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Review article
12 2024
:17;
106018
doi:
10.1016/j.arabjc.2024.106018

A comprehensive review of in vitro precursor feeding strategies for the overproduction of high-value plant secondary metabolites

,
 Department of Biology, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia

⁎Corresponding author at: Department of Biology, Institut Teknologi Sepuluh Nopember, Kampus ITS Sukolilo, 60111 Surabaya, Indonesia. nuruljadid@bio.its.ac.id (Nurul Jadid)

Received: , Accepted: ,
© The Author(s) 2024
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

Plant tissue culture techniques have revolutionized various aspects of modern agriculture, horticulture, and pharmaceutical industries. This method contributes to enhancing the production of plant secondary metabolites, which have noteworthy applications in pharmaceuticals, agriculture, and industry. Presently, over 50,000 plant metabolites have been identified and categorized into three primary groups: terpenoids, flavonoids, and alkaloids. Numerous studies have elucidated the metabolic pathways involved in the biosynthesis of plant compounds to better understand the metabolic flux, with the aim of identifying and engineering the production of valuable constituents. In vitro precursor feeding is one phytochemical strategy that helps increase the accumulation of plant compounds. Therefore, this review focuses on investigating the application of in vitro precursor feedings to the overproduction of a high-value phytochemicals. Moreover, it evaluates the influence of type and concentration of precursors, plant species and culture conditions as well as the phytochemical products providing insights into optimization strategies. Additionally, the potential application of accumulated terpenoids, flavonoids, and alkaloid-derived compounds for agricultural, pharmaceutical, and industrial purposes are discussed. Finally, perspective challenges and limitations related to in vitro precursor feeding strategy are addressed, including production stability, cytotoxicity effect, and uptake efficiency. The overall data presented might serve as an up-to-date report on the application of in vitro precursor feedings for enhancing plant secondary metabolite production.

Keywords

Metabolic pathway
Phytochemical
In vitro precursor feeding
Pharmaceuticals
Plant secondary metabolites
1

1 Introduction

Plants are renewable sources, providing materials (biomass) and phytochemicals (both primary and secondary metabolites) (Guerriero et al., 2018). Moreover, plants serve as bio-factories, manufacturing human nutrition (Jadid et al., 2018). Diverse plant secondary metabolites (PSMs) have been elucidated including terpenoids, flavonoids, and alkaloid-derived compounds (Dasari et al., 2020). The PSMs are beneficial for medicinal purposes, food additives, flavorings, agriculture and other industrial ingredients. As an example, numerous ethnobotanical studies have indicated that tropical plants could be explored as alternative reservoirs for the production of medicinal drugs, owing to their varied plant secondary metabolites (PSMs) (Jadid et al., 2023). In addition, these organic substances also self-guard the plants from many adverse environmental conditions, including protecting the plants from pathogenic microorganisms, insects, and herbivore attacks. Moreover, in some cases, they protect the ecological equilibrium in nature by facilitating plant-animal pollinator contact (Slavković and Bendahmane, 2023).

Rigorous use of crop products creates a large gap between demand and availability. Moreover, PSM are available in small quantities, and harvesting at current demand scales may be environmentally unpleasant and impractical (Marchev et al., 2020; Wilson and Roberts, 2012). Furthermore, the economic impracticality of chemical synthesis for plant secondary metabolites is frequently attributed to their structural complexity (Dziggel et al., 2017; Staniek et al., 2014). Hence, finding alternatives to sourcing plant materials from natural reserves, as well as suitable systems and approaches are necessary. Plant cell cultures offer a great advantage to address these challenges, especially for propagating high commercial or medicinal crops (Fig. 1) (Jadid et al., 2024a). However, the widespread adoption of plant cell culture in commercial settings is constrained, and only a tiny fraction of secondary metabolites can be produced. A few key reasons include inadequate yields, instability in biosynthesis, and challenges in scaling up production (Sevón and Oksman-Caldentey, 2002). Notably, the prevalent issue is the inefficiency of the biosystem in producing high concentrations of the desired compounds (Qu et al., 2011). This predicament arises from the empirical approach in selecting cultures with high and consistent yields and limited information on the PSM biosynthetic pathways (Isah et al., 2018).

In vitro culture of medicinal and industrial crops. 1) Efficient shoot proliferation and callus induction in Gynura pseudochina in vitro culture (Anjalani et al. (2024), 2) callus and organogenesis of Stevia rebaudiana by Jadid et al., (2024a). 3) Callus culture and organogenesis in Pogostemon cablin by Jadid et al. (2024c) after being cultured in an optimized medium supplemented with plant growth regulators (PGR) and elicitor methyl jasmonate, resulting in enhanced growth, increased metabolic gene expression, and changes in phytochemical composition.
Fig. 1
In vitro culture of medicinal and industrial crops. 1) Efficient shoot proliferation and callus induction in Gynura pseudochina in vitro culture (Anjalani et al. (2024), 2) callus and organogenesis of Stevia rebaudiana by Jadid et al., (2024a). 3) Callus culture and organogenesis in Pogostemon cablin by Jadid et al. (2024c) after being cultured in an optimized medium supplemented with plant growth regulators (PGR) and elicitor methyl jasmonate, resulting in enhanced growth, increased metabolic gene expression, and changes in phytochemical composition.

A good biosystem in plant cell culture for boosting the PSMs could be seen in how the system consistently ensures substantial amounts of the desired compounds. Rigorous selection of productive plant cell types and careful control of culture conditions can lead to higher levels of specific products within cultured cells. Numerous approaches have been employed to stimulate biosynthetic activities, focusing on optimizing the production of phytochemicals (Rao and Ravishankar, 2002). Typically, culture cell systems amass significant quantities of secondary compounds under specific circumstances. Over time, diverse strategies have emerged to augment PSMs production using plant cell culture, including selecting high-productive cell lines (Tripathi et al., 2019). Moreover, many studies revealed that enhancing secondary metabolites could be achieved by elicitation using both abiotic (environmental stress or chemical compounds) (Anjalani et al., 2024) and living organism-based elicitors (Tothong et al., 2023). Recently, the use of metal-based nanoparticles has also been applied to the plant in vitro medium to induce the synthesis of commercially important substances. Previous work has been demonstrated that plants develop a fascinating physiological and molecular strategies to cope with adverse environmental conditions, including the over-production of secondary metabolites (Jadid et al., 2017). However, the environmental inducers, in some cases, might also alter the genetic profile of the plants.

Another technique involves the addition of precursor feeding of the targeted PSM into the in vitro culture medium. Precursor feeding refers to supplementation of the primary building block that initiates the biosynthetic pathway leading to the production of specific plant metabolites. Generally, precursors are derived from primary metabolites including amino acids, fatty acids, sugars or other organic acids. This in vitro supplementation can result in higher yields of the desired end products (Hussain et al., 2012; Rao and Ravishankar, 2002). A critical aspect of this method involves a comprehensive exploration of the entire biosynthetic pathway. This inclusive approach considers multiple examples that might influence the production of the compound (Espinosa-Leal et al., 2018). Particularly, this method is advantageous when the precursors are cost-effective (Namdeo et al., 2007). Many studies, including those by Marchev et al., Dasari et al., Skrzypczak-Pietraszek et al., Guerriero et al., and Kundu et al., have effectively isolated specific compounds using the precursor feeding approach (Dasari et al., 2020; Guerriero et al., 2018; Kundu et al., 2018; Marchev et al., 2020; Skrzypczak-Pietraszek et al., 2018).

