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Growth Kinetics, Metabolites Production and Expression Profiling of Picrosides Biosynthetic Pathway Genes in Friable Callus Culture of Picrorhiza kurroa Royle ex Benth

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Abstract

The rising demand for picrosides commercially and over-exploitation of Picrorhiza kurroa from natural habitat has to initiate alternative strategies for sustainable production of metabolites. In the present research, wild leaf explant of P. kurroa was used to produce friable callus under different culture condition, i.e., dark and light with two temperature variants (15 °C and 25 °C). Afterward, callus cell lines were screened based on growth biomass and metabolites content accumulation. The results revealed, maximum callus growth index along with antioxidant potential (IC50–40.88 μg/mL) and total phenol content (41.35 μg/mg) were observed under dark 25 °C. However, under light 15 °C, highest accumulation of picroside II (0.58 μg/mg), cinnamic acid (0.15 μg/mg), p-hydroxy acetophenone (0.30 μg/mg), total flavonoids (77.30 μg/mg), nitrogen (7.06%), carbohydrates (18.03%), and protein (44.12%) were detected. Major reported metabolite in callus was picroside I (1.63 μg/mg) under dark 15 °C. For the first time, picroside III content (range 0.15–0.56 μg/mg) was also detected and quantified in leaf-derived calli. Expression profiling of picroside biosynthetic pathway genes showed a positive correlation with the observed metabolites. Furthermore, an optimized protocol of metabolites enriched callus biomass could be used as potential strategy for sustainable production of picrosides at commercial scale.

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Abbreviations

AA:

Ascorbic acid

ACT:

Anthocyanin 5-aromatic acyltransferase

AEAC:

Ascorbic acid equivalent antioxidant capacity

ANOVA:

Analysis of variance

ARP:

Antiradical power

CA:

Cinnamic acid

CAF:

Caffeic acid

C4H:

Cinnamate-4-hydroxylase

CAM:

Caffeic acid 3-o-methyltransferase

CM:

Chorismate mutase

CAR:

Carbohydrates

DAHPS:

3-Deoxy-D-arabinoheptulosonate 7-phosphate synthase

DPPH:

2,2-Diphenyl-1-picrylhydrazyl

EC50 :

Half maximal effective concentration

GI:

Growth index

G10H:

Geraniol 10-hydroxylase

GIFW:

Growth index fresh weight

GIDW:

Growth index dry weight

IBA:

Indole-3-butyric acid

IC50 :

Half maximal inhibitory concentration

ISPD:

2-C-methyl D-erythritol 4-phosphate cytidylyltransferase

MDS:

Multidimensional scaling

MS:

Murashige and Skoog medium

N:

Nitrogen

P. kurroa :

Picrorhiza kurroa

P-I:

Picroside I

P-II:

Picroside II

P-III:

Picroside III

PCA:

Principal component analysis

PHA:

p-Hydroxy acetophenone

PMK:

Phosphomevalonate kinase

PPM:

Plant preservative mixture

PR:

Protein

RT-qPCR:

Quantitative real-time polymerase chain reaction

TDZ:

Thidiazuron

TFC:

Total flavonoid content

TPC:

Total phenolic content

UHPLC-PDA:

Ultra-high-performance liquid chromatography-photometric diode array

References

  1. Chettri, N., Sharma, E., & Lama, S. D. (2005). Non-timber forest produces utilization, distribution and status in a trekking corridor of Sikkim, India. Lyonia, 8(1), 89–101.

    Google Scholar 

  2. Debnath, P., Rathore, S., Walia, S., Kumar, M., Devi, R., & Kumar, R. (2020). Picrorhiza kurroa: A promising traditional therapeutic herb from higher altitude of western Himalayas. Journal of Herbal Medicine, 23, 100358. https://doi.org/10.1016/j.hermed.2020.100358.

    Article  Google Scholar 

  3. Soni, D., & Grover, A. (2019). “Picrosides” from Picrorhiza kurroa as potential anti-carcinogenic agents. Biomedicine & Pharmacotherapy, 109, 1680–1687.

    Article  CAS  Google Scholar 

  4. Kumar, V., Chauhan, R. S., & Tandon, C. (2017). Biosynthesis and therapeutic implications of iridoid glycosides from Picrorhiza genus: The road ahead. Journal of Plant Biochemistry and Biotechnology, 26(1), 1–13.

    Article  CAS  Google Scholar 

  5. Kant, K., Walia, M., Agnihotri, V. K., Pathania, V., & Singh, B. (2013). Evaluation of antioxidant activity of Picrorhiza kurroa (leaves) extracts. Indian Journal of Pharmaceutical Sciences, 75(3), 324–329.

