Bacopa floribunda (R.Br.) Wettest. (Family: Plantaginaceae) is indigenous to Madagascar, Africa, and Asia. In Ayurveda, it has been utilized as a brain tonic and antiaging and also to treat psychological disorders. It is an excellent source of natural medicine due to its cholinergic enzyme-inhibiting, anti-inflammatory, and antioxidant properties. Because of its potent nootropic effects, which decrease acetylcholinesterase (AChE) inhibition, it is used in traditional remedies to improve memory (Olatunji et al., 2019; Adetuyi et al., 2022). Triterpenoid saponin glycosides are said to be the active chemical components in the genus Bacopa and categorized as pseudojujubogenin (bacoside II and bacosaponin C) and jujubogenin glycosides (bacoside A3 and bacopaside X) and to exhibit memory-improving effects (Mathur et al., 2016; Nuengchamnong et al., 2016; Watcharatanon et al., 2019; Bhandari et al., 2020). Triterpenoid saponin glycosides from B. monnieri (Brahmi) are the well-known marker compounds used to improve nerve impulse transmission. Triterpenoid saponin glycosides help injured neurons’ recovery by promoting kinase activity, neuronal synthesis, and the restoration of synaptic function. It ultimately results in restoration of nerve impulse transmission and an increase in de novo synthesis of proteins in the brain. Nerve impulse transmission plays a crucial role in promoting healthy cognitive functions such as attention span, focus, concentration, learning, and memory (Mathur et al., 2016; Singh and Dhawan, 1997).

Due to its usage in the production of pharmaceuticals and herbal extracts, B. monnieri (Brahmi) is in very high demand in India, which promotes massive collection from their natural habitats and the extinction of the species. The greatest obstacle to the use of Brahmi in several industrial applications is the high variability in the levels of bioactive principles (Christopher et al., 2017). Since ancient times, B. monnieri (Brahmi) has been used as a brain tonic or memory booster. Bacopa formulations have been employed for therapeutic purposes by numerous academics and pharmaceutical firms worldwide, including those in India, New Zealand, the United States, and Australia. It has been estimated that approximately 10,000 t of fresh plant material was harvested from natural habitats each year that caused a significant decline in wild populations. Due to the presence of bioactive compounds, Brahmi has unique medicinal utility. Low yields, geographical and environmental variability, and other factors are linked to the synthesis and accumulation of secondary metabolites in the field-grown plants of Brahmi (Christopher et al., 2017; Watcharatanon et al., 2019; Asadollahei et al., 2022). Considering the problems associated with the growth and importance of the species, it is necessary to find out alternative sources and their mass multiplication, conservation, and production of bioactive metabolites. In vitro propagation makes it possible for cells, tissues, and organs to grow aseptically without the presence of microbes, which is crucial for the development of therapeutic plants (Ali et al., 2021; Bhattacharyya et al., 2022). The demand of providing high-quality-stock plants for conservation and pharmaceutical needs through micropropagation has remarkably increased (Komakech et al., 2020). In vitro produced cells, tissues, and organ cultures offered a good source of homogenous, sterile, and suitable materials for the conservation of plant species, their biochemical characterization, and the identification of bioactive constituents (Patil et al., 2013). In vitro propagation techniques have been used earlier to conserve several commercially important, ornamental, medicinal, and RET species (Nikam et al., 2009; Gupta et al., 2016; Chandra et al., 2020).

Recent studies have shown numerous methods for large-scale production; the synthesis of secondary metabolites includes strain improvement, optimization, elicitations, medium and culture environment manipulation, and nutrient and precursor feeding (Murthy et al., 2014). Precursors and elicitors can trigger morphological and physiological alterations that result in the accumulation of phytoalexins. Elicitation with abiotic and biotic treatments can also boost the production of bioactive metabolites (Watcharatanon et al., 2019). Elicitors have the ability to activate a cell immune system and produce secondary metabolites as a protection mechanism (Asadollahei et al., 2022). It is believed that adding precursors into the plant biosynthesis pathway will facilitate the production of secondary metabolites (Watcharatanon et al., 2019). The use of elicitors like methyl jasmonate (MJ), salicylic acid (SA), and precursors like squalene (SQ) for production of secondary metabolites in in vitro culture systems has been reported earlier (Largia et al., 2015; Baek et al., 2019; ). Several reports are available on micropropagation and enhanced metabolite production in B. monnieri (Banerjee and Shrivastava, 2008; Parale and Nikam, 2009; Parale et al., 2010; Largia et al., 2015; Sharma et al., 2015; Muzynska et al., 2016; Watcharatanon et al., 2019).