In this review, we emphasize the precursor feeding method as an advanced approach for augmenting the biosynthesis of PSMs through in vitro plant systems. Our review gathered some elements supporting the biosystem, including the plant species, plant organs used in the culture system, methods, target compounds, and precursors used in the experiments. This review also briefly describes the potential applications of PSMs in agriculture, pharmacy, and industries. Finally, we discussed precursor feeding methods' future prospects and limitations, including production stability, cytotoxicity effect, and uptake efficiency. This review offers an overview of the current application of precursor feeding techniques via in vitro culture for augmenting the production of PSMs. This review also serves as valuable information for future research direction on the synthesis of commercial plant metabolites in vitro using combinatorial strategies with biotic and abiotic elicitors.

2

2 In vitro precursor feeding

2.1

2.1 Precursor feeding method and common pathways in plant

The core idea behind the in vitro precursor feeding involves incorporating intermediate metabolites in the bioactive molecule synthesis at the beginning or during the plant cell cultivation process. This inclusion acts as extra substrates, effectively enhancing the yield of metabolites within plant tissues, cells, and organs undergoing cultivation (Isah et al., 2018; Rasche et al., 2016). The term “precursor” pertains to substances originating externally or internally that can be changed into secondary compounds by in vitro cell cultivation via biosynthesis pathways. Generally, the levels of these compounds are comparatively lower in plant cell cultures when compared to differentiated plant tissues. This discrepancy elucidates the correlation between cellular development and increased production of pharmaceutical-based natural products in various micropropagation methods involving plant tissues and organs (Constabel and Kurz, 1999). The use of transgenic cell lines and the incorporation of these compounds into the culture medium show promise as strategies to expedite the synthesis of molecules in plant cell cultures (Namdeo et al., 2007).

It is essential to ascertain the production rates and the kinetics of biosynthetic precursor utilization while attempting to increase pharmaceutical output by choosing a precursor molecule. Identifying the best conditions for these processes is also imperative (Isah et al., 2018). Critical considerations in choosing and introducing a precursor molecule into the system encompass the timing of its introduction, the concentration at which it is added, and its seamless integration into the intended biosynthetic pathway (Jackson & Attalla, 2010). Furthermore, it is crucial to consider the potential impact of feedback inhibition when determining the precise quantity to modify the culture medium for plant tissues, cells, and organs.

Shikimic acid, phenylpropanoid, and mevalonic acid pathways represent extensive biosynthesis routes frequently investigated in plant cell culture research to enhance the production of phytochemicals using the precursor feeding method. The shikimic acid pathway is particularly significant for synthesizing a range of essential aromatic compounds, including vitamins, amino acids, and other phytochemicals (Cheynier et al., 2013). It is pivotal in primary and secondary plant metabolisms (Macheroux et al., 1999). The initiation of this pathway involves the condensation of two compounds of phosphoenolpyruvate (PEP) and one molecule of erythrose-4-phosphate (E4P), both are derived from glycolysis and the pentose phosphate pathways. Following this, the pathway advances through a sequence of enzymatic reactions, yielding a crucial intermediate molecule known as shikimic acid. The ultimate step in the pathway culminates in the production of chorismate (Dewick, 2002). It acts as a branching point in the pathway and provides the precursor for various important biochemicals (see Fig. 2) (Mesnage et al., 2021). Numerous studies employing precursor feeding methods utilize intermediate compounds from this pathway as supplementary precursors. For instance, shikimic acid is added to enhance the production of 3-O-glucosyl-resveratrol and 4-(3,5-dihydroxyphenyl)-phenol from V. vinifera (Riedel et al., 2012), shikimate is introduced to increase the production of sanguinarine from P. somniferum (Verma et al., 2014a), and it is used to produce vincamine from V. minor (Verma et al., 2014b) (Fig. 2).

Common precursors (red circles) are used in precursor feeding method. (A) Phenylalanine and tyrosine are potential amino acids that could be used in flavonoid-derived compounds (B) Acetyl-CoA and mevalonic acid are organic acid precursors in the biosynthesis of isoprenoids (C) The shikimate serves as precursors in the shikimate-derived compounds. (D) Tryptophan and tryptamine serve as precursors in the biosynthesis of strictosidine-derived compounds.
Fig. 2
Common precursors (red circles) are used in precursor feeding method. (A) Phenylalanine and tyrosine are potential amino acids that could be used in flavonoid-derived compounds (B) Acetyl-CoA and mevalonic acid are organic acid precursors in the biosynthesis of isoprenoids (C) The shikimate serves as precursors in the shikimate-derived compounds. (D) Tryptophan and tryptamine serve as precursors in the biosynthesis of strictosidine-derived compounds.

The phenylpropanoid metabolic pathway produces diverse phytochemicals, relying on the products generated in the shikimate pathway (Vogt, 2010). Phenylalanine acts as the starting point for phenylpropanoid biosynthesis. The core of the phenylpropanoid pathway involves specific enzymatic steps, including (i) phenylalanine deamination resulting in trans-cinnamic acid, (ii) hydroxylation of trans-cinnamic acid to 4-coumarate, and (iii) conversion of 4-coumarate to 4-coumaroyl-CoA (Biała and Jasiński, 2018). Coumaroyl-CoA is a crucial intermediate in the phenylpropanoid pathway, serving as a precursor for various compounds, including stilbene, monolignol, isoflavonoid, and coumarins biosynthesis (Buchanan et al., 2015). In the context of the precursor feeding method, many studies employ phenylalanine as an additional substrate in the initial stages of this pathway. For example, it is used in the production of wedelolactone from S. calendulacea (Kundu et al., 2018), daidzein from P. corylifolia (Shinde et al., 2009), quercetin from I. tinctoria (Miceli et al., 2023), and silymarin from S. marianum (Firouzi et al., 2013) (Fig. 2).

In living organisms, the mevalonate pathway is predominantly present in eukaryotes and primarily occurs within the cytoplasm. Initially, two molecules of acetyl CoA are enzymatically transformed to create 3-hydroxy-3-methyl-glutaryl-CoA. This compound is then transformed into mevalonic acid (MVA), and subsequently, isopentenyl diphosphate (IPP) is produced. A portion of IPP undergoes isomerization resulting in the formation of dimethylallyl diphosphate (DMAPP) (Xiao and Zhong, 2016). Ultimately, the remaining IPP and DMAPP are utilized by polyisoprene pyrophosphate synthase to generate precursors for triterpenes, sesquiterpenes, and steroids. In the context of the precursor feeding method, numerous studies employ mevalonic acid, an intermediate compound from this pathway, to enhance the production of various phytochemicals. For example, mevalonic acid is used in the production of whitanolide A from W. somnifera (Sivanandhan et al., 2014), curcubitacin E from C. colocynthis (Dasari et al., 2020), artemisinin from A. annua (Baldi and Dixit, 2008), and bacoside A from B. monnieri (Hegazi et al., 2017) (Fig. 2).

