Introduction

Capsaicinoids are compounds that produce pungency in chillies and capsaicin and dihydrocapsaicin are the main pungent principles that account for most of the pungency in chillies. The site for capsaicinoid synthesis is mainly in the placenta and interlocular septum, where they are produced by epidermis cells and are secreted through glandular vesicles called ‘‘blisters’’ located on the surface of placental tissue (Iwai et al. 1979; Rowland et al. 1983). Some amount of capsaicinoids has also been shown to accumulate in the pericarp tissue of the fruits (Tanaka et al. 2016; Sugiyama 2017; Park et al. 2019). Capsaicin has many pharmacological and physiological properties and is commonly used for its analgesic, anticancer, anti-inflammation, anti-obesity and antioxidant effects (Deal et al. 1999; Moore and Moore 2003; Kempaiah et al. 2005; Sawynok 2005). Since capsaicinoids are also used in food and other industries, the demand for capsaicinoids is increasing and cell and tissue cultures of highly pungent chili cultivars may serve as an alternative potential source of capsaicinoids. To sustain a stable production of capsaicinoids, a number of earlier studies have reported the production of capsaicinoids from cell suspension cultures established from hypocotyl explants obtained from seedlings germinated in vitro or immobilized placental tissues of different Capsicum cultivars (Lindsey 1985; Johnson et al. 1990, 1996; Kehie et al. 2014). The capsaicin content in cell cultures established from callus obtained from the hypocotyl explants were low (Holden et al. 1987) and the immobilized placenta cultures of different Capsicum species, on the other hand, showed higher capsaicin accumulation using various enhancement strategies (Johnson et al. 1990; Johnson and Ravishankar 1996; Aldana-Iuit et al. 2015). However, since the immobilized placenta does not provide a consistent source and capsaicinoids biosynthesis occurs mainly in the placental tissue of fruits, the use of placental tissue as explants for establishing in vitro cultures are expected to accumulate higher amount of capsaicinoids and may provide an alternative source for production of capsaicinoids (Aza-Gonzalez et al. 2011; Ancona-Escalante et al. 2013). Callus induction and assessment of capsaicinoids production potential of callus derived from placental tissues of Capsicum annuum have been reported only by Umamaheswari and Lalitha (2007). Since callus derived from placental tissue explant showed higher capsaicinoid accumulation than other explants of Capsicum annuum L. (Umamaheswari and Lalitha 2007) and the average capsaicinoid content of Capsicum chinense Jacq cv. ‘Umorok’ (synonym-Naga jolokia, Naga King Chilli, Bhut jolokia) fruits is high (Sanatombi and Sharma 2008), the cultures derived from placental explants of this highly pungent cultivar may have the potential for higher capsaicinoid production if the descendant cells retain their potential.

Further, capsaicinoid biosynthesis involves two converging pathways, phenylpropanoid pathway for synthesis of vanillylamine and the pathway for synthesis of fatty acids (Fujiwake et al. 1980). Numerous enzymes are involved in capsaicinoid biosynthesis pathway and phenylalanine ammonia-lyase (PAL) is the first enzyme in the phenylpropanoid pathway (Perucka and Materska 2001) followed by cinnamate-4-hydroxylase (C4H), 4-coumarate: Coenzyme A Ligase (4CL), hydroxycinnamoyl transferase (HCT), coumarate-3-hydroxylase (C3H), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) and a putative aminotransferase (pAMT), which catalyses the formation of vanillylamine. In the branched-fatty-acid synthesis pathway, 8-methyl-6-nonenoyl-CoA is formed (Suzuki et al. 1981), which finally undergoes condensation reaction with vanillylamine from the phenylpropanoid pathway to form capsaicinoids catalysed by acyl-transferase encoded by Pun1 (Stewart et al. 2005). Most of the structural genes of capsaicinoid biosynthesis pathway have been identified and their expression pattern in different chilli cultivars has been determined (Curry et al. 1999; Liu et al. 2012; Tanaka et al. 2016; Zhang et al. 2016) and non-pungent cultivars have been shown either to accumulate very few capsaicinoids or to accumulate another group of compounds called capsinoids (Yazawa et al. 1989; Kobata et al. 1998). Although several studies have been conducted to assess the capsaicinoid production potential of tissue cultures obtained from different explants and the studies reported lower production of capsaicinoids in cell cultures compared to chilli fruits, the genes involved in capsaicinoid synthesis responsible for lower capsaicinoid accumulation in cultures have not been studied. Therefore, the present study aimed at investigating the capsaicinoids production potential of callus cultures established using placental tissue explants of the highly pungent chilli cultivar and to investigate the change in expression patterns of genes involved in capsaicinoid synthesis during callus formation so that the down-regulated genes may be identified for future metabolic engineering considerations.