Although B. floribunda is a versatile medicinal herb, there are no reports available on its in vitro propagation and elicited production of bioactive compounds. Taking all of this into consideration, the objectives of the current study were to develop a micropropagation protocol and production of secondary metabolites. To achieve these goals, MS media supplemented with different plant growth regulatory substances (PGRs), precursors, and elicitors and their optimal concentrations were studied for the production of total saponins, four triterpenoid saponin glycosides, and stigmasterol.

Bacopa species are mainly found in wetland areas of tropical and subtropical countries, and its growth under natural conditions is limited. In order to increase mass multiplication of B. floribunda and secondary metabolites including triterpenoid saponin glycosides and sterols, it is necessary to develop an efficient in vitro micropropagation protocol. In order to accomplish this goal, we focused on micropropagation and augmented production of stigmasterol and triterpenoid saponin glycosides from the unexplored plant species known as B. floribunda. Owing to their enormous potential in medicinal purposes, most of B. monnieri have been collected extensively from their native habitat, resulting in the species’ extinction and threat. Search of alternate herbs and their mass multiplication is also needed to reduce the overexploitation of B. monnieri. To achieve this goal, we aimed at micropropagation and elicited production of triterpenoid saponin glycosides as well as stigmasterol in B. floribunda. Seeds of B. monnieri had poor germination, low viability, and frequent two-leaf seedling deaths, but it can be propagated efficiently using stem cuttings (Parale and Nikam, 2009; Parale et al., 2010). In the study, fresh nodal segments were used as explants and cultures were established using MS medium supplemented with varied concentrations of auxins and cytokinins. The present investigation revealed that MS supplemented with PGRs (BAP, KIN, IAA, IBA, and NAA) alone and in combination revealed the formation of multiple shoots and roots in B. floribunda. Our study revealed optimal regeneration responses when the MS medium was supplemented with cytokinins such as KIN and BAP. These findings are in accordance with earlier reports where researchers suggested that the highest concentrations of cytokinins promoted the regeneration of B. monnieri through axillary nodes and internodes (Banerjee and Shrivastava, 2008). In addition, several researchers also reported that BAP was found to be the most effective in cytokinin, which played an important role in induction of multiple shoots in B. monnieri (Tiwari et al., 2001; Ceasar et al., 2010). According to Banerjee and Shrivastava (2008), internode explants of B. monnieri revealed a significantly higher number of shoots when inoculated into MS media supplemented with 1.0 mg/l BAP and 0.5 mg/l KIN. Our findings are in good agreement with Parale and Nikam (2009) who observed that BA and KIN at concentrations of 1 to 10 μM improved the shoot regeneration in B. monnieri. In all the auxin treatments, incorporation of 1.0 mg/l NAA in nutrient medium showed optimum root regeneration (Banerjee and Shrivastava, 2008). Researchers reported that roots of B. monnieri showed optimum growth when MS medium was supplemented with 1.5 mg/l NAA. It is a fact that PGRs such as cytokinins (BAP and KIN) stimulate the initiation and further elongation of shoots whereas auxins (IAA, IBA, and NAA) promoted the root initiation, growth, and development. In addition, the composition of nutrient media used in in vitro propagation also affected the plant growth and development (Komakech et al., 2020). Plant cell culture growth is mainly affected by nutrient concentration, stress variables, photoperiod, light quality, genotype, and concentration as well as combinations of plant growth regulators (Chavan et al., 2010).