2.2

2.2 Current reports on precursor feeding strategy in plant in vitro cultures

Many studies report the use of in vitro precursor feeding to promote plant secondary metabolite synthesis. The supplementation of precursors into the culture medium is commonly employed in callus and cell suspension culture. We have updated the information provided by Namdeo et al. (2007) in their previous review. We collected the data from 2007 to 2024, we found that out of 56 studies, 48 were conducted on medicinal plants (Table 1). Meanwhile, 4 of the studies focused on industrial plants such as Vitis vinifera, Isatis tinctoria, Papaver somniferum, and Argania spinosa (Riedel et al., 2012; Miceli et al., 2023; Verma et al., 2014a; Hegazi et al., 2020). The remaining 4 studies used ornamental plants as research subjects, including Solenostemon scutellarioides, Dionaea muscipula, Drosera capensis, Aronia melanocarpa (Michx.) Elliott, Aronia arbutifolia (L.) Pers., and Dendrobium fimbriatum (Dewanjee et al., 2014; Królicka et al., 2008; Szopa et al., 2020; Paul and Kumaria, 2020) (Suppl. 2).

Table 1 Studies of the usage of precursor feeding method to enhance phytochemicals productions.
No. Compound Class Plant Species Explant Culture Media Precursors Products References
1 Steroidal lactones Withania somnifera root derived callus 30 ml of MS + 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5 % sucrose cholesterol,
mevalonic acid,
squalene		 withanolide A,
withanolide B, withaferin, withanone,
deoxy withanstramonolide, withanoside I, withanoside V		 (Sivanandhan et al., 2014)
2 Ecdysteroid Achyranthes aspera seed derived callus MS + 1 mg/l 2, 4-dichlorophenoxyacetic acid and 1 mg/l α-naphthaleneacetic acid cholesterol, 7-dehydrocholesterol, 20-hydroxyecdysone (John et al., 2018)
3 Polyprenylated acylphloroglucinol Hypericum perforatum shoot 20 ml MS + 0.1 mg/l BA, 30 g/l sucrose, 0.1 g/l myo-inositol and 0.1 g/l ascorbic acid l-isoleucine, l-threonine, l-valine hyperforin, adhyperforin (Karppinen et al., 2007)
4 Phytosteroid Digitalis purpurea L. nodal, internodal, and leaf MS + 7.5 μM BA, and MS + 15 μM IAA progesterone, cholesterol, and squalene cardiotonic glycosides, digitoxin, and digoxin (Patil et al., 2013)
5 Flavonoids and phenolic acids. Vitex agnus castus L. shoots MS + BAP 1 mg/L; NAA 0.5 mg/L; GA3 0.25 mg/L and BAP 2 mg/L;NAA 0.5 mg/L. L-phenylalanine neochlorogenic acid, p-coumaric acid, rutin, caffeic acid, cinaroside, (Skrzypczak-Pietraszek et al., 2018)
6 Terpenoid Citrullus colocynthis leaves, shoot apex and nodal callus MS + 2.0 mg/L BAP + 0.5 mg/L NAA squalene, mevalonic acid cucurbitacin E (Dasari et al., 2020)
7 Terpenoid Artemisia annua seed derived callus 50 ml MS + 3 % (w/v) sucrose + 0.1 mg/l NAA + 0.1 mg/l Kn. mevalonic acid lactone Artemisinin (Baldi and Dixit, 2008)
8 Coumestan Sphagneticola calendulacea shoot derived HR ½ MS + 100 μM SA phenylalanine wedelolactone (Kundu et al., 2018)
9 Lignan Larrea divaricata leaves derived callus MS + 9 μM 2,4-D + 5 μM BA l-phenylalanine, cinnamic acid, ferulic acid, and sinapic acid nordihydroguaiaretic acid (Palacio et al., 2011)
10 Terpenoid Bacopa monnieri aerial parts MS (liquid) l-alanine and l-phenylalanine saponin glycosides (Watcharatanon et al., 2019)
11 Ester Rhodiola rosea seeds MS + 25 g/L sucrose + 6.5 g/L agar cinnamyl alcohol, cinnamaldehyde Cinnamyl alcohol glycosides (Javid et al., 2021)
12 Stilbenes Morus alba L. root MS + 1 mg/L NAA L-tyrosine mulberroside A, oxyresveratrol, and resveratrol (Inyai et al., 2021)
13 Phenol Vitis vinifera callus 25 ml of B5VIT basal medium shikimic acid, phenylalanine 3-O-glucosyl-resveratrol and 4-(3,5-dihydroxyphenyl)-phenol (Riedel et al., 2012)
14 Isoflavones Psoralea corylifolia stem derived hairy root 50 mL MS phenylalanine daidzein, genistein (Shinde et al., 2009)
15 Flavonoids Isatis tinctoria shoot MS + 1.0/1.0 mg/L BAP/NAA L-Phenylalanine and L-Tyrosine apigetrine, quercetin, apigenin, quercitrin (Miceli et al., 2023)
16 Amino acid Mucuna pruriens seed derived callus 50 mL MS + 3 % sucrose + 0.5 mg/L picloram tyrosine, phenylalanine L-DOPA (Rakesh and Praveen, 2022)
17 Alkaloids Solanum lyratum seed derived callus 30 ml MS + 0.75 mg/l 2,4-D + 3 % sucrose cholesterol, stigmasterol α-solanine, solanidine, and solasodine (Lee et al., 2007)
18 Naphthoquinone Drosera burmannii Vahl and Drosera indica L.
 whole plantlets 30  mL MS sodium acetate plumbagin (Boonsnongcheep et al., 2019)
19 Alkaloids Rauwolfia serpentina nodal segments and shoot Hoagland solution tryptamine reserpine (Panwar and Guru, 2015)
20 Caffeic acid ester Solenostemon scutellarioides whole plantlets MS L −phenylalanine, L −tyrosine rosmarinic (Dewanjee et al., 2014)
21 Flavonoids Silybum marianum whole plantlets 50 mL MS + 3 g./L picloram + 0.4 g/L kinetin phenylalanine silymarin (Firouzi et al., 2013)
22 Terpenoid Centella asiatica leaf and petiole derived HR 100 mL MS + 50 g/L sucrose squalene and pyruvic acid madecassoside, asiaticoside, madecassic acid, asiatic acid (Baek et al., 2020)
23 Alkaloids Rauwolfia tetraphylla L. leaf, stem, root derived callus MS + 2, 4-D (2.0 mg/L) Tryptophan reserpine (Rohela et al., 2021)
24 Alkaloids Papaver bracteatum cell suspension 50 mL MS liquid L-tyrosine thebaine (Zare et al., 2014)
25 Phenolic Decalepis hamiltonii leaf derived callus MS + BA + Kn + NAA + 2.4 D ferulic acid vanillin, 2H4MB, vanillic acid (Matam et al., 2017)