Materials and methods

Callus culture induction from placental tissue explants

Fresh and healthy fruits of Capsicum chinense Jacq cv. ‘Umorok’ at matured green stage were washed with running tap water and then treated with 0.1% Carbendazim for about 10 min and then rinsed with distilled water for 3 times. The fruits were then surface sterilized by flaming for 2–3 s after dipping in 70% ethanol under aseptic condition followed by treatment with 0.5% HgCl2 solution for 5 min. After 5 min, the fruits were washed for about 5 times using autoclaved distilled water. The fruits were then cut open with a sterile surgical blade; the placental tissues were taken out and trimmed to about 0.5 cm long pieces. These explants were then inoculated on MS medium supplemented with 0.5 mg L−1 Kinetin (Kin) along with 1, 2 or 3 mg L−1 2,4-Dichlorophenoxyacetic acid (2,4-D); 0.5 mg L−1 Kin along with 2 mg L−1 indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA); 0.5 mg L−1 Benzylaminopurine (BAP) alone or 2 mg L−1 2,4-D alone. The culture tubes were incubated in dark condition at 25 ± 2 °C. The biomass growth and capsaicinoid production of the placental callus cultures were studied for a period of six consecutive growth cycles, each comprising of 32 days. The cultures were subcultured by transferring about a gram of callus in 25 mL of medium after every 32 days. The growth curve for the callus was established by measuring FW, DW and capsaicinoids (viz. capsaicin and dihydrocapsaicin) content at 4 days interval. The experiments were repeated thrice with ten replicates each.

Extraction and quantification of capsaicinoids

Capsaicinoids were extracted from callus and placental tissue by following the method of Collins et al. (1995). The capsaicinoid content of the extracts were measured by UV/Vis Spectrophotometer at 296 nm by following the methods of Salgado-Roman et al. (2008). Capsaicinoid content was further confirmed by high performance liquid chromatography (HPLC) using Agilent 1260 infinity series equipped with Zorbax Eclipse Plus C18 (4.6 × 100 mm, 3.5 micron). The mobile phase consisted of a binary mixture of solvent A (methanol)–B (water) at 50%:50% ratio for 3 min followed by an increasing gradient to 100% solvent B till 10th min and further maintained at 100% solvent B for another 5 min. Detection was at 230 nm, and the flow rate was maintained at 1 mL min−1.

