Enhancement of Vindoline and Catharanthine Accumulation, Antioxidant Enzymes Activities, and Gene Expression Levels in Catharanthus roseus Leaves by Chitooligosaccharides Elicitation
Abstract
:1. Introduction
2. Results and Discussion
2.1. Growth and Morphological Characteristics
2.2. Vindoline and Catharanthine Content
2.3. Antioxidant Enzymes Activities
2.4. Expression Determination of Key Enzymatic Genes
3. Materials and Methods
3.1. Plant Material, Chitooligosaccharides and Chemicals
3.2. Treatments of Catharanthus Roseus with Chitooligosaccharides
3.3. Determination of the Physiological Properties
3.4. HPLC Analysis of Vindoline and Catharanthine Content
3.5. Assays of Antioxidant Enzymes Activities
3.6. Gene Expression Profiling and Analysis
3.6.1. Primers
3.6.2. RNA Extraction and cDNA Synthesis
3.6.3. qRT-PCR Analysis
3.7. Statistical Analysis
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Kulkarni, R.N.; Baskaran, K.; Jhang, T. Breeding medicinal plant, periwinkle Catharanthus roseus (L.) G. Don: A review. Plant Genet. Resour. 2016, 14, 283–302. [Google Scholar] [CrossRef]
- Aslam, J.; Khan, S.H.; Siddiqui, Z.H.; Fatima, Z.; Maqsood, M.; Bhat, M.A.; Nasim, S.A.; Ilah, A.; Ahmad, I.Z.; Khan, S.A.; et al. Catharanthus roseus (L.) G. Don. an important drug: Its applications and production. Pharm. Glob. 2010, 4, 1–16. [Google Scholar]
- Ababaf, M.; Omidi, H.; Bakhshandeh, A. Changes in antioxidant enzymes activities and alkaloid amount of Catharanthus roseus in response to plant growth regulators under drought condition. Ind. Crops Prod. 2021, 167, 113505. [Google Scholar] [CrossRef]
- Madsen, M.L.; Due, H.; Ejskjær, N.; Jensen, P.; Madsen, J.; Dybkær, K. Aspects of vincristine-induced neuropathy in hematologic malignancies: A systematic review. Cancer Chemother. Pharmacol. 2019, 84, 471–485. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Eltayeb, N.M.; Ng, S.Y.; Ismail, Z.; Salhimi, S.M. Anti-invasive effect of Catharanthus roseus extract on highly metastatic human breast cancer MDA-MB-231 cells. J. Teknol. 2016, 78, 35–40. [Google Scholar] [CrossRef] [Green Version]
- Mariadi; Prasetyo, B.E.; Adela, H.; Wiladatika, W. Formulation and characterization of nanoemulsion of tread leave ethanol extract (Catharanthus roseus (L.) G. Don) as antihyperglycemic. Indones. J. Pharm. Clin. Res. 2019, 2, 24–30. [Google Scholar] [CrossRef]
- Goboza, M.; Aboua, Y.G.; Chengou, N.; Oguntibeju, O.O. Vindoline effectively ameliorated diabetes-induced hepatotoxicity by docking oxidative stress, inflammation and hypertriglyceridemia in type 2 diabetes-induced male Wistar rats. Biomed. Pharmacother. 2019, 112, 108638. [Google Scholar] [CrossRef]
- Oguntibeju, O.O.; Aboua, Y.; Goboza, M. Vindoline-a natural product from Catharanthus roseus reduces hyperlipidemia and renal pathophysiology in experimental type 2 diabetes. Biomedicines 2019, 7, 59. [Google Scholar] [CrossRef] [Green Version]
- Wang, Y.Y.; Yang, B.R.; Zhang, M.X.; Jia, S.S.; Yu, F. Application of transport engineering to promote catharanthine production in Catharanthus roseus hairy roots. Plant Cell Tissue Organ Cult. 2019, 139, 523–530. [Google Scholar] [CrossRef]