26 Anthocyanins Panax sikkimensis root derived callus MS + 3 % sucrose, 0.01 % myoinositol, 0.33 μM thiamine HCL, 2.5 μM pyridoxine hydrochloride, 4.0 μM nicotinic acid, 5.4 μM NAA, and 1.2 μM Kinetin. phenylalanine anthocyanins content (Biswas et al., 2020)
27 Furanocoumarins Psoralea corylifolia L. cotyledon derived callus MS + 10 µM BA + 5 µM IBA umbelliferone, cinnamic acid, NADPH psoralen (Mohammadparast et al., 2015)
28 Alkaloid Mitragyna speciosa petiole and leaves derived callus 25  mL of liquid WPM Tryptophan, loganin mitragynine (Mohamad Zuldin et al., 2013)
29 Anthraquinones Cassia angustifolia Vahl leaves, nodes, roots MS + NAA + IBA + 3 % (w/v) sucrose α-keto glutaric acid and pyruvic acid sennoside A and B (Chetri et al., 2016)
30 Terpenoid Bacopa monnieri leave derived callus MS + 9 µM 2,4-D and 2.3 µM KIN mevalonic acid Bacoside A (G. Hegazi et al., 2017)
31 Phenylpropanoid glycoside Rhodiola imbricata leaf and root cells suspension MS + 0.5 mg/L TDZ + 1 mg/L NAA tyrosol salidroside (Rattan et al., 2022)
32 Phenylpropanoids Spilanthes acmella Murr. leaf derived callus MS + 15 µM BA + 5 µM 2,4-D casein hydrolysate, L-phenylalanine scopoletin (Abyari et al., 2016)
33 Amino acid Hybanthus enneaspermus (L.) leaf derived HR MS + 300 mg/L of cefotaxime L-tyrosine L-DOPA (Sathish et al., 2023)
34 Phenolic Dionaea muscipula,Drosera capensis whole planlets modified ½ MS + 2 % sucrose l-phenylalanine, trans-cinnamic acid naphthoquinones, quercetin, myricetin (Królicka et al., 2008)
35 Alkaloid Papaver somniferum shoot derived callus MS (aq) + 1 ppm 2,4-D shikimate sanguinarine (Verma et al., 2014a)
36 Vitamins Argania spinosa leaf derived callus 100 ml MS + 4.5 µM 2,4-D + 5 µM NAA tyrosine α-tocopherol (Hegazi et al., 2020)
37 Flavonoids Silybum marianum root derived callus 50 ml MS + 4.55 μM 2,4D + 4.44 μM BA L-phenylalanine Silymarin (Hassanen et al., 2021)
38 Stilbenoid Morus alba L. leaf derived callus 30 ml MS + 0.1 mg/lTDZ + 1 mg/l NAA L-phenylalanine, L-tyrosine mulberroside A (Pongkitwitoon et al., 2020)
39 Terpenoid Picrorhiza kurroa shoots MS + 3 mg/L indole-3-butyric acid + 1 mg/L kinetin cinnamic acid (CA) and catalpol (CAT) picroside-I (P-I) (Kumar et al., 2016)
40 Flavonoid Cassia occidentalis L. cotyledon derived callus MS + 2.4D + Kin + NAA Phenylalanine, Methionine Rotenoids (Vats and Kamal, 2014)
41 Phenolic compounds Aronia melanocarpa (Michx.) Elliott, Aronia arbutifolia (L.) Pers. shoot 90 mL MS phenylalanine, cinnamic acid, benzoic acid, caffeic acid neochlorogenic, chlorogenic, cryptochlorogenic, isochlorogenic, rosmarinic acids, and syringic (Szopa et al., 2020)
42 Terpenoid Bacopa floribunda shoots and roots MS + 2.0 mg/l BAP + 2.0 mg/l KIN and MS + 0.5 mg/l IAA + 0.5 mg/l IBA + 1.0 mg/l NAA squalene bacoside A3, bacopaside X, bacopaside II, and bacosaponin C (Otari et al., 2023)
43 Phenolic compounds Arnebia euchroma bud derived callus MSA + 1 mg/L kinetin and 0.3 mg/L IAA L-phenylalanine naphthoquinones (Sykłowska-Baranek et al., 2012)
44 Alkaloid Catharanthus roseus leaf derived callus MS + 1 mg/l kin L-tryptophane; L-glutamine; L-asparagine; L-cystine and L-arginine vinblastine and vincristine (Taha et al., 2009)
45 Amino acid Mucuna pruriens leaf derived callus 20 ml MS + BAP 0.88 μM + NAA 11.41 μM L-tyrosine L-Dopa (Raghavendra et al., 2011)
46 Phenolic, alkaloid, flavonoid, and tannins Dendrobium fimbriatum protocorm-like bodies MS + 3 % sucrose + 0.7 % agar + BAP + Picloram caffeic acid, ferulic acid and p-coumaric acid phenolic, alkaloid, flavonoid compounds (Paul and Kumaria, 2020)
47 Alkaloids Rauwolfia
tetraphylla L. 		leaf, stem, and root derived callus MS + 2, 4-D2.0 mg/L tryptophan reserpine (Rohela et al., 2021)
48 Phenylpropanoid Rhodiola rosea L. leaf derived callus MS + 4.5 g/l agar + 30 g/l sucrose + 1 mg/l NAA + 0.5 mg/l BAP tyramine, 4-hydroxyphenylpyruvate and tyrosol salidroside (Mirmazloum et al., 2019)
49 Flavonoid Moringa oleifera leaf and stem derived callus MS + 1.0 mg/L BAP + 1.5 mg/L IBA Phenylalanine Niazirin, Benzylcarbamate, Vincosamide (Mahood et al., 2018)
50 Coumarins,glucosinolates, phenols, flavonoids Nasturtium officinale microshoots MS (solid) + 3 % (w/v) sucrose + 1 mg/L BA + 1 mg/L NAA L-phenylalanine, L-tryptophan coumaric, ferulic acids, rutoside, and glucosinolates (Klimek-Szczykutowicz et al., 2021)
51 Organosulfur Allium sativum L. crown derived callus MS (liquid) + 0.3 mg/L 2.4 D and 0.5 mg/L kin glutathione allicin, ajoene, alliin, dithiin groups, and allyl sulfide groups (Setiowati et al., 2022)
52 Indole terpenoid alkaloids (TIAs) Catharanthus roseus L. leaves derived callus ½ B5 medium (Gamborg's B5) + sucrose 2 % (w/v) L-phenylalanine, L-tyrosine vincristine, vinblastine (Vu et al., 2022)
53 Alkaloid Mitragyna speciosa petioles and leaves derived callus WPM + 4 mg/L 2,4-D tryptophan and loganin mitragynine (Mohamad Zuldin et al., 2013)
54 Amino acids Mucuna prurita leaf derived callus 20 ml MS (liquid) + IAA (11.41 μM) and BAP (0.88 μM) L-tyrosine L-Dopa (Raghavendra et al., 2018)
55 Indole alkaloids Vinca minor leaf derived HR ¼ Gamborg's B5 (liquid) shikimate, tryptophan, tryptamine, loganin, and secologanin vincamine (Verma et al., 2014b)
56 Alkaloid Corylus avellana L. cotyledons derived callus MS (liquid) + 3 % sucrose/lactose/fructose phenylalanine and vanadyl sulfate Paclitaxel (Rahpeyma et al., 2015)

