How to cite:
Mohan SN, Hemanthakumar AS, Preetha TS (2023) A Novel System for the Production of the Bioactive N-alkylamide Research Article
A Novel System for the Production of the Bioactive N-alkylamide ‘Spilanthol’
Through Somatic Embryogenesis in Acmella ciliata Kunth (Cass.)
S Neethu Mohan1, AS Hemanthakumar2, TS Preetha1*
1 Plant Tissue Culture Laboratory, Department of Botany, University College, Thiruvananthapuram, PIN - 695034, Kerala, India; Research Centre, University of Kerala
2 Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Thiruvananthapuram, PIN - 695562, Kerala, India
Article history:
Submission May 2021 Revised May 2021 Accepted July 2021
ABSTRACT
Acmella ciliata Kunth (Cass.), a medicinally important plant in the family Aster- aceae, has high commercial value because of its traditional phytomedicinal uses.
The plant contains many phytochemicals like alkyl amides, alkaloids, tannins, sap- onins and flavonoids accountable for most of its pharmacological applications.
The study presented here reports the callus culture and somatic embryogenesis of this plant thereby raising a novel system for the subsequent production of the N- alkyl amide ‘spilanthol’, the valuable secondary metabolite presents in it. Mu- rashige and Skoog (MS) medium supplemented with auxins either alone or in combination with cytokinins were used for the induction and maturation of so- matic embryos. MS medium supplemented with 2,4-D (0.5, 1.0 and 2.0 mg.L-1) produced black friable callus whereas, 1.0 mg.L-1 NAA in combination with 0.5 mg.L-1 BA induced white, slightly purple coloured friable callus which on further subculture to fresh medium induced somatic embryos that germinated into plant- lets upon transfer to MS basal medium. The mode of regeneration via somatic embryogenesis was confirmed by histological analysis through free-hand section- ing and stereomicroscopic observation. The plantlets raised through somatic em- bryogenesis after a short hardening period, were found to acclimatise in the field at 83.33% efficiency and exhibited genetic uniformity with 96.6% similarity in the ISSR analysis. HPLC analysis of in vitro raised embryogenic callus showed 239.512 µg.g-1 spilanthol content which was comparatively higher than the mother plants (92.19 µg.g-1). The bioproduction of the N-alkylamide ‘spilanthol’ through embryogenic callus can be extended for the scale-up production of this bioactive compound using bioreactor technology for the formulation of phytodrugs.
Keywords: Acmella ciliata, Embryogenic callus, HPLC, ISSR analysis, Somatic embryogenesis Spilanthol
*Corresponding author:
E-mail: [email protected]
Introduction
Acmella ciliata (Kunth) Cass., a medicinally important plant belonging to the family Aster- aceae, is widely distributed in the tropical and sub- tropical regions of the world. Spilanthes is also mentioned as Acmella in some of the literature.
However, monographs have been written about both genera Acmella and Spilanthes and the
“toothache plant” was placed in the genus Acmella [1]. Phytochemicals like alkaloids, tannins, an- thraquinones, glycosides, flavonoids, saponins
and cardiac glycosides have been reported in Ac- mella calva [2]. N-alkyl amides or alkamides are the most abundant phytochemicals in the genus Spilanthes/Acmella, which account for its biologi- cal activity. Spilanthol is a high-value bioactive al- kamide in this taxa; currently, about twenty-three pharmaceutical companies manufacture spilan- thol-containing products. The different species of Acmella are well known in folklore remedies as a potential medicinal plant used for culinary pur-
JTLS | Journal of Tropical Life Science 462 Volume 13 | Number 3 | September | 2023
poses by the tribes in various parts of the world.
Apart from its traditional usage, it is commercially cultivated and marketed in different parts of the world, which indicates the increasing economic potential of this herbaceous medicinal species.
However, the genetic diversity of traditional me- dicinal plants is under continuous threat due to factors such as exploitation, injudicious harvest- ing, unmonitored trade and the loss of growth hab- itat by climate change. Also, poor seed germina- tion capacity and continuous exploitation for phar- macological preparations have depleted this valu- able plant genetic resource. Therefore, adopting alternate means of production and conservation having rapidity and limitless potentialities for fill- ing the gap between demand and supply needs to be undertaken. In this scenario, plant tissue-based interventions provide complementary conserva- tion options for plant species having limited repro- ductive capacity, ensuring rapid mass propagation for large-scale cultivation and enhancing the pro- duction of secondary metabolites.