The present study reported that among the precursor and elicitors used, utilization of 1.5 μM SQ in MS medium showed optimum regeneration responses. In accordance with our studies, Baek et al. (2019) also reported that squalene at different concentrations (up to 10 mM) increased the growth index of hairy roots of Centella asiatica. Likewise, elicitors at various concentrations altered the growth performance and the best response was observed when 80 μM MJ was added to the nutrient medium. Furthermore, cultures of B. floribunda grown on MS nutrient medium with methyl jasmonate, salicylic acid, potassium chloride, and magnesium chloride also flourished well. In the same way, Sharma et al. (2015) reported that abiotic elicitors (jasmonic acid, copper sulfate, and salicylic acid) stimulated the biomass production in in vitro shoot cultures of B. monnieri. Moreover, Largia et al. (2015) studied the influence of elicitors (methyl jasmonate, salicylic acid) on growth, biomass, and morphology of B. monnieri. Patil et al. (2013) also found that elicitation feeding improved the in vitro growth of Digitalis purpurea. Elicitors are compounds well known to increase the metabolic activities of phytochemicals and further improve plant performance. The plant physiology and biochemistry of the tissues are also affected by the type and/or concentration of the elicitor. Responses of the species also depend on the elicitor and its concentration added in the medium. In accordance with our findings, in plant cell cultures, the use of elicitors has been found to be beneficial (Guru et al., 2022). Additionally, a number of precursors and elicitors have been used earlier to induce morphological and physiological alterations in in vitro cultures (Patil et al., 2013; Watcharatanon et al., 2019). Optimal concentrations of PGRs, precursor, and elicitors alone and in combination were used to study the growth responses in B. floribunda. The goal of this experiment was to increase the production of in vitro biomass of B. floribunda. From the present investigations, we found that optimum cytokinin concentrations, i.e., MS + 2.0 mg/l KIN + 2.0 mg/l BAP, demonstrated the highest growth responses (average shoot length, number of leaves per shoot, FW, and DW). Martins et al. (2022) found that the combination of two cytokinins can be more advantageous than using only one, which is consistent with our findings. In our studies, also the combination of auxins (MS + 0.5 IAA mg/l + 0.5 IBA mg/l + 1.0 mg/l NAA) promoted the root initiation, elongation, and number of roots per shoot. According to earlier reports, rooting of the in vitro produced shoots of B. monnieri was higher when auxin-rich MS medium was used (Banerjee and Shrivastava, 2008; Sudheer et al., 2022). Selection of PGRs plays a crucial role in in vitro propagation of the plant. Cytokinins and auxins were commonly used in culture media to influence physiological reactions that led to promotion of the growth of shoots, roots, and whole plant. A number of researchers have investigated the impact of auxins (NAA, IAA) and cytokinins (BAP, KIN) on in vitro growth and secondary metabolites from the plant (Amoo and Staden, 2012; Martins et al., 2022). The highest concentrations of cytokinins offered the best outcomes, which may be due to the ability of BAP to promote cell proliferation and lateral bud development. It also plays a key role in altering the axillary bud dormancy. BAP exhibits a more sustained stimulation of cell division due to its stable nature and resistance to oxidation characteristics. In addition, it cannot easily broken down by plants (Komakech et al., 2020; Okello et al., 2021; Martins et al., 2022).

Well-developed plantlets were successfully acclimatized to the field conditions with a good survival rate of 95%, which was in agreement with earlier reports (Tiwari et al., 2001; Sharma et al., 2016; ). These plantlets were morphologically similar to the field-grown plants. Acclimatized plants may have aberrant anatomy, physiology, or morphology, which takes longer to adapt and lowers growth. From various tested concentrations of PGRs, precursor, and elicitors, a reliable amount of total saponin content was noted. In cytokinins, MS + 1.0 mg/l BAP showed the highest TSC (31.79 ± 0.011 mg DE/g DW), whereas, among the tested auxins, incorporation of IBA in the medium MS + 0.5 mg/l IBA proved to be the best (32.0 ± 0.001 mg DE/g DW). In our studies, addition of precursor SQ (1.5 μM) revealed the highest TSC. Likewise, among all the elicitors tested, incorporation of KCl (80 mM) to the nutrient medium offered the highest content of total saponins. Additionally, combination of cytokinins and squalene (MS + 2.0 mg/l BAP + 1.5 μM SQ) demonstrated the best results as compared with other treatments. Our findings are in good agreement with Phrompittayarat et al. (2011). Triterpenoid saponin glycosides of B. monnieri were earlier reported from the aerial parts as well as shoots. The saponin content may vary depending on several variables, including the plant components, environment, season, and harvesting time. Watcharatanon et al. (2019) reported improvement in total triterpenoid saponin glycosides when in vitro cultures of B. monnieri were fed with the precursors and exposed to light-emitting diode (LED). The study suggested that saponin content may be enhanced as a result of the feeding of precursors and LED light exposure to in vitro cultures of B. monnieri.

