3.1. Selection of elite plant part
Synthesis and accumulation of bioactive compounds in plants are influenced by biotic (pathogens, herbivores, etc.) and abiotic (temperature, light, water, soil, etc.) factors including geographical location, temporal and seasonal variation (Page et al. 2005), genotypes (Shamloo et al. 2017) and developmental stages (Popović et al. 2020) of the plant. These factors can significantly affect the plant growth and development, even their ability to synthesize secondary metabolites, consequently leading to the change in the overall phytochemical profile (Yang et al. 2018). Therefore, before proceeding further induction of callus and elicitation of bioactive compounds, the selection of an elite plant part yielding maximum content of luteolin and rutin is highly desirable. In the present study, significant difference in luteolin and rutin contents was seen in various in vivo plant parts viz. roots, stem and leaves of R. hastatus collected from Mandi, Himachal Pradesh, India. HPLC chromatogram of standard compounds such as rutin and luteolin showed the peaks at retention time at 19.39 and 28.35 min, respectively (Fig. 1b). HPLC analysis of plant samples revealed that roots had highest content of luteolin (747.32 ± 20.71d µg/g d.w.) and rutin (1689.43 ± 25.43a µg/g d.w.) compared to other plant parts (Supplementary Table 2; Fig. 1c). The amount of luteolin was 528.95 ± 13.75e µg/g d.w. and 322.51 ± 8.39f µg/g d.w. in leaf and stem, respectively (Fig. 1c). However, respective content of rutin in leaf and stem was 1315.00 ± 22.39b and 1193.34 ± 45.43c µg/g d.w. (Fig. 1c). As roots had maximum luteolin and rutin content compared to other plant parts and thus it was used for callus induction and regeneration.
3.2. Establishment of aseptic cultures
Surface sterilized nodal segments were taken from in vivo grown R. hastatus and utilized for establishment of aseptic cultures. The nodal segments cultured on MS basal medium (without growth regulator) exhibited single axillary shoot per explant within 7–14 d of culture. These in vitro shoots were used for preparing nodal explants for future experiments. Besides, roots organized at the basal cut end of nodal segments were excised and used for callogenesis.
3.3. Induction of callus from in vitro roots
Based on the above data, optimum level of luteolin and rutin was present in roots of R. hastatus. Thus, root explants were used for further in vitro regeneration through callus cultures (Fig. 1d). Initiation of callus was started within two weeks of inoculation, but the variation in nature and amount of the calluses were recorded after 30–d of culture. Calluses induced on different auxins such as IAA, IBA and NAA were whitish, friable and non–morphogenic with numerous root hairs on the surface of growth medium (Supplementary Table 3). Similarly, it was observed that 2, 4–D formed moderate amount of creamy and friable callus. Such friable calluses are not ideal for differentiation into shoots, hence these calluses were not further used for sub–culturing. Contrary to this, scanty to nil callus production was observed in root explants cultured on MS medium fortified with BAP. Interestingly, best callusing was observed on 5.0 and 10.0 µM TDZ after 30–d of culture (Supplementary Table 3; Fig. 1e). These calluses were greenish brown, compact and morphogenic which were considered as ideal for further shoot bud differentiation (Fig. 1e). TDZ, a thiadiazole–substituted phenylurea (N-phenyl-1,2,3-thidiazol-5-yl urea) has cytokinin like activity and proved to be an effective plant growth regulator in various culture systems (Mok et al. 1982; Huetteman and Preece 1993; Sharma and Agrawal 2018). TDZ promotes the conversion of purine–based cytokinins to more biologically active nucleosides and also stimulates accumulation and translocation of auxin (Mok et al. 1982; Ahmed and Anis 2012). There are several other reports regarding callus mediated organogenesis using 5.0 µM TDZ in various plants such as Dendrobium nobile (Bhattacharyya et al. 2014), Rumex thyrsiflorus (Ślesak et al. 2015), Plumbago zeylanica (Sharma and Agrawal 2018), Pluchea lanceolata (Kher and Nataraj 2019) and Atropa acuminata (Rajput and Agrawal 2020). Besides, several other reports revealed the use of some other plant growth regulators for callogenesis. For example, callus from nodal segments of Rumex nepalensis was induced with picloram (Bhattacharyya et al. 2017), a combination of BA and NAA was found optimum for leaf callus induction in Rumex cyprius (Al Khateeb et al. 2017). In recent reports, Manoj et al. (2019) have revealed that BA-mediated induction of leaf callus after 3 weeks of culture in Rumex vesicarius and El-Shafey et al. (2019) have reported that a combination of 2, 4–D and BAP was effective for induction of callus from leaf explant in Rumex pictus.