Somatic embryogenesis, one of the plant tissue culture techniques, has been proven to be a useful and efficient method for mass clonal propagation of selected species, production and plant germ- plasm conservation many years back [3, 4]. More- over, plant regeneration through somatic embryo- genesis is a reliable alternative to produce high- frequency plantlet production with clonal fidelity [5]. There are several studies regarding in vitro propagation in Spilanthes acmella [6, 7], but only limited information on somatic embryogenesis in Spilanthes mauritiana [8] and no reports yet in A.
ciliata. In this paper, we report the callus culture and somatic embryogenesis in A. ciliata, the sub- sequent production of valuable secondary metab- olites ‘spilanthol’, and the clonal assessment of the regenerates using molecular markers.
Material and Methods Plant material
A. ciliata (Kunth) Cass. (Herbarium Voucher Nos. TBGT 32710-32711) collected from Aru- vikkara, Thiruvananthapuram, Kerala, India, maintained in the greenhouse of the Department of Botany, University College, Thiruvananthapuram, Kerala, India served as the source of explants for the present study.
Shoot culture establishment
Explants like shoot tips, leaves and nodal seg-
ments (first, second, third and fourth) collected from the plant source grown in the Department of Botany, University College were washed under running tap water for 20 minutes and then with 0.5% (w/v) Teepol for 10 minutes. They were sur- face sterilised with 0.1% (w/v) HgCl2 at various time intervals (5-10 minutes) and subsequently rinsed 2-3 times with sterile distilled water. The sterilised explants were inoculated on Murashige and Skoog (MS) medium [9] supplemented with various plant growth regulators like BA/ Kinetin (0.5. 1.0 and 2.0 mg.L-1), NAA/IAA (0.1 mg.L-1) either alone or in combination for in vitro shoot production and multiplication. Subculturing was performed at an interval of 4 weeks each.
Callus induction
For callus induction, in vitro, leaves were cut into appropriate sizes (0.5 - 1 cm) and inoculated on MS medium supplemented with 0.5, 1.0, 2.0 mg.L-1 NAA/ IAA/ IBA/ 2,4-D either individually or in combination with 0.5 mg.L-1 BA.
Induction of somatic embryogenesis
The friable calli induced during the callus in- duction phase were subcultured in fresh medium containing the same or reduced/increased concen- trations of plant growth regulators (NAA/ IAA/
IBA/ 2, 4-D/ BA) either alone or in combination with BA for the induction of somatic embryos.
Medium devoid of plant growth regulators were also tested for this purpose. The embryoids were transferred to MS basal medium to evoke somatic embryo maturation and their conversion into plantlets. After two weeks of hardening in the greenhouse, the completely grown plantlets were transferred to the field. For hardening, somatic embryogenesis-derived plantlets were transferred to paper cups filled with garden soil and river sand (1:1) and covered with polythene bags for three days. Holes were punched on the polythene bags to facilitate efficient evapotranspiration. After two weeks of this type of hardening in the greenhouse, the established plants were transferred to soil taken in earthen pots.
For assessing the regeneration pathway, the embryogenic calli and embryoids were subjected to free-hand sectioning followed by safranine staining and observed under a stereomicroscope (Magnus). The data on somatic embryo induction and plantlet conversion was statistically analysed using ANOVA and the means were compared by
Duncan’s multiple range test (p < 0.05) using the computer software SPSS/ PC + version 4.0 (SPSS Inc., Chicago, USA).
Genetic uniformity analysis of the regenerated plants via ISSR markers
A total of ten genomic DNA samples, one col- lected from the mother plant (P10) and nine from somatic embryo-derived plants (P1-P9), were iso- lated using the cetyltrimethylammonium bromide (CTAB) method [10]. ISSR assay was carried out in 25 µL reaction mixture containing 0.2 mM dNTP’s, 10 mM Tris-HCL,1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 1.0 U Taq DNA pol- ymerase (Finzymes, Helsinki, Finland), 15 pmol primers (IDT, Coralville, USA) and 50 ng of ge- nomic DNA. The amplification was performed in a thermal cycler (Eppendorf ESP-S). After the in- itial cycle of 2 min at 93°C, 2 min at 50 to 55°C*, and 2 min at 72°C, a total of 39 cycles of 1 min at 93°C, 1 min at 50 to 55°C and 1 min at 72 °C were performed. The last cycle was performed by 10 min extension at 72°C. Amplified products were resolved in 1.40% agarose gel (1×TBE) followed by EtBr staining. The *annealing temperature of the primers ranges from 50 to 55°C for the differ- ent primers used in this study. The ISSR primers selected and their sequences are given in Table 1.