The type of compounds produced in these 56 studies were diverse, including phenolic compounds (14), followed by alkaloids (13), flavonoids (9), terpenoids (8), amino acids (4), phenylpropanoids (3), steroids (3), esters (2), stilbenes (2), steroidal lactones (1), polyprenylated acylphloroglucinol (1), coumestan (1), lignan (1), isoflavones (1), naphthoquinone (1), furanocoumarins (1), anthraquinones (1), and vitamins (1). As for the type of media used, 47 studies employed suspension culture with liquid medium, and 9 studies used solid agar medium in the precursor feeding process, as seen in studies by Javid et al. (2021), Panwar and Guru (2015), Dewanjee et al. (2014), Mohammadparast et al. (2015), Chetri et al. (2016), Królicka et al. (2008), Otari et al. (2023), Mirmazloum et al. (2019), and Mahood et al. (2018).

The types of precursor compounds used were also varied, with seven different categories: amino acids (9), sterols (6), phenolic compounds (9), shikimic pathway compounds (5), intermediates in metabolic pathways (4), other nitrogenous compounds (4), and other organic compounds (2) (Suppl. 1). Meanwhile, some types of explants have been employed. Callus induced from different parts of the plant was used in 32 studies, while 5 studies used the hairy root method, 18 studies used direct organ culture, and 1 study used protocorm-like bodies (Table 1). For the culture medium, 50 studies used Murashige and Skoog (MS) supplemented also with PGR, while 2 studies used WPM medium, 2 used Gamborg’s B5 medium, and 1 used Hoagland medium (Table 1). The general step by step technique used in in vitro precursor feeding is briefly described in Fig. 3.

General step by step technique used in in vitro precursor feeding and elicitor addition for enhancing plant secondary metabolites. 1–2) Plant parts commonly used as explants in culture include leaves, apical and axillary meristem shoots, cotyledons, hypocotyls, and meristematic roots. 3–4) Culture can be performed through indirect organogenesis by inducing callus growth, or directly by growing specific organs such as hairy roots, adventitious roots, and protoplasts. 5) The culture type can be done using suspension methods or growth on solid agar medium. 6–7) Precursor feeding compounds and elicitor molecules can be added to the culture medium. 8) Changes in phytochemical composition can be analyzed using spectrometry techniques and desired compounds can be isolated to obtain pure compounds.
Fig. 3
General step by step technique used in in vitro precursor feeding and elicitor addition for enhancing plant secondary metabolites. 1–2) Plant parts commonly used as explants in culture include leaves, apical and axillary meristem shoots, cotyledons, hypocotyls, and meristematic roots. 3–4) Culture can be performed through indirect organogenesis by inducing callus growth, or directly by growing specific organs such as hairy roots, adventitious roots, and protoplasts. 5) The culture type can be done using suspension methods or growth on solid agar medium. 6–7) Precursor feeding compounds and elicitor molecules can be added to the culture medium. 8) Changes in phytochemical composition can be analyzed using spectrometry techniques and desired compounds can be isolated to obtain pure compounds.

Precursor feeding enhances the production of secondary metabolites in in vitro cultures by supplying essential building blocks directly into the plant's biosynthetic pathways (Cheng et al., 2024). This increased availability of precursors boosts the metabolic flux toward desired compounds, upregulates key biosynthetic enzymes, and alleviates bottlenecks in the pathways. As a result, there is a significant enhancement in the yield of targeted secondary metabolites (Lu et al., 2022 ). Furthermore, precursor feeding treatments can be combined with elicitor compounds. Both can have a positive effect on cell growth and the production of secondary metabolite compounds (Rakesh and Praveen, 2022).

Different precursors have distinct mechanisms for entering the plant cells. Many of them are facilitated by transporters. The AAT (Amino Acid Transporter) plays a role in introducing amino acid precursor compounds (Dong et al., 2024). The ABC (ATP-binding cassette) transporter is involved in introducing lipid-derived compounds and organic compounds that are intermediates in secondary metabolite biosynthesis pathways (Kang et al., 2011). Other precursor molecules can enter the cell through passive diffusion. In addition to serving as building blocks in phytochemical biosynthesis, precursors may also influence the expression of growth genes and hormones (Inyai et al., 2021), resulting in increased biomass growth, which correlates with a higher production of secondary metabolite compounds. For elicitors, their interaction with cell receptors triggers a defence response by activating genes encoding secondary metabolite compounds (Namdeo, 2007; Rakesh and Praveen, 2022). The combined use of precursor feeding, and elicitors can yield optimal results in the production of secondary metabolite compounds (Fig. 4).

Predicted mechanism of intake and the role of precursor feeding and elicitor compounds in plant cells. Various types of precursor additive compounds can enter the cell through different pathways, including Amino Acid Transporters (AATs) such as LHT (Lysine-Histidine Transporters), AAP (Amino Acid Permease), and POT (Proton-dependent Oligopeptide Transporters). Additionally, precursors can enter the cell via ABC Transporters, facilitated diffusion, and passive diffusion. Meanwhile, elicitors interact directly with receptors or target molecules. Both applications influence the regulation of growth genes and the expression of secondary metabolite genes. The synergy between these two applications can enhance the production of desired phytochemicals.
Fig. 4
Predicted mechanism of intake and the role of precursor feeding and elicitor compounds in plant cells. Various types of precursor additive compounds can enter the cell through different pathways, including Amino Acid Transporters (AATs) such as LHT (Lysine-Histidine Transporters), AAP (Amino Acid Permease), and POT (Proton-dependent Oligopeptide Transporters). Additionally, precursors can enter the cell via ABC Transporters, facilitated diffusion, and passive diffusion. Meanwhile, elicitors interact directly with receptors or target molecules. Both applications influence the regulation of growth genes and the expression of secondary metabolite genes. The synergy between these two applications can enhance the production of desired phytochemicals.

3

3 Potential applications of plant secondary metabolites

Plant phytochemicals are typically classified into three main groups based on their biosynthetic pathways: terpenes, phenolics, and alkaloids (Bourgaud et al., 2001). These three clusters of phytochemicals have a very diverse range of applications, especially in the fields of pharmaceuticals, agriculture, the food industry, and others. Many reports explain the biological effects of phytochemical groups. This section provides a summary of the applications of plant phytochemicals.

Phenolic compounds, including flavonoids, are often recommended for inclusion in dietary supplements and nutraceuticals due to their perceived significance in the human diet. Apart from their role as antioxidants, these compounds possess a diverse range of biological capabilities (Pengfei et al., 2009). For instance, several research has shown that phenolic compounds have ability as anticancer (Tavsan and Kayali, 2019; Rosa et al., 2018), microcirculation-improving (Mastantuono et al., 2018), antihypertensive (Hou et al., 2012), hypo-lipidemic agents (Bae et al., 2014), and anti-inflammatory agents (Jang et al., 2020). They have also demonstrated potential as natural colorants (Rose et al., 2018) and active ingredients in the cosmetic industry (Chiocchio et al., 2021; Lianza et al., 2020; Shin et al., 2013).

Phenolic acids, which naturally occur in fruits and vegetables, encompass a diverse array of bioactivities, including antihyperglycemic (Sanchez et al., 2017), neuroprotective (Zaitone et al., 2019), antihypertensive (Agunloye et al., 2019), antidepressant (Barauna et al., 2018), anti-inflammatory (Zaitone et al., 2019), anticancer, and antidiarrheal (Frauches et al., 2016). In the veterinary area, tannins are employed as anthelmintic and antibacterial agents (del Carmen Acevedo-Ramirez et al., 2019; Redondo et al., 2014). Additionally, their tanning properties make phenolic acids useful in the hide industry (Mutiar et al., 2019). Nonetheless, caution is warranted when using tannins, as alongside their health-promoting attributes, certain detrimental effects have been observed (Labieniec and Gabryelak, 2003). Even though tannin-rich substances are frequently used in bovine feed, little is known about how hydrolyzable tannins affect the ruminant intestinal bacteria. It is well-recognized that several metabolites generated from hydrolyzable tannins, such as pyrogallol, have negative effects on the host animal's intestinal microorganism (Lotfi, 2020).