3.4. Shoot bud differentiation and elongation
The root–derived callus obtained earlier on MS + 5.0 µM TDZ was further sub–cultured on same but fresh medium augmented with different concentrations (0.1, 1.0, 5.0 or 10.0 µM) of TDZ to induce morphogenesis. Incidentally, differentiation of shoot buds appeared from callus masses cultured on MS + 5.0 or 10.0 µM TDZ after 15–d culture (Fig. 1f). Maximum shoot bud differentiation was achieved on MS + 5.0 µM TDZ, where 75% cultures induced an average of 10.50 ± 1.29a shoot buds per explant, followed by 10.0 µM TDZ, where 54.16% of callus cultures differentiated an average of 6.00 ± 0.90b shoot buds, after 30–d of cultures (Table 1). However, concentrations (0.1 or 1.0 µM) of TDZ and MS basal medium failed to induce any shoot buds. The shoot buds differentiated on MS medium fortified with TDZ remained stunted till 30–d (Fig. 1g), so in order to elongate them, the callus pieces with shoot buds were sub–cultured on MS basal medium. Incidentally, these shoot buds elongated into healthy shoots within 15–d of transfer (Fig. 1h). It was observed that TDZ at higher concentrations promoted the differentiation of callus into shoot buds. However, elongation of shoot could not be obtained at the same concentrations of TDZ. Induction and differentiation of shoot buds by supplementing TDZ to growth medium has also been observed in Vitex trifolia (Ahmed and Anis 2012), Rumex thyrsiflorus (Ślesak et al. 2015), Plumbago zeylanica (Sharma and Agrawal 2018) and Atropa acuminata (Rajput and Agrawal 2020).
Table 1
Effect of various concentrations of thidiazuron (TDZ) on shoot bud differentiation in root callus of R. hastatus.
Growth regulator
|
Concentration
(µM)
|
Percent shoot bud differentiation
(%)
|
Average number of shoot buds*
(Mean ± SE)
|
TDZ
|
0.0
|
0.0
|
0.0c
|
0.1
|
0.0
|
0.0c
|
1.0
|
0.0
|
0.0c
|
5.0
|
75.00
|
10.50 ± 1.29a
|
10.0
|
54.16
|
6.00 ± 0.90b
|
Different alphabets on the values are significantly different from each other as determined by Duncan’s multiple range test at P ≤ 0.05. Each value indicates mean of 12 replicates ± SE (n = 3). |
3.5. Induction of roots in the in vitro excised shoots
The root callus–derived shoots (2–3 cm) were then excised and inoculated separately on the MS medium fortified with different concentrations (0.1, 1.0, 5.0 or 10.0 µM) of IAA, IBA and NAA. It was recorded that 0.1 µM IBA was the most suitable for root formation, where 100% of culture induced an average of 12.03 ± 2.49a (Fig. 1i) roots per shoot explant, respectively having an average length of 5.75 ± 0.53a cm, after 30–d of culture (Table 2). The roots were healthy and long with numerous lateral roots and induced directly from the base of cut ends of the shoot without intervening callus. Both the number of roots and root length were reduced with increasing concentrations of IBA. However, at higher concentrations of IAA and NAA, formation of callus at the basal cut ends was observed which inhibited the development of roots. Recently, studies on rhizogenesis also reported that IBA at lower concentrations were proved to be effective for the formation of roots such as Gupta et al. (2020) reported maximum mean number of roots (14.1 ± 2.9) on half-strength MS medium augmented with 0.2 mg/L IBA in Moringa oleifera, whereas, Kher and Nataraj (2019) observed maximum average number of roots (19.8 ± 0.9) on half strength MS with 2 mg/L IBA within 28 d of culture in Pluchea lanceolata. There are several other investigations, where, IBA was found to be effective for the rhizogenesis in various plant systems such as Cucumis sativus (Sangeetha and Venkatachalam 2014), Exacum bicolor (Ashwini et al. 2015), Plumbago zeylanica (Sharma and Agrawal 2018), Polygonum multiflorum (Ho et al. 2018). Several studies of exogenous application of IBA in genus Rumex has been reported which improved the rooting response in Rumex vesicarius (Kakarla et al. 2014; Manoj et al. 2019), Rumex thyrsiflorus (Ślesak et al. 2015), Rumex cyprius (Al Khateeb et al. 2017), Rumex pictus (El-Shafey et al. 2019), Plumbago europaea (Beigmohamadi et al. 2021) and Lippia javanica (Mood et al. 2022).
Table 2
Effect of different concentrations of auxins on in vitro roots formation in root callus-derived excised shoots of R. hastatus cultured on MS medium for 30 d.