HPLC analysis confirmed the presence of four triterpenoid saponin glycosides (bacoside A3, bacopaside X, bacopaside II, and bacosaponin C) from the in vitro grown biomass treated with various PGRs, precursor, and elicitors as well as acclimatized plantlets. On the other hand, several researchers identified and quantified bacoside saponins from B. monnieri and also reported potent biological activities like anti-inflammatory, antioxidant, anti-hepatotoxic, and analgesic and superoxide inhibition (Bhandari et al., 2006; Sudheer et al., 2022). It is a classified and well-known drug used in the improvement of memory or mental power (Ganzera et al., 2004; Deepak et al., 2005; Bhandari et al., 2006; Naik et al., 2012; Saini et al., 2012; Bansal et al., 2014; Christopher et al., 2017; Bhandari et al., 2020).

Bacosides are the mixture of four saponin glycosides, viz., bacoside A3, bacopaside II, bacosaponin C, and bacopaside X, generally referred to as bacoside A (Nuengchamnong et al., 2016). Additionally, LC-ESI-QTOF-MS-based screening of the mixture of the authenticated saponin (bacoside A) also revealed that each of the individual compounds was identified individually by its mass as bacoside A3, bacopaside II, bacopaside X, and bacosaponin C. Additionally, total ion chromatogram (TIC) of the authenticated compounds proved that the bacoside mixture includes bacoside A3, bacopaside II, bacopaside X, and bacosaponin C (Nuengchamnong et al., 2016).

