INTRODUCTION Plant tissue culture is also referred to as culturing of cells or tissues under sterile conditions to propagate different plant species and conserve their genetic resources on a large scale. However, many plants cannot successfully propagate using standard plant tissue culture techniques and require complex combinations of different media and hormones to improve in-vitro propagation. This will provide better and more efficient growth in plants. Several studies showed the potential of different hormones to achieve successful plant propagation. This compilation of studies illustrated the use of phloroglucinol and melatonin acting as growth-promoting regulators in plants (Table 1 and 2), as an efficient way to enhance the growth of plants under lab conditions. This research will also provide reasonable solutions for certain problems that restrict the current plant sciences research such as promoting secondary metabolite production, genetic conservation, and transformation studies. Significance and use of Phloroglucinol in plant tissue culture. The compound Phloroglucinol (PG) has been used as an additional supplement with other plant growth regulators, yet the growth-promoting properties of this hormone in plants is still under. Phloroglucinol also known as 1,3,5-trihydroxy benzene or phloroglucin is a naturally occurring secondary metabolite extensively present in various plant families like Lauraceae, Cannabinacaeae, Rosaceae and some marine plants such as brown alga species (da Silva et al., 2013). In the medicinal and pharmaceutical sector, PG possesses antibacterial, antioxidant, and anti-inflammatory bioactivities including anti-ulcer and cytotoxic effects against tumor cell lines (Abdel et al., 2016; Singh et al., 2009, Singh and Sharma 2020). This compound was isolated from organic waste such as lemon peels and showed bactericidal properties against oral bacteria like Streptococcus mutans, and Porphyromonas gingivalis causing period on it is in the mouth which gives an edge in evaluating PG as an antimicrobial agent and exploring its use for plants. Moreover, various studies in plant tissue culture illustrated hyper-hydricity and poor recovery of cryopreserved tissue that gave negative results (da Silva et al., 2013). Hyper-hydric stress illustrates different biochemical activities showing the oxidative stress and morphological abnormalities in the plant (da Silva et al., 2013). This hyper-hydric or humid environment in plant is linked to low production of enzymes responsible for lignin production. Hence, addition of phloroglucinol in the media can resolve hyper-hydricity by increasing the production of the enzymes and hence, increasing lignin synthesis in plants (Phan and Hegedus, 1986). Phloroglucinol with other growth hormones like IAA, resulted in 2 times growth of new rootsin the sugarcane plant and later, in the ex-vitro acclimatization phase, results recorded the formation of root hairs and high survival rate percentage (Gómez-Kosky et al., 2021). Various researches have shown the use of PG in propagation of plants with in vitro tissue culturing of J. curcas where PG added with IBA promoted the growth frequency of roots up to 76.7% (Kumar et al., 2010). PG in combination with IBA promoted root induction and with BA enhanced the shoot induction (Petti, 2020). Furthermore, regeneration from nodal plants of W. coagulans Dunal evaluated supplementation of 3.9uM PG with 2.2uM BA and 2.3uM Kn in MS media, which enhanced the propagation of nodal plants within the shoot region. In addition, a two-step method was used with pulse treatment for elongation of roots where 71.6uM choline chloride (CC) and 3.9 uM PG was given. The roots were transferred to the media and showed enhanced roots elongation (Jain et al., 2011). Studies also show the association of phloroglucinol with other growth hormones to enhance root and shoot induction to multiple folds (Gómez-Kosky et al., 2021; Kumar et al., 2010; Petti, 2020; Jain et al., 2011). The effect of PG on direct rooting of apple rootstocks with different concentrations of PG were evaluated for 0, 0.5, 1.0 and 2 mg•L−1 concentrations respectively. Induction of a low concentration of phloroglucinol in root media resulted in early rooting with an 80% high rooting percentage after the third week of culturing whereas high concentrations of phloroglucinol up to 1 mM induced rooting up to 100% from the second week of culturing (Kim et al., 2020). The broader applications of the use of Phloroglucinol are very limiting and yet to be explored as there are several drawbacks of using a high concentration of PG in the media where higher concentrations up to 2mM resulted in reduced rooting efficiency of 80%. Several studies also illustrated PG as a sterilizing agent in tissue culture