Auxin
|
Concentration
(µM)
|
Percentage of morphogenic cultures*
(%)
|
Average number of roots*
(Mean ± SE)
|
Average root length in cm*
(Mean ± SE)
|
Control
|
0.0
|
75.00
|
5.67 ± 2.08abcd
|
5.80 ± 0.55a
|
IAA
|
0.1
|
100.00
|
10.50 ± 1.60ab
|
5.21 ± 0.76ab
|
1.0
|
83.34
|
4.66 ± 1.76bcd
|
4.36 ± 1.08abc
|
5.0
|
66.00
|
2.16 ± 1.66cd
|
2.83 ± 1.03cde
|
10.0
|
100.00
|
10.00 ± 4.09ab
|
1.58 ± 0.34ef
|
IBA
|
0.1
|
100.00
|
12.03 ± 2.49a
|
5.75 ± 0.53a
|
1.0
|
83.34
|
8.50 ± 2.56abc
|
4.21 ± 0.43abc
|
5.0
|
100.00
|
10.83 ± 2.60ab
|
3.63 ± 0.75bcd
|
10.0
|
66.00
|
4.66 ± 1.45bcd
|
1.81 ± 0.14def
|
NAA
|
0.1
|
100.00
|
10.66 ± 2.72ab
|
4.85 ± 0.94abc
|
1.0
|
33.00
|
0.83 ± 0.44d
|
0.40 ± 0.20f
|
5.0
|
16.67
|
0.67 ± 0.67d
|
0.10 ± 0.10f
|
10.0
|
00.00
|
00.00d
|
00.00f
|
*Different alphabets on the values of average number and length of roots are significantly different from each other as determined by Duncan’s multiple range test at P ≤ 0.05. Each value indicates mean of 12 replicates ± SE (n = 3). |
3.6. In vitro hardening and field acclimatization
MS medium augmented with 0.1 µM IBA induced long and thick roots, which apparently increased the uptake of nutrients and facilitated the hardening and acclimatization of regenerants. In vitro rooted plantlets were successfully transferred to paper cups containing soil–rite mixture for hardening (Fig. 1j). The plants attained an average height of 60–68 cm after 3 month and morphologically similar to in vivo plants. Almost 96% of root callus-derived regenerants (RDR) were effectively acclimatized and raised in pots containing garden soil in green house condition (Fig. 1k).
3.7. Genetic fidelity analysis employing SCoT and SRAP markers
Genetic fidelity of mother plant (M) and root callus-derived regenerants (RDR1–RDR10) was analyzed using SCoT and SRAP markers. Initially, a total of 34 SCoT and 52 SRAP primers were screened, out of which 10 of each were selected on the basis of scorable amplification bands on agarose gel. SCoT primers resulted in 48 reproducible scorable bands between 200–2200 bp, with an average of 4.8 bands per primer in RDR (Supplementary Table 4; Fig. 2a). Besides, SRAP marker analysis of RDR yielded a total of 54 distinct scorable bands between 50–1100 bp with an average of 5.4 bands per primer (Supplementary Table 4; Fig. 2b). The pooled SRAP and SCoT matrix data set produced a total of 102 bands, out of which 94 bands were monomorphic with an average of 4.7 bands per primer (Supplementary Table 4). The combined similarity matrix was obtained from both SRAP and SCoT markers based on Jaccard's similarity coefficients which showed the coefficient range between 0.931 to 1.000 (Supplementary Table 5). The dendrograms obtained from UPGMA showed 95% similarity between the RDR and mother plant (Fig. 2c). This high monomorphism in the banding profile obtained by both the markers confirmed the genetic stability of the micropropagated plants. Interestingly, the present investigation revealed that the plants generated through root–derived callus have very less variation. The possible reason could be due to the exogenous application of phytohormone during callus induction, shoot regeneration and prolonged culture passage or incubation period (Devarumath et al. 2002; Bhattacharyya et al. 2017). Type of explant used and regeneration pathway may also attribute towards the genetic variations among regenerants. Differentiated tissues such as root, stems and leaves generally produce more polymorphism as they undergo an intermediate callus-phase which leads to changes in genome quantitatively and qualitatively during cell reprogramming (Leva et al. 2012). However, such genetic changes cannot be observed visually as the structural variations in the gene product do not always alter its biological activity of the genome to an extent for phenotypic expression (Bhattacharyya et al. 2017). Molecular markers such as SCoT and SRAP employed in the current study are known to be highly reproducible, reliable and efficient in detecting genetic variation in in vitro raised plants. Start codon targeted polymorphism (SCoT) markers are based on the short-conserved region adjoining the initiation codon (ATG) present in plant genes and can be correlated to functional genes and their corresponding traits (Collard and Mackill 2009). Besides, sequence-related amplified polymorphism (SRAP) markers designed for the amplification of open reading frames which is based on two-primer (forward and reverse primers) amplification (Li and Quiros 2001). Similar study has also been reported by Bose et al. (2016) in which genetic variability was 4.38% and 7.14% in direct shoot organogenesis and callus mediated organogenesis derived plants, respectively, in Nardostachys jatamansi using SCoT and intron splice junction (ISJ) markers. The percent polymorphism between mother and micropropagated plants were also reported in Aconitum violaceum employing RAPD (3.22%) and ISSR (5.88%) markers (Rawat et al. 2013), Dendrobium nobile (3%) using RAPD and SCoT markers (Bhattacharyya et al. 2014), Plumbago zeylanica (3%) using ISSR and SCoT markers (Sharma and Agrawal 2018). In contrast, Bhattacharyya et al. (2017) found a very high polymorphism such as 6.06% and 15.62% in regenerants from direct and indirect shoot organogenesis, respectively in Rumex nepalensis by using SCoT and RAPD markers. Recently, Dhiman et al. (2021) have been observed 8.03% polymorphism in micropropagated plants of Nardostachys jatamansi by employing SCoT and Directed Amplification of Minisatellite DNA (DAMD) markers.
