Quantification of endogenous phytochemicals and determination of their exogenous effects in somatic embryogenesis pathways of white and water yams

In vitro propagation of yam via organogenesis is constrained with low multiplication rate. Somatic embryogenesis (SE) has shown rapid multiplication potentials in yam. However, it has not been adopted by practical seed system scenarios due to genotype specificity. Reports have shown that SE is regulated endogenously by phytochemicals, but this is yet to be elucidated for yam. This study identified, quantified endogenous, and evaluated effects of exogenous application of selected identified phytochemicals in yam SE. Callus was induced from in vitro axillary bud explants of three Dioscorea rotundata genotypes in Murashige and Skoog (MS) medium containing 9.1 µM 2,4-dichlorophenoxyacetic acid and 5.4 µM naphthaleneacetic acid. Plantlets were regenerated using MS medium containing 4.4 µM benzylaminopurine and 34.0 µM uniconazole-P. Endogenous phytochemicals associated with axillary bud, calluses, and plantlets were identified and quantified using GC/MS. Effect of selected identified phytochemicals on the genotypes was investigated in a 5 × 6 factorial in completely randomized design (r = 3). Data taken on plantlet regeneration was analyzed using ANOVA at α0.05. A total of 27, 22, and 35 phytochemicals were identified in Kpamyo, Ekiti2a, and Asiedu, respectively. Hexamethylcyclotrisiloxane (36.4%, Kpamyo), Tris-tert-butyldimethylsilyloxy-arsane (59.3%, Ekiti2a), and 4-methyl-2-trimethylsililoxy-acetophenone (52.7%, Asiedu) were highest in callus. N-Methyl-1-adamantaneacetamide (31.8%, Kpamyo) and Tris-tert-butyldimethylsilyloxy-arsane (52.7%, Ekiti2a, Asiedu) were highest in plantlets while Tris-tert-butyldimethylsilyloxy-arsane (41.2%, Kpamyo), hexamethylcyclotrisiloxane (55.8%, Ekiti2a), and erythro-9,10-dibromopentacosane (38.9%, Asiedu) were highest in axillary bud. Plantlet regeneration differed significantly among phytochemicals and ranged from 0.7 ± 0.3 (control) to 4.5 ± 0.5 (40.5 µM phenylacetic acid). Also, genotype × phytochemical interactions on number of plantlets regenerated were significant, and mean values ranged from 0.0 ± 0.0 (TDa2014, 4.8 µM decamethyltetrasiloxane) to 7.0 ± 1.7 (TDa2014, 40.5 µM phenylacetic acid). The application of 40.5 µM phenylacetic acid enhanced plantlet regeneration in Kpamyo and TDa2014 by 5.39% and 343.04%, respectively.


Introduction
Yam is a multi-species crop that is mostly propagated using the tuber part, which is also the edible part (Adeigbe et al. 2015). Despite the importance of yam in alleviating food insecurity in Africa, the availability of clean planting material remains a major challenge due to its low propagation ratio, coupled with the long periods of tuber dormancy . Various technologies have been investigated for increase in yam propagation rates. These include the advanced yam minisett technology (planting cut yam tubers of 25 to 50 g size range), rooted vine cuttings, the temporary immersion bioreactor system of tissue culture, and aeroponics and hydroponics system Balogun et al. 2017Balogun et al. , 2021.
Tissue culture technique has the advantage of cleaning infected propagules from pathogens hitherto accumulated during cultivation in the informal seed system (Coyne et al. 2009;Balogun et al. 2014). However, the use of tissue culture technique through organogenesis is still constrained with low multiplication ratio (1:4). Somatic embryogenesis (SE) (the production of embryos from somatic cells and subsequent conversion of the embryos to plantlets) has been successfully used by many researchers to increase this limited multiplication in different crops (Junaid et al. 2013;Ossai et al. 2018). However, rates of SE are affected by genotype, type and concentration of growth regulators in culture medium, photoperiod, type, and stage of explants and endogenous phytochemicals that mediate hormone actions in various plant developmental stages and their morphogenic processes (Kakkar and Sawhney 2002).
