Silicon Supplementation regulates Gallic acid Accumulation in In Vitro cultures of Ipomea batatus
Dhavalaganga Acharya, Narasimhan S*
Department of Biotechnology, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka 576104 India.
*Corresponding Author E-mail: narasimhan.s@manipal.edu
ABSTRACT:
In vitro cultures raised from leaf segments of the tuber crop Ipomea batatus exhibited gallic acid variation in response to presence of silicon. Supplementation of silicon in the form of K2SiO3 at a concentration varying from 2-5 ml/l exhibited significant enhancement of the phenol gallic acid. However, cell growth was not significantly affected. Silicon supplementation in the form of Na2SiO3 (0.5-5 mg/l) was less efficient in inducing gallic acid accumulation when compared to K2SiO3. Enhanced cell growth was observed in presence of Na2SiO3. The results indicates that the secondary metabolism leading to the biosynthesis of soluble plant phenolics such as gallic acid can be regulated by regulating the supply of silicon. Thus silicon proved to be a one of the potential elicitor in in vitro plant cell and tissue cultures.
KEYWORDS: Gallic acid, Ipomea batatus, Plant cell cultures, Plant phenols, Silicon.
INTRODUCTION:
Research studies indicates a beneficial role of silicon in several aspects of plant tissue culture such as in vitro growth, morphogenesis and metabolism. However these studies are limited to medicinal plants [10], and cereals [11,12]. Only a few studies has been conducted in tuber crops [13]. Therefore the current study is aimed at understanding the effect of silicon on callus growth and phenolic acids accumulation in the root tuber, sweet potato (Ipomea batatus).
Plants are the main sources of natural gallic acid [14,15,16]. It is one of the most important plant phenol. Chemically it is 3, 4, 5 trihydroxybenzoicacid [17]. Gallic acid is one of the main constituent of ayurvedic drugs such a triphala churna [18], Vatari guggulu and Kaishore guggulu [17], Draksharishta [19], Drakshaswara [20], Ashwagandharishta [21], Drakshadi gutika [22] Bhuvneshvara vati [23] and hepatoprotective formulations [24]. Apart from this, gallic acid is considered as a standard chemical for the analysis of plant phenols [17, 25].
MATERIAL AND METHODS:
Plant material:
Stock plants of sweet potato was raised from the tubers obtained from the local market. The plants were maintained at the greenhouse conditions.
Sterilization of explants and media:
Young leaf segments were pre-sterilized with running tap water for 15 minutes and chemically sterilized with 0.1% (w/v) mercuric chloride. The media used for the current study is woody plant medium (WPM) [26] containing 1mg/l of 2,4-D and 0.1mg/L each of NAA and BAP. The media was sterilized at 12 psi at a temperature of 1210C for 15 minutes. To find out effect of silica, two different silica sources were used (i) Potassium silicate, K2SiO3 and (ii) Sodium silicate, Na2SiO3 . Silica source was added before pH adjustment to 5.80 of the media.
Quantitative analysis of gallic acid [27]:
For this the fine ground powder of callus tissue was extracted with 70% acetone. The extract was centrifuged at 10000 RPM for 10 minutes at 40C. The supernatant was saved and diluted to 10ml in methanol. To 200 ml of of supernatant 600 ml of 0.2 N sulphuric acid and 900 ml of rhodanine solution (0.667%) was added. After 9 minutes 600 ml of 0.5N KOH was added and after 6 minutes 12.9 ml of distilled water was added. Absorbance was measured at 520 nm after allowing a reaction time of 25 minutes against blank. Quantification was done against a standard curve prepared from reference gallic acid sample.
Instrumentation:
For measuring absorbance, Shimadzu UV-visible spectrophotometer (model UV-1800 series) was used. All the precision weighing was done by using a Shimadzu balance (AUW 120D). Inoculation was done in a horizontal laminar airflow chamber.
RESULTS AND DISCUSSION:
The maximum amount of callus was obtained with 0.5 -1.0 ml/l for K2SO3 and 0.5-1.0 mg/l for Na2SiO3. Silica didn’t exhibited a significant effect on callus growth as revealed from the statistical analysis (Table 1). Higher amount of silica concentration exhibited a significantly retarding effect on callus growth. Callus cultures of Passiflora edulis also reported no significant effect of silicon on callus growth. Here the silicon was supplemented in the form of H4SiO4 [28]. However the beneficial effect of silicon has been experimentally proved in in vitro culture of Phoenix dactylifera [29], Phragmites australis [30] and rice [31].