3.8. Phytochemical evaluation of mother plant and regenerants
Amongst all the plant extract used, the highest phenolic content was observed in methanolic root extract of RDR (89.24 ± 1.24a mg GAE/g d.w.), followed by methanolic root extract of mother plant (74.57 ± 4.94b mg GAE/g d.w.) (Fig. 3a). Contrary, the lowest phenolic content of 1.49 ± 0.22h mg GAE/g d.w. was seen in the hexane leaf extract of mother plant (Fig. 3a). Our findings revealed the varying amount of phenolic content in the different solvent system used. Similarly, maximum flavonoid content was also observed in methanolic root extract of RDR (384.11 ± 3.22a mg QE/g d.w.), followed by extract of mother plant (341.11 ± 1.70b mg QE/g d.w.) (Fig. 3b). The results indicated that in R. hastatus, regenerants possessed maximum phenolic and flavonoid contents in general when compared to the mother plant. Polyphenols exhibit high solubility in polar solvents such as acetone, ethanol, ethyl acetate and methanol (Barchan et al. 2014; Do et al. 2014). The maximum phenolic and flavonoid contents were observed in methanolic root extract of RDR compared to extract of other plant parts. As root extract prepared in methanol proved best in terms of maximum TPC and TFC in mother plant and regenerants of R. hastatus, it was chosen for further analysis and quantification of luteolin and rutin content through HPLC. Maximum content of luteolin was also seen in RDR (1042.15 ± 19.30c µg/g d.w.), followed by mother plant (747.32 ± 20.71d µg/g d.w.) (Fig. 3c). Similarly, maximum quantity of rutin was observed in RDR (1914.49 ± 45.99a µg/g d.w.), followed by mother plant (1689.43 ± 25.43b µg/g d.w.) (Fig. 3c). The HPLC analysis thus has revealed that a significant higher amount of luteolin and rutin present in root extract of RDR. Significantly high quantity of secondary metabolites has been reported due to endogenous physiological changes, organ type or hormonal contents during culture conditions in micropropagated plants (Surveswaran et al. 2010). Addition of synthetic plant growth regulators alters the expression of genes involved in the biosynthesis of secondary metabolites resulting in the significantly higher production of these compounds under culture conditions (Pourebad et al. 2015). The above results are in consonance with the findings of Bose et al. (2016) and Bhattacharyya et al. (2017), where micropropagated plants of Nardostachys jatamansi and Rumex nepalensis, respectively, possessed a significantly higher amount of phenolics and flavonoids as compared to the mother plant. Micropropagated plants of Rumex cyprius showed enhanced quantity of phenols as well as luteolin and rutin content (Al Khateeb et al. 2017).
3.9. Analysis of antioxidant activity of mother plant and regenerants
Of all the different organic solvents tried (such as methanol, ethyl acetate and hexane), methanolic extract found to be the most useful in terms of maximum TPC, TFC and content of luteolin and rutin. Hence, methanolic extract of different parts (root, stem and leaf) of R. hastatus was selected for further determination of antioxidant activity as evaluated through phosphomolybdenum and DPPH free radical scavenging assays.
3.9.1. Total antioxidant capacity (TAC)
Phosphomolybdenum (PM) assay is based on the ability of a reducing agent (antioxidant) to convert molybdenum (VI) into the phosphomolybdate (V) complex, which is evaluated spectrophotometrically at 765 nm (Ahmed et al. 2015). In the present investigation, higher antioxidant capacity was seen in methanolic root extract of RDR (300.05 ± 8.45a µg/mL AAE d.w. of extract), followed by mother plant (280.71 ± 10.70b µg/mL AAE d.w. of extract) (Fig. 3d). Leaf and stem extract showed less but significant antioxidant capacity. In the current study, a strong correlation has been seen between antioxidant capacity and increasing concentration of plant extract. Higher antioxidant activity in these regenerated plants corresponds to the presence of higher amount of secondary metabolites as compared to the mother plant. Plants produce a wide range of secondary metabolites such as phenols, flavonoids, tannins, alkaloids, etc. which act as a source of natural antioxidants with multiple biological activities. The strong antioxidant activity of phenols and flavonoids is attributed to their ability to play a role of free radical scavengers which prevent the production of reactive oxygen species (Panche et al. 2016). Higher antioxidant activity in micropropagated plants can be directly correlated to the occurrence of higher amount of phenolics and flavonoids compared to mother plant (Sen et al. 2013; Piątczak et al. 2014). Methanolic leaf extracts of micropropagated plants of Rehmannia glutinosa showed strong antioxidant activity (Piątczak et al. 2014). Kousalya and Bai (2016) also reported that methanol extract of callus derived from in vitro regenerants of Canscora decussate possessed potent antioxidant activity. Recently, Rebelo et al. (2022) have also been studied that in vitro micropropagated plants had high activity of antioxidant enzymes compared with the mother plants of Tuberaria lignosa.