RNA isolation for cDNA synthesis and qPCR analysis

To compare the expression levels of genes involved in capsaicinoid biosynthetic pathway during callus formation, qRT-PCR analysis was performed. The placental tissue of mature dark green fruits of C. chinense Jacq cv. ‘Umorok’, callus cultures from the first  six months and 1 year-old callus tissue collected on the day with maximum capsaicinoids content of every growth cycle studied were taken for RNA extraction. Total RNA was extracted using ZR Plant RNA MiniPrep (Zymo Research, Germany) and the RNeasy Mini spin column (Qiagen). The RNA extracted from the samples used for RT-PCR were treated with DNase I prior to cDNA synthesis to remove DNA contamination. Four hundred nanograms of RNA were converted into cDNA using the iScript™ Reverse Transcription Supermix for RT-qPCR (BIO-RAD). Quantitative PCR was performed using the SYBRR Green JumpStart™ Taq Ready Mix™ (Sigma–Aldrich, USA) on the QuantStudio™ 5 System according to the manufacturer’s instructions. The thermal cycle used was 94 °C for 2 min followed by 39 cycles of 94 °C for 15 s, 60 °C for 1 min and a melting curve analysis was performed at 95 °C for 15 s, 60 °C for 1 min and 95 °C for 1 s. The dissociation temperature range extends from 65 to 95 °C. Actin gene was used as the reference gene for normalization and the Livak (ΔΔCT) method was used to analyse the expression levels (Livak and Schmittgen 2001). The RNA extraction was conducted with three biological repeats and each data represent the mean relative expression of three biological repetitions with standard error bars. The primer sequences used for qRT-PCR are listed in Table 1.

Table 1 Nucleotide sequences of primers used for qRT-PCR
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Statistical analysis

All the tissue culture experiments were repeated thrice, each consisting of three technical replicates and data were analyzed using one-way analysis of variance (ANOVA, p < 0.05). The significant differences among the means were determined by Duncan’s multiple range tests.

