolic core groups (Chaurasiya et al. 2009; Sangwan et al. 2017). Such markers provided clues in establishing genetic diversity of collection at intra- and interspecies levels. The levels of secondary metabolites such as wihanolidal diversity in terms of qualitative as well as quantitative variations were also elucidated. In vitro clonal plants were also established for their clonal stability and also evaluation for distinctness of secondary phytochemicals (Sabir et al. 2007). High throughput analytical techniques of metabolomics and a combination of markers together further substantiated large numbers of core collections for metabolites in an integrated manner (Dhar et al. 2006; Chaurasiya et al. 2009).

3 Status of Polyploidy and Its Impact on Secondary Metabolites

Polyploidy is a phenomenon of two processes, i.e., endomitosis and endoreduplication. Other processes like fusion of nuclei, ineffective mitosis, or the emergence of multinucleated cells are also involved in creating polyploid cells. Unlike normal mitosis, the endomitosis is a process in which the cell membrane is not destroyed, and mitosis takes place within the nucleus, chromosome numbers double and sister chromatids are likely to separate and return to the interphase mode, except those chromosomal spindles are not created and as a result sister chromatids are not stretched to the side of the cell and polyploidy cells are not created. The endometriosis process mostly occurs in animal cells and is rare in angiosperms (flowering plants). The endoreduplication nucleus may replicate DNA without going through the mitotic process, resulting in 4n, 8n, 16n, and so on (Ghasemi et al. 2021). Diploidy refers to the presence of two complete set of chromosomes in somatic cells, one from each parent, whereas polyploidy refers to the presence of three or more complete sets of chromosomes in somatic cells (Park et al. 2021). Each organism has a specific number of chromosomes that are divided into different groups. Ploidy refers to the number of chromosomal groupings. In this case, the number of chromosomes may alter. Euploidy and aneuploidy are the two types of alterations that occur. Polyploidy is formed by two mechanisms: somatic doubling and the creation of unreduced gametes (2n).

