**3. Effect of gene regulation and modification in flavonoid research and production in crop breeding: recent advances and applications**

#### **3.1 Engineering of flavonoid pathway**

The flavonoid pathway has been extensively employed in the industry with the goal of accumulating compounds on purpose. Plant species like gerbera, petunia, rose, lisianthus, torenia, and carnation have been genetically modified for the production of novel flower colors. This was achieved by modification of the flavonoid biosynthesis pathway, either via transcriptional down-regulation, inactivation of key anthocyanin pathway enzymes, or by heterologous expression of key enzymes.

There are two significant, possible ways for improving flavonoid biosynthesis. The first is based on the discovery of TFs as a viable alternative to multi-step engineering, while the second is based on the use of inducible promoters to avoid the negative consequences of a constitutive production system. Virus-induced gene silencing has also been proven to be a simple and rapid method of functionalizing TF genes [88].

A few years ago, it was discovered that the pathway to pelargonidin might be opened by transferring a gene encoding DFR from a species where the enzyme does not really exhibit substrate selectivity into a petunia line Lacking F3′5′H activity. In another study, Brick red petunia flowers were produced using the maize gene *A1* and petunia lines with vivid orange blooms obtained from an ornamental plant *Gerbera hybrida* [89]. Now various initiatives are being undertaken to boost anthocyanin concentrations beyond those found naturally. According to a previous short study undertaken, the high-anthocyanin tomatoes have been found to slow tumor development in cancer-prone rats. However, the consequences on human health still require additional research. In this subject, basic proof-of-concept research has

been undertaken on a variety of vegetable and fruit species, including apple, grape, tomato, and cauliflower.

### **3.2 Natural flavonoids variation in horticultural species and horticulture breeding**

The molecular basis of various flavonoid production focuses on the activation of genes along with respective pathways by diverse means. The majority of the important structural enzymes, part of the central flavonoid metabolism is encoded by single-copy genes, although some, such as PAL, CHS, F3H, or FLS, are encoded by several genes. The expression of biosynthetic (structural) genes varies significantly between species [19].

Activation of flavonoid and anthocyanin biosynthetic genes in response to light has been reported in most horticultural types. Accumulation of anthocyanin is regarded as one of the most investigated mechanisms in potatoes. This is because colored potato varieties are considered to be a strong source of phytochemicals at levels similar to cranberries, blackberries, blueberries, and grapes. Potato, like other species, has numerous genetic loci that influence anthocyanin production. StAN1, StAN2, StMYBA1, and StMYB113 are important regulators of the phenylpropanoid and anthocyanin pathways. However, bHLH co-factors also play a role, since StAN1 and StAN2 associates with StbHLH1 and StJAF13 in diverse organs, such as the tuber and leaf. A WD40-repeat gene, namely StAN11 has been recently postulated as a regulator of the system via modulating the expression of DFR among other TFs encoding genes, impacting anthocyanin accumulation in potatoes [90]. Apart from potatoes, few other horticulture species have also been exploited due to their antioxidant property via molecular plant breeding techniques [15].

Recently, a variety of red-fleshed and high-flavonoid containing apple genetic resources had been embodied in the complexities of the control of flavonoid production. In one of the studies, red-fleshed apple flavonoid metabolism has been found to get influenced by both hereditary and environmental variables. Numerous flavonoid biosynthesis cascade genes have also been discovered and cloned, so as to identify the flavonoid metabolism that get affected by several environmental factors and genetic variabilities [91].

All these current researches make it evident that flavonoids play a significant role in both food and primary agriculture development and will soon be an intriguing target for molecular plant breeding.

#### **3.3 Flavonoids in tomato breeding**

Tomatoes (*Solanum lycopersicum*) are the most abundant dietary source of carotenoids (lycopene), polyphenols, and flavonoids, which are key bioactive compounds favorable to human health. The flavonoids that are mainly produced in tomatoes are predominantly produced mostly in the peels. Naringenin chalcone and rutin (quercetin-rutinoside) are the two primary flavonoids found in tomato fruit so far [92, 93]. To date, three approaches have been made to engineer the flavonoid pathway in tomatoes (*S. lycopersicum*) with the goal to alter its agronomical traits such as its nutritional value, its flower and fruit color as well as its ability to build resistance against insects [94]. They are as follows:

a.Use of structural or regulatory genes to increase endogenous tomato flavonoids; Structural genes are the genes that encode enzymes that directly engage in the

synthesis of flavonoids. On the other hand, regulatory genes are the ones that influence the expression of structural genes.


Knowing the fact that there is a lack of flavonoid expression in tomatoes, to date, several attempts have been previously undertaken to generate transgenic tomatoes (**Table 1**).

For example:



#### **Table 1.**

*Genes responsible for different flavonoid production in tomato.*


In one of the studies, it was concluded that despite having intense debate over the advantages, disadvantages, and risks of genetically modified food, around 96% of the customers showed interest in purchasing high flavonoid containing tomatoes. It is considered that this changing mindset of people will prove to be crucial for the development of transgenic vegetables in the future [94]. Moreover, various other studies are in process to make transgenic tomato breeding more productive and nutritional in the coming future.