- Vázquez-Flota, F.; Hernández-Domínguez, E.; Ma, M.H.; Monforte-González, M. A differential response to chemical elicitors in Catharanthus roseus in vitro cultures. Biotechnol. lett. 2009, 31, 591–595. [Google Scholar] [CrossRef]
- Zhong, Z.H.; Liu, S.Z.; Han, S.L.; Li, Y.H.; Tao, M.L.; Liu, A.M.; He, Q.; Chen, S.X.; Dufresne, C.; Zhu, W.; et al. Integrative omic analysis reveals the improvement of alkaloid accumulation by ultraviolet-B radiation and its upstream regulation in Catharanthus roseus. Ind. Crops Prod. 2021, 166, 113448. [Google Scholar] [CrossRef]
- Liu, J.W.; Zhu, J.H.; Tang, L.; Wen, W.; Lv, S.S.; Yu, R.M. Enhancement of vindoline and vinblastine production in suspension-cultured cells of Catharanthus roseus by artemisinic acid elicitation. World J. Microbiol. Biotechol. 2014, 30, 175–180. [Google Scholar] [CrossRef]
- Karla, R.E.; Heriberto, V.L.; Diego, H.; Elisabeth, M.; Marta, G.; Rosa, C.; Javier, P. Elicitation, an effective strategy for the biotechnological production of bioactive high-added value compounds in plant cell factories. Molecules 2016, 21, 182. [Google Scholar]
- Ali, E.F.; El-Shehawi, A.M.; Ibrahim, O.H.M.; Abdul-Hafeez, E.Y.; Moussa, M.M.; Hassan, F.A.S. A vital role of chitosan nanoparticles in improvisation the drought stress tolerance in Catharanthus roseus (L.) through biochemical and gene expression modulation. Plant Physiol. Biochem. 2021, 161, 166–175. [Google Scholar] [CrossRef]
- Hassan, F.A.S.; Ali, E.; Gaber, A.; Fetouh, M.I.; Mazrou, R. Chitosan nanoparticles effectively combat salinity stress by enhancing antioxidant activity and alkaloid biosynthesis in Catharanthus roseus (L.) G. Don. Plant Physiol. Biochem. 2021, 162, 291–300. [Google Scholar] [CrossRef]
- Wang, W.Q.; Meng, Q.Y.; Li, Q.; Liu, J.B.; Zhou, M.; Jin, Z.; Zhao, K. Chitosan derivatives and their application in biomedicine. Int. J. Mol. Sci. 2020, 21, 487. [Google Scholar] [CrossRef] [Green Version]
- Mourya, V.K.; Inamdar, N.N.; Choudhari, Y.M. Chitooligosaccharides: Synthesis, characterization and applications. Polym. Sci. 2011, 53, 583–612. [Google Scholar] [CrossRef]
- Li, Y.Q.; Kong, D.X.; Fu, Y.; Sussman, M.R.; Wu, H. The effect of developmental and environmental factors on secondary metabolites in medicinal plants. Plant Physiol. Biochem. 2020, 148, 80–89. [Google Scholar] [CrossRef]
- Kumar, M.; Rajput, M.; Soni, T.; Vivekanand, V.; Pareek, N. Chemoenzymatic production and engineering of chitooligosaccharides and N-acetyl glucosamine for refining biological activities. Front. Chem. 2020, 8, 469. [Google Scholar] [CrossRef]
- Zhang, X.Q.; Li, K.C.; Liu, S.; Xing, R.E.; Yu, H.H.; Chen, X.L.; Li, P.C. Size effects of chitooligomers on the growth and photosynthetic characteristics of wheat seedlings. Carbohydr. Polym. 2016, 138, 27–33. [Google Scholar] [CrossRef]
- Jia, Y.J.; Ma, Y.L.; Zou, P.; Cheng, G.G.; Zhou, J.X.; Cai, S.B. Effects of different oligochitosans on isoflavone metabolites, antioxidant activity and isoflavone biosynthetic genes in soybean (Glycine max) seeds during germination. J. Agric. Food Chem. 2019, 67, 4652–4661. [Google Scholar] [CrossRef]