Table 1: Response of callus growth and gallic acid accumulation in presence of varying concentrations of silicone
|
Treatment No. |
Growth media (WPM) |
Callus growth (g dry wt) |
Gallic acid (mg /g dry wt) |
|
|
K2SO3 (ml/l) |
Na2SiO3 (mg/l) |
|||
|
1 |
0 |
0 |
0.735ab+ 0.214 |
26.385a+9.853 |
|
2 |
0.5 |
0 |
1.230abc+0.403 |
14.847a+4.297 |
|
3 |
1.0 |
0 |
1.030ab+0.604 |
26.315a+10.583 |
|
4 |
2.0 |
0 |
0.935ab+0.744 |
57.868ab+28.960 |
|
5 |
3.0 |
0 |
0.347a+0.089 |
51.289ab+16.359 |
|
6 |
4.0 |
0 |
0.639ab+0.423 |
52.132ab+22.719 |
|
7 |
5.0 |
0 |
0.281a+0.158 |
92.846b+36.597 |
|
8 |
0 |
0.5 |
2.346cd+0.494 |
7.798a+2.160 |
|
9 |
0 |
1.0 |
2.651d+0.0262 |
5.880a+.060 |
|
10 |
0 |
2.0 |
0.971a+0.608 |
31.309a+14.003 |
|
11 |
0 |
3.0 |
1.751bcd+0.102 |
9.231a+0.431 |
|
12 |
0 |
4.0 |
1.001ab+0.198 |
16.863a +3.100 |
|
13 |
0 |
5.0 |
0.735a+0.214 |
30.966a +5.475 |
Values followed by the same letter are not significantly different (p < 0.05) by Duncan's multiple range test
K2SO3 induced a high amount of gallic acid accumulation compared to Na2SiO3. Maximum yield of gallic acid accumulation was observed when callus cultures were incubated in presence of 5ml/l K2SO3. Higher amount of silica was found to be favorable for the synthesis of gallic acid in in vitro cultures of I. batatus. In all concentrations tested, K2SiO3 was found to be more effective than Na2SiO3. Potassium is a stress tolerant [32]. However, the results of the present study reveals more accumulation of gallic acid when potassium silicate was used compared to sodium silicate.
Another interesting observation was that gallic acid accumulation was inversely proportional to callus growth (Fig 1). This fact leads to the conclusion that gallic acid accumulation is stress associated and is related to the enhanced silicon concentration in the media. Thus the current study reveals the possible use of silicon as an elicitor in regulating plant phenol metabolism. Stress has been linked to activate phenol metabolism in plants [33]. A study in rose plants revealed the active role of silicon in disease resistance of rose plants because silicone induces phenol metabolism [34].
Figure 1: Graph showing the relationship between callus growth and accumulation of gallic acid in 13 treatments
CONCLUSION:
The results of the present study revealed the effect of silicon on the accumulation of gallic acid in in vitro cultures of I. batatus. The results generated here are interesting as it is possible to regulate cell growth and metabolic yield using varying concentrations of silicon. Further, it is significant towards further research in molecular role of silicon in regulating plant metabolism, especially in a agriculturally relevant tuber crop.
ACKNOWLEDGEMENT:
Authors acknowledge the support of Director, Manipal Institute of Technology, Manipal Academy of Higher Education for the facilities provided.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
REFERENCES:
1. Narasimhan S, Nair GM. Release of berberine and its crystallization in liquid medium of cell suspension cultures of Coscinium fenestratum (Gaertn.) Colebr. Current Science, 2004; 86:1369-1371.
2. Babu VS, Narasimhan S, Nair GM. Enhanced accumulation of triterpenoids and flavonoids in cell suspension cultures of Azadirachta indica (A. Juss.) with an extended stationary phase. Indian Journal of Biotechnology, 2008; 7:270-272.
3. Babu VS, Narasimhan S, Nair GM. Bioproduction of azadirachtin-A, nimbin and salannin in callus and cell suspension cultures of neem (Azadirachta indica A. Juss.). Current Science, 2006; 9:22-24.