media that will sterilize the media without the process of autoclaving (Cardoso and Teixeira da Silva 2012). Significance and use of Melatonin in plant tissue culture. Melatonin (N-acetyl-methoxy tryptamine) is a ubiquitous molecule having pleiotropic actions with some multi-functional properties like growth and development in plants including root architecture, leaf senescence, seed germination, fruit ripening, flowering time, and biomass production, and also fights oxidative stresses such as reactive oxygen species and NO to provide tolerance to the plants. Melatonin was first discovered in the bovine pineal gland in the year 1958 and was known for its role as a neurotransmitter in animals (Bose and Howlader 2020). Melatonin contributes to regulating many physiological events such as cardiac rhythms, sleep, body temperature, mood, appetite, immunological systems in animals, and retina physiology in animals (Arnao and Hernández 2015; Tousi et al., 2020). It was until 1995 that melatonin was discovered in higher plants by by Dubbels et al. (1995) and Hattori et al. (1995) and was called Phyto-melatonin (Arnao and Hernández 2015; Liu et al., 2020). This discovery led to the growing study of this hormone in plants and its presence was reported in several variety vegetables, fruits, seeds, cereals, and medicinal herbs (Paredes et al., 2009). Further research on melatonin was focused to determine the physiological role in plants and as a result, melatonin was reported as a growth promoter and rooting agent followed by strong shreds of evidence (Zhang et al., 2015). Apart from growth and development processes, melatonin was also seen to participate in biotic and abiotic response regulation in plants (Zhang and Zhang 2021). Melatonin being a plant growth regulator has come forward as a bio stimulant of choice for an eco-friendly and safe method to boost plant resistance against severe stress conditions. This is because the plant’s natural defense system fails to give adequate protection against extreme conditions (Sharma et al., 2020). The amount of phyto-melatonin varies which makes extraction and quantification techniques difficult in different parts of the same plant (Ghosh, 2021). Several studies provided pieces of evidence suggesting that plants just do not synthesize their melatonin but also it can be stored in plant’s certain parts such as fruits, dry seeds, etc. Melatonin is synthesized in intracellular organelles such as mitochondria and chloroplast. Regulation of circadian time management is one common function of melatonin in plants and animals (Reiter et al., 2015). Melatonin has a very significant influence on improving root regeneration and growth and it has been observed that at low concentrations it promotes root growth whereas at high concentrations it inhibits the root growth (Liu et al., 2022). Exogenous melatonin as a growth stimulator shows a similar role as auxin as they share tryptophan as a precursor (Bhattacharya and Jha 2020). In horticulture, melatonin alleviates damages due to cold stress in many plants, one such example is tea, where melatonin enhanced cold stress tolerance by promoting redox homeostasis and anti-oxidant defenses in plants (Li et al., 2018). APPLICATIONSOF PHLOROGLUCINOL IN PLANT TISSUE CULTURE Hyper-Hydricity. In plant tissue culture, a problem arises of accumulation of excess water in which plant roots result in poor development and growth. This phenomenon is termed hyper-hydricity and is linked with reduced production of several enzymes responsible for the synthesis of lignin precursors and their polymerization due to humid environment, high illuminance, and high PGR levels (Kevers et al., 2004). This causes stress conditions in plants which leads to several morphological changes in plants. These conditions develop several abnormalities in the plant such as a reduction in lignin oxidative stress (Rogers and Campbell 2004). The possible solution to this problem can be controlling the PGR levels or light conditions and changing the gelling agents. Different enzymes like CoA ligase resulted in lesser levels and expressions in hyper-hydric tissues and explants. When phloridizin and PG were added to media for Malus domestica and Helianthus resulted in increased activity of lignin synthesis enzymes (Phan and Hegedus 1986). Another study was conducted showing identical results where different plant Achyrocline flaccida was used in which shoots were grown in a liquid medium with phloroglucinol resulted in better and increased lignification and improved xylem development (Ross and Castillo 2010). Growth and development of plants using Phloroglucinol. There are several methods of tissue culturing protocols in different studies given in the literature that can be most effective in the micropropagation