**3.1 Secondary metabolites being extracted and applied directly in cotton production**

Cotton secondary metabolites have been recognized as natural pesticides [79]. The simplest way to utilize them is to extract them and apply the extract directly in cotton production.

Ref. [90] extracted secondary metabolites by water from different plant parts of *G. hirsutum* plant and used them to suppress weed. Among the various water extracts of the plant parts, leaf extract was found to impart maximum inhibitory effect on the germination indices of the wheat (*T. aestivum*) followed by the stem extracts. The concentrated extract led to wheat germination percentage and seedling vigor index reduction of 46% and 62%, respectively. The research did not reach into the active ingredients of the cotton leaf extract. As discussed earlier, cotton leaf is rich in secondary metabolites such as condensed tannins (up to 20% dry weight), terpenoids, non-tannin flavonoids, and phenolic acids. As terpenoids are not soluble in water, they might not be the main active ingredients of the extract except the extract contained a significant number of solid particles (our HPLC analysis showed leaf water leachate contained less than 1 mg/L gossypol). Hence, the most probable active ingredients are non-tannin flavonoids and phenolic acids. Phenolic acids have been explored for cotton allelopathy [74]. Flavonoids, however, have not been investigated for cotton allelopathy. As listed in **Table 1**, cotton contains a rich array of flavonoids, some of which have been reported to be allelopathic. For example, Ref. [87] reported quercetin from *Fagopyrum esculentum* roots to inhibit the radicle growth of *Phelipanche ramose*. The data also suggest that two ortho-free hydroxy groups of the C (B?) ring could be essential to impart allelopathic activity. In contrast, the carbon skeleton of the B ring and substituents of both the A and B rings are unnecessary. According to this rule, lots of flavonoids in **Table 1** are allelopathic such as Gossypetin, Gossypin,

Gossypitrin, Gossypetin 8-O-rhamnoside, Catechin, Luteolin, Luteolin 8-C-hexosyl-- O-hexoside, Hyperoside, Quercetin-3-sophoroside, Quercemeritrin, Gallocatchin, Delphinidin, Cyanidin, Hirsutrin, Butin, Isoquercitrin Hyperoside, Isotrifoliin, Rhamnetin, etc. Hence, allelopathy spread by cotton flavonoids is expected to be confirmed soon [91].

In this way of extraction and application, we can extract secondary metabolites from other allelopathic plants and apply them to cotton production. For example, Ref. [92] used water extracts from sorghum, sunflower, and brassica for preemergent application in the cotton field. It was observed that the best treatment produced dry biomass reduction by 40% of weeds *Trianthema portulacastrum* and *Cyperus rotundus* and an increase in seed cotton yield (12%). Similar extraction and application can be extended to different allelopathic plants. For example, we used sicklepod seed methanol extract (replaced with water after extraction) and applied it to soybean to prevent deer browsing [93].

## **3.2 Screening and breeding for high secondary metabolites cultivars**

Since Cook [67] observed the better resistance to insect herbivores of Guatemalan cotton, numerous efforts have been made to increase U.S. cotton resistance to various biotic and abiotic stresses in cotton production [6]. So far, secondary metabolites were reported to contribute cotton resistance include condensed tannins [47, 89], terpenoids/gossypol [51–54, 63, 94], phenolic acids [49, 50, 74], and bacterial endotoxins [95, 96]. All these secondary metabolites have been screened and bred for higher secondary metabolite levels. Their common basis is that selection among cultivars or species for higher secondary metabolite levels is an attainable goal [63, 97].

## **3.3 Cotton secondary metabolite regulators or elicitors**

As discussed before, cotton secondary metabolites (gossypol and flavonoids) were shown to increase under salinity stress [81]. The salinity stress was imposed by salt water as irrigation; foliar application of the salt water also increased root gossypol concentrations in our lab. The saltwater can be regarded as a secondary metabolite regulator or elicitor to increase gossypol and flavonoid production for defense. In addition, Ref. [43] showed that oligosaccharides filtrates of *Fusarium oxysporum* f. sp. *vasinfectum* could stimulate phenolic compounds production in cotton plants. Furthermore, some herbicides [98, 99] were reported to cause an increase in gossypol concentration in cotton plants. Since the introduction of GMO cotton in the mid-1990s, herbicide application has become a routine and indispensable practice in cotton production. Tank-mix of herbicide with secondary metabolites regulators or elicitors, such as the salt, as mentioned earlier solution or pretreatment of the cotton seeds by the herbicides as mentioned above can increase the gossypol concentration of the seedling roots, a potential practice to prevent nematode damage to cotton plants.

#### **3.4 Metabolic engineering method to modify cotton**

Contrary to the exogenous metabolite regulator, metabolic engineering optimizes genetic and regulatory processes within cells to increase the cell's production of a specific substance. Compared to general metabolic engineering, where the target substance is commercial, we would increase specific substances (secondary metabolites) in situ for cotton plant defense or profit.

Cotton is a multibillion-dollar industry where engineering is often employed to improve profitability. In a study conducted by John and Keller in 1996 [100], they utilized particle bombardment to introduce phbB and phbC genes into cotton. This genetic modification led to the development of cotton plants with fibers containing poly-(3R) hydroxybutanoate (PHB), thereby enhancing the insulating properties of the fibers. Genetic mapulations of production of trienoic fatty acids in cotton seeds have improved low-temperature seed germination, plant photosynthesis, and fiber quality [101].

Most recently, Ref. [102] used a selective gene editing method to remove the toxic enantiomer ()-gossypol while the (+)-gossypol and other structurally related terpenoids remained, keeping their resistance to insect herbivores and pathogens. The modified genes (including the current widely accepted GMO cotton genes) may flow into wild species in nature, which is a concern.