Terpenes are employed as medicines, food supplements, flavors, scents, biopesticides, and other things thanks to their broad spectrum of bioactivities in the cosmetic, food, cosmetic, pharmaceutical, agriculture, and perfumery (Chiocchio et al., 2021). Within the pharmaceutical industry, terpenes play a crucial role as therapeutic factors with a wide array of bioactivities. These include chemo-preventive, antifungal, anti-inflammatory, antimicrobial, antihyperglycemic, antiviral, analgesic, and antiparasitic activities (Anthoni et al., 2006; Hammer et al., 2003; Jesch and Carr, 2017). Terpenes are also utilized as excipients in the pharmaceutical industry to optimize the intake of active ingredients across the skin (Fox et al., 2011).

This family of chemicals is crucial for food, aromatherapy, and fragrances due to the unique scent of many monoterpenes, which are mostly found in aromatic plant essential oils. Monoterpenes derived from essential oils, including carvacrol, thymene, and thymol (commonly found in plants of the Lamiaceae family), possess not only aromatic properties but also a diverse array of biological activities. For example, they have the potential to treat diseases of the nervous, cardiovascular, and respiratory systems as well as act as antimicrobial and antioxidant agents (Salehi et al., 2018).

Furthermore, diterpenes of the labdane type are valuable in the industry of perfumes and serve as fixative agents in premium fragrances. Essentially, a fixative agent is a low-volatility substance that prolongs the longevity of a fragrance in perfumes, provides a lasting aroma, and enables harmonious blending with other components. Resin gathered from C. ladanifer (a member of the Cistaceae family) is a significant origin of labdanum-type diterpenes utilized as a fixing agent in perfumery (Raimundo et al., 2018).

Saponins, belonging to the subclass of terpenoids, hold immense significance in the food industry. Processed foods like desserts, ice creams, baked goods, sauces, and beverages often incorporate dispersions such as emulsions and foams. These dispersions play a vital role in defining, stabilizing, and managing the consistency and flow-related characteristics of these products. Saponins, because of their amphiphilic characteristics, have demonstrated the capability to maintain the stabilization of emulsions in food. Importantly, they exhibit lower sensitivity to factors such as ionic concentration, pH, and elevated temperatures (approximately 90 °C) in comparison to the emulsifiers presently utilized (McClements & Gumus, 2016).

Additionally, saponins demonstrated several biological actions crucial for human healthcare at modest doses (del Hierro et al., 2018; Rehan et al., 2020; Singh et al., 2017). The cholesterol-lowering, anticancer, and antiviral characteristics are the most pertinent among them (Jesch and Carr, 2017; Marrelli et al., 2016; Vinarova et al., 2015; Zhao et al., 2008). Boswellic and betulinic acids are two examples of the medicinal potential of triterpenic saponins. Betulinic acid exhibits a broad spectrum of biological functions, notably displaying potent antiviral effects (Alakurtti et al., 2006; Singh and Sharma, 2015). Another compound derived from the resin of incense trees (Banksia serrata Roxb.), boswellic acid, is utilized as an anti-inflammatory drug (Anthoni et al., 2006). Furthermore, clinical tests utilizing the gum resin from B. serrata have shown a decrease in signs among patients coping with rheumatoid arthritis and osteoarthritis (Poeckel et al., 2005, 2006).

Saponins are antibacterial substances that are effective against fungi and bacteria that attack plants (Hoagland et al., 1996; Moses et al., 2014). The probable mechanism underlying these effects is the capability of saponins to interact with sterols found in bacterial membranes, resulting in membrane disruption (Augustin et al., 2011; Sreij et al., 2019). Along with their allelopathic effects on certain plant species, saponins also have insecticidal, and molluscicidal effects (Nielsen et al., 2010; Huang et al., 2003). Saponins possess potential as natural biopesticides for agricultural applications because of their inherent characteristics and biological role in plant defense. For instance, Trdá et al. (2019) reported that a specific saponin called aescin not only exhibits antifungal properties against crop diseases but also can stimulate the immunity of B. napus and A. thaliana against fungus and bacteria, due to aescin contribution in salicylic acid-dependent resistance.

Alkaloids boast a longstanding history of utilization in medicinal applications and continue to play a crucial role in modern medicine (Newman and Cragg, 2020). An example of their significance is morphine derived from Papaver somniferum, which stands as among the most widely utilized pain relievers today. In their overview (Debnath et al., 2018), Debnath et al. listed the biological features of the major subclasses of alkaloids. Examples include the historically significant antimalarial drugs quinidine and quinine, which are produced from the bark of C. officinalis L. Another notable alkaloid is an adrenergic amine sourced from plants within the Ephedra genus (Ephedraceae family), ephedrine. Ephedrine finds application in various drug formulations, serving as respiratory dilators for individuals with asthma and allergy issues, as well to prevent low blood pressure during intrathecal anesthesia (Ma et al., 2007). Additionally, the Apocynaceae plant C. roseus (L.) is a significant source of anticancer drugs, specifically vinblastine and vincristine (Ronghe et al., 2001).

Other alkaloids have garnered attention for their potential bioactivity. For instance, a tropane alkaloid named catuabine present in the bark of T. catigua A. Juss., demonstrated effects similar to an antidepressant in a rodent depression model (Campos et al., 2005). Another compound, berberine, found in the stem bark and roots of various Berberidaceae, has displayed anti-diabetic effects in rodent models of insulin resistance induction (Turner et al., 2008). Moreover, berberine showcases a range of bioactivities including antidepressant, anti-inflammatory, antioxidant, hepatoprotective, antihypertensive, and anti-cancer features (Amritpal et al., 2010).

Additionally, certain alkaloids are vital in spices and well-known drinks because they have psychoactive characteristics, such as the caffeine found in coffee, which plays a part in social and ceremonial events (Crozier et al., 2006). Coffea arabica L. and Camelia sinensis (L.) are the two most significant biological sources. Caffeine is classified as a methylxanthine alkaloid. It finds applications in various domains, including cosmetics, analgesics, anti-cold medications, and slimming products.

Numerous alkaloids also exhibit insecticidal and fungicidal properties, aligning with their protective function (Liu et al., 2012; Yang et al., 2002). For instance, the phytopathogen Puccinia recondita is highly vulnerable to the potent fungicidal activity of the piperidine alkaloid pipernonaline. This alkaloid is purified from the hexane fraction of P. longum (Yogendra et al., 2017). P. grisea, E. graminis, B. cinerea, P. recondita, R. solani, and P. infestans, are among the phytopathogens that the Coptis japonica Makino extracts (berberine chloride, coptisine chloride, palmatine iodide, and isoquinoline alkaloids,) and its alkaloid content exhibit fungicidal activity against in an in vivo plant model (Ju-Hyun, 2005).

4

4 Prospects and limitations of in vitro precursor feeding methods for boosting the production of plant secondary metabolites

We have successfully compiled and analyzed the precursor feeding studies to elevate the biosynthesis of various types of PSMs via in vitro. These data unequivocally demonstrate the positive impact of incorporating diverse feeding precursors on the synthesis of secondary metabolite compounds. The strategic utilization of amino acids (organic chemicals) to the culture media, has been validated to be an effective method for augmenting the in vitro production of several therapeutic plant byproduct compounds. Notably, in Nasturtium officinale microshoot cultures, amino acid supplementation, including L-Phenylalanine and L-Tryptophan, resulted in heightened production of coumaric acid, ferulic acid, rutoside, and glucosinolates (Klimek-Szczykutowicz et al., 2021). Furthermore, the leaf callus of Hypericum perforatum, when cultivated in a growth medium enriched with 200 mM/L L-tyrosine, demonstrated a substantial enhancement in the production of L-Dopa, increasing by 36.36 folds (Raghavendra et al., 2018).