of plants. This effectiveness of growth and development is given using different combinations of chemicals such as PGRs-illuminance with several abiotic and biotic factors. Furthermore, the most effective way to achieve a considerable growth of plants is chemical manipulation in plant tissue culture, where new and novel chemicals can be used and these can become the alternative for currently used plant growth hormones. Different phenolic compounds reactions with other hormones are already being used in PTC to enhance cellular multiplication, callus formation, formation of adventitious shoots and roots, and promoting the development and proliferation of shoots upto certain folds. The most important reaction to phenolic compounds is mainly with IAA which includes regulation of IAA levels and oxidative catabolism of IAA can be the modification which resulted in the inactivation of auxins (Normanlya et al., 2004). When the phenolic compounds are added externally, they act as alternatives for oxidative enzymes and prevent oxidative catabolism of auxins. The phenolic compounds act as alternates to several and wide range of plant hormones that are normally not used in plant tissue culture as their oxidation can cause several problems like the browning of media and necrosis of cells (Benson, 2000; Reis et al., 2008). Effects of PG on shoot proliferation. Shoot proliferation is the most important part of plant tissue culture where the development of shoots takes place by elongation of axillary buds. Due to its easy and simple protocol, it is an important development for mass-production of plants. Several studies illustrated the improved proliferation of shoots under lab conditions after induction of phloroglucinol. A study showed different linked interactions of PG and sucrose in shoot tips depending on their genotypes. PG when supplemented in media containing Capsicum annuum showed an increase in bud induction response by 18% (Kumar et al., 2005). Another study revealed that the addition of PG for Coccoloba uvifera spp. has no effect on in vitro proliferation of shoots when compared to control, but significantly increased the shoot elongation with the combination of BAP (1.0 mg L-1) and NAA (0.5 mg L-1). The results recorded an increase in shoot length up to 8.2 cm within 4 weeks. The concentration of PG in this study revealed that lower and higher concentrations affected the morphometric traits in comparison to the control (Manokari et al., 2021). Effects of PG on cryopreservation. The PG when added to media increased the recovery and survival rate of cryopreserved plants named as Dendrobium nobile, Dendrobium protocorms and hybrid seeds, Cattleya walkeriana seeds, Oncidium flexuosum seeds, and Catasetum atratum seeds. (Pereira et al., 2021; Vendrame and Faria 2011; Galdiano et al., 2012, 2017, 2013; Prenzier et al., 2018). Several other researchers reported a reduction in tissue browning by PG in culture media that was tested along with leaf segments of Ficus carica where PG provided an increased rate of morphogenesis with a high survival rate for cryopreserved samples (Kim et al., 2007). Effects on the initiation of root development. Phloroglucinol acts as a potential hormone that promotes stimulation of callus induction and organogenesis in shoot and micro bulbs region with enhancement of root region. The concentrations can vary from 1-10 mg/L where the rate of induction ranges from 25-37% (Petti, 2020). Phloroglucinol acts as the root promoting hormone along with several other factors like auxin type and concentration, quality of shoots and roots, age, and temperature. Recent literature revealed that PG promoted rooting in different plant species like Jatropha curcas L. (Daud et al., 2013) and apple cultivars (Dobránszki and Silva 2010). The growth of roots was significantly affected by induction of PG in plant media with a concentration of p<0.05 PG in Carica papaya spp (Al-Shara et al., 2020). The development of roots significantly increased up to 5 folds when IAA was induced with PG and auxin oxidative catabolism was decreased from 100% in control to 21% in PG-induced media. There were several hypothetical observations made by the researchers like IAA has a much higher role in inducing roots in explants than PG but PG enhances the activity and expression of IAA (Dobránszki and Silva 2010). Somatic embryogenesis. From single cells, identical clones with similar genetic makeup can be achieved by the technique named somatic embryogenesis. There are several challenges to somatic embryogenesis such as somatic clones cannot always be obtained using regular plant growth hormones and manipulation of physiological conditions. Hence, the use of PG as potential PGR can induce somatic embryogenesis. When PG added to media it