Feijoa (Acca sellowiana (O. Berg) Burret), also known as pineapple guava, is a member of the Myrtaceae family and is native to South America. Feijoa is widely grown in regions like New Zealand, Australia and some parts of the USA for its highly aromatic fruit. Recently, research into the bioactivity of feijoa fruit has significantly appreciated its pharmacological value. Patents have been published on the commercial application of extracts of feijoa for dietary supplements, which alleviate inflammation and pain (Nair et al., 2004) as well as the use of feijoa extract as a lipid antioxidant (Ishigami et al., 1998) and suppress the absorption of cholesterol (Onodera et al., 1998). These research works indicate the production of useful bioactive compounds from different parts of the feijoa plant. New triterpene acids were described by Verardo et al. (Verardo et al., 2019) while analysing callus developed from the fruit pulp of feijoa could have potential applications in human health. Mokhtari et al. (2018) identified high-value antifungal compounds from the peel of feijoa fruit, which inhibit chitin synthesis after screening several feijoa varieties and have been effective against Candida species which cause most fungal infections in humans. Montoro et al. (2020) also identified bioactive compounds from feijoa flowers that can be used to prepare nutraceuticals and dietary supplements. Recently, Amer et al. (2023) identified antithrombotic, anti-oxidant and anti-inflammatory properties from feijoa extracts isolated from callus and cell biomass from cell cultures in a bioreactor using biological assessments through in-vivo studies. The fruit extract has also shown bactericidal activity against gram-negative bacteria such as Pseudomonas, Enterobacter and Salmonella (Vuotto et al., 2000). There have been reports about a significantly higher concentration of abscisic acid than other fruit plants, which has been identified as a novel nutraceutical for controlling glycaemic index (Zocchi et al., 2017). These properties of feijoa are already being exploited by commercial entities for marketing feijoa fruit extract as a potential immune-boosting agent against diseases such as type II diabetes.

However, the practicality of using the identified bioactive compounds towards their intended use is restricted since these compounds are found in very low concentrations, and their production depends on many biotic and abiotic factors. Furthermore, these compounds tend to be complex, which could require highly complex extraction and purification processes. Recently, a lot of focus has been on developing alternative methods of generating biomass for extraction of high-value compounds, such as the application of large bioreactors (to the scale of 1000L and more) and utilisation of the biomass for various purposes such as extraction of high-value compounds, as food substitutes with the aim of reducing environmental footprint and maintaining the ecological balance (Nordlund et al., 2018; Rischer et al., 2020). Private investment in this technology has resulted in the successful production of drugs such as Paclitaxel and Docetaxel by Phyton Biotech, and Taliglucerase Alfa by Protalix Biotherapeutics (Häkkinen et al., 2020). Plant cell and organ cultures have been considered as a potential source to produce biomass for the synthesis of bioactive compounds, which can be used in various applications, including pharmaceuticals, nutraceuticals and cosmeceuticals production and tissue culture techniques such as cell cultures in bioreactors. Plant tissue culture is a perpetual source for producing industrially important bioactive compounds (Chandran et al., 2020). The production of ginsenoside from Panax ginseng (Sivakumar et al., 2005), stevioside from Stevia rebaudiana cell suspension culture (Mathur and Shekhawat, 2013), ajmalicine from Catharanthus roseus (Lee and Shuler, 2000) shikonin from Lithospermum erythrorhizon, berberine from Coptis japonica (Fujita, 2007) rosmarinic acid from Orthosiphon stamineus and Coleus blumei (Ulbrich et al., 1985; Liang et al., 2006) and sanguinarine from Papaver somniferum (Eilert et al., 1985) are some examples where different types of plant tissue culture systems have been used to generate biomass for the extraction of bioactive compounds. These techniques provide options to circumvent the limitations for producing useful bioactive compounds (Robles-Martínez et al., 2016; Krasteva et al., 2021). Advances in plant genomics and metabolomics have also made it possible to better understand bioactive compounds’ dynamics in terms of their properties controlling factors which could result in improved production systems. This paper focuses on the effect of different parameters investigated in the generation of in vitro cell suspension culture system for feijoa floral buds, which could be used to generate cell biomass to synthesise secondary metabolites. This study also describes identifying bioactive compounds of significant importance from feijoa tissue and cell cultures, which could be explored for their potential application in various fields.

In this paper we demonstrate a possible route to explore the production of bioactive compounds from feijoa through the generation of cell biomass using an in vitro cell suspension culture method.