3.9.2. DPPH free radical scavenging activity
DPPH assay is the most widely accepted method to determine free radical scavenging activity due to its stability, rapidity, sensitivity and less expensive. In the presence of an antioxidant molecule, the violet solution of DPPH is converted to DPPH–H (diphenylhydrazine) with discoloration. This discoloration implies the radical scavenging activity of the plant extract (Aksoy et al. 2013). A dose-dependent increase in the free radical scavenging activity with increasing concentrations of extracts was recorded. The maximum radical scavenging activity was shown by RDR (93.81 ± 0.50a %), followed by mother plant (87.31 ± 0.21c %) at 1000 µg/ml of methanolic root extract (Table 3). Leaf extract showed significantly moderate DPPH free radical scavenging activity (Table 3). However, stem extract showed the least DPPH free radical scavenging activity in all mother plant and regenerants (Table 3). Thus, the results revealed that regenerants showed strong free radical scavenging activity compared to the mother plant. The lower free radical scavenging activity of mother plant extract might be due to the presence of lower amount of phenols and flavonoids (Bagnazari et al. 2018). Similar results were also obtained in Nardostachys jatamansi (Bose et al. 2016), Rumex nepalensis (Bhattacharyya et al. 2017) and Atropa acuminata (Rajput and Agrawal 2020), where micropropagated plants possessed strong radical scavenging activity compared to the mother plant. A positive correlation has been reported between TPC and DPPH free radical scavenging activity of plant extract (Li et al. 2009).
Table 3
Comparative percent DPPH radical scavenging activity of methanolic extract of different plant parts (root, stem and leaf) of mother plant and root-derived regenerants of R. hastatus.
|
Concentration (µg/ml)
|
DPPH radical scavenging activity (%)
|
Root
|
Stem
|
Leaf
|
Mother plant
|
62.5
|
36.94 ± 0.00k
|
7.92 ± 0.16i
|
12.12 ± 0.96j
|
125
|
57.92 ± 2.31h
|
14.64 ± 1.34g
|
16.69 ± 0.26hi
|
250
|
67.53 ± 1.61f
|
20.24 ± 0.16f
|
27.51 ± 0.16f
|
500
|
83.02 ± 0.64d
|
31.71 ± 0.32d
|
46.07 ± 0.10d
|
1000
|
87.31 ± 0.21c
|
48.22 ± 0.16c
|
77.70 ± 0.16b
|
Root–derived regenerants (RDR)
|
62.5
|
45.94 ± 2.08i
|
13.15 ± 1.91gh
|
15.45 ± 1.49ij
|
125
|
64.59 ± 1.44fg
|
13.05 ± 1.12gh
|
22.36 ± 2.43g
|
250
|
75.53 ± 1.63e
|
21.20 ± 0.92f
|
33.84 ± 3.19e
|
500
|
89.02 ± 0.06bc
|
30.80 ± 0.79de
|
52.40 ± 1.65c
|
1000
|
93.81 ± 0.50a
|
49.87 ± 2.31a
|
83.53 ± 1.00a
|
Different alphabets on the values are significantly different from each other as determined by Duncan’s multiple range test at P ≤ 0.05. Each value indicates Mean ± SE (n = 3). |
3.10. In vitro elicitation of luteolin and rutin compounds with various elicitors
The root calluses raised on MS + 5 µM TDZ were augmented with different elicitors such as chitosan (0, 1, 5, 25, 50, 100 and 200 mg/L), phenylalanine (0, 1, 5, 25, 50, 100 and 200 mg/L), proline (0, 1, 5, 25, 50, 100 and 200 mg/L), salicylic acid (0, 1, 5, 25, 50, 100 and 200 µM) and yeast extract (0, 1, 5, 25, 50, 100 and 200 mg/L) for the elicitation of luteolin and rutin contents after 30 d of treatment and analyzed through HPLC.