    Article  CAS  Google Scholar 

  6. Bhandari, P., Kumar, N., Singh, B., & Ahuja, P. S. (2010). Online HPLC-DPPH method for antioxidant activity of Picrorhiza kurroa Royle ex Benth. and characterization of kutkoside by ultra-performance LC-electrospray ionization quadrupole time-of-flight mass spectrometery. Indian Journal of Experimental Biology, 48(3), 323–328.

    CAS  PubMed  Google Scholar 

  7. Kumar, R., Bhandari, P., Singh, B., & Ahuja, P. S. (2012). Evaluation of Picrorhiza kurrooa accessions for growth and quality in north western Himalayas. Journal of Medicinal Plants Research, 6(13), 2660–2665.

    Google Scholar 

  8. Pandit, S., Shitiz, K., Sood, H., & Chauhan, R. S. (2012). Differential biosynthesis and accumulation of picrosides in an endangered medicinal herb Picrorhiza kurroa. Journal of Plant Biochemistry and Biotechnology, 22(3), 335–342.

    Article  Google Scholar 

  9. Hussain, M. S., Fareed, S., Ansari, S., Rahman, M. A., Ahmad, I. Z., & Saeed, M. (2012). Current approaches toward production of secondary plant metabolites. Journal of Pharmacy and Bioallied Sciences, 4(1), 10–20.

    Article  Google Scholar 

  10. Eibl, R., Meier, P., Stutz, I., Schildberger, D., Hühn, T., & Eibl, D. (2018). Plant cell culture technology in the cosmetics and food industries: Current state and future trends. Applied Microbiology and Biotechnology, 102(20), 8661–8675.

    Article  CAS  Google Scholar 

  11. Lee, E. K., Jin, Y. W., Park, J. H., Yoo, Y. M., Hong, S. M., Amir, R., Yan, Z., Kwon, E., Elfick, A., Tomlinson, S., Halbritter, F., Waibel, T., Yun, B. W., & Loake, G. J. (2010). Cultured cambial meristematic cells as a source of plant natural products. Nature Biotechnology, 28(11), 1213–1217.

    Article  CAS  Google Scholar 

  12. Anonymous, (2019). Global cell culture market size by product, by end user, by application, by geographic scope and forecast, https://www.verifiedmarketresearch.com/product/global-cell-culture-market-size-and-forecast-to-2025/. [Accessed 26 Dec 2019].

  13. Gahlan, P., Singh, H. R., Shankar, R., Sharma, N., Kumari, A., Chawla, V., Ahuja, P. S., & Kumar, S. (2012). De novo sequencing and characterization of Picrorhiza kurrooa transcriptome at two temperatures showed major transcriptome adjustments. BMC Genomics, 13(126), 126.

    Article  CAS  Google Scholar 

  14. Thakur, K., Partap, M., Kumar, D., & Warghat, A. R. (2019). Enhancement of picrosides content in Picrorhiza kurroa Royle ex Benth. mediated through nutrient feeding approach under aeroponic and hydroponic system. Industrial Crops and Products, 133, 160–167.

    Article  CAS  Google Scholar 

  15. Kawoosa, T., Singh, H., Kumar, A., Sharma, S. K., Devi, K., Dutt, S., Vats, S. K., Sharma, M., Ahuja, P. S., & Kumar, S. (2010). Light and temperature regulated terpene biosynthesis: Hepatoprotective monoterpene picroside accumulation in Picrorhiza kurroa. Functional & Integrative Genomics, 10(3), 393–404.

    Article  CAS  Google Scholar 

  16. Sharma, N., Kumar, V., Chauhan, R. S., & Sood, H. (2016). Modulation of picroside-I biosynthesis in grown elicited shoots of Picrorhiza kurroa in vitro. Journal of Plant Growth Regulation, 35(4), 965–973.

    Article  CAS  Google Scholar 

  17. Rawat, B., Rawat, J. M., Mishra, S., & Mishra, S. N. (2013). Picrorhiza kurrooa: Current status and tissue culture mediated biotechnological interventions. Acta Physiologiae Plantarum, 35(1), 1–12.

    Article  CAS  Google Scholar 

  18. Adil, M., Ren, X., & Jeong, B. R. (2019). Light elicited growth, antioxidant enzymes activities and production of medicinal compounds in callus culture of Cnidium officinale Makino. Journal of Photochemistry and Photobiology B: Biology, 196, 111509. https://doi.org/10.1016/j.jphotobiol.2019.05.006.