Various studies have shown that feijoa plants produce different compounds that can benefit human well-being. Weston (2010) published a comprehensive review of the application of feijoa bioactive compounds for various purposes. Feijoa extracts have been tested for their efficacy against various human ailments. More recently, bioactive compounds were identified and assessed from feijoa callus and cell suspensions for their anticoagulant properties, which resulted in decreased platelet aggregation (Amer et al., 2023). The source of these extracts has so far been the fruit or the fruit peel. Cell suspension cultures offer an alternative method to produce high-value compounds in large quantities and high purity in contained environments where biotic and abiotic factors responsible for the production of these compounds can be monitored and regulated. In vitro cultures, such as cell suspensions, provide a continuous and reliable source of bioactive compounds making the process standardised and economically sustainable (Davies and Deroles, 2014). However, biomass accumulation and synthesis of bioactive compounds by the cultured cells is a two-stage process as proposed by Murthy et al. (Murthy et al., 2014), which involves: stage 1) the establishment of a cell suspension system and stage 2) the elicitation of the bioactive compounds, which are controlled by various factors.

Friable callus induction is a prerequisite to generate cell suspensions. Different plant groups respond differently to the callus induction process, which depends on many factors, such as types and concentrations of auxins and cytokinins, the tissue and the physiological state of the tissue. During earlier research studies on feijoa, where application of callus has been majorly restricted to achieve somatic embryogenesis, callus had been induced from stamen filaments, petals and ovaries (Stefanello et al., 2005), whole intact cotyledonary zygotic embryos (Reis et al., 2008), fruit pulp (Verardo et al., 2019) and leaves (Amer et al., 2023). In this study, we used floral buds without separating stamen filaments, petals, or ovaries but used the regions of floral buds as explants (Figure 1). Callus was induced in 10 days’ time from all regions of the floral buds, which is similar to the results obtained by Stefanello et al. (2005). As highlighted in many other studies the auxin 2,4-D is one of the key exogenous auxins to evoke a response from explants for callus induction. Menbari et al. (2021) used 2,4-D and BA to induce callus from Granny Smith apples, which were later used to generate cell suspension cultures. Soleimani et al. (2012) also reported the highest callus induction from Arctium lappa using 2,4-D and BAP. Our study results support those findings. However, we also observed callus induction in our control treatment without 2,4-D and BAP (Figure 3). This could be attributed to the highly active metabolic state of the explants, which responded to the wounding process. The callus in our control treatment could be classified as a wound-induced callus, which can be derived from various cell types such as vascular-associated cells, cortical cells and pith cells (Ikeuchi et al., 2013). This response of callus induction without any exogenous hormones could have also been influenced by the transitioning phase of the floral bud, where the stage includes a complex system of factors which interact and involve various biochemical signals, including plant hormones, polyamines, gibberellic acids and cytokinins (Meijón et al., 2011).

Friable callus was selected for initiation of suspension cultures. The friable property of callus helps initiate suspension cultures when cultured in a liquid medium, which helps easy dispersion and growth of the cells in a liquid medium when agitated. The biomass accumulation in the medium depends on multiple factors such as the salt concentrations, inoculum density and the type and concentration of the sugars used in the medium (Murthy et al., 2014). When we cultured the cell suspension using different strengths of MS medium to understand the requirement of salt concentration for optimal growth of biomass, the highest biomass growth was achieved using half-strength MS medium as compared to three-fourth and full-strength medium (Figure 7). The salt requirements in the medium have been shown to vary in cells from different plants (Wu et al., 2006; Nagella et al., 2011; Naik et al., 2017). It has also been reported that during Panax ginseng root biomass production, three-fourth strength MS medium produced better results (Sivakumar et al., 2005). Improving the growth and biosynthetic capacity of the in vitro generated plant cell mass by developing optimal culture conditions through the choice of medium and salt concentrations is critical (Isah et al., 2018). During in vitro culture of Gloriosa superba, higher concentrations of phosphates and calcium have resulted in the inhibition of alkaloid biosynthesis (Pandurangan and Philomina, 2010), which highlights the importance of the concentrations of media nutrients for achieving productivity in in vitro culture system. It has been observed in earlier studies that the sucrose concentration and osmoticum of the medium affect the physiological and metabolic activity of the cells and influence fresh weight and dry weight of the cells (Kim et al., 2001). The lower percentage growth with higher dry weight seen in our sucrose and inoculum size experiments could influence a combination of factors, including sucrose concentration, the osmolarity of the medium and the inoculum size.

Inoculum density is one of the important factors that could influence both biomass production and the productivity of the bioactive compound. This has been shown by Lee and Shuler (2000) while working with Catharanthus roseus. While working with Stevia rebaudiana Mathur and Shekhawat (2013) observed differences in cell biomass production at different inoculum densities. Our results also highlight that the inoculum density influences biomass production at the end of the growth period. The results (Figure 7) show that 3 g FW/100 mL of MS medium was the most suitable inoculum density for achieving the best biomass production.