HPLC Analysis
HPLC analysis was carried out for the estima- tion of spilanthol content in the in vitro raised em- bryogenic callus, keeping the in vivo mother plant as the control. The 60-day-old callus induced and
subcultured in MS medium supplemented with 1.0 mg.L-1 NAA and 0.5 mg.L-1 BA was dried, pow- dered and the extract was made by maceration technique using methanol as a solvent in the ratio 1:25. Quantification of spilanthol was carried out on Varian Prostar HPLC system (Varian, USA) consisting of Ultraviolet (UV) detector, a pro star binary pump and a 20-lL injection loop. Hypersil BDS RP-18 column (Thermo, USA) of dimen- sions 4.6 9 250 mm was used with acetonitrile:
water (93 : 7) as mobile phase at a flow rate of 0.5 mL.min-1. The eluted samples were detected at 237 nm. The spilanthol peaks obtained in HPLC were identified by comparing with published data and were tentatively quantified on the basis of the reference compound, dodeca-2(E), 4(E)-dienoic acid isobutylamide (Chromadex, USA). Stock so- lutions (1,000 µg.mL-1) of dodeca-2(E), 4(E)- dienoic acid isobutylamide, were prepared by dis- solving 5 mg of the compound in 5 mL of HPLC- grade methanol. The solution was then stored at 20°C. Quantification was carried out using five levels of external standards obtained by serial di- lutions of stock solutions at a concentration range of 250–15 µg.mL-1. Each concentration of stand- ard was filtered through a 0.22 µm nylon mem- brane filter (Millipore, USA) before HPLC analy- sis. Method linearity was demonstrated by deter- mining a calibration curve, injecting standard at different concentrations, and calculating the re- gression coefficient (r2). The slope equation ob- tained was used to calculate the amount of the spilanthol in unknown samples and was reported as µg.g-1 DW of the sample.
Table 1. ISSR primers selected and their sequences
Sl. No. Primer Code Primer Sequence
5 → 3’ Annealing Temper-
ature (°C) 1. 808 5'-AGA GAG AGA GAG AGA GC-3' 52 2. 815 5'-CTC TCT CTC TCT CTC TG-3' 52 3. 834 5'-AGA GAG AGA GAG AGA GYT-3' 50 4. 836 5’-AGA GAG AGA GAG AGA GYA-3’ 50 5. 840 5’- GAG AGA GAG AGA GAG AYT*-3’ 50 6. 841 5'-GAG AGA GAG AGA GAG AYC-3' 53 7. 844 5'-CTC TCT CTC TCT CTC TRC-3' 53 8. 845 5'-CTC TCT CTC TCT CTC TRG-3' 53 9. 847 5’-CAC ACA CAC ACA CAC ARC-3’ 53 10. 848 5’-CAC ACA CAC ACA CAC ARG**-3’ 50
JTLS | Journal of Tropical Life Science 464 Volume 13 | Number 3 | September | 2023
Results and Discussions Shoot culture establishment
The nodal explants produced maximum of 4.4
± 0.40 shoots during the first subculture in MS me- dium supplemented with 0.5 mg.L-1 BA. Further subculture passages resulted in the multiple pro- duction of healthy shoots (data not shown).
Callus induction
The present study noticed callus formation in leaf segments after 14-20 days of inoculation in MS medium supplemented with different concen- trations and combinations of 2,4-D, NAA, IAA, IBA and BA (Table 2). Callus formation depends upon several factors viz., the culture environment, nature of explants and hormonal and nonhormonal regulators, which may act synergistically in deter- mining the proper induction, proliferation and re- generation of callus into plantlets [11]. A defined auxin–cytokinin ratio was required to achie-ve maximum callus induction in A. ciliate as in Chlo- rophytum arundinacem, Catharanthus roseus and Brassica napus [12, 13, 14]. When auxins like NAA and IAA, IBA (0.5, 1.0, 2.0 mg.L-1) were supplemented individually, the leaf segment showed callusing from the incised margins after the 14th day of inoculation and rhizogenesis oc- curred from the callus on the 20th day. In MS me- dium fortified with 0.5, 1.0 and 2.0 mg.L-1 2,4–D, cream friable callus was formed after 14 days, turning brown within 20 days and then black after 24 days of culture. The production of mucilagi- nous fragile callus (embryonic callus) was re- ported in S. acmella in MS medium supplemented with 2.25 mg.L-1 BA and 1.0 mg.L-1 2,4-D indicat- ing the presence of higher level of BA and 2,4 -D for embryonic callus induction [6]. MS medium supplemented with 10.0 mgl-1 BA induced callus formation and resulted in the eventual death of ex- plants in S. acmella suggesting that higher cyto- kinin concentration is detrimental to callus induc- tion.