3.10.1. Chitosan
A significant increase in luteolin and rutin content as well as callus biomass proliferation was observed in the root callus when treated with increasing concentrations (0, 1, 5, 25, 50, 100 and 200 mg/L) of chitosan (Table 4). Maximum callus growth of 273.50 ± 12.03a mg d.w. (76.59%) was found with 50 mg/L chitosan over control of 154.87 ± 12.37b mg d.w. Interestingly, the highest luteolin synthesis of 106.73 ± 2.77a µg/g d.w. (3.82–fold) was also recorded with 50 mg/L chitosan over control (22.10 ± 0.57g µg/g d.w.) (Table 4; Fig. 4a). Contrary to this, the highest rutin synthesis of 1169.07 ± 46.49a µg/g d.w. (2.74–fold) was recorded at low concentration, i.e. 25 mg/L chitosan over control (312.41 ± 12.42e µg/g d.w.) (Table 4; Fig. 4b). A decline in rutin and luteolin content was observed beyond 25 mg/L and 50 mg/L of chitosan concentrations, respectively. Chitosan (1,4-linked glucosamine) is a deacetylated derivative of chitin, which is the component of many pathogenic fungi (Katiyar et al. 2015). It is a potent elicitor having bio-degradable properties and also known for inducing the defense responses through production of secondary metabolites in plants (Muxika et al. 2017; Hidangmayum et al. 2019). Besides, chitosan causes transient accumulation of second messenger molecules such as cAMP, Ca2+ and NO, ion uptake and transportation, activation of NADPH oxidase and G-protein, mitogen-activated protein kinases (MAPKs) phosphorylation and salicylic acid-related signaling pathways (Ramirez-Estrada et al. 2016; Singh et al. 2020b). Chitosan had positive effect on callus growth up to 50 mg/L concentration in R. hastatus. Similar reports demonstrated the positive effect of chitosan on callus biomass in Rumex vesicarius (Sayed et al. 2016), Fagonia indica (Khan et al. 2019), Cullen corylifolium (Singh et al. 2020a) and Plumbago zeylanica (Singh et al. 2020b). However, chitosan at higher concentrations is inhibitory for callus growth and cells shifted their energy expenditure to the production of secondary metabolites (Golkar et al. 2019). There are several studies reported elicitation of various bioactive compounds using chitosan as an elicitor. The increased production of formononetin (12.45-fold) and calycosin (6.17-fold) has been achieved with 100 mg/L chitosan in hairy root cultures of Astragalus membranaceus (Gai et al. 2019). Similarly, Singh et al. (2020a) have also reported elicitation in contents of daidzein (11.2-fold) and psoralen (7.2–fold) at 50 mg/L chitosan and genistein increased to 6.21-fold by augmenting 100 mg/L chitosan to callus cultures of Cullen corylifolium. In another report of Singh et al. (2020b), 4.58-fold increment in plumbagin yield has been achieved with 50 mg/L chitosan compared to control in root callus cultures of Plumbago zeylanica. The content of lepidine has been enhanced up to 19.87-fold with 250 mg/L chitosan over control in callus cultures of Lepidium sativum (Bakhtiari and Golkar 2021). In addition, Bavi et al. (2022) have also been reported that chitosan at 40 mg/L promoted the synthesis of cafeic acid, benzoic acid, 4-hydroxy benzoic acid, epicatechin, and apigenin in the cell suspension culture of Zataria multifora. Chitosan caused oxidative burst which induced the expression of genes pertaining to MAPKs cascades which activates the transcription factors involved in the biosynthesis of secondary metabolites (Gai et al. 2019).
Table 4
Effect of different elicitors on callus biomass production and quantity of luteolin and rutin content in root callus of R. hastatus after 30 d of treatment.
Elicitors
|
Concentration
|
Callus biomass
(mg d.w.)
|
Quantity
|
Luteolin (µg/g d.w.)
|
Luteolin (Fold increase)
|
Rutin
(µg/g d.w.)
|
Rutin (Fold increase)
|
Chitosan
|
Control
|
154.87 ± 12.37b
|
22.10 ± 0.57g
|
0.0
|
312.41 ± 12.42e
|
0.0
|
1 mg/L
|
168.50 ± 11.96b
|
59.74 ± 1.55f
|
1.70
|
781.98 ± 31.10c
|
1.50
|
5 mg/L
|
169.16 ± 14.94b
|
68.46 ± 1.77e
|
2.09
|
962.11 ± 38.26b
|
2.07
|
25 mg/L
|
245.32 ± 13.41a
|
97.98 ± 2.54c
|
3.43
|
1169.07 ± 46.49a
|
2.74
|
50 mg/L
|
273.50 ± 12.03a
|
106.73 ± 2.77a
|
3.82
|
771.24 ± 30.67c