Yam is rich in different levels of hormonally active phytochemicals (Polycarp et al. 2012) which could play different roles in their competence to SE as seen in other plants. Plants typically release some of these phytochemicals into the environment in response to alterations in the ambient conditions, whether harmful or beneficial, a condition known as plasticity (Loretta 2014). According to Graf et al. (2007), plant response to tissue culture is controlled largely by the epigenetic changes (methylation or demethylation) exhibited by the plants at different stages of regeneration pathway (organogenesis or somatic embryogenesis).
In somatic embryogenesis pathway, several genes that regulate oxidative stresses in plants (Santos et al. 2018) characterize the somatic embryo induction stage. This is because environmental stressors stimulate the callus induction stage, which leads to the production of reactive oxygen species (ROS) or pro-oxidants. These ROS under normal conditions are controlled by antioxidants, which are endogenic (phytochemicals) in plants (Joseph and Jini 2010). However, in a stress condition, there is an imbalance between the production and elimination of ROS in plants, and the capability to balance the redox varies among and within plant species, a situation that could necessitate the exogenic application of the antioxidants to balance the system experimentally. Proteomics is a veritable tool to understand these phytochemicals and their control mechanisms (Ahmad et al. 2016). Knowledge of these phytochemicals is important in adjusting the media compositions to enhance SE in otherwise incompetent genotypes. This study identified, quantified endogenous, and exogenically evaluated effects of selected identified phytochemicals during stages of SE in white (Kpamyo, Ekiti2a and Asiedu) and water (TDa2014 and Swaswa) yams.

Study Location
The tissue culture experiments were carried out at the Cell Biology Unit of the Bioscience Center, International Institute of Tropical Agriculture (IITA), while the identification of expressed phytochemicals during somatic embryogenesis was carried out at the Central Research Laboratory, Tanke Road, Ilorin, Nigeria. The equipment were sourced from the central store, supply chain unit of IITA, Ibadan, Nigeria, while the PGRs and phytochemicals were sourced from Bristol Scientific, a Sigma-Aldrich distributor in Lagos, Nigeria.

Identification of Endogenous Phytochemicals During SE
Stages of Three Genotypes of White Yam (D. rotundata Poir) (Fig. 1) Organogenesis-derived axillary bud 4-wk-old callus and 8-wk-old callus cultures at plantlet regeneration stage of Asiedu, Ekiti2a, and Kpamyo were collected for phytochemical profiling and quantification following the protocol of Saleethong et al. (2016) and Bradford (1976-Bradford protein assay), respectively. The protein samples were lyophilized and derivatized by the addition of 5 µL hexadecane, 73.0 µL dimethylformamide, 20.0 µL N-tert-butyldimethylsilyl-N-methyltriflouroacetamide, and 5.0 µL of triethylalanine (catalyst) at a temperature of 60 °C and left for 30 min. The derivatized samples were injected into a gas chromatograph, heated up to 300 °C where the material was volatilized to enable separation of components that flow through the column by size for phytochemical detection. The data generated were searched against the National Institute of Standards and Technology (NIST) and the National Center for Biotechnology Information (NCBI) databases for phytochemical identification and the respective percentage of the sample covered by each phytochemical detected.
Decamethyltetrasiloxane (DT), hexamethylcyclotrisiloxane (HC), phenylacetic acid (PAA), and glutaric acid (GLA) were quantified prior to investigation of effects of their exogenous application on SE. In endogenous determination in tissue cultures, 1 L of callus induction medium was dispensed into 60 Petri plates of 16.7 mL each. Five (5) axillary bud explants were cultured in 16.7 mL of callus induction medium for callus formation. Hence, weight (g) of phytochemical / liter = detected weight of phytochemical (g) per callus × 5 × 60 (Table 1). Selected DT and HC were found across the three genotypes in the 4-wk-old callus culture, and PAA was detected across the three genotypes in the 8-wk-old callus cultures, while GLA was detected across the genotypes in the organogenesis-derived axillary bud. The phytochemicals were sourced from Sigma-Aldrich and added to the callus induction and plantlet regeneration media for Asiedu, Ekiti2a, Kpamyo, Swaswa, and TDa2014 genotypes.