    Article  CAS  Google Scholar 

  19. Kapoor, S., Raghuvanshi, R., Bhardwaj, P., Sood, H., Saxena, S., & Chaurasia, O. P. (2018). Influence of light quality on growth, secondary metabolites production and antioxidant activity in callus culture of Rhodiola imbricata Edgew. Journal of Photochemistry and Photobiology B: Biology, 183, 258–265.

    Article  CAS  Google Scholar 

  20. AOAC International. (2012). Official methods of analysis of AOAC international (19th ed.). Gaitherburg, MD: Association of Official Analytical Chemists. pp. 121-130.

  21. Thani, P. R., Sharma, Y. P., Kandel, P., & Nepal, K. (2018). Standardization of extraction techniques of picroside-I and picroside-II from “Kutki” (Picrorhiza kurroa Royle ex Benth.). Global Journal of Science Frontier Research: C Biological Science, 18(1), 51–56.

    Google Scholar 

  22. Partap, M., Gupta, R. C., & Pradhan, S. K. (2019). Comparative analysis of morphology and phytochemical constituents in different populations and morphotypes of Datura innoxia mill. And Datura metel L. from Punjab plains. Asian Journal of Pharmaceutical and Clinical Research, 12(1), 193–199.

    Article  CAS  Google Scholar 

  23. Dhar, P., Bajpai, P. K., Tayade, A. B., Chaurasia, O. P., Srivastava, R. B., & Singh, S. B. (2013). Chemical composition and antioxidant capacities of phytococktail extracts from trans-Himalayan cold desert. BMC Complementary and Alternative Medicine, 13(1), 259.

    Article  Google Scholar 

  24. Kumar, D., Kumar, R., Singh, B., & Ahuja, P. S. (2016). Comprehensive chemical profiling of Picrorhiza kurroa Royle ex Benth using NMR, HPTLC and LC-MS/MS techniques. Combinatorial Chemistry & High Throughput Screening, 19(3), 200–215.

    Article  CAS  Google Scholar 

  25. Shitiz, K., Sharma, N., Pal, T., Sood, H., & Chauhan, R. S. (2015). NGS transcriptomes and enzyme inhibitors unravel complexity of picrosides biosynthesis in Picrorhiza kurroa Royle ex. Benth. PLoS One, 10(12), e0144546.

    Article  Google Scholar 

  26. Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3(6), 1101–1108.

    Article  CAS  Google Scholar 

  27. Tauno, M., & Jaak, V. (2015). Clustvis: A web tool for visualizing clustering of multivariate data using principal component analysis and heatmap. Nucleic Acids Research, 43, W566–W570.

    Article  Google Scholar 

  28. Sood, H., & Chauhan, R. S. (2009). High frequency callus induction and plantlet regeneration from different explants of Picrorhiza kurroa - a medicinal herb of Himalayas. African Journal of Biotechnology, 8(9), 1965–1972.

    CAS  Google Scholar 

  29. Jan, A., Thomas, G., Shawl, A. S., Jabeen, N., & Kozgar, M. I. (2010). Improved micropropagation protocol of an endangered medicinal plant Picrorhiza kurrooa Royle ex Benth. promptly through auxin treatments. Chiang Mai Journal of Science, 37(2), 304–313.

    CAS  Google Scholar 

  30. Patil, A. A., Sachin, B. S., Shinde, D. B., & Wakte, P. S. (2013). Supercritical CO2 assisted extraction and LC-MS identification of picroside-I and picroside-II from Picrorhiza kurroa. Phytochemical Analysis, 24(2), 97–104.

    Article  CAS  Google Scholar 

  31. Helena, D. S., Kannojia, G., & Gaur, A. K. (2015). In vitro regeneration of Picrorhiza kurroa Royal ex Benth for ex situ conservation and sustainable utilization. Annals of Biological Research, 6(5), 7–14.

    CAS  Google Scholar 

  32. Ganeshkumar, Y., Ramarao, A., & Veeresham, C. (2017). Picroside I and picroside II from tissue cultures of Picrorhiza kurroa. Pharmacognosy Research, 9, S53–S56.

    Article  Google Scholar 

  33. Parihar, D., Bameta, A., Kumari, A., Kannojia, G., & Gaur, A. K. (2018). Metabolite profiling of in vitro established cultures of Picrorhiza kurroa Royle ex Benth in different growth regime (s). International Journal of Pure and Applied Bioscience, 6(2), 1663–1671.