In the present study in A. ciliata, the better re- sponse of explants for callus formation was ob- served in MS medium supplemented with 1.0 mg.L-1 NAA along with 0.5 mg.L-1 BA, which produced white slightly purplish friable callus (Figure 1a & b) and the amount of callogenesis was also comparatively better in this combination.
In contrast, the highest percentage of callus induc- tion (80%) was observed in the medium supple- mented with 3.0 mg.L-1 of 2,4-D and 3.0 mg.L-1
BA in Spilanthes acmella [7]. In another study, maximum callus formation occurred in the pres- ence of 2,4-D at the concentration of 6.78 µmol.L-
1 and the callus produced were fragile and yellow- ish green in colour [15]. MS medium supple- mented with 0.15 mg.L-1 NAA and 1.5 mg.L-1 of BA was found to be the best treatment for callus induction in Spilanthes paniculata [16]. The callus produced in this was light yellowish green. Be- sides, an efficient micropropagation protocol has been developed earlier in S. acmella using seed- ling leaf explants through callus organogenesis, wherein green and compact callus was obtained in 1 µmol.L-1 NAA along with 10 µmol.L-1BA on the 15th day, which differentiated an average of 12.90
± 0.32 shoot buds in 50% cultures [17]. Variations in colour and the differences in morphogenic re- sponses during callogenesis according to the type and concentration of plant growth regulators indi- cate their crucial role in dedifferentiation, cell ag- gregation and subsequent development stages dur- ing in vitro culture of A. ciliata.
Induction of Somatic embryos
The de novo organogenesis via somatic em- bryogenesis is a multi–step regeneration process starting with the formation of proembryogenic masses, followed by the formation of somatic em- bryos, their maturation and plant regeneration. A complete reorganisation of the cellular state, in- cluding physiology, metabolism and gene expres- sion, is characteristic of this developmental path- way, thereby beginning a new life cycle [18]. So- matic embryogenesis has many advantages for mass propagation and genetic improvement over micropropagation via organogenesis. Moreover, the embryoids are more suitable for in vitro han- dling for commercial applications as they are rela- tively small, uniform, and have a high regenera- tion efficiency with long-term storage potential.
The process of somatic embryogenesis is prefera- bly controlled by the concentration and combina- tion of growth regulators and also depends on sev- eral other factors such as genotype, explant sources and medium components.
In the study conducted here in A. ciliata, the induced calli, when subcultured to a reduced con- centration of auxin–cytokinin does not produce many variations in the morphology of the callus.
Nevertheless, they showed an increase in the num- ber of cells. Similar was the observation noticed in a medium devoid of any plant growth regulators.
Interestingly, the callus subcultured to MS me- dium augmented with 1.0 mg.L-1 NAA in combi- nation with 0.5 mg.L-1 BA turned embrogenic, which upon further subculture in the fresh medium of the same PGR concentration induced the for- mation of somatic embryos after 16 days (Table 3). The influence of cytokinin and auxin on em- bryo development and maturation is explained in Catharanthus tinctorious [19] thus supporting our
studies, wherein auxin-cytokinin combination re- sulted in the formation of somatic embryos. Direct somatic embryogenesis in Farfugium japonicum occurred in the medium containing NAA and Ki- netin indicating that auxin type, concentration and combination with cytokinins could influence so- matic embryogenesis according to plant species in the Asteraceae family [20]. The auxin/cytokinin ratio also influenced the intensity of embryo for- Table 2. Callus induction in A. ciliata
Plant growth regulators (mg.L-1) % of callus
induction Morphology of
callus Colour Amount of
callus BA 2,4-D NAA IAA IBA
- 0.5 - - - 65.24±0.12h Friable Black ++