Callus induction medium (CIM) was prepared and the selected phytochemicals were added to give six (6) treatments (T): T1: CIM, T2: CIM plus 2.1 µM GLA, T3: CIM plus 28.5 µM PAA, T4: CIM plus 95.9 µM HC, T5: CIM plus 9.6 µM DT, and T6: yam multiplication medium (YMM). The six culture medium regimes were autoclaved at 121 °C and 15 Psi for 15 min and dispensed into Petri plates (16.7 mL each) under the laminar flow hood. Five (5) in vitro axillary buds (0.1 to 0.5 cm) of Asiedu, Kpamyo, Ekiti2a, Swaswa, and TDa2014 were excised and inoculated into Petri plates containing 16.7 mL of the different callus induction media and replicated three times per genotype. The cultures were kept in the dark for callus formation and proliferation (Manoharan et al. 2016).
The calluses were transferred to PGR-free YMM and kept in a 16-h photoperiod at 25 ± 1 °C condition for 3 wk. During this period, the cultures were assessed for the formation and maturation of somatic embryos by  In each of callus induction and plantlet regeneration studies, a factorial 5 (genotypes) by 6 (medium regimes) in completely randomized design (CRD) with 3 replicates was used. Data were collected on the number of days to callus induction, percentage callus formation, days to plantlet regeneration, number of plantlets regenerated per callus, and number of roots formed. Data were analyzed using correlation (between quantity of identified phytochemicals by number of plantlets regenerated) and ANOVA (SAS 9.0 version), and treatment means were separated using the Duncan multiple range test (DMRT) at p ≤ 0.05.

Discussion
The induction of callus is a response of explants to stress, such as wounding and high salt concentration, and is associated with increased formation of ROS at some point during exposure, which may result in the unspecific reduction of proteins and membrane lipids (Joseph and Jini 2010). Similar effects are also achieved through the inclusion of phytochemicals like auxins (2,4-D, NAA, IAA, and picloram) at high concentrations in the culture environment (Momoko et al. 2013). At the callus formation stage, two organosilicons (decamethyltetrasiloxane and hexamethylcyclotrisiloxane) and a carboxylic compound (phenylacetic acid (PAA)) that were identified across the genotypes in this study suggest that they are part of the defense systems to ROS. Amino acids, catalases, peroxidases, and enzymatic scavengers of activated oxygen which can be endogenic in the plants or exogenically applied in the SE culture environment have been reported in other crops (Joseph and Jini 2010).
Organosilicons are linked to antioxidant activities that are essential in plant defense mechanism in response to stress (Ethela et al. 2017), while PAA, being a naturally occurring auxin, has been used to maintain callus proliferation in tobacco, sunflower, and pea although at higher concentrations of 25 to 500 µM (Leuba and Letourneau 1990). Organosilicons have antioxidant properties, as demonstrated in Origanum vulgare (Galehassadi et al. 2014), and siliconderived amino acids have also been described as a potent antioxidant (Annaliese and Sean 2013). Antioxidants are essential in callus cultures to reduce ROS (Momoko et al. 2013) because too much oxidation can cause the callus to turn brown and eventually die back. However, the optimum concentration and the time of exogenous application during callus induction are yet to be determined in order to avoid its being counterproductive in the case of SE since the interest is to produce more callus.
More phytochemicals were identified at the plantlet regeneration stage while the callus induction stage had the least number of phytochemicals. This could be that the plantlet regeneration stage is more complex, requiring more phytochemicals or antioxidants for the conversion of somatic embryos into plants. This finding could be an insight to the recommendation of Gaj (2004) for the hormonal characterization of somatic embryogenesis environment and responsive cells in plants. In the axillary bud explant used, out of the 26 phytochemicals discovered, only glutaric acid belonging to the carboxylic group of compounds was identified across the three genotypes. Glutaric acid is a dicarboxylate analog of glutamic acid, and aspartic acids are amino acids that have been reported in yam (Doss et al. 2019). Other phytochemicals belonging to the carboxylic group are dlaspartic acid and phenylacetic acid. Aspartic acid is a precursor of four essential amino acids: asparagine, methionine, threonine, and lysine (Johnson 2017), and both glutaric acid and aspartic acid have been shown to have significant positive correlation with the sprouting of Saffron corm (Bagri et al. 2017), which is a vegetative propagative part of Saffron like the axillary buds of in vitro yam plantlets. Another carboxylic compound identified in the axillary bud explant of yam in this study is l-tryptophan, an essential plant-derived amino acid needed for the in vivo biosynthesis of proteins (Mendel 2018) and a precursor for the production of indole-3-acetic acid (Lalit 2002) which is an auxin that plays a role in callus formation (Harish et al. 2010). The incorporation of tryptophan in callus induction and plantlet-regenerating media of rice favored callus induction and plantlet regeneration through somatic embryogenesis (Shahsavari 2011).