|
1.46
|
100 mg/L
|
178.55 ± 14.86b
|
101.00 ± 2.62ab
|
3.57
|
746.69 ± 29.69c
|
1.39
|
200 mg/L
|
165.07 ± 10.41b
|
76.88 ± 1.99d
|
2.47
|
477.73 ± 19.00d
|
0.52
|
Phenylalanine
|
Control
|
154.87 ± 12.37c
|
22.10 ± 0.57e
|
0.0
|
312.41 ± 12.42f
|
0.0
|
1 mg/L
|
167.24 ± 11.83c
|
39.20 ± 1.01d
|
0.77
|
399.66 ± 15.89e
|
0.27
|
5 mg/L
|
167.33 ± 10.54c
|
66.02 ± 1.71c
|
1.98
|
585.98 ± 23.30d
|
0.87
|
25 mg/L
|
210.00 ± 15.35b
|
67.03 ± 1.73bc
|
2.03
|
703.28 ± 27.97c
|
1.25
|
50 mg/L
|
217.33 ± 10.71ab
|
80.17 ± 2.08b
|
2.62
|
868.53 ± 34.54b
|
1.78
|
100 mg/L
|
250.00 ± 14.47a
|
407.18 ± 10.58a
|
17.42
|
1019.80 ± 40.55a
|
2.26
|
200 mg/L
|
210.25 ± 9.74b
|
71.43 ± 1.85bc
|
2.23
|
866.82 ± 34.47b
|
1.77
|
Salicylic acid
|
Control
|
154.87 ± 12.37abc
|
22.10 ± 0.57f
|
0.0
|
312.41 ± 12.42d
|
0.0
|
1 µM
|
165.61 ± 12.13abc
|
83.16 ± 2.16d
|
2.76
|
341.91 ± 13.59d
|
0.09
|
5 µM
|
184.52 ± 10.83ab
|
128.17 ± 3.33b
|
4.79
|
452.98 ± 18.01c
|
0.44
|
25 µM
|
187.37 ± 12.29a
|
224.03 ± 5.82a
|
9.13
|
1133.74 ± 45.09a
|
2.62
|
50 µM
|
148.27 ± 11.38bc
|
110.06 ± 2.86c
|
3.98
|
801.09 ± 31.85b
|
1.56
|
100 µM
|
145.32 ± 13.01c
|
109.68 ± 2.85c
|
3.96
|
787.68 ± 34.31b
|
1.52
|
200 µM
|
78.10 ± 9.51d
|
51.54 ± 1.33e
|
1.13
|
737.01 ± 29.31b
|
1.35
|
Yeast extract
|
Control
|
154.87 ± 12.37b
|
22.10 ± 0.57f
|
0.0
|
312.41 ± 12.42e
|
0.0
|
1 mg/L
|
167.41 ± 11.83ab
|
55.89 ± 1.45e
|
1.52
|
400.44 ± 15.92d
|
0.28
|
5 mg/L
|
178.54 ± 11.83ab
|
90.11 ± 2.34d
|
3.07
|
541.50 ± 21.53c
|
0.73
|
25 mg/L
|
183.37 ± 10.29ab
|
102.93 ± 2.67c
|
3.65
|
783.31 ± 31.15a
|
1.50
|
50 mg/L
|
187.32 ± 10.67ab
|
167.51 ± 4.35b
|
6.57
|
835.92 ± 33.24a
|
1.67
|
100 mg/L
|
197.44 ± 12.08a
|
219.36 ± 5.70a
|
8.92
|
861.94 ± 34.28a
|
1.75
|
200 mg/L
|
165.83 ± 12.42ab
|
167.00 ± 4.34b
|
6.55
|
625.56 ± 24.87b
|
1.00
|
Different alphabets on the values are significantly different from each other as determined by Duncan’s multiple range test at P ≤ 0.05. Each value indicates mean of 12 replicates ± SE (n = 3). |
3.10.2. Phenylalanine
A slight increment in callus biomass production as well as in luteolin and rutin contents were seen in root calluses after treatment with increasing concentrations (0, 1, 5, 25, 50, 100 and 200 mg/L) of phenylalanine. Maximum callus growth of 250.00 ± 14.47a mg d.w. (61.42%) was observed at 100 mg/L phenylalanine over control of 154.87 ± 12.37c mg d.w. (Table 4). A tremendous enhancement in luteolin content of 407.18 ± 10.58a µg/g d.w. (17.42–fold) was achieved with 100 mg/L phenylalanine treatment over control of 22.10 ± 0.57e µg/g d.w. (Table 4; Fig. 4c). However, rutin accumulation was increased upto 1019.80 ± 40.55a µg/g d.w. (2.26–fold) at 100 mg/L phenylalanine compared to control of 312.41 ± 12.42f µg/g d.w. (Table 4; Fig. 4d). Subsequently, a sharp decline in content of luteolin and rutin beyond 100 mg/L phenylalanine concentration was also observed. Phenylalanine is an aromatic amino acid, which acts as a precursor molecule for the synthesis of numerous secondary metabolites (Yang et al. 2020). To enhance the production of secondary metabolites, various precursors of their biosynthetic pathways have already been employed in in vitro cultures. An increase in flavonoids content (1.6–fold) and antioxidant capacity have been reported in lentil sprouts treated with phenylalanine (Świeca et al. 2014). Similarly, Kumar et al. (2018) have also reported that phenylalanine at 200 mg/L concentration improved the conessine content (7.0–fold) in green bark–derived callus cultures of Holarrhena antidysenterica. When phenylalanine was supplemented with lactalbumin hydrolysate led to 3.5–fold increment in anthocyanin yield compared to control in cell suspension cultures of Panax sikkimensis roots and also upregulation of enzyme activity and gene expression of phenylalanine ammonia lyase (PAL) and UDP-glucose: flavonoid 3-O-glycosyltransferase (UFGT) was observed at same treatment (Biswas et al. 2020). The results of current study suggest the use of in vitro elicitation supported by precursor feeding and it is an effective, reliable and cheap tool for ameliorating the bioactive compounds.