- 1.0 - - - 72.36±0.58g Friable Black ++
- 2.0 - - - 25.41±0.40k Friable Black +
0.5 0.5 - - - 63.28±0.57i Friable Black ++
0.5 1.0 - - - 58.43±0.64j Friable Black ++
0.5 2.0 - - - --- --- --- ---
- - 0.5 - - 91.54±0.45c Compact,
Rhizogenic White ++
- - 1.0 - - 95.24±0.84b Compact,
Rhizogenic White ++
- - 2.0 - - 92.48±0.36c Compact,
Rhizogenic White +
0.5 - 0.5 - - 75.63±0.84f Compact Off white ++
0.5 - 1.0 - - 97.28±0.53a Friable White
purplish +++
0.5 - 2.0 - - 95.36±0.64b Friable White
purplish +++
- - - 0.5 - 89.57±0.72d Compact more
Rhizogenic White ++
- - - 1.0 - 92.42±0.84c Compact more
Rhizogenic White ++
- - - 2.0 - 88.63±0.45d Compact more
Rhizogenic White ++
0.5 - - 0.5 - 72.27±0.94g Compact White ++
0.5 - - 1.0 - 80.18±0.49e Compact
Caulogenic
White,
purplish +++
0.5 - - 2.0 - 82.43±0.63e Compact White,
purplish +++
- - - - 0.5 91.68±0.62c Compact
Rhizogenic Cream ++
- - - - 1.0 94.47±0.35b Compact
Rhizogenic Cream ++
- - - - 2.0 90.18±0.17c Compact
Rhizogenic Cream ++
0.5 - - - 0.5 81.34±0.24e Compact Cream ++
0.5 - - - 1.0 76.49±0.15f Compact Cream +++
0.5 - - - 2.0 79.25±0.48e Compact Cream +++
Notes: + ~ 0.25 mg of callus; ++ ~ 0.5 mg of callus; +++ ~ 1.0 mg of callus; - no response. Data represent mean values of ten replicates repeated thrice, recorded after 4 weeks of culture. The mean values fol- lowed by the same letter in the superscript in a column do not differ significantly based on ANOVA and Duncan’s multiple range test at p ≤ 0.05.
JTLS | Journal of Tropical Life Science 466 Volume 13 | Number 3 | September | 2023
mation. The callus grown in 2,4-D supplemented MS medium derived from petiole and stem ex- plants of Cichorium intybus, showed intensive em- bryogenesis in comparison to IAA and NAA, however, the root-derived callus from all three treatments was not embryogenic [21]. All these re- ports agree with our finding that combinations of cytokinin and auxin have a significant role in so- matic embryo induction in A. ciliata.
Embryogenic callus of A. ciliata when subcul- tured to MS medium supplemented with 1.0 mgl-1 NAA and 0.5 mg.L-1 BA evoked the formation of 15.21 ± 0.13 embryoids (Table 2) during the in- duction phase (Figure 1c). In the second phase, i.e., during the expression phase, the embryoids expressed their embryogenic competence, under- went maturation and developed into plantlets (Fig- ures 1d-f).
Anatomical study
The histological sections under a stereomicro- scope revealed the presence of several embryonic/
embryogenic cells that have completed the transi- tion from a somatic state to an embryogenic state.
These embryogenic cells resemble the meriste- matic cells, generally smaller, more isodiametric in shape with densely staining nuclei and nucleoli and have denser cytoplasm. The structural disor- ganisation of the tissue observed after 14 days of
culture is characteristic of the indirect embryogen- esis process involving the formation of somatic embryos from callogenic masses, as observed in Passiflora edulis [22]. Two distinct groups of cells were noticed; one formed of large, non-embryonic cells at the periphery and the other group was lo- calised centrally composed of small cells with meristematic resemblance (Figure 1g). A similar structural pattern was observed in Carica papaya [23]. Somatic embryogenesis observed in A. cil- iata is not synchronised as globular embryos and embryoids in the late stages of development were visualised during the induction and maturation phase. The lack of embryogenic synchronisation has been reported in passion fruit [24]. The embry- oids developed into later embryological stages viz.
heart (Figure 1h) and torpedo, not depending on the explant tissue exhibiting a closed vascular sys- tem (Figure 1h). This study is the first report of somatic embryogenesis on A. ciliata. It high- lighted the influence of plant growth regulators in the induction and maturation of embryoids and the significant role of auxins in the embryogenesis process.