At times, the introduction of a precursor of biosynthetic to the growth media can be used to stimulate the formation of metabolites without impeding biomass accumulation. However, there are instances where the use of biosynthetic precursors alone might not lead to high yield of bioactive compounds in in vitro cultures. In such cases, employing a combination of biosynthetic precursors with enhancement techniques may offer a workaround. This approach is particularly useful when there is a scarcity of the precursor or constraints in its absorption by the cells from the culture medium, and integration into metabolic pathways (Isah et al., 2018).

To achieve the necessary productivity, it is essential to employ various augmentation strategies. One of the most frequently utilized methods is synergistic precursor and elicitor application. This strategy has efficiently proven in boosting the accumulation of PSMs. For instance, in callus cultures originating from leaves of Cassia augustifolia, combining elicitation with precursor addition yielded in a notable increase in the production of sennoside A and B (Chetri et al., 2016). Similarly, the incorporation of both elicitation and precursor feeding into cell suspension cultivation of Mucuna pruriens resulted in a noteworthy increase in the production of L-Dopa (Raghavendra et al., 2011). Notably, in this context, precursor feeding demonstrated greater success in boosting production compared to elicitation.

Given that many of the biochemical processes involved are cross-linked in cells, in certain cases a precursor might demonstrate an impact on the creation of more than one pathway of certain biochemical. For instances, Glutathione addition in the culture media of Allium sativum L. promoted allyl sulfide group (allyl methyl disulfide; allyl propyl disulfide; 1-propenyl allyl disulfide; 2-propenyls-1-propenyl disulfide; allyl trisulfide; diallyl heptasulfide; allyl methyl trisulfide;) and allicin, alliin, dithiin, ajoene, groups (3-vinyl 1,2-dithiin; 2-vinyl 1,3-dithiin) (Setiowati et al., 2022). The synthesis of asiatic acid, madecassoside, asiaticoside, and madecassic acid was enhanced by adding squalene to the Centella asiatica calli culture media (Baek et al., 2020). In cell cultures of Solanum lyratum, the production of insolasodine, solasonidine, and solanine was significantly boosted when exogenous sterols like cholesterol, stigmasterol, or mixed sterols were introduced (Lee et al., 2007). However, there was no influence on the growth of biomass. Precursor feeding strategy in plant in-vitro culture hold promising prospects and provide a variety of possible uses and advantages. These techniques have a lot of promise for several companies and fields of study. The following are some significant prospects of the precursor feeding application:

4.1

4.1 Enhanced phytochemical production

Combined in vitro plant propagation and precursor feeding enables the targeted and regulated augmentation of secondary metabolite synthesis. This is especially useful for getting larger yields of certain bioactive compounds, such as phenolics, flavonoids, and alkaloids that exhibit drug-related applications, nutraceutical, and industrial industries. Numerous studies previously indicated that adding precursors to the growth medium yields in a rise in the formation of secondary metabolite chemicals. The production of mulberroside A compounds with addition of L-Phenylalanine and L-Tyrosine, the increase in L-Dopa compounds with L-Tyrosine precursors, and the production of Silymarin compounds with L-Phenylalanine precursors are a few examples (Hassanen et al., 2021; Pongkitwitoon et al., 2020; Raghavendra et al., 2018).

4.2

4.2 Applications in pharmacology

Many secondary metabolites made by plants because of feeding on precursors have pharmacological characteristics. These metabolites can be further isolated and used as a source of chemicals for the synthesis of new medications or for the creation of innovative pharmaceuticals. A regulated method to maximize the generation of these beneficial chemicals is precursor feeding. Most of the secondary metabolite compounds are intended for application as medicines. Examples of applications of precursor feeding to increase compound production are vincristine, vinblastine, Paclitaxel, reserpine, silymarin, and salidroside, which have implications for cancer therapy drugs (vincristine and vinblastine), chemotherapy agents, hypertension, hepatoprotective, nerve protection (Mirmazloum et al., 2019; Rahpeyma et al., 2015; Rohela et al., 2021; Vu et al., 2022).

4.3

4.3 Nutraceutical and functional food development

By feeding precursors, secondary metabolites can be used to optimize the nutritional and functional qualities of food items. Bioactive ingredients can be added to functional meals or dietary supplements to promote health and wellness while addressing a variety of medical issues. An example of a compound that is useful in the food and beverage industry is 2-hydroxy-4-methoxybenzaldehyde (2H4MB) with the addition of ferulic acid as a precursor (Matam et al., 2017).

4.4

4.4 Crop improvement and stress tolerance

Utilizing precursor feeding for enhancing the synthesis of plant phytochemicals involved in stress response can aid plants development, increasing their tolerance to environmental stressors and pests. This has potential implications for improving crop resilience and productivity, especially to faceclimate change and changing environmental conditions. Rotenoids and solanine are examples of secondary metabolite compounds that can be utilized as fungicides and pesticides. These compounds can be produced by adding feeding precursors such as phenylalanine and methionine and exogenous sterols (cholesterol, stigmasterol) respectively (Lee et al., 2007; Vu et al., 2022).

4.5

4.5 Flavour and fragrance industries

Specific phytochemicals are needed for the industries of flavour and fragrance. The creation of certain compounds contributing to the flavour and scent of various goods, such as perfumes, essential oils, drinks, and food products, can be optimized using precursor feeding. An example of the application of precursor feeding is the vanillin compounds, 2H4MB, and Vanillic acid production, coumarins with ferulic acid, L-phenylalanine, and L-tyrosine as the precursors (Klimek-Szczykutowicz et al., 2021; Matam et al., 2017).

4.6

4.6 Bioremediation and environmental applications

The capacity to detoxify or break down contaminants exists in several secondary metabolites. These metabolites, which might be used in phytoremediation or bioremediation techniques to clean up polluted settings, can be stimulated by precursor feeding in plant cultures. For example, bacteria that can degrade Polychlorinated Biphenyls (PCBs) utilize coumarin and myricetin as carbon sources (Klimek-Szczykutowicz et al., 2021; Singer et al., 2003). In these cases, myricetin demonstrated the highest efficacy in PCB degradation. Burkholderia cepacia LB400, for instance, was able to degrade 16 out of 19 tested congeners using myricetin, while coumarin led to the degradation of 13 congeners by Corynebacterium sp. MB1, surpassing the efficacy of PCB degradation (Donnelly et al., 1994).

4.7

4.7 Customized metabolite profiles

The reaction mixture and concentration of the precursors can be changed to create specific metabolite profiles using precursor feeding techniques. The focused creation of metabolites following industrial, or research requirements is made possible by this level of customization. for example, according to Kiong et al., (Kiong et al., 2005), the addition of Farnesyl pyrophosphate (FPP), squalene, leucine, and Isopentenyl pyrophosphate (IPP) into Centella asiatica cell culture medium, can enhance triterpenoids productions.