#### **3.4 Flavonoids in rice breeding**

Rice is staple food in many Asian countries. Even though white rice is the most popular, Asian cuisine often includes colored rice. Several pieces of evidence reveal that pigmented rice has important biological properties, including antioxidants, antiallergic, and neuro-protective properties. The rich flavonoid and nutritional content of colored rice warrants enhancement flavonoid content in rice by implementation of different breeding strategies (**Table 2**).

The functional activities of TFs influence the color of rice grains. In black rice, the *Kala3* gene, which codes for R2R3-Myb, and the *Kala4* gene, which codes for basic helix–loop–helix (bHLH), activate the flavonoid biosynthesis genes *ANS CHS*, and *DFR* resulting in anthocyanin pigment buildup in the grain. In red rice, the *Rc* gene expressing bHLH activates *CHS*, *DFR*, and *LAR*, resulting in the buildup of proanthocyanidin pigment in the grain. The promoter of *Kala4* in white rice differs from that in pigmented rice, and loss of 14 base pairs inside the *Rc* open reading frame, resulted in lack of color in the grain. In addition, the gene *CYP75B3* is strongly expressed in pigmented rice grains, along with other flavonoid pathway genes. This explains why leucocyanidin-derived anthocyanin and proanthocyanidin pigments are abundant in colored rice grains [97].

Recently, in order to create flavone, isoflavone, and flavonol in rice grain, the flavonol (*AtF3H/AtFLS*), isoflavone (*GmIFS*), and flavone (*PoFNSI/GmFNSII*) biosynthetic enzyme genes, as well as *OsPAL* and *OsCHS*, were expressed in a


#### **Table 2.**

*Genes responsible for different flavonoid production in rice.*

*Flavonoids: Recent Advances and Applications in Crop Breeding DOI: http://dx.doi.org/10.5772/intechopen.107565*

seed-specific way. These biosynthetic genes were expressed in seed using the *GluB-1* promoter and the 18-kDa oleosin promoter [99]. In another study, the *OsCOP1* gene, an ortholog of *Arabidopsis thaliana* constitutive photomorphogenic 1 (COP1) was introduced in rice using CRISPR-Cas9. This not only turned the pericarp of the rice variety yellowish but also caused embryonic death. Moreover, this also reduced the size of the transgenic seeds [98]. According to a study, a total of 82 flavonoids have been chemically identified in transgenic rice seeds. Moreover, exogenous enzymes produced flavonoids in rice seeds that were later altered by endogenous enzymes and transported, causing persistent accumulation in PB-I and/or PB-II. Based on these results, the heterologous and ectopic expression of biosynthetic enzymes in rice seeds not only serves as a productive platform for the production of flavonoids but can also be used to broaden the structural diversity of flavonoids and hence open up a new, untapped source of bioactive substances [100].

Increasing the flavonoid biosynthesis and its accumulation in rice have been found to contribute to the enhanced heat tolerance under stress, as well as plays a regulatory role in the activation of the antioxidant enzyme system [101].

#### **3.5 Flavonoid research in maize**

Obtaining security of grain supply in the twenty-first century with limited arable land is a big challenge because of the constantly changing environment and increasing global population [102–104]. Maize plays a very important role in global grain production. Drought is a significant factor restricting plant development and productivity. Drought stress affects growth and development of plants, which is directly related to yield. Under drought stress, *doi57* gene is observed to play an important role in maintaining the plant to grow and survive in order to give good yield. *doi57* gene is one of the key genes involved in biosynthesis of flavonoid. With less soil water content (SWC), doi57 guard cells can accumulate more flavonols and less hydrogen peroxide (H2O2). Furthermore, under drought conditions, *doi57* seedling extracts had a stronger potential to scavenge oxygen free radicals than B73 maize genome. Moreover, in terms of transpiration rates, photosynthetic rates, water consumption efficiency, and stomatal conductance, *doi57* seedlings outperformed B73, resulting in high biomass and enhanced root/shoot ratios in *doi57* mutant plants [105].

#### **3.6 Flavonoid profile in millets breeding**

Millet polyphenols protect the neurological system by lowering oxidative stress, and vitexin is a crucial component of millet polyphenols. Vitexin, a flavonoid derived from millet, is present in many foods such as millet, mung bean, and others. It is also known chemically as apigenin-8-C-glucoside. According to research, vitexin contains potent free radical scavenging and antioxidant enzyme protection properties that may protect cells from oxidative damage [106].

#### **3.7 Flavonoids in olive breeding**

With almost 1200 olive varieties listed, the olive has a great genetic diversity. High heterozygosity, prolonged juvenile phase, and a paucity of information on trait heritability have all been major limiting factors in olive breeding. Attempts to obtain new varieties have concentrated on improving olive response to varied growing situations through systematic breeding. New olive breeding methods uses two

varieties "Picual" and "Arbequina" as controls along with other varieties. The phenolic compound metabolism in the olive tree is quite complex, and is controlled by environmental and genetic factors that regulate the final phenolic composition of olive fruits. The unique breeding selection UCI2–68 demonstrated an optimal phenolic profile, resulting in good agronomic performance [107].