4. Narasimhan S, Trichome structure of Coscinium fenestratum (Gaertn.) colebr., a critically endangered medicinal liana suggests a role in defense mechanism. Medicinal Plants, 2018, 10:151-154. DOI: 10.5958/0975-6892.2018.00024.2
5. Narasimhan S, Ultrastructural and histochemical analysis of the trichomes of Cyclea peltata (Menispermaceae). Medicinal Plants, 2019, 1:422-426. DOI: 10.5958/0975-6892.2019.00055.8
6. Andresen E, Peiter E, Küpper H, Trace metal metabolism in plants, Journal of Experimental Botany, 2018; 69:909-954. DOI: 10.1093/jxb/erx465
7. Cooke J, Jane L, De Gabriel, Hartley SE. The functional ecology of plant silicon: geoscience to genes. Functional Ecology, 2016; 30:1270-1276. DOI: 10.1111/1365-2435.12711
8. Epstein E. Silicon, Annual Review of Plant Physiology and Plant Molecular Biology, 1999; 50:641-664.
9. Sahebi M, Hanafi MM, Azizi P Application of silicon in plant tissue culture, In Vitro Cellular and Developmental Biology – Plant, 2016; 52:226–232. DOI :10.1007/s11627-016-9757-6 Sahebi M, Hanafi MM, Akmar ASN, Rafii MY, Azizi P, Tengoua F,Nurul Mayzaitul Azwa J, Shabanimofrad M. Importance of silicon and mechanisms of biosilica formation in plants. BioMed Research International, 2015:396010. DOI: 10.1155/2015/396010
10. Soundararajan P, Sivanesan I, Jo EH, Jeong BR. Silicon promotes shoot proliferation and shoot growth of Salvia splendens under salt stress in vitro. Horticulture, Environment and Biotechnology, 2013; 54:311–318.
11. Balakhnina T, Borkowska A. Effects of silicon on plant resistance to environmental stresses: review. International Agrophysics, 2013; 27:225–232. DOI: 10.2478/v10247-012-0089-4
12. Islam MM, Ahmed M, Mahaldar D. In vitro callus induction and plant regeneration in seed explants of rice (Oryza Sativa L.). Research Journal of Agriculture and Biological Sciences, 2015; 1:72–75. DOI: 10.3126/ijasbt.v2i2.10313
13. Qing W, Huiying H, Jinwen Z. Effect of exogenous silicon and proline on potato plantlet in vitro under salt stress. China Vegetables 2005; 9:16–18.
14. Rakesh SU, Salunkhe VR, Dhabale PN, Burade KB. HPTLC method for quantitative determination of gallic acid in hydroalcoholic extract of dried flowers of Nymphaea stellata Willd. Asian Journal of Research in Chemistry, 2009; 2:131-134.
15. Bairagi VA, Shinde PR, Senthikumar KL, Sandu N. Isolation and Characterizations of phytoconstituents from Quisqualis indica Linn. (Combretaceae). Research Journal of Pharmacognosy and Phytochemistry 2012; 4: 229-233.
16. Hima V, Kumar RS, Duganath N, Devanna N. A novel validated stability indicating chromatographic method for the simultaneous estimation of ascorbic acid and gallic acid in the ayurvedic capsule dosage form of Amla by UFLC. Asian Journal of Research in Chemistry 2013; 6:826-831.
17. Krishna KVVS, Sankar GK, Vardhan SM, Krishna RS, Tamizhmani T. Simultaneous estimation of gallic acid and rutin in Kaishore guggulu and Vatari guggulu by HPTLC. Research Journal of Pharmacognosy and Phytochemistry, 2018: 10:221-225. DOI: 10.5958/0975-4385.2018.00036.5
18. Mahajan AD, Pai NR. Simultaneous determination of eight phytoconstituents in Triphala churna by HPLC–DAD. Research Journal of Pharmacognosy and Phytochemistry 2011:3-62-66.
19. Tiwari P, Sen DJ, Patel RK. Development and validation of HPTLC Method for quantification of gallic acid and catechin from Draksharishta. Asian Journal of Research in Chemistry 2013; 6:248-253.