Another critical condition for achieving optimum biomass generation in cell suspensions and at the same time, maintaining the productivity of the desired secondary metabolite, is that the growth medium is provided with the optimum ratio of auxin and cytokinin. The application of some of these plant growth regulators is limited by their classification as harmful to the environment and human health, especially the synthetic growth regulators, which are considered pesticides and their use is regulated by law around the world, such as in Europe (https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/public/?event=homepage&language=EN). Häkkinen et al. (2020) reported only trace amounts of free growth regulators are present in the cells at the harvesting point. At the same time, the relationship between auxin/cytokinin and the production of secondary metabolites has been highlighted in various studies (Baque et al., 2010; Kurepa et al., 2023). Auxins and cytokinins are closely linked with the production of reactive oxygen species (ROS) in cells which in turn is linked to secondary metabolite production (Huang et al., 2019). In feijoa cell suspensions, we observed that 2,4-D at 2 mg/L and 4 mg/L concentration, along with BAP at 0.2 mg/L, were the best concentrations for optimal growth (Figure 8). On the other hand, no growth of cell suspensions was observed in the absence of both the auxin and cytokinin, indicating that the growth of feijoa cell suspension depended on the availability of an exogenous supply of auxins and cytokinins. Further studies on the effect of 2,4-D and BAP on accumulating secondary metabolites in feijoa cell suspensions need to be investigated. Also, the possibilities of applying safer plant growth regulators such as coconut water and indole acetic acid or strategies that would minimise the application of harmful synthetic growth regulators to acceptable levels in combination with natural PGR should be investigated. Häkkinen et al. (2020) reported a two-phase culture system where the maintenance of cells could be with the original medium (which might include any synthetic plant growth regulators), whereas the scaling up of cells could be in plant growth regulator free medium.

Sugars act as energy sources and signal molecules that affect cultured cells’ growth, development, and metabolism (Rolland et al., 2006). Sugars are also classified as important regulatory molecules with signalling functions in plants. Earlier work (Wang and Weathers, 2007) has indicated that different plants have different abilities towards the uptake of different forms of sugars such as hexoses, glucose and fructose, and disaccharide sucrose, which can result in differential growth patterns even though in most cases of plant cell suspension cultures, sucrose is the most commonly used sugar. When cultured with fructose, Phaseolus vulgaris cell suspensions had a higher growth rate than glucose or sucrose (Botha and O’Kennedy, 1998). Cynara cardunculus cell suspension cultures could use glucose better than fructose (Oliviero et al., 2022). Cell wall invertase, cytoplasmic invertase and vacuolar invertase are the key enzymes regulating the breakdown of sucrose and its availability in downstream processes. These enzymes generate high levels of extracellular glucose and fructose that are taken up by hexose transporters (Rolland et al., 2006). The higher growth rate seen with glucose and fructose could be due to the higher affinity of the feijoa cells towards hexose transporters, as Rolland et al. (2006) suggested. Our experiments with different sucrose concentrations revealed that at 3% sucrose, the growth of feijoa cells was optimum. Further studies are required to understand carbohydrate assimilation, which would help optimise the medium for optimum biomass growth. Expression of secondary metabolites is intricately associated with their complex pathways. Numerous primary and secondary metabolites, such as products of the glyoxylate pathway, anthocyanin and artemisin, have responded differently to sugars (Arsenault et al., 2010). In other species, such as the Brazilian Hancornia speciosa (Gomes) cell suspension cultures, adding 3% glucose increased fresh weight and production of polyphenols (Dantas et al., 2021). The differences seen in the period for achieving the maximum growth during the growth curve between Amer et al. (2023) and the current study, 14 and 30 days, respectively, could be attributed to the differences in the varieties of feijoa, the different source tissue used for induction of callus as well as the differences in the set of growth conditions. Even though applications of feijoa cell suspensions for bioreactor setup have been mentioned recently (Amer et al., 2023) investigating the anticoagulant properties of feijoa, there has been a lack of data on the effect of different parameters involved in generating cell suspensions from feijoa. The current study has investigated and presented for the first time the effect of different parameters involved in establishing feijoa cell suspension cultures aimed at generating biomass for the extraction of bioactive compounds. The differences observed in the percentage growth rates under MS half strength, 3% sucrose and 2 mg/L of 2,4-D could be due to sucrose’s dual role, which serves as an energy source and an osmotic stabiliser. Sugars have been considered as signalling molecules that affect the growth, development, and metabolism of plant cell cultures (Murthy et al., 2014).