promoted SE development into plantlets and PG helped in increasing the proliferation rate of SE in many cases though some cases illustrated inhibitory effects by inhibiting rooting of Prunus cerasus shoots up to 50% when PG was added at 1.28mM concentration (Snir, 1983). The technique of somatic embryogenesis is the process of producing an embryo using somatic cells. This process of embryo production is explained by Strasburger in 1878 as a form of apomixis, also known as adventitious embryony (Merkle et al., 1995). Somatic embryogenesis is considered a model system to study various events and processes that occur during the growth and development of a plant, and not restricted to limiting factors when observed in variable conditions (Quiroz-Figueroa et al., 2006). The application of PG in somatic embryogenesis of plantshas shown a positive growth result as in the case of Feijoa Sellowiana Berg, the addition of PG and phloridzin gave rise to 3 replicates of 15-20 zygotic embryos in each treatment. The observation showed that the highest number of embryos were obtained at 79µM concentration of PG, and at 197.5µM, nearly 94% of the total explants produced somatic embryos but inhibits the germination of embryos when added in a higher concentration (Reis et al., 2008). In Ornithogalum dubium, 37.5% induction rate was obtained when 4mg/l PG is added to the media showing positive effects in callus induction within a concentration range of 1-4 mg/L (Petti, 2020). The addition of 2.5µM PG gave highest number of globular embryos and influences the change in the physiological process of endogenous cytokinin pool for in vitro development of Tulbaghia simmleri, (Kumari et al., 2018). When PG was used along with nutrient media for the somatic embryogenesis of Lachenalia viridiflora within a concentration range of 2.5-5µM, the highest percentage of embryo germination was obtained in addition to 5µM of PG with full strength MS media (Kumar et al., 2016). The positive influence of PG on the development and growth of somatic embryos in the case of different plant species has been observed and reported (Reis et al., 2008; Kaur et al., 2018). APPLICATIONS OF MELATONIN IN PLANT TISSUE CULTURE Melatonin: Role in stimulating secondary metabolites production: In literature, melatonin has been reported to increase the secondary metabolites production in several plant species such as Lepidium sativum, Vitis labrusca, Salvia rosmarinus, and Citrus aurantium. In Lepidium sativum, exogenous melatonin treatment and UV-C radiation were used for the enhanced production of secondary metabolites i.e., polyphenolic compounds. The results revealed that for melatonin at 20 µM concentration among nine quantified compounds, the accumulation of secondary metabolites increased by almost three times (Ullah et al., 2019). In Vitis labrusca, about 27 metabolites were observed which are known to be accumulated because of melatonin treatment. Furthermore, among the total of the present 464 metabolites, exogenous melatonin treatment significantly increased the production of 27 metabolites. The underlying reason lies in the fact that melatonin promotes the synthesis of ethylene which regulates certain metabolic pathways as well as plant hormone signal transduction and in turn regulates the expression of certain genes like VvLAR2, VvDRF, etc. and these genes promote the production of secondary metabolites production. In the grape berry, melatonin significantly changes the outline in terms of secondary metabolites production by encouraging the VvMYB-14 effectuated biosynthesis of ethylene. This VvMYB-14 engages in the MT signaling pathway which is ultimately responsible for the management of the secondary metabolites production (Ma et al., 2021). Exogenous melatonin treatment of 50 µM in Salvia rosmarinus assisted them to overcome arsenic stress by building up their anti-oxidant machinery and their osmoregulation capacity which in turn improved the production of secondary metabolites in the herb. Rosemary is a medicinal plant that secretes essential oils that contain flavonoids which are helpful to treat various brain, blood, and heart-related diseases. On foliar exogenous melatonin treatment of 50µM, the percentage of the essential oil increased by 100% (Farouk et al., 2019). In Citrus aurantium, there is a variety of bioactive compounds production such as phenolic compounds, essential oils, and certain flavonoids which are known to have promising antiallergic, cardioprotective and vasodilatory effects. 