Hardening and field transfer
Somatic embryogenesis-derived plantlets after a short hardening period acclimatised in the field at 83.33% efficiency. The plantlets were geneti- Table 3. Somatic embryo induction in A.a ciliata
Concentration of PGRs ( mg.L-1 )
% Response Number of embryoids induced
Number of embryoids converted in to
plantlets NAA BA 2,4-D Kinetin Glycine Sucrose
(%)
0.0 0.0 - - - - 29.42±0.27j 2.00±0.00f 1.00±0.00g
0.5 0.5 - - - - 66.36±0.24e 8.00±0.32e 3.42±0.12f 1.0 0.5 - - - - 75.51±0.34a 15.21±0.13a 12.54±0.24a 2.0 0.5 - - - - 73.33±0.15b 10.54±0.27c 9.12±0.42b 0.5 0.1 - - - - 56.35±0.46g 12 .36±0.02b 9.46±0.11b 1.0 0.1 - - - - 53.45±0.12h 8.17±0.20e 5.28±0. 42e 2.0 0.1 - - - - 48.61±0.28i 11.71±0.72b 9.22±0.32b 0.5 1.0 - - - - 71.34±0.32d 8.13±0.54e 5.27±0.13e 1.0 1.0 - - - - 58.21±0.26f 9.27±0.24d 7.28±0.11c 2.0 1.0 - - - - 71.07±0.27d 8.21±0.26e 6.54±0.25d
- - 0.5 - - 5.0 --- --- ---
- - 1.0 - - 5.0 --- --- ---
0.2 - - 0.5 - 5.0 --- --- ---
1.0 0.5 - - 25 - --- --- ---
1.0 0.5 - - 50 - --- --- ---
1.0 0.5 - - 100 - --- --- ---
--- No response
Notes: Data represent mean values of ten replicates repeated thrice, recorded after 4 weeks of culture. The mean values followed by the same letter in the superscript in a column do not differ significantly based on ANOVA and Duncan’s multiple range test at p≤0.05.
cally uniform expressing 96.6% similarity in the banding pattern upon genetic fidelity analysis us- ing ISSR markers (Figures 2-4). All nine micro- propagated plants exhibited a monomorphic band- ing pattern with the mother plants, while the fidel- ity was assessed by ISSR markers (Figure 2). A
pair-wise similarity value among all the samples ranged from 0.94 to 0.98 (Figure 3). The dendro- gram generated through Nei's unbiased genetic distance matrix [25] revealed mean similarity across the samples as 0.96, exhibiting 96% simi- larity amongst them, thus confirming the genetic Figure 1. Somatic embryogenesis in A. ciliata. (a) Cream friable callus induced in MS+1.0 mg.L-1 NAA + 0.5
mg.L-1 BA, (b) Callus turning embryogenic arrow mark showing the formation of white globular embryos, (c) Bipolar somatic embryos (arrow mark) after 16 days, (d-f) maturation of somatic em- bryos and their germination into plantlets, (g) longitudinal section of embryogenic callus showing centrally composed small meristematic cells, (h) Longitudinal section of heart-shaped somatic em- bryo exhibiting closed independent vascular system.
Figure 2. ISSR banding pattern of somatic embryo-derived plants of A.ciliata. M: Marker, 1: mother plant, 2- 10: somatic embryo-derived plants.
JTLS | Journal of Tropical Life Science 468 Volume 13 | Number 3 | September | 2023 P1 1
P2 0.95 1
P3 0.97 0.94 1 P4 0.95 0.97 0.97 1 P5 0.97 0.94 0.95 0.94 1
P6 0.96 0.96 0.96 0.98 0.96 1 P7 0.96 0.98 0.96 0.96 0.96 0.94 1 P8 0.98 0.94 0.97 0.95 0.97 0.96 0.96 1
P9 0.97 0.97 0.97 0.97 0.95 0.96 0.96 0.97 1 P10 0.98 0.95 0.97 0.95 0.97 0.96 0.96 1 0.97 1
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
Mean Genetic Similarity = 0.96
Figure 3. Pair-wise Similarity Matrix of 10 samples (P1-P10; P1-P9 Regenerants, P10 Control mother plant) based on ISSR analysis
Figure 4. Dendrogram based on similarity indices from ISSR data showing the genetic similarity of somatic embryo regenerates and mother plants of A. ciliata. P1-P9 - Regenerants, P10 – Control mother plant.
stability of the in vitro clones.
To determine the genetic relationship among the 10 samples, clustering was carried out using Nei's unbiased genetic distance matrix [25].
The dendrogram clearly shows two groups. Six populations were clustered in one group (Group I).
Within group I, four samples (P3, P10, P1, and P8) clustered into a subgroup. Four samples (P2, P7, P4 and P6) clustered into another group (Group II) within which P2 and P7, as well as P4 and P6
showed further similarities (Figure 4).
HPLC Analysis
Spilanthol content in vitro raised embryogenic callus was quantified using HPLC, and a characte- ristic chromatogram of spilanthol was obtained at a retention time of 1.013 minutes for the embryo- genic callus sample, while in the control in vivo plant, it was 0.984 minutes. Using Dodeca- 2(E),4(E)-Dienoic acid Isobutylamide as standard, a
b
Figure 5. HPLC chromatogram of spilanthol in the embryogenic callus of A. ciliate (a), and HPLC chromato- gram of spilanthol in the in vivo plant of A. ciliata (control) (b).