- Tang, W.Z.; Lei, X.T.; Liu, X.Q.; Yang, F. Nutritional improvement of bean sprouts by using chitooligosaccharide as an elicitor in germination of soybean (Glycine max L.). Appl. Sci. 2021, 11, 7695. [Google Scholar] [CrossRef]
- Zou, P.; Tian, X.Y.; Dong, B.; Zhang, C.S. Size effects of chitooligomers with certain degrees of polymerization on the chilling tolerance of wheat seedlings. Carbohydr. Polym. 2017, 160, 194–202. [Google Scholar] [CrossRef]
- Smith, S.E.; Smith, F.A. Fresh perspectives on the roles of arbuscular mycorrhizal fungi in plant nutrition and growth. Mycologia 2012, 104, 1–13. [Google Scholar] [CrossRef] [PubMed]
- Steenackers, W.; Houari, I.E.; Baekelandt, A.; Witvrouw, K.; Dhondt, S.; Leroux, O.; Gonzalez, N.; Corneillie, S.; Cesarino, I.; Inze, D.; et al. cis-Cinnamic acid is a natural plant growth-promoting compound. J. Exp. Bot. 2019, 70, 6293–6304. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tabassum, N.; Ahmed, S.; Ali, M.A. Chitooligosaccharides and their structural-functional effect on hydrogels: A review. Carbohydr. Polym. 2021, 261, 117882. [Google Scholar] [CrossRef]
- Gonçalves, C.; Ferreira, N.; Lourenço, L. Production of low molecular weight chitosan and chitooligosaccharides (COS): A review. Polymers 2021, 13, 2466. [Google Scholar] [CrossRef] [PubMed]
- Yin, H.; Du, Y.G.; Dong, Z.M. Chitin oligosaccharide and chitosan oligosaccharide: Two similar but different plant elicitors. Front Plant Sci. 2016, 7, 522. [Google Scholar] [CrossRef] [Green Version]
- Li, K.C.; Xing, R.E.; Liu, S.; Li, P.C. Chitin and chitosan fragments responsible for plant elicitor and growth stimulator. J. Agric. Food Chem. 2020, 68, 12203–12211. [Google Scholar] [CrossRef]
- Mall, M.; Verma, R.K.; Gupta, M.M.; Shasany, A.K.; Khanuja, S.P.S.; Shukla, A.K. Influence of seasonal and ontogenic parameters on the pattern of key terpenoid indole alkaloids biosynthesized in the leaves of Catharanthus roseus. S. Afr. J. Bot. 2019, 123, 98–104. [Google Scholar] [CrossRef]
- Mukarram, M.; Naeem, M.; Aftab, T.; Khan, M.M.A. Radiation-Processed Polysaccharides: Emerging Roles in Agriculture: Chitin, Chitosan, and Chitooligosaccharides: Recent Advances and Future Perspectives; Naeem, M., Aftab, T., Khan, M.M.A., Eds.; Academic Press: New York, NY, USA, 2021; pp. 339–353. [Google Scholar]
- Pliankong, P.; Suksa-Ard, P.; Wannakrairoj, S. Chitosan elicitation for enhancing of vincristine and vinblastine accumulation in cell culture of Catharanthus roseus (L.) G. Don. J. Agric. Sci. 2018, 10, 287. [Google Scholar] [CrossRef] [Green Version]
- Mengibar, M.; Mateos-Aparicio, I.; Miralles, B.; Heras, A. Influence of the physico-chemical characteristics of chito-oligosaccharides (COS) on antioxidant activity. Carbohydr. Polym. 2013, 97, 776–782. [Google Scholar] [CrossRef]
- Dhayanithy, G.; Subban, K.; Chelliah, J. Diversity and biological activities of endophytic fungi associated with Catharanthus roseus. BMC Microbiol. 2019, 19, 22. [Google Scholar] [CrossRef]