MeJA is considered a major secondary signalling molecule able to trigger signal transduction, leading to the generation of secondary signals that stimulate regulatory proteins, which coordinate the expression of biosynthetic genes (Zhao et al., 2005). Earlier studies have revealed that amongst the elicitors and concentrations, the use of 100 µM MeJA was found optimum for secondary metabolite enhancement in the majority of the experiments (Giri and Zaheer, 2016). The production of secondary metabolites typically occurs in the late stationary phase of the growth curve and is associated with growth inhibition and the production of enzymes for secondary metabolism (Ochoa-Villarreal et al., 2016). In our study, we evaluated 100 and 50 µM MeJA concentrations and the treatment of the feijoa cell suspension with 50 µM MeJA during the stationary phase after 30 days of growth, increasing the number of secondary metabolites and plant hormones. This increase in secondary metabolites could be due to multiple factors, as discussed by several authors.

In this study, we also reported an increased number of metabolites in cell cultures versus floral buds as compared to the previous work reported by Amer et al. (2023), in which they reported fewer total phenolic and flavonoids in the cell cultures from bioreactors when compared to the leaves. Interaction of signalling compounds such as jasmonic acid generates increased ROS, contributing to gene expression and the production of requisite transcripts to synthesise increased secondary metabolites (Giri and Zaheer, 2016). It is also known that chemical elicitors have the multitasking ability to drive a number of pathways at the biochemical and genetic levels (Boatwright and Pajerowska-Mukhtar, 2013). During the mechanism of elicitor action such as jasmonic acid, the key control points, which play a significant role, could be elicitor perception by the plasma membrane, a signal from organelles such as chloroplasts and mitochondria and participation of the 26S proteasomal degradation pathway facilitated by JA (Giri and Zaheer, 2016). Peng et al. (2022) identified 10 phenolic compounds in feijoa extracts, including phenolic acids, flavonoids and procyanidins, which play a significant role in the anticancer activity of the feijoa extract. Our study’s UPLC – QTOF MS analysis of floral buds and cell suspensions identified arctigenin, a high-value bioactive compound with pharmaceutical applications for treating various diseases. Arctigenin is a dibenzylbutyrolactone lignan, a phenolic compound and has been attributed with several therapeutic properties, including anti-tumor, anti-inflammatory, anti-colitis and anti-microbial effects (Shabgah et al., 2021). The identification of arctigenin in feijoa floral buds and cell cultures also compliments the strong anticancer activity of feijoa extract in earlier studies (Peng et al., 2022). Arctigenin has often been used as a lead compound for drug development because of its novel structure, pharmacological properties, and great developmental potential (Wu et al., 2022). Further evaluation with respect to the yield of arctigenin in the cell suspensions undergoing experiments with different types of elicitors and precursors, needs to be investigated.

We have identified the key parameters that could influence the establishment of feijoa cell suspension cultures from the various experiments conducted. Our experiments have identified the optimal salt concentrations in the medium, optimum inoculum density, the optimum 2,4-D concentrations in the medium and the best sugar sources and their concentrations for initiating and growing feijoa cell suspension cultures.

However, more studies are required to scope for alternative strategies with suitable plant growth regulators that would be readily acceptable for biomass generation for extracting high-value bioactive compounds at an industrial scale, considering the negative effects of 2,4-D on human health and the environment.

The phytochemical analysis of elicitor treated cell suspensions revealed increased phenolic compounds and secondary metabolites at 50µM MeJA. Our study’s identification and confirmation of arctigenin, a natural anticancer agent, also strongly supports exploring feijoa in in vitro cell suspension cultures for generating biomass to extract arctigenin and other high-value compounds. The presence of arctigenin supports previous studies in which anticancer properties in feijoa extracts have been identified. The presence of these types of high-value compounds in feijoa provides the foundation for the development of biomass production in bioreactors, which would lead to reduced environmental and ecological footprint and, at the same time, will allow harvest of these compounds year-round independently of biotic and abiotic factors and supply chain issues. Future studies will investigate establishing feijoa cell suspension cultures in bioreactors with acceptable plant growth regulators. Studies should also focus on identifying key elicitors which would further increase the production of arctigenin from the cells. More efforts should also be focused on revealing other pharmacological properties of the cell extracts derived from feijoa cell suspensions. The possibilities of extracting arctigenin from feijoa cell suspensions from bioreactors should also be explored.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

SR: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Resources, Visualization, Writing – original draft, Writing – review & editing. II: Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – review & editing. SP: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review & editing. HN: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – review & editing. SH: Funding acquisition, Writing – review & editing.