Results and discussion

Callus culture induction

The placental segment explants cultured on callus induction medium showed increase in size accompanied by swelling and bulging with slight callusing on the margins in the first month of culture. When these explants were subcultured after 32 days, the explants cultured on callus induction medium containing 2 mg L−1 2, 4- D and 0.5 mg L−1 K in produced friable callus after about 15 days. The effect of different auxins and cytokinins on callus induction from placental tissue explants at the end of the second month is shown in Table 2. Among the different combinations and concentrations of auxins with cytokinins used, MS medium containing 2 mg L−1 2, 4- D and 0.5 mg L−1 Kin was found to be the most effective for callus induction producing rapidly growing friable calli (Fig. 1a–d) while the explants cultured on the media containing other combinations and concentrations of auxins with cytokinins produced compact calli with low proliferation rate. Similar effectiveness of the use of 2 mg L−1 2, 4-D and 0.5 mg L−1 Kin together in callus induction from placenta explants of Capsicum annum L. was reported by Umamaheswari and Lalitha (2007). When the friable callus obtained from placental tissue explants cultured on medium containing 2 mg L−1 2,4-D with 0.5 mg L−1 Kin were subcultured on fresh medium supplemented with the same combination of growth regulators after every 32 day, the callus tissue proliferated rapidly on the medium showing a growth curve characteristic of cultured plant cells. From the third month onwards, the biomass growth of the callus cultures were maintained in a similar manner till the sixth month of cultures studied (Fig. 2a). The growth curve of the callus cultures consisted of an initial slow lag phase comprising of the first 4 days followed by an exponential growth phase ending at day 15. The cultures then entered a linear growth phase lasting from day 16 to day 23 followed by a stationary phase of growth from day 24 onwards. The maximum increase in callus biomass were obtained on day 24 of every cycle reaching up to 7.8 g FW culture−1 and 0.56 g DW culture−1 on the 4th month which was about 16-fold over the initial explant FW. During the first growth cycle comprising of 32 days, the amount of capsaicinoids in the placental tissue gradually decreased from 19 mg g−1 FW capsaicin and 8.8 mg g−1 FW dihydrocapsaicin in fresh placental tissue explants to 12 mg g−1 FW capsaicin and 5.3 mg g−1 FW dihydrocapsaicin respectively in the callus cultures (Fig. 2b, c). The decrease in capsaicin and dihydrocapsaicin content further continued in the second month showing maximum content of 6.3 mg g−1 FW and 2.8 mg g−1 FW at day 20 as the placental cells undergo dedifferentiation to form callus tissue. The optimum capsaicinoid accumulation were obtained on 20th day of every growth cycle and in the third growth cycle, the amount of capsaicin was 1.9 mg g−1 FW and the amount of dihydrocapsaicin was 0.9 mg g−1 FW, which further decreased a little to 1.65 mg g−1 FW and 0.8 mg g−1 FW in the fourth month. However, from the fourth month onwards, the capsaicin and dihydrocapsaicin content of the callus on 20th day of every growth cycle remained stable up to 6 months at an average of 1.6 mg g−1 FW (equivalent to 12.5 mg culture−1) and 0.78 mg g−1 FW, respectively (Fig. 2b, c). The average content of capsaicinoid is maintained in the 12 months-old cell line maintained for further studies. In vitro capsaicinoid production from different explants of Capsicum species using various enhancement strategies have been reported earlier by several workers as reviewed by Kehie et al. (2014). However, the earlier reported capsaicin content in suspension cultures after enhancement using different strategies were in the range of 0.0307 mg culture−1 in Capsicum frutescens Mill (Sudha and Ravishankar 2003) to 3.83 mg culture−1 in Capsicum frutescens Mill (Rao and Ravishankar 2000), which is lower than the content (12.5 mg culture−1) reported in the present study. Similarly, suspension cultures established from callus derived from seedling explants of Capsicum chinense Jacq. ‘Umorok’ syn. ‘Naga King Chilli’ were shown to accumulate maximum of 1.6 mg g−1 FW capsaicin after enhancement, (Kehie et al. 2012) which is comparable to the capsaicin content in the callus (1.6 mg g−1 FW) without enhancement being reported in the present study. Earlier, several studies have shown the use of immobilized placental culture to accumulate higher amount of capsaicin when treated with elicitors of precursors. Capsaicin production was increased to 0.593 mg g−1 FW after enhancement in immobilized cell culture derived from seedlings of Capsicum annuum by Ravishankar et al. (1988) while Johnson and Ravishankar (1996) reported the accumulation of 5.78 mg g−1 DW capsaicin from cells of placenta of Capsicum frutescens Mill following immobilization and enhancement treatment. Similarly, in case of Capsicum chinense Jacq. (Habanero peppers), Aldana-Iuit et al. (2015) reported maximum capsaicinoid production from immobilized placental tissue of 1.56 mg g−1 DW. Thus, the capsaicinoids content in the present stable callus cultures reported in this study is higher than the content in most of the earlier reports of capsaicin production by cultures derived from seedling explants of different Capsicum species. Although the content in immobilized placental tissues of different Capsicum species after enhancement is comparable to the content in the callus cultures obtained in the present study, the callus cultures have the advantage of rapid proliferation compared to the immobilized tissues. Thus, the present study reports the production of high capsaicinoid producing callus from the placental tissue explants of the highly pungent chilli cultivar C. chinense Jacq cv. ‘Umorok’ that have the potential to serve as an alternative source of capsaicinoids.

Table 2 Effects of plant growth regulators in callus induction from the placental tissue explants of Capsicum chinense Jacq. cv. ‘Umorok’
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Fig. 1
figure 1

Induction of callus cultures from placental tissue explants of Capsicum chinense Jacq. cv. ‘Umorok’ on MS medium containing 2 mg L−1 2, 4-D and 0.5 mg L−1 Kin: a placental explant inoculated on MS medium; b compact callus at 1st month; c semi friable callus at 2nd month; d friable callus at 3rd month and e fully grown friable callus at 4th month culture

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Fig. 2
figure 2

Growth cycles for placental callus of Capsicum chinense Jacq. cv. ‘Umorok’: a biomass growth; b capsaicin and c dihydrocapsaicin accumulation during the growth cycles for 6 months after subsequent subcultures at 32nd day. Error bars shows standard errors

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Expression patterns of capsaicinoid biosynthesis genes in callus cultures