15µM exogenous melatonin treatment was observed to give 1.5 times higher phenolic and flavonoid content in the plant whereas 1µM exogenous foliar treatment increased the yield of essential oils by 0.46% (Sarrou et al., 2015). In the past decades, the potential use of melatonin has shown extraordinary results in terms of regeneration and secondary metabolites production (Li et al., 2020). Role of melatonin in cell differentiation. After the meticulous analysis of the data related to this hormone’s influence on photosynthesis, growth to cell differentiation process in a given plant, it is observed that it is a multifunctional hormone that affects various sets of aspects that combine individual benefits for plant growth. The most common problems faced by a plant during cell differentiation are photosynthesis, metal/chemical phytotoxicity, chlorophyll content, etc. Experiments by Tousi et al. (2020), exogenous pre-treatment of melatonin alleviates cadmium phytotoxicity and improves growth in mallow plants. Cadmium accumulates in different parts of plants like roots, shoots and edible parts which leads to a lowering of the quality of plants by synthesis of Reactive Oxygen Species causing damage to plant membranes and destroying cellular organelles (Farouk et al., 2019). To eliminate this problem, pre-treatments of melatonin are given exogenously reducing the accumulation when concentrations were scrutinized. This was due to the effects of melatonin during cadmium stress made to reduce the translocation factor of cadmium from roots to other parts of the plant. Results show that more cadmium is found in roots than in any other part of the plant. The assumptions by researchers illustrate that this is due to stimulation of carbohydrates metabolism and reduced cadmium-induced oxidative stress. When this hormone is provided exogenously at different concentrations of 15uM, 50uM & 100uM results were varying comparing to the control plant. However, the higher composition of hormones gave negative results by inhibiting the biochemical/physiological activities. 15uM & 50uM concentrations resulted in almost equal with little variation in shoot length, relative water content, and stomatal conductance (Tousi et al., 2020). When ROS and RNS are formed and accumulated in plants it leads to electron leakage, lipid peroxidation, and membrane damage along with nucleic acid and protein damage (Kabiri et al., 2018). NO is another signaling molecule that has several roles in physiological functions so they play a key role in responding to multiple abiotic stresses. The signaling pathway that is mediated directly by NO, melatonin has been observed to be involved in such signaling pathways. One study was conducted on pea plants on the parts of the foliate application and seed soaking activity using melatonin hormone. Rather than seed soaking, foliate application of hormone enhanced/regulated the growth biomarkers like root growth, shoot growth, leaf area, and fresh/dry mass of plants (Yusuf et al., 2020). Results showed that this hormone influenced all mentioned biomarkers i.e., giving concentrations of 10µM, and 50µM only 100µM concentration gave better results in length, weight, and height of plant (Zhang et al., 2013). Coming to the chlorophyll content aspect, seeds soaked with melatonin for 100µM resulted to have better chlorophyll and carotenoid contents of pea plants. Along with these, nitrate reductase and carbonic anhydrase which are the most important photosynthetic enzymes are regulated and stimulated by the application of melatonin as foliar spray (Yusuf et al., 2020). This enhances the process and leads to the growth and development of plants without delay. Net photosynthetic rate and stomatal conductance are also raised during analyzation of data collected (Yusuf et al., 2020). When melatonin hormone was applied to soybean seeds by soaking them with a coagulating agent with different concentrations of hormone-like 10µM, 50µM, 100µM, 200µM, it was observed that at the end of the 3rd and the 4th day, seeds are germinated which are treated with 50µM and 100µM concentration of hormone (Wei et al., 2015). Apart from this, it was also noted that the leaf size of plants with melatonin treated is larger than control plants. This entitles the effect of hormone signaling on photosynthesis and growth factor stimulation in plant development. After the fifth week, plants are observed to be taller than controls and a trifoliate leaf is appeared to signify the hormone present in the inputs for plant growth and development (Wei et al., 2015). After analyzing the agronomic traits of soybean, it revealed that the size and number of pods and seeds are more/better than control plants during the field test. The positive results showed that hormone did not affect the aspect of test weight (Wei et al., 2015). Among the above-mentioned concentrations, 200µM of hormone didn’t provide desired results and inhibited some of the mechanisms from occurring. Stress tolerant activity. A plant faces several stresses depending on its physical or