According to their origin, polyploidies can be classified as auto-polyploidies and allopolyploids (Ghasemi et al. 2021). Polyploidy can arise as a result of autopolyploidy, which describes the occurrence of polyploidy inside a species, or allopolyploidy, which is described as the development of polyploidy as a result of hybridization between two different species (Park et al. 2021). Polyploidy refers to the duplication of the same genome in autopolyploids or the duplication of separate genomes in allopolyploids (Pradhan et al. 2018). The growth of chromosomes in one kind of diploid results in two (or more) homologous chromosomal pairs, resulting in auto-polyploidies. Allopolyploids are the result of chromosomal growth following the hybridization of two distinct species. In general, the terms euploid and aneuploid are used to refer to organisms with different ploidy a status, which is discussed below. In euploidy, the total number of chromosomes is the exact multiple of the base chromosome series. Euploids can be divided into three groups: monoploids, diploids, and polyploids. But most euploids have two sets of chromosomes and are diploid. However, some euploid species have more than two chromosomal classes and are polyploidy (Ghasemi et al. 2021). According to Talebi et al. (2016) autotetraploidy polyploidization increases the number of chromosomes, gene dosage, concentration, and activity of several enzymes and amino acids. An increased number of genes elevates phenylalanine and tyrosine levels, which are further required for the biosynthesis of phenylpropanoids. It is worth mentioning that geneticists use the letter x to denote the number of base chromosomes, which are collections of single chromosomes that combine to form a full set. The number of chromosomes in each gamete or ploidy surface is also indicated by the letter n. New and modified cultivars of commercially significant species have been developed in recent decades by inducing artificial polyploidies with mutagenic agents. However, polyploidy is not as simple as genome multiplication, and it generates a wide variety of molecular and physiological modifications (Ghasemi et al. 2021). Polyploidy in angiosperms is stable, implying that this genome status has adaptive value and is positively selected (Iannicelli et al. 2020). The majority of polyploidies (natural and artificial) have distinct characteristics from their ancestor (Ghasemi et al. 2021). Polyploidy can disclose novel properties that are not found in either of the diploid progenitors, or it can outperform the progenitor’s characteristics. Prior investigations suggest that polyploidy can alter the qualitative or quantitative features of plant metabolites, which has implications for plant metabolism (Park et al. 2021). In most plant species, artificial polyploidy boosted the vitality of determinate plant portions. It has the potential to improve the production of vegetative organs and plant biomass. Polyploidy can be employed to improve the quality and quantity of essential therapeutic substances in plants, as well as to boost secondary metabolites. In flowering plants, chromosome doubling may alter secondary chemistry. DNA methylation, histone changes, and antisense RNA, as well as silence and up- or down regulation of genes involved in secondary metabolite biosynthesis, are all affected by genome doubling (Talebi et al. 2016). Normally, polyploids exhibit an increase in secondary metabolites or enlargement of different plant sections, however, there have been instances of no effect or a negative effect on the number of secondary metabolites. Polyploids make use of their resources through shorter metabolic pathways. They also have a larger cell size and methylation of cytosine (Pradhan et al. 2018). Some of these features, such as bigger organ size (leaf, flower, etc.), a wider biomass range, dryness tolerance, disease tolerance, and a variety of flowering times, might help polyploids adapt to new environmental changes (Ghasemi et al. 2021). Polyploidy alters physical and physiological alterations, which affect habitat, geographical distribution, reproductive systems, and breeding systems (Talebi et al. 2016). Furthermore, these different polyploidy features increase the likelihood of them being selected for agricultural uses (Ghasemi et al. 2021). It is mostly observed that polyploidy enhances the agronomic traits and attributes such as size of plant leaf and flowers as well as the increased tolerance owing to doubling of genome sets (Iannicelli et al. 2020). Polyploidy promotes growth in secondary metabolites in medicinal plants, as well as more and better adaptation to the environment, and increases the thickness of petals and the size of flower, among other things, in ornamental plants. In reality, artificial polyploidy allows for rapid genetic improvement of plants, making polyploidization one of the most important technologies in plant breeding (Ghasemi et al. 2021). Cell size enlargement is the most well-known and widely recognized result of polyploidization in plants. In reality, the generations of synthetic polyploids allow for rapid genetic improvement in plants, making polyploidization one of the most essential and common technologies employed in plant breeding (Iannicelli et al. 2020). Ploidy induction is an effective strategy for improving the genetics of many plants (Talebi et al. 2016). These distinctive polyploidy cells have almost double the volume of diploid predecessors and a 1.5-fold increase in cell surface area. Polyploids differ from their diploid status in their morpho-physiological parameters, due to the rise in cell size of enlarged flowers, leaf, and fruit sizes. The phenotypic differences are due to two polyploidization-related difficulties that contribute to the most constant impacts of the “polyploid phenotype”: an increase in gene dosage. In addition to the reported increases in secondary metabolite production in autopolyploids, the finding in different MAP’s suggests that the “polyploid effect” may influence pathways in diverse ways. It has been hypothesized that changes in ploidy cause changes in the control of metabolite production. As a result, while some metabolites are increased, others are reduced in favor of the former (mono and sesquiterpenoids in A. annua, morphine/thebaine and codeine in Papaver somniferum, and Scopolamine/hyoscyamine in Centella asiatica) (Iannicelli et al. 2020). For example, tetraploids exhibited higher levels of sesquiterpenoids in Artemisia annua and scopolamine alkaloid accumulation in H. muticus plants, presumably through increasing the expression of pathway genes (Talebi et al. 2016).

4 Conclusion

Secondary metabolites are of immense importance for the plants and human beings. Their production in plants is under the influence of environmental and genetic constitutions. Ploidy level of plants affects the secondary metabolite accumulation under tight genetic control. In some plants, induction of polyploidy leads to improvement in metabolite accumulation. Various reports are available showing the genetic variability in different species or cultivars of various medicinal and aromatic plants. These variations also contribute in alteration of secondary metabolites accumulation pattern. Various tools and techniques like genetic markers and QTL mapping can be utilized for identification of genetic diversity in plants and further phytochemical analysis of these plants confer the impact of genetic variance on the chemistry of associated plant up to significant level. Therefore, often plants differing at genetic level also vary in their metabolites either qualitatively or quantitatively. Such studies are very helpful for identification and characterization of improved varieties. Medicinal plants with improved metabolic content will have impact on global trading.