JTLS | Journal of Tropical Life Science 470 Volume 13 | Number 3 | September | 2023
a calibration curve was generated at different con- centrations (250-15 µg.mL-1). The linear curve ob- tained was Y = 0.85x+2.82 with regression coeffi- cient (r2) (0.99), (x is the concentration of standard and y is the total peak area). The amount of spilan- thol was quantified by a calibration curve gener- ated from the standard. High spilanthol content was noticed in callus cultures established in NAA- BA medium combination (239.512 µg.g-1) (Figure 5a) compared to 92.19 µg.g-1 in in vivo plant (con- trol) (Figure 5b). The spilanthol content was 2703 µgg-1 in in vivo leaves, 3294.36 µg.g-1 in in vitro leaves, 998.03 µg.g-1 in callus and 91.4 µg.g-1 in cell suspension cultures of S. acmella [26]. Varia- tion in the values noticed in the study presented here may be attributed to the difference in species as well as extraction procedures. High tempera- tures and low pressures were essential for obtain- ing high content of spilanthol in the extracts [27], whereas the extracts taken by supercritical CO2 of Acmella oleracea, which are lyophilised showed maximum spilanthol content (1.07%) than the dried samples [28]. However, the present study re- ported a more effective bioproduction system for the biosynthesis of spilanthol in A. ciliata via cal- lus culture for the first time.
Conclusion
Somatic embryogenesis is a cloning technique for the rapid and exponential multiplication of par- ticular genotypes and a suitable method to main- tain genetic variability. The study presented here describes a novel method for the induction of so- matic embryogenesis in A. ciliata. The embryo- genic callus helps in the enhanced production of the N-alkylamine ‘spilanthol’ present in this plant.
This technique can be extended for the production of this bioactive compound using bioreactor tech- nology for the formulation of phytodrugs.
Acknowledgement
We are thankful to the Director, JNTBGRI, Thiruvananthapuram, Kerala, India for providing the facilities for herbarium authentication and ISSR analysis. Also, we thank Dr. Ajayakumar G, Department of Chemistry, Government College for Women, Thiruvananthapuram, Kerala, India for HPLC analysis of the samples.
References
1. Jansen RK (1981) Systematics of Spilanthes (Composi- tae: Heliantheae). Systematic Botany 6(3): 231-257. doi:
10.2307/2418284.
2. Shanthi P, Amudha P (2010) Evaluation of the phyto- chemical constituents of Acmella calva (DC). RK Jansen.
International Journal of Pharmacy and Biological Science 1(4): 308-314.
3. Gonzalez-Arnao MT, Panta A, Roca WM et al. (2008) Development and large scale application of cryopreserva- tion techniques for shoot and somatic embryo cultures of tropical crops. Plant Cell Tissue and Organ Culture 92: 1- 13. doi: 10.1007/s11240-007-9303-7.
4. Hasbullah NA, Taha RM, Saleh A (2013) Physiological response of callus from Gerbera jamesonii Bolusex.
Hook.f. to gamma irradiation. Brazilian Archives of Biol- ogy and Technology 55: 411-416.
5. Chen K, Wu HJ, Chen JF et al. (2012) Somatic embryo- genesis and mass spectrometric identification of proteins related to somatic embryogenesis in Eruca sativa. Plant Biotechnology Reports 6: 113-122. doi: 10.1007/s11816- 011-0203-2.
6. Haw AB, Keng CL (2003) Micropropagation of Spilan- thes acmella L., a bioinsecticide plant, through prolifera- tion of multiple shoots. Journal of Applied Horticulture 5(2): 65-68.
7. Yadav K, Singh N (2010) Micropropagation of Spilanthes acmella Murr. -An Important Medicinal plant. Nature and Science 8(9): 5-11.
8. Sharma S, Shahzad A, Jan N, Sahai A (2009) In Vitro Studies on Shoot Regeneration through Various Explants and Alginate-Encapsulated Nodal Segments of Spilanthes mauritiana DC., an Endangered Medicinal Herb. Interna- tional Journal of Plant Developmental Biology 3(1): 56- 61.
9. Murashige T, Skoog F (1962) A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cul- ture. Physiologia Plantarum 15: 473-497. doi:
10.1111/j.1399-3054.1962.tb08052.x.
10. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Re- search 8: 4321-4325. doi: 10.1093%2Fnar%2F8.19.4321.
11. Arumugam S, Chu Fu, Wang SY, Chang ST (2009). In vitro plant regeneration from immature leaflets derived callus of Acacia confusa Merr via organogenesis. Journal of Plant Biochemistry and Biotechnology 18(2): 1-5. doi:
10.1007/BF03263319.