20. Tiwari P, Patel RK. Quantification of Gallic acid and Catechin in Drakshasava by Validated HPTLC Densitometry. Asian Journal of Research in Chemistry 2019; 5:1033-1037.
21. Tiwari P, Patel RK. Development and validation of HPTLC method for quantification of gallic acid and ellagic acid in Ashwagandharishta. Asian Journal of Research in Chemistry 2012; 5:759-764.
22. Jain V, Jain T, Saraf S, Saraf S. HPTLC method for routine quality control of Ayurvedic formulation Drakshadi gutika. Asian Journal of Pharmaceutical Analysis. 2013; 3:111-114.
23. Jain V, Daharwal SJ, Jain T, Saraf S, Saraf S. HPTLC methods for quantification of gallic acid in Bhuvneshvara vati for routine quality control. Asian Journal of Research in Chemistry. 2012; 5:193-196.
24. Arivukkarasu R, Rajasekaran A, Hussain SHB, Ajnas M. Simultaneous detection of Rutin, Quercetin, Gallic acid, Caffeic acid, Ferulic acid, Coumarin, Mangiferin and Catechin in hepatoprotective commercial herbal formulations by HPTLC technique. Research Journal of Pharmacognosy and Phytochemistry. 2018; 10:59-62. DOI: 10.5958/0975-4385.2018.00009.2.
25. Darshan D, Kamlesh D. Standardization of ‘Avipathikara Churna’: A HPLC Approach. Asian Journal of Research in Chemistry. 2012; 5:1415-1418.
26. Lloyd G, McCown BH. Commercially-Feasible micropropagation of mountain laurel (Kalmia latifolia), by shoot tip culture. Proceeding of the International Plant Propagation Society, 1981; 30:421-427.
27. Vazirian M, Khanvi M, Amanzadeh Y, Hajimehdipoor H. Quantification of gallic acid in fruits of three medicinal plants. Iranian Journal of Pharmaceutical Research, 2011; 10:233-236. DOI:10.22037/ijpr.2011.917
28. Dias GDMG, Anjos DCD, De Souza BN, Pasqual M, Homem BGC, Costa IDJS. Silicon in the embryogenic potential of callus in vitro of Passiflora edulis. Journal of Agricultural Science, 2018; 10:345-352. DOI:10.5539/jas.v10n5p345
29. Fadl AE and Reda E. Effect of silicon on somatic embryogenesis and shoot regeneration of dry date palm (Phoenix dactylifera) cv bartamuda. Egyptian Journal of Desert Research, 2014; 64:65-82. DOI: 10.21608/EJDR.2014.5810
30. Máthé C, Mosolygó Á, Surányi G, Beke A, Demeter Z, Tóth VR, Beyer D, Mészáros I, M-Hamvas M. Genotype and explant-type dependent morphogenesis and silicon response of common reed (Phragmites australis) tissue cultures. Aquatic Botany, 2012; 97:57-63. DOI: 10.1016/j.aquabot.2011.11.005
31. He C, Wang L, Liu J, Liu X, Li X, Ma J, Lin Y, Xu F. Evidence for ‘silicon’ within the cell walls of suspension‐cultured rice cells. New Phytologist, 2013;200:700-709. DOI: 10.1111/nph.12401
32. Hasanuzzaman M, Bhuyan MHMB, Nahar K, Hossain MS, Mahmud JA, Hossen MS, Masud AAC, Moumita M, Fujita M. Potassium: A vital regulator of plant responses and tolerance to abiotic stresses. Agronomy, 2018; 8: 31 DOI: 10.3390/agronomy8030031
33. Ibrahimn KM, Musbah HM. Increasing polyphenols in Coleus blumei at the cellular and intact plant levels using PEG stress. Research Journal in Pharmacy and Technology, 2018; 11:321-327. DOI: 10.5958/0974-360X.2018.00059.8
34. Shetty R, Jense B, Shetty NP, Hansen M, Hansen CW, Starkey KR, Jorgensen HJL. Silicon induced resistance against powdery mildew of roses caused by Podosphaera pannosa. Plant Pathology, 2012; 61: 120-131. DOI: 10.1111/j.1365-3059.2011.02493.x
Received on 18.02.2020 Modified on 06.04.2020
Accepted on 09.05.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(12):6124-6127.
DOI: 10.5958/0974-360X.2020.01068.9