To understand the gene expression patterns during callus formation from the placental tissue explants, the expression levels of genes involved in capsaicinoids biosynthetic pathway were determined by RT-qPCR analysis. Overall, all the candidate genes are down-regulated in callus cultures compared with the placental tissue. Nine structural genes involved in capsaicinoids biosynthetic pathways, including Phenylalanine ammonia-lyase (PAL), cinnamatc 4-hydroxylase (C4H), hydroxycinnamoyl transferase (HCT), coumarate3-hydroxylase (C3H), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), NADH-Glutamine oxoglutarate aminotransferase (NADH-GOGAT), glutathione S-transferase (GST), pAMT, and Pun1 were studied for comparative gene expression analysis and the expression pattern of each candidate gene is shown in Fig. 3. Among the nine candidate genes analysed, Pun1 and pAMT showed decreased expression during the first  two months of culture compared to the placental tissue and were highly down regulated in the further growth cycles of callus cultures (Fig. 3h, i). Pun1 encodes an acyltransferase enzyme, which is believed to catalyze the last step in the capsaicinoid biosynthetic pathway (Stewart et al. 2005; Lang et al. 2006) and loss-of-function in Pun1 gene results in non-pungency (Stewart et al. 2005). The expression pattern of Pun1 gene is consistent with capsaicinoid accumulation in callus cultures during the growth cycles. The expression of Pun1 gene was 3 and  4-fold lower respectively than the placental tissue in the first and second callus growth cycles and the expression was greatly reduced in the further growth cycles (Fig. 3h). A similarity was also observed for pAMT gene expression pattern, which showed approximately 9- and 13-fold lower expression in the first two months and the expression level was almost negligible from the third month onwards (Fig. 3i). These expression patterns in callus cultures correspond with the capsaicinoid accumulation during the growth cycles. pAMT gene catalyses the conversion of vanillylamine from vanillin in phenylpropanoid pathway, which is an important step in capsaicinoid biosynthesis (Lang et al. 2009; Tanaka et al. 2015). Earlier, pAMT and Pun1 genes were reported to play crucial roles in capsaicinoid accumulation in chilli peppers including C. Chinense (Stewart et al. 2005; Ogawa et al. 2015; Sarpras et al. 2016), and silencing of the pAMT gene expression or loss-of-function in a particular mutant (CH-19 Sweet) has been shown to result in significant reduction in capsaicin production in placental tissues (Lang et al. 2009). Similarly, in the present study, the expression pattern of these genes are observed to correspond with capsaicinoid accumulation in callus cultures and down-regulation of these two putative genes (pAMT and Pun1 gene) in the friable callus cultures may be responsible for the lower capsaicinoid accumulation in callus cultures compared to placental tissues. For the other candidate genes (PAL, C4H, HCT, C3H, CCoAOMT, NADH-GOGAT and GST), the expression patterns were not consistent with the capsaicinoid accumulation during the growth cycles (Fig. 3a–g). The reason of this inconsistency might be that they are involved in early stages of the capsaicinoid biosynthesis pathway and also participate in the production of other secondary metabolites such as flavonoids, coumarins and lignins (Vogt 2010; Fraser and Chapple 2011). Thus, the present study shows, for the first time, the regulation of capsaicinoid accumulation during callus formation from placental tissue explants of C. chinense Jacq. and observed marked down-regulation in the expression of two candidate genes (pAMT, and Pun1) in the callus cultures and these two genes may be considered as potential targets for enhancement of in vitro capsaicinoid production using different strategies including metabolic engineering. Studying the mechanism underlying the down-regulation of these capsaicinoid biosynthesis pathway genes in cells cultures will be useful in controlling the in vitro capsaicinoid production potential of cell cultures.

Fig. 3
figure 3

Differential gene expression levels of capsaicinoid biosynthesis genes during callus formation. The relative expression levels of 9 structural genes were determined by comparative qRT-PCR analysis in the placental tissue and 1st–6th and 12th months-old callus cultures: a PAL, b NADH-GOGAT, c C4H, d HCT, e C3H, f CCoAOMT, g GST, h pAMT and i Pun1. All data are presented as means of three repeats. Error bars shows standard errors

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