- Ochoa-Meza, L.C.; Quintana-Obregón, E.A.; Vargas-Arispuro, I.; Falcón-Rodríguez, A.B.; Aispuro-Hernández, E.; Virgen-Ortiz, J.J.; Martínez-Téllez, M.Á. Oligosaccharins as elicitors of defense responses in wheat. Polymers 2021, 13, 3105. [Google Scholar] [CrossRef]
- Sepasi Tehrani, H.; Moosavi-Movahedi, A.A. Catalase and its mysteries. Prog. Biophys. Mol. Biol. 2018, 140, 5–12. [Google Scholar] [CrossRef]
- Lan, W.Q.; Wang, W.; Yu, Z.M.; Qin, Y.X.; Luan, J.; Li, X.Z. Enhanced germination of barley (Hordeum vulgare L.) using chitooligosaccharide as an elicitor in seed priming to improve malt quality. Biotechnol. Lett. 2016, 38, 1935–1940. [Google Scholar] [CrossRef]
- Sikanyika, M.; Aragão, D.; McDevitt, C.A.; Maher, M.J. The structure and activity of the glutathione reductase from Streptococcus pneumoniae. Acta Crystallogr. F 2019, 75, 54–61. [Google Scholar] [CrossRef] [Green Version]
- Müller-Schüssele, S.J.; Wang, R.; Gütle, D.D.; Romer, J.; Rodriguez-Franco, M.; Scholz, M.; Buchert, F.; Luth, V.M.; Kopriva, S.; Dörmann, P.; et al. Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis. Plant J. 2020, 103, 1140–1154. [Google Scholar] [CrossRef]
- Misra, N.; Gupta, A.K. Effect of salinity and different nitrogen sources on the activity of antioxidant enzymes and indole alkaloid content in Catharanthus roseus seedlings. J. Plant Physiol. 2006, 163, 11–18. [Google Scholar] [CrossRef]
- Adak, S.; Datta, A.K. Leishmania major encodes an unusual peroxidase that is a close homologue of plant ascorbate peroxidase: A novel role of the transmembrane domain. Biochem. J. 2005, 390, 465–474. [Google Scholar] [CrossRef] [Green Version]
- Kidwai, M.; Ahmad, I.Z.; Chakrabarty, D. Class III peroxidase: An indispensable enzyme for biotic/abiotic stress tolerance and a potent candidate for crop improvement. Plant Cell Rep. 2020, 39, 1381–1393. [Google Scholar] [CrossRef]
- Maria, K.; Yogeshwar, V.D.; Neelam, G.; Madhu, T.; Iffat, Z.A.; Mehar, H.A.; Debasis, C. Oryza sativa class III peroxidase (OsPRX38) overexpression in Arabidopsis thaliana reduces arsenic accumulation due to apoplastic lignification. J. Hazard. Mater. 2018, 362, 383–393. [Google Scholar]
- Nabaei, M.; Amooaghaie, R. Melatonin and nitric oxide enhance cadmium tolerance and phytoremediation efficiency in Catharanthus roseus (L.) G. Don. Environ. Sci. Pollut. Res. 2020, 27, 6981–6994. [Google Scholar] [CrossRef]
- Trist, B.; Hilton, J.B.; Crouch, P.J.; Hare, D.J.; Double, K.L. Superoxide dismutase 1 in health and disease: How a front-line antioxidant becomes neurotoxic. Angew. Chem. Int. Ed. 2021, 60, 9215–9246. [Google Scholar] [CrossRef]
- Khataee, E.; Karimi, F.; Razavi, K. Alkaloids production and antioxidant properties in Catharanthus roseus (L.) G. Don. shoots and study of alkaloid biosynthesis-related gene expression levels in response to methyl jasmonate and putrescine treatments as eco-friendly elicitors. Biol. Futura 2019, 70, 38–46. [Google Scholar] [CrossRef]