Precursor feeding techniques in plant in-vitro culture provide a lot of benefits, but they also have certain drawbacks and difficulties that need to be considered before using them. The primary limitations of the precursor feeding strategy employed in plant in vitro culture are as follows:

The metabolic processes involved in the synthesis of phytochemicals are often complicated and may vary among different plant species, organs, and even individual plants. Comparing plant metabolic networks to those of other living things, they are considerably complex. This is caused by several interconnected characteristics of plant life, including its sessileness, ectothermic, and autotrophic nature, as well as its extensive chemical repertory and high levels of subcellular compartmentation (Allen et al., 2009). Moreover, the required secondary metabolites may not always be produced in high or consistent quantities because of precursor feeding (Palacio et al., 2011; Srivastava & Srivastava, 2014). The production and quality of the phytochemicals are affected by a broad spectrum of elements, including the stage of plant development, culture conditions, and genetic diversity (Verma and Shukla, 2015). Therefore, studies of gene expressions involved in secondary metabolite biosynthesis are also important (Jadid et al., 2016). Due to this intricacy and diversity, precursor feeding may not always provide predictable results.

Additionally, secondary metabolite biosynthesis pathways and the involved enzymes are poorly understood. The accurate implementation of precursor feeding techniques, and the optimization of results may be hampered by this lack of understanding. Metabolic pathway studies use a combination of several approaches such as: metabolic and bioinformatics pathway databases (e.g., metaCyc, cathaCyc, and KEGG), metabolite identification (GC–MS, HPLC–MS, and NMR), RNA-seq, Metabolic Flux Analysis (MFA), and structural interaction study of several enzymes implying in secondary metabolite pathways can help in studying phytochemicals pathways (Caspi et al., 2020; Jadid et al., 2024b; Marguerat and Bähler, 2010; Shih and Morgan, 2020; P. Zhang et al., 2005).

The production of a variety of phytochemicals by plant cells in response to precursor feeding might make it difficult to target the biosynthesis of a particular molecule. The targeted metabolite may not be a single reaction, resulting in a variety of chemicals in the culture. Several strategies such as optimizing co-culture condition, substrate channeling, CRISPR-Cas9 genome editing, and inhibiting undesired pathways with artificial microRNA, can be utilized for enhancing the specificity of precursor feeding (Endo et al., 2019; Hidalgo et al., 2017; Marchev et al., 2020; Y. Zhang and Fernie, 2021).

Using high precursor concentrations can be perilous for plant cells cultivated in vitro, potentially leading to cell death or reduced growth rates. Striking a delicate balance is crucial to optimize metabolite synthesis while mitigating potential cell damage caused by high precursor concentrations. An illustration of this intricate balance is evident in the use of sodium acetate as a precursor for Azadirachtin production in the hairy root culture of Azadirachta indica. Studies suggest that the inclusion of sodium acetate in the medium can potentially damage plant cells and trigger the release of oligogalacturonides from the plant cell (Srivastava and Srivastava, 2014). This signaling can subsequently trigger an upregulation in phytoalexin production, which are defensive-related secondary metabolites (Davis et al., 1986). In a different investigation by Palacio et al., (2011), focusing on nordihydroguaiaretic acid (NDGA) production from Larrea divaricata, the supplementation of l-phenylalanine at varying concentrations (3, 1, and 0.5 mM) resulted in a notable increase in NDGA levels, reaching of up to 190.53 ± 19.50, 285.23 ± 28.44, and 301.35 ± 1.19 μg/g DW, respectively. However, the introduction of 0.5 μM cinnamic acid promoted cell propagation but did not influence NDGA accumulation. On the other hand, when cinnamic acid (at 1 and 1.5 μM), ferulic acid (at 0.1, 0.5, and 1 mM), and sinapic acid (at 0.1, 0.5, and 1 mM) was supplemented, the medium became excessively toxic, leading to suppression of phenylpropanoid and NDGA production because of the high toxicity levels.

The cost corelated with obtaining and incorporating purified precursors into the culture medium can be relatively high, especially for specialized or rare precursors. This cost factor can significantly impact the economic feasibility and scalability of precursor feeding methods, particularly for mass production. To optimize the production of specific compounds in the culture system, it is essential to establish procedures that maximize the use of a precursor, especially when it is the most cost-effective byproduct of other processes (Isah et al., 2018). In modern scale-up production studies, there is a notable shift towards focusing on cell suspension cultures rather than differentiated tissues such as hairy roots and somatic embryos, which are used less frequently (Ferrie, 2010; Ziv, 2010). This change is probably attributed to the benefits of cell suspension cultures in comparison to tissue and organ cultures, particularly in terms of simplicity, predictability, and ease of extracting metabolites from the biomass or growth medium (Park and Paek, 2014). Furthermore, the biosynthesis of plant chemicals can be scaled up by employing a combination of various methods, including cell permeabilization, immobilization, and elicitation (Choi et al., 1995).

Beside of many advantageous of the in vitro precursor feeding for accelerating the quantity of PSMs, these techniques also genetic instability of the plant and production of the targeted compounds as well as cytotoxicity. Genetic instability in tissue cultures over extended periods often detrimental. Plant cells grown in vitro are often subject to somaclonal variation, leading to genetic mutations and altered metabolic profiles, reducing the reliability and consistency of metabolite production (Kang et al., 2011). Additionally, precursor uptake and transport can be also inefficient due to mechanic barriers of the plant like cell walls or the absence of specific transporters. This will end up with an inefficient uptake of the precursors into the cells. Moreover, high concentrations of precursors can induce stress or cytotoxicity, leading to cellular damage or abnormal metabolic responses. Therefore, suitable concentration of the precursors, re-checking the metabolic profile and additional genetic fidelity confirmation using molecular markers such as random Amplified Polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) should also be performed (Andriyani and Jadid, 2021).

5

5 Conclusion

In summary, precursor feeding stands as a promising in-vitro culture technique, offering a scalable and sustainable approach for producing secondary metabolites without relying on traditional agricultural processes. It provides a flexible and effective strategy for controlling and enhancing the production of valuable phytochemicals. These applications present intriguing subjects for study and advancement in contemporary plant biotechnology, with far-reaching implications for various industries and research domains. To entirely harness the potential of precursor feeding strategies and optimize the formation of phytochemicals in plant in-vitro cultures, a deep understanding of the involved constraints is required. Enhancing the efficacy and efficiency of secondary metabolite synthesis necessitates focused attention on these aspects. Integration between precursor feeding and cutting-edge biotechnological methods like synthetic biology and metabolic engineering shows potential for substantially enhancing the precision and efficiency of phytochemical synthesis in plant cultures. However, it's important to acknowledge the existing limitations that affect the production of PSMs through this method including potential genetic instability, cytotoxicity and altered metabolic profile. Therefore, additional molecular-based approaches including RAPD, ISSR and ScoT as well as metabolomic assessment should also performed to complement the results of the study. Finally, continued research and progress in biotechnology have the potential to surmount these challenges, offering opportunities to enhance the application of precursor feeding for optimized phytochemical synthesis in plants.

CRediT authorship contribution statement

Muhammad Rifqi Nur Ramadani: Data curation, Visualization, Writing – original draft. Nurul Jadid: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.

Acknowledgments

The authors gratefully acknowledge the Ministry of education, culture, research and technology of the Republic of Indonesia for financially supporting this project through fundamental research scheme No. 1770/PKS/ITS/2024.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2024.106018.

Appendix A

Supplementary data

The following are the Supplementary data to this article:

Supplementary Data 1

Supplementary Data 1


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