The addition of the identified phytochemicals to callus induction medium successfully induced calluses in the different genotypes within a 3-wk period, except for the addition of decamethyltetrasiloxane to callus induction medium that could not induce callus in TDa2014. A higher or lower concentration than what was applied could have produced the desired effect if its stability varies significantly with environment (Mathe et al. 2012). However, this was not significantly different from the medium without the phytochemicals. The baseline callus induction medium gave the highest percentage callus induction (69%) in all genotypes except Swaswa where addition of glutaric acid to callus induction medium improved callus formation. Callus induction from the treatments containing the two (2) organosilicons (decamethyltetrasiloxane and hexamethylcyclotrisiloxane) was less than the 22% induction rate from the PGR-free yam multiplication medium, probably due to habituation (Ossai 2014). Studies have shown that an appropriate combination of NAA and 2,4-D gave the best condition for callus induction in yams (Belarmino and Gonzales 2008;Ossai et al. 2018). Both decamethyltetrasiloxane and hexamethylcyclotrisiloxane as detected from the callus explants of Asiedu, Ekiti2a, and Kpamyo might not be playing an active role in callus formation but on the protection of the plants from free radicals (Galehassadi et al. 2014). This can be confirmed using more specific methods like radioactive labeling (Khosrav et al. 2019).
The Dioscorea alata genotypes responded more favorably than the Dioscorea rotundata genotypes in terms of gain in SE from baseline to the realized plantlet regeneration. This could be that the D. alata genotypes respond more or are more affected by the changes in environmental conditions. Fluctuations in the concentration of endogenous hormones in water yam were reported to be high (Matsumoto et al. 2013). The white yams on the other hand are more stable, thereby having little changes in SE protocol alterations. Despite the superior effect of baseline callus induction medium on callus formation relative to others, addition of 2.1 µM GLA gave above 50% callus induction rate in the three (3) genotypes of white yam. Although there is a dearth of information on the use of glutaric acid to induce callus in yam, the application of ketoglutaric acid at 100 mgL −1 was reported to improve the formation of embryogenic callus from Zoysia japonica seeds (Asano et al. 1996).
The use of 4.4 µM BAP plus 34.0 µM UP for plantlet regeneration had been reported by Ossai et al (2018) as the best treatment combination for plantlet regeneration in yam through somatic embryogenesis. In this study, an addition of 40.5 µM PAA to PRM also gave improved plantlet regeneration from the somatic embryos of Kpamyo and TDa2014 by 5.39% and 343.04%, respectively. It also supported better root formation from the somatic embryos of the five (5) yam genotypes than other treatments. Phenylacetic acid has been used in the direct regeneration of chickpea (Ghanti et al. 2009). Auxins like phenylacetic acid at high concentrations in culture media are mostly used to induce callus by causing an oxidative stress to the wounded explants. However, PAA has been reported to possess moderate levels of free radical scavenging activities in Ilex aquifolium (Nahar et al. 2005;Kazan and Manners 2009). Akram et al. (2016) also found that PAA elicits induced systemic resistance (IRS: resistance mechanism in plants activated by infection or injuries) in plants, which is accompanied by the expression of defenserelated genes and proteins (Zamioudis and Pieterse 2012).

Conclusion
A total of 27, 22, and 35 different phytochemicals were identified in callus, axillary buds, and plantlets of white and water yam. Out of the tested exogenously, phenylacetic acid improved plantlet regeneration significantly while decamethyltetrasiloxane and hexamethylcyclotrisiloxane were detrimental. The implications of these to the seed system development are the need for further standardization of the timing of application and different concentrations of phytochemicals towards the adoption of the SE system. This is expected to lead to significant increase in the multiplication ratio of yam leading to its availability all season. However, it is necessary to confirm the genetic integrity of the plantlets regenerated since somaclonal variation is indispensable during SEs.