- Liaqat, F.; Eltem, R. Chitooligosaccharides and their biological activities: A comprehensive review. Carbohydr. Polym. 2018, 184, 243–259. [Google Scholar] [CrossRef]
- Verma, P.; Mathur, A.K.; Khan, S.A.; Verma, N.; Sharma, A. Transgenic studies for modulating terpenoid indole alkaloids pathway in Catharanthus roseus: Present status and future options. Phytochem. Rev. 2015, 16, 19–54. [Google Scholar] [CrossRef]
- Liu, J.Q.; Cai, J.J.; Wang, R.; Yang, S.H. Transcriptional regulation and transport of terpenoid indole alkaloid in Catharanthus roseus: Exploration of new research directions. Int. J. Mol. Sci. 2016, 18, 53. [Google Scholar] [CrossRef] [Green Version]
- Sharma, A.; Verma, P.; Mathur, A.; Mathur, A.K. Overexpression of tryptophan decarboxylase and strictosidine synthase enhanced terpenoid indole alkaloid pathway activity and antineoplastic vinblastine biosynthesis in Catharanthus roseus. Protoplasma 2018, 255, 1281–1294. [Google Scholar] [CrossRef]
- Liu, Y.L.; Patra, B.; Singh, S.K.; Paul, P.; Zhou, Y.; Li, Y.Q.; Wang, Y.; Pattanaik, S.; Yuan, L. Terpenoid indole alkaloid biosynthesis in Catharanthus roseus: Effects and prospects of environmental factors in metabolic engineering. Biotechnol. Lett. 2021, 43, 2085–2103. [Google Scholar] [CrossRef]
- Liu, J.Q.; Gao, F.Y.; Ren, J.S.; Lu, X.J.; Ren, G.J.; Wang, R. A novel AP2/ERF transcription factor CR1 regulates the accumulation of vindoline and serpentine in Catharanthus roseus. Front. Plant Sci. 2017, 8, 2082. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Khataee, E.; Karimi, F.; Razavi, K. Different carbon sources and their concentrations change alkaloid production and gene expression in Catharanthus roseus shoots in vitro. Funct. Plant Biol. 2020, 48, 40–53. [Google Scholar] [CrossRef] [PubMed]
- Pandey, S.S.; Singh, S.; Babu, C.S.V.; Shanker, K.; Srivastava, N.K.; Shukla, A.K.; Kalra, A. Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis. Sci. Rep. 2016, 6, 26583. [Google Scholar] [CrossRef] [Green Version]
- Zhu, W.; Yang, B.X.; Komatsu, S.; Lu, X.P.; Li, X.M.; Tian, J.K. Binary stress induces an increase inindole alkaloid biosynthesis in Catharanthus roseus. Front Plant Sci. 2015, 6, 00582. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pan, Y.J.; Liu, J.; Guo, X.R.; Zu, Y.G.; Tang, Z.H. Gene transcript profiles of the TIA biosynthetic pathway in response to ethylene and copper reveal their interactive role in modulating TIA biosynthesis in Catharanthus roseus. Protoplasma 2015, 252, 813–824. [Google Scholar] [CrossRef] [PubMed]
- Available online: https://www.jinke-chitin.com/ (accessed on 27 February 2022).
- Pérez-Harguindeguy, N.; Díaz, S.; Garnier, E.; Lavorel, S.; Poorter, H.; Jaureguiberry, P.; Bret-Harte, M.S.; Cornwell, W.K.; Craine, J.M.; Gurvich, D.E.; et al. New handbook for standardised measurement of plant functional traits worldwide. Aust. J. Bot. 2013, 61, 167–234. [Google Scholar] [CrossRef]
- Karimi, F.; Khataee, E. Aluminum elicits tropane alkaloid production and antioxidant system activity in micropropagated Datura innoxia plantlets. Acta Physiol. Plant. 2012, 34, 1035–1041. [Google Scholar] [CrossRef]