12. Latto SK, Bamotra S, Sapuradhar R et al. (2006) Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker – an endangered medicinal herb. Plant Cell Reports 25: 499-506. doi: 10.1007/s00299-005-0103-4.
13. Junaid A, Mujib A, Bhat MA et al. (2007) Somatic em- bryogenesis and plant regeneration in Catharanthus roseus. Biologia Planatrum. 51: 641-646. doi:
10.1007/s10535-007-0136-3.
14. Burbulis N, Kupriene R, Blinstrubiene A (2008) Callus induction and plant regeneration from somatic tissue in spring rapeseed (Brassica napus L.). Biologia 54: 258- 263. doi: 10.2478/v10054-008-0053-1.
15. Sahu J, Jain K, Jain B, Sahu RK (2011) A Review on Phy- topharmacology and micropropagation of Spilanthes ac- mella. Pharmacology online Newsletter 1105-1110.
16. Kulathilaka PS, Senarath WTPSK (2013) Determination of cytotoxicity and chemical identities in natural plants and callus cultures of Spilanthes paniculata Wall. ex DC.
International Journal of Herbal Medicine 1(3): 135-141.
17. Pandey V, Agarwal V (2009) Efficient micropropagation protocol of Spilanthes acmella L. possessing strong anti- malarial activity. In Vitro Cellular and Developmental Bi- ology Plant 45: 491-499. doi: 10.1007/s11627-008-9184- 4.
18. Von Arnold S, Sabala I, Bozhkov P et al. (2002) Devel- opmental pathways of somatic embryogenesis. Plant Cell Tissue and Organ Culture 69: 233-249. doi:
10.1023/A:1015673200621.
19. Kumar PS, Kumar BDR (2011) Factors affecting on so- matic embryogenesis of safflower (Carthamus tinctorius L.) at morphological and biochemical levels. World Jour- nal of Agricultural Sciences 7(2): 197-205.
20. Lee SY (2006) Plant regeneration via somatic embryo- genesis in Farfugium japonicum. New Zealand Journal of Crop and Horticultural Science 34: 349-355. doi:
10.1080/01140671.2006.9514425.
21. Abdin MZ, Illah A (2007) Plant regeneration through so- matic embryogenesis from stem and petiole explants of Indian chicory (Cichorium intybus L.). Indian Journal of Biotechnology 6: 250-255.
22. Pinto DLP, Almeida AMR, Rego MM et al. (2011) So- matic embryogenesis from mature zygotic embryos of commercial Passion fruit (Passiflora edulis Sims) geno- types. Plant Cell Tissue and Organ Culture 107: 521-530.
doi: 10.1007/s11240-011-0003-y.
23. Cipriano JLD, Cruz ACF, Mancini KC et al. (2018) So- matic embryogenesis in Carica papaya as affected by
auxins and explants and morphoanatomical related as- pects. Annals of the Brazilian Academy of Science doi:
10.1590/0001-3765201820160252.
24. Silva GM, Cruz ACF, Otoni WC et al. (2015) Histochem- ical evaluation of induction of somatic embryogenesis in Passiflora edulis Sims (Passifloraceae). In Vitro Cellular and Developmental Biology Plant 51: 539-545. doi:
10.1007/s11627-015-9699-4.
25. Nei M (1973) Analysis of gene diversity in subdivided populations. Proceedings of National Academy of Sci-
ences USA 70: 3321-3323. doi:
10.1073/pnas.70.12.3321.
26. Singh M, Chaturvedi R (2012) Evaluation of nutrient up- take and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilan- thes acmella Murr. Bioprocess and Biosystems Engineer- ing 35: 943-951. doi: 10.1007/s00449-012-0679-3.
27. Dias AMA, Santos P, Seabra IJ et al. (2012) Spilanthol from Spilanthes acmella flowers, leaves and stems ob- tained by selective supercritical Carbondioxide extrac- tion. The Journal of Supercritical Fluids 61: 62-70. doi:
10.1016/j.supflu.2022.105652.
28. Barbosa AF, Carcalho de MG, Smith RE, Sabaa-srur AU (2016) Spilanthol: Occurrence, extraction, chemistry and Biological activities. Revista Brasileira de Farmacogno- sia 26: 128-133. doi: 10.1016/j.bjp.2015.07.024.
JTLS | Journal of Tropical Life Science 472 Volume 13 | Number 3 | September | 2023 This page is intentionally left blank.