    Article  Google Scholar 

  34. Chaudhary, V., Singh, S., Sharma, R., Singh, A., & Sharma, N. (2019). Conservation of the endangered medicinal plant Picrorhiza kurroa through in vitro multiple shoot regeneration, BioTechnologia. Journal of Biotechnology Computational Biology and Bionanotechnology, 100(3), 209–217.

    CAS  Google Scholar 

  35. Narayani, M., & Srivastava, S. (2018). Elicitation: A stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary metabolite production. Phytochemistry Reviews, 16, 1227–1252.

    Article  Google Scholar 

  36. Ibrahim, M. H., Jaafar, H. Z. E., Karimi, E., & Ghasemzadeh, A. (2012). Primary, secondary metabolites, photosynthetic capacity and antioxidant activity of the malaysian herb kacip fatimah (Labisia Pumila Benth) exposed to potassium fertilization under greenhouse conditions. International Journal of Molecular Sciences, 13(12), 15321–15342.

    Article  CAS  Google Scholar 

  37. Efferth, T. (2019). Biotechnology applications of plant callus cultures. Engineering, 5(1), 50–59.

    Article  CAS  Google Scholar 

  38. Singh, H., Gahlan, P., & Kumar, S. (2013). Cloning and expression analysis of ten genes associated with picrosides biosynthesis in Picrorhiza kurrooa. Gene, 515(2), 320–328.

    Article  CAS  Google Scholar 

  39. Kumar, V., Shitiz, K., Chauhan, R. S., Sood, H., & Tandon, C. (2016). Tracking dynamics of enzyme activities and their gene expression in Picrorhiza kurroa with respect to picroside accumulation. Journal of Plant Biochemistry and Biotechnology, 25(2), 125–132.

    Article  CAS  Google Scholar 

  40. Pandit, S., Shitiz, K., Sood, H., Naik, P. K., & Chauhan, R. S. (2013). Expression pattern of fifteen genes of non mevalonate (MEP) and mevalonate (MVA) pathways in different tissues of endangered medicinal herb Picrorhiza kurroa with respect to picrosides content. Molecular Biology Reports, 40(2), 1053–1063.

    Article  CAS  Google Scholar 

  41. Kumar, P., Partap, M., Ashrita, Rana, D., Kumar, P., & Warghat, A.R. (2020). Metabolite and expression profiling of steroidal alkaloids in wild tissues compared to bulb derived in vitro cultures of Fritillaria roylei – high value critically endangered Himalayan medicinal herb. Industrial Crops and Products https://doi.org/10.1016/j.indcrop.2019.111945.

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Acknowledgments

The authors are thankful to the Director, Dr. Sanjay Kumar, CSIR-IHBT, for providing necessary facilities. The authors are also thankful to Ms. Sampa das for help in conducting primary metabolite analysis. CSIR-IHBT publication number for this manuscript is 4535.

Funding

This study is financially supported by the Council of Scientific and Industrial Research (CSIR), Government of India, under the project “Biotechnological interventions for sustainable bio-economy generation through characterization, conservation, prospection, and utilization of Himalayan bioresources (MLP-0201) and Phytopharma mission project (HCP-0010).” MP is supported by the CSIR, New Delhi, through Junior Research Fellowship and Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India for Ph.D. enrolment.

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Authors and Affiliations

  1. Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, 176061, Himachal Pradesh, India

    Mahinder Partap, Pankaj Kumar,  Ashrita & Ashish R. Warghat

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

    Mahinder Partap,  Ashrita, Dinesh Kumar & Ashish R. Warghat

  3. Chemical Technology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, 176061, Himachal Pradesh, India

    Pawan Kumar & Dinesh Kumar

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  1. Mahinder Partap
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Contributions

Conceptualization: Mahinder Partap and Ashish R. Warghat; methodology: Mahinder Partap and Ashish R. Warghat; data analysis and investigation: Mahinder Partap, Pankaj Kumar, Pawan Kumar, Ashrita, Dinesh Kumar, and Ashish R. Warghat; writing-original draft preparation: Mahinder Partap and Ashish R. Warghat; writing-review and editing: Mahinder Partap, Ashish R. Warghat, Pankaj Kumar, and Dinesh Kumar.

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Correspondence to Dinesh Kumar or Ashish R. Warghat.

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Partap, M., Kumar, P., Ashrita et al. Growth Kinetics, Metabolites Production and Expression Profiling of Picrosides Biosynthetic Pathway Genes in Friable Callus Culture of Picrorhiza kurroa Royle ex Benth. Appl Biochem Biotechnol 192, 1298–1317 (2020). https://doi.org/10.1007/s12010-020-03391-x

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