- Soltani, N.; Nazarian-Firouzabadi, F.; Shafeinia, A.; Sadr, A.S.; Shirali, M. The expression of Terpenoid Indole Alkaloid (TIAs) pathway genes in Catharanthus roseus in response to salicylic acid treatment. Mol. Biol. Rep. 2020, 47, 7009–7016. [Google Scholar] [CrossRef]
- Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
Treatment | COS Concentration (μg/mL) | Root Weight | Stem Weight | Leaf Weight | Plant Height |
---|---|---|---|---|---|
Control | 0 | 155.56 ± 51.14 h | 73.56 ± 12.58 h | 297.222 ± 37.31 g | 3.75 ± 0.36 f |
1 kDa COS | 0.01 | 199.84 ± 4.09 fg | 84.58 ± 3.81 fgh | 375.96 ± 10.60 f | 4.39 ± 0.15 de |
0.1 | 293.30 ± 17.84 c | 102.90 ± 2.83 f | 434.72 ± 12.78 e | 4.76 ± 0.15 cd | |
1 | 187.38 ± 5.85 fgh | 81.11 ± 4.60 gh | 366.24 ± 9.49 f | 4.13 ± 0.16 ef | |
10 | 180.54 ± 13.19 fgh | 75.68 ± 1.79 h | 307.76 ± 6.12 g | 3.99 ± 0.05 ef | |
2 kDa COS | 0.01 | 255.19 ± 6.64 d | 185.75 ± 15.70 d | 475.73 ± 6.78 d | 4.58 ± 0.07 cd |
0.1 | 333.91 ± 4.13 b | 212.13 ± 12.10 c | 557.72 ± 16.71 c | 4.95 ± 0.13 c | |
1 | 239.56 ± 4.00 de | 175.44 ± 11.53 d | 409.22 ± 5.13 e | 4.83 ± 0.06 cd | |
10 | 214.53 ± 3.73 ef | 90.64 ± 6.38 fgh | 318.81 ± 5.64 g | 4.41 ± 0.16 de | |
3 kDa COS | 0.01 | 332.00 ± 12.46 b | 230.66 ± 4.79 b | 621.57 ± 24.02 b | 5.49 ± 0.37 b |
0.1 | 416.26 ± 17.46 a | 264.57 ± 10.57 a | 702.33 ± 13.10 a | 6.57 ± 0.16 a | |
1 | 300.46 ± 19.80 bc | 175.44 ± 6.80 e | 548.48 ± 18.34 c | 5.70 ± 0.19 b | |
10 | 172.40 ± 5.33 gh | 99.16 ± 5.20 fg | 355.60 ± 12.66 f | 4.84 ± 0.22 cd |
Primer Name | Sequence (5′ → 3′) |
---|---|
SLS-F | GTTCCTTCTCACCGGAGTTG |
SLS-R | CCCATTTGGTCAACATGTCA |
STR-F | AAAATTCCCGATACTCCG |
STR-R | ACCAATGGGCACTTCCTT |
SGD-F | TCACAAAGCTGCTGTGGAAG |
SGD-R | CACCCGTTGTTAATGGCTCT |
T16H-F | AGGACCTTGTTGATGTGCTAC |
T16H-R | CATTGCCCAATCGACTGTTG |
D4H-F | TACCCTGCATGCCCTCAACC |
D4H-R | TTGAAGGCCGCCAATTGAT |
DAT-F | AAACCCTCTTCTCCAACCCCTC |
DAT-R | CTTCCACGAACTCAATTCCATC |
PRX1-F | CTGCTGCTTCTTCTCATTTCC |
PRX1-R | CAACATCGTTTTGGAAGACCT |
ORCA3-F | CGAATTCAATGGCGGAAAGC |
ORCA3-R | CCTTATCTCCGCCGCGAACT |
RSP9-F | GAGGGCCAAAACAAACTTGA |
RSP9-R | CCCTTATGTGCCTTTGCCTA |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Tang, W.; Liu, X.; He, Y.; Yang, F. Enhancement of Vindoline and Catharanthine Accumulation, Antioxidant Enzymes Activities, and Gene Expression Levels in Catharanthus roseus Leaves by Chitooligosaccharides Elicitation. Mar. Drugs 2022, 20, 188. https://doi.org/10.3390/md20030188
Tang W, Liu X, He Y, Yang F. Enhancement of Vindoline and Catharanthine Accumulation, Antioxidant Enzymes Activities, and Gene Expression Levels in Catharanthus roseus Leaves by Chitooligosaccharides Elicitation. Marine Drugs. 2022; 20(3):188. https://doi.org/10.3390/md20030188
Chicago/Turabian StyleTang, Wenzhu, Xiaoqi Liu, Yuning He, and Fan Yang. 2022. "Enhancement of Vindoline and Catharanthine Accumulation, Antioxidant Enzymes Activities, and Gene Expression Levels in Catharanthus roseus Leaves by Chitooligosaccharides Elicitation" Marine Drugs 20, no. 3: 188. https://doi.org/10.3390/md20030188