In the present findings, HPLC analysis confirmed the presence of four triterpenoid saponin glycosides in the in vitro grown biomass and acclimatized plantlets of B. floribunda. MS + 2.0 mg/l KIN alone highlighted the highest triterpenoid saponin glycosides, in which bacoside A3 (0.013 ± 0.001 mg/g DW), bacopaside II (9.934 ± 0.276 mg/g DW), bacopaside X (1.972 ± 0.101 mg/g DW), and bacosaponin C (0.060 ± 0.038 mg/g DW) contents were recorded. Similar to cytokinins tested, MS + 1.5 mg/l NAA revealed the highest contents of bacoside A3 (0.004 ± 0.001 mg/g DW), bacopaside II (6.16 ± 0.05 mg/g DW), bacopaside X (1.40 ± 0.044 mg/g DW), and bacosaponin C (0.043 ± 0.001 mg/g DW). Our results clarified that the higher concentrations of cytokinins (2 mg/l) proved to be the best and offered the highest triterpenoid saponin glycoside content. Our results are in agreement with reported results of Watcharatanon et al. (2019) who reported the highest triterpenoid saponin glycosides from in vitro grown cultures of B. monnieri (17.35 ± 0.13 mg/g DW). Likewise, Muzynska et al. (2016) also reported that shoots grown on MS medium supplemented with 1.0 mg/l BAP and 0.2 mg/l NAA revealed the highest bacoside A3 (2.18 ± 0.15 mg/g DW), bacopaside II (19.19 ± 1.1 mg/g DW), bacopaside X (0.67 ± 0.05 mg/g DW), and bacosaponin C (4.75 ± 0.01 mg/g DW) contents. Kamonwannasit et al. (2008) reported that shoots produced from internodal explants using MS + 0.5 mg/l TDZ showed remarkable pseudojujubogenin glycoside content (30.62 ± 1.29 mg/g DW). Among the different concentrations of the precursor used, MS medium enriched with 1.5 μM SQ revealed the highest total bacoside content (6.4 ± 0.10 mg/g DW). In accordance with our studies, Baek et al. (2019) also reported the enhancement of targeted triterpenoids in Centella asiatica hairy root cultures fed with 2.5 mM SQ. Dasari et al. (2020) also revealed that productivity of bioactive compounds depends on the concentration of chemicals used in the nutrient medium and also the exposure time. In in vitro grown biomass of B. floribunda, elicitors such as MJ, SA, KCl, and MgSO₄ were found to be the most effective for the production of triterpenoid saponin glycosides. In line with our findings, elicitors like methyl jasmonate and salicylic acid enhanced the bacoside production in shoot cultures of B. monnieri (Largia et al., 2015). Sharma et al. (2015) noted enhancement of triterpenoid saponins from B. monnieri by utilizing methyl jasmonate as the key elicitor. Researchers also studied the influence of different elicitors (jasmonic acid, copper sulfate, and salicylic acid) on stimulation of bacoside production in shoot cultures of B. monnieri. Biotic (chitosan) and abiotic (salicylic acid, methyl jasmonate) elicitors directly affected the metabolite(s) production in medicinal plants (Kaur et al., 2022). Among all various studied elicitors, MJ and SA have been well documented for the increased production of triterpenoid saponins in plant cell cultures (Largia et al., 2015). As compared with previous reported results, we found satisfactory results from in vitro grown biomass of B. floribunda treated with MJ and SA. Elicitation is a technique of inducing or increasing the production of secondary metabolites in plant tissue culture (Kaur et al., 2022). According to the studies, elicitors may serve as signaling molecules in proper quantities, and when perceived by a plasma membrane receptor, they trigger a complex signal transduction network that regulates gene expression and leads to the production of the targeted compound(s). The complexity of elicitor signal transduction, the diversity and specificity of connections between elicitor signals and plant cell receptors, and subsequent plant cell defense responses may play a role in the variation in yield of targeted compounds (Chavan et al., 2010). As most of the elicitors are easily available in the market or can be produced and given to cell cultures in the laboratory, they can be used to produce secondary metabolites on a large scale (Chavan et al., 2010). Based on the aforesaid findings, an optimal level of the precursor was combined with the optimal concentrations of PGRs to get the maximum amount of metabolite accumulation. Surprisingly, we discovered that the combination of SQ and PGRs used in the medium promoted the bacoside content (46.04 ± 0.10 mg/g DW). When cytokinin (BAP) and precursor (SQ) were used in combination, it significantly enhanced total Bacopa saponins. Additionally, among all tested concentrations of elicitors and precursors, squalene was discovered to be the best. As SQ was found in the production route of saponins, it considerably improved the triterpenoid saponin glycosides. The mevalonate pathway is a crucial metabolic pathway with end products such as 3,3-dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) that play a key mediating role in the formation of various secondary metabolites in plants. In order to produce different glycosylated triterpenoids, several enzymes participate in the structural alteration of these intermediates by substitution, oxidation, and glycosylation. Farnesyl pyrophosphate (FPP) molecules are converted to squalene by the enzyme squalene synthase, which is a key regulator in the biosynthesis of sterols and triterpenoids (Jeena et al., 2017; Murthy, 2022). According to earlier reports, B. monnieri was found to be an excellent source of bacosides. The results of our investigations showed that in vitro grown biomass of B. floribunda may serve as an alternate source of bacosides.

Additionally, stigmasterol is a subclass of phytosterols and has been shown to be preventative against cancer, diabetes, and hepatic and cardiovascular disorders. These sterols can reduce cancer risk by 20%, if included into the diet (Shahzad et al., 2017). Phytosterols have attracted the attention of researchers due to its diverse medicinal properties. In the current studies, cytokinin alone (2.0 mg/l BAP) showed the highest stigmasterol (7.69 ± 0.102 mg/g DW) content as compared with other levels of cytokinins. Similarly, MS supplemented with IBA alone (1.5 mg/l) showed the highest stigmasterol content (2.31 ± 0.665 mg/g DW). In precursor and elicitor studies, incorporation of squalene (1.5 μM) and methyl jasmonate (80 μM) in the nutrient medium revealed the maximum stigmasterol content (1.41 ± 0.13 and 1.51 ± 0.10 mg/g DW, respectively). In optimal concentrations of PGRs, elicitors, precursor, and their combinations (MS + 1.0 mg/l NAA + 2.0 mg/l KIN) denoted the maximum (2.56 ± 0.03 mg/g DW) stigmasterol content. These findings are in good agreement with Ghosh et al. (2011). The authors reported stigmasterol from the aerial portions of B. monnieri. It has been demonstrated that phytosterols have the ability to decrease cholesterol and also possess anticancer properties (Ghosh et al., 2011). Numerous secondary metabolites including stigmasterol also demonstrated potential biological functions and play a significant role in the food and pharmaceutical industries. Different plant parts have medicinal potential due to the presence of secondary metabolites such as phenolics, flavonoids, alkaloids, terpenoids, and sterols (Otari et al., 2022; Patel et al., 2022; Ghane et al., 2018; Lekhak et al., 2021). Many bioactive substances appear to have a variety of health benefits; thus, the demand for them in the food and pharmaceutical industries is high (Bhattacharyya et al., 2022; Otari et al., 2022). B. floribunda was also identified as a potential candidate with an additional source of bioactive constituents including triterpenoid saponin glycosides and stigmasterol.