physiological or biological deficiencies affecting the growth and developmental phases. Regarding these aspects, there are some experiments and research available playing an evidential role in the usage of melatonin hormone preparing the plant and culture to be tolerant of certain stresses of the plant. Experiments were done on basil (Ocimum basilicum L.) under salt stress, despite not affecting the root growth and shoot growth, but have shown enhanced results in growth, and antioxidant activity with the exogenous application of 10µM melatonin (Bahcesular et al., 2020). Experiments conducted on cucumber seeds with 100µM of melatonin resulted in a better seed germination rate than control plants (Zhang et al., 2013). Moreover, it was stated that 150µM of NaCl along with 1% melatonin also gave the expected rate of seed germination in cucumber seeds (Zhang et al., 2013). This enhanced the assimilation rate under salt stress water provided to the plants and salinity did not affect the growth and germination activities. The same conditions are repeated with the chilling stress (Posmyk et al., 2008). It was recorded that use of melatonin also resulted in a better germination rate, seedling growth, and vigor index under the cadmium stress (Nabaei and Amooaghaie 2019). Fewer studies showed that cadmium or other metal stresses make the plant weak in assimilation and developmental phases, but in the case of Catharanthus, results were a contrast in nature with no defective qualities roseus (Nabaei and Amooaghaie 2019). Researchers recently recognized this hormone as a bio-stimulant and plant signaling regulator (Liang et al., 2018) based on the study and research conducted in a few experiments on Catharanthus roseus and Moringa oleifera. Under abiotic stress, this hormone regulates the signaling pathways of proteins to enhance the growth of roots, shoot, and even in flowering stages also in Moringa oleifera (Sadak et al., 2020). Especially, under drought conditions, in cucumber plants, exogenous application of 100µM hormone in plant raised the root-shoot growth ratio along with the enhanced root development later (Zhang et al., 2014). Increased levels of ROS and RNS inside a cell are one of the primary responses to stress. Abiotic stresses make plants perform down-regulation of growth factors and upregulation of suppression factors; this results in plant death gradually. But the use of melatonin hormone between the concentrations of 50µM-100µM boosts the plant growth gene regulation like psa A, F, G, H, and others for photosynthesis. It could also regulate the ATPase activity for plant development. This is possible only when different combinations of NaCl and H2O are collectively presented to plants (Wei et al., 2015). Growth regulatory properties of melatonin in higher plants. One study revealed the role of melatonin as a growth promoter in etiolated lupin (Lupinus albus) where it shows growth-promoting effects of hypocotyls at micromolar concentrations and inhibitory effects at higher concentrations. Melatonin is 63% similar to indole-3-acetic acid which makes it an auxin-like hormone (Hernandez-Ruiz et al., 2004). In red cabbage (Brassica oleracea rubrum) and mustard, the concentration-dependent action of melatonin is there ranging from promoting to inhibitory effect (Posmyk et al., 2008; Chen et al., 2009). Another effect of melatonin on organogenesis was seen as a histogenesis inducer and later it was confirmed in cucumber (Cucumis sativus) (Zhang et al., 2013). The effect of melatonin in the formation of adventitious roots is considerable where melatonin in combination with auxins such as indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) shows potential effect on rooting and is a topic of interest (Pacurar et al., 2014). Melatonin treatment altered the levels of abscisic acid (ABA) and gibberellins (GA). In saline conditions, melatonin provoked GA biosynthetic genes (GA20ox and GA30x) in cucumber seedlings. This promoted salt inhibited the germination process due to the high-level activation of GAs as GA4 (Zhang et al., 2014). Melatonin also upregulated ABA catabolism genes (two CYP707 monooxygenases) and down regulated a key enzyme in the biosynthesis of ABA called 9-cis-epoxy carotenoid dioxygenase (NCED). This decreased ABA levels rapidly under salt stress during seed germination (Zhang et al., 2014). A study was done to see the effect of exogenous melatonin treatment on tomatoes to understand the difference in the quality of the fruit, ethylene metabolism, and post-harvest ripening proved that 501M melatonin for 2hrs brings a considerable change in fruit ripening parameters such as ethylene signaling, lycopene levels, fruit softening, flavor, and biosynthesis of enzymes as compared to untreated tomatoes (Sun et al., 2015).