3.10.3. Salicylic acid
When root callus was exposed to various concentrations (0, 1, 5, 25, 50, 100 and 200 µM) of salicylic acid, a significant increase in luteolin and rutin content was observed. Salicylic acid at 25 µM concentration found to be effective for callus growth as well as enhancement of luteolin and rutin contents. Maximum root callus proliferation of 187.37 ± 12.29a mg d.w. (20.98%) was seen at 25 µM salicylic acid over control of 154.87 ± 12.37abc mg d.w. (Table 4). Interestingly, maximum luteolin synthesis of 224.03 ± 5.82a µg/g d.w. (9.13–fold) and rutin accumulation of 1133.74 ± 45.09a µg/g d.w. (2.62–fold) were also observed at 25 µM salicylic acid over their respective controls (Table 4; Fig. 4e, f). However, it was also observed that a sharp decrease in the callus growth and bioactive compounds accumulation at higher concentrations (> 25 µM). Salicylic acid act as an endogenous signaling molecule that induces the defense system in plants through activation of phenylalanine ammonia lyase synthesis pathway and other major antioxidant enzymes involved in metabolic pathways ultimately resulted to enhanced yield of secondary metabolites (Rahimi et al. 2014; Fesel and Zuccaro 2016). Supplementing the growth medium with salicylic acid led to the increased production of luteolin (9.13–fold) and rutin (2.62–fold) in R. hastatus root callus cultures. Similarly, several studies are also documented where salicylic acid improved the synthesis of secondary metabolites in various plant taxa. The increased contents of hypericin (7.98-fold) and pseudohypericin (13.58-fold) has been achieved in shoot cultures of Hypericum hirsutum treated with 50 µM salicylic acid (Coste et al. 2011). Cell suspension cultures of Gymnema sylvestre supplemented with 200 µM salicylic acid has elicited 4.9-fold gymnemic acid content (Chodisetti et al. 2015). Plumbagin accumulation has also been enhanced to 3.4-fold with 25 µM salicylic acid added to root callus cultures of Plumbago zeylanica (Sharma and Agrawal 2018). Enhancement in quantity of psoralen, daidzein and genistein up to 14.8-fold, 4.8-fold and 5.5-fold has been achieved in cotyledon callus cultures of Cullen corylifolium fortified with 5 µM salicylic acid (Singh et al. 2020a). Rajan et al (2020) have reported that 1 mM salicylic acid enhanced the production of rhamnetin to 2.7-fold compared to control in cell suspension culture of Vernonia anthelmintica.
3.10.4. Yeast extract
A gradual increase in luteolin and rutin content as well as callus biomass proliferation were analyzed with increasing dose (0, 1, 5, 25, 50, 100 and 200 mg/L) of yeast extract. Increment of 27.48% (197.44 ± 12.08a mg d.w.) in callus biomass was observed at 100 mg/L yeast extract over control (154.87 ± 12.37b mg d.w.) (Table 4). Optimum luteolin quantity of 219.36 ± 5.70a µg/g d.w. (8.92–fold) was achieved at 100 mg/L yeast extract over control (22.10 ± 0.57f µg/g d.w.) (Table 4; Fig. 4g). At the same concentration of yeast extract, rutin content was enhanced upto 861.94 ± 34.28a µg/g d.w. (1.75-fold) over control of (312.41 ± 12.42e µg/g d.w.) (Table 4; Fig. 4h). However, a slight decrease in the callus growth production along with luteolin and rutin content were observed at 200 mg/L yeast extract. Yeast extract is a mixture of chitin, N-acetylglucosamine oligomers, β–glucan, amino acid residues, glycopeptides, and vitamins (Pitta-Alvarez et al. 2000; Baenas et al. 2014). El-Sharabasy et al. (2012) reported that callus biomass production has been promoted by amino acid residues and glycopeptides. Besides, plant cells having pattern recognition receptors for chitin and β–glucan which acts as microbe-associated molecular patterns, and modulate the expression of certain defense-related genes consequently resulted in enhanced synthesis of secondary metabolites (Jumali et al. 2011; Fesel and zuccaro 2016). Saponin content was increased to 20–fold with yeast extract treatment in cell cultures of Panax ginseng (Lu et al. 2001). Enhanced production of plumbagin up to 3.5–fold was achieved with yeast extract in root cultures of Drosera burmanii (Putalun et al. 2010). Yeast extract was also observed to enhance total isoflavonoids (4.5–fold) compared to control in hairy root cultures of Pueraria candollei (Udomsuk et al. 2011). Maqsood and Abdul (2017) have also reported that when yeast extract added to the growth medium improved vinblastine and vincristine productivity by 22.74% and 48.49%, respectively, over control in Catharanthus roseus. Sharma and Agrawal (2018) have reported increased synthesis of plumbagin to 6.5-fold with yeast extract in root callus cultures of Plumbago zeylanica. Application of yeast extract to cell cultures of Trigonella foenum-graecum has also led to 1.66-fold increased content of diosgenin (İlgar et al. 2021). In a recent report of Bavi et al. (2022) where yeast extract at very high concentration (1200 mg/L) has been increased the contents of phenylpropanoids gallic acid, vanillin, salicylic acid, catechin, rutin and terpenoids carvacrol and thymol in cell suspension culture of Zataria multifora.