In the present investigation, the correlation among studied optimal mixed concentrations with their respective regeneration responses along with bioactive metabolites (total saponins, triterpenoid saponin glycosides, and stigmasterol) was studied using a data reduction tool, i.e., PCA. The optimum concentration of cytokinin (MS + 2.0 mg/l BAP + 2.0 mg/l KIN) was identified as the best treatment that showed the highest shoot length, higher number of leaves per shoot, FW, and DW. Similarly, optimum mixed combinations of cytokinin with the precursor (MS + 2.0 mg/l BAP + 1.5 μM SQ) contributed toward the triterpenoid saponin glycosides. Similarly, the combination of auxins (MS + 0.5 mg/IAA + 0.5 mg/IBA + 1.0 mg/l NAA) revealed the highest root length and number of roots per shoot. According to the above findings, it can be concluded that cytokinins (BAP and KIN) were responsible for shoot growth whereas root initiation and multiple roots per shoot were mainly controlled by the combined treatment of optimal auxins (NAA, IBA, and IAA). Significant amounts of bioactive compounds were found in the optimal mixed combinations of cytokinin and precursor (MS + 2.0 mg/l BAP + 1.5 μM SQ). Jauhari et al. (2019) reported a strong correlation between total bacosides and phytochemical content due to incorporation of elicitors in the nutrient medium. It was noted that precursors play a vital role in boosting the bioactive potential in plants. Previous reports stated that PCA is the most powerful statistical method for evaluating the relationship between a large number of variables and condensing the data into small elements (Otari et al., 2022; Patel and Ghane, 2021; Ghane et al., 2018).

A thorough analysis confirmed the remarkable amount of the desired metabolites (total saponins, triterpenoid saponin glycosides, and stigmasterol) in in vitro grown biomass of B. floribunda. The favorable outcomes also underlined the goal of improving the production of secondary metabolites in the in vitro cultures of B. floribunda by using precursor and elicitor feeding. The current research work provides an appropriate approach for mass multiplication and also validates the biosynthesis and accumulation of secondary metabolites in in vitro grown biomass of B. floribunda. It forms the first report on in vitro propagation and elicited production of total saponins, triterpenoid saponin glycosides, and stigmasterol contents from B. floribunda. It could be a reliable source of nootropic drugs as well as an important substitute to B. monnieri.

From the studies, we conclude that MS medium fortified with 2.0 mg/l BAP + 2.0 mg/l KIN was found to be the best for shoot development. Similarly, MS + 0.5 mg/l IAA + 0.5 mg/l IBA + 1.0 mg/l NAA promoted the root initiation and number of roots per shoots. The in vitro regenerated B. floribunda plants showed a survival rate of up to 95% after acclimatization and was found to be identical to the mother plant. The presence of triterpenoid saponin glycosides and stigmasterol in in vitro grown biomass was confirmed by HPLC, wherein MS + 2.0 mg/l BAP + 1.5 μM SQ offered the highest content of total pseudojujubogenin glycosides. Similarly, biomass obtained from MS medium fortified with 2.0 mg/l BAP exhibited the highest content of stigmasterol. The species must be conserved in order to produce valuable pharmaceuticals commercially (such as bacosides and stigmasterol), and its sustainable utilization is required. It may serve as a potential candidate and alternate substitute to the Indian Pennywort. Further, studies on detailed phytochemical diversity, pharmacological activities, and elicited production of bioactives using biotechnological tools are needed.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

SGG designed the experiment. The experiment was carried out by SSO, SPD, and SBP performed all the experiments. SO drafted the manuscript and had it critically reviewed by SGG. SSO, SBP and SGG took part in the statistical analysis and assisted in refining the manuscript. All authors contributed to the article and approved the submitted version.