Sains Malaysiana 50(3)(2021): 667-676

http://doi.org/10.17576/jsm-2021-5003-09

 

Enhanced in vitro Shoot Regeneration and Biochemical Properties of Stevia rebaudiana using Chitosan

(Penambahbaikan Penjanaan Semula Pucuk Secara in vitro Serta Sifat Biokimia pada Stevia rebaudiana oleh Kitosan)

 

CHU-NIE TANG1, DHILIA UDIE LAMASUDIN1, WAN MUHAMAD ASRUL NIZAM WAN ABDULLAH1, CHEW-LI MOO1, MIAO-SI CHIEW1, QIAN-YEE CHAI1, JANNA ONG-ABDULLAH1 & KOK-SONG LAI2*

 

1Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

 

2Health Sciences Division, Abu Dhabi Women’s College, Higher Colleges of Technology, 41012 Abu Dhabi, United Arab Emirates

 

Received: 5 September 2019/Accepted: 13 August 2020

 

ABSTRACT

Stevia rebaudiana is a herbaceous perennial plant with great global demand due to its beneficial steviol glycosides (SGs) content. Current conventional breeding technique is unable to cater the need for more S. rebaudiana planting materials. Therefore, an improved in vitro shoot regeneration protocol was developed for S. rebaudiana by using chitosan. The highest fresh weight of plant (0.586 ± 0.176 g/explant), dry weight of plant (0.056 ± 0.02 g/explant) and plant height (4.94 ± 1.17 cm/explant) with maximum number of leaves (25.33 ± 6.95 /explant) were observed on explants grown in optimun treatment of MS basal medium supplemented with 1.0 mg/L of 6-benzyaminopurine (BAP) and 60 mg/L of low molecular weight (MW) chitosan after 4 weeks of culture. Scanning electron microscopy (SEM) analysis showed that new shoot primordia and shoot bud formation can be seen as early as day 3 and 5 of cultures on optimun treatment. Further biochemical assays showed that total phenolic acid content, total protein content and total hydrolyzed sugar content were recorded higher in explants cultured in optimun treatment as compared to control media. In contrast, total chlorophyll content and total flavonoids content were reduced in optimum treatment. Meanwhile, no significant difference in antioxidant activity was observed. All cultures from the optimal treatment were successfully regenerated, acclimatized and grew well with 100% survival rate. Taken together, an enhanced and efficient shoot regeneration protocol of S. rebaudiana was successfully developed which will be useful for rapid and large-scale micropropagation in future.

 

Keywords: Chitosan; micropropagation; scanning electron microscope (SEM); stevia; tissue culture

 

ABSTRAK

Stevia rebaudiana ialah sejenis tumbuhan herba saka yang mempunyai permintaan antarabangsa yang tinggi dengan kandungan steviol glikosid (SGs) yang berfaedah. Kaedah biakbaka konvensional tidak dapat memenuhi permintaan anak pokok S. rebaudiana yang semakin meningkat. Oleh itu, satu protokol penambahbaikan penjanaan semula pucuk S. rebaudiana secara in vitro dengan menggunakan kitosan telah dibangunkan. Tumbuhan dengan berat basah maksimum (0.586 ± 0.176 g/eksplan), berat kering maksimum (0.056 ± 0.02 g/eksplan), ketinggian maksimum (4.94 ± 1.17 cm/eksplan) dan bilangan daun tertinggi (25.33 ± 6.95 /eksplan) telah diperhatikan dalam media bes MS yang ditambah dengan 1.0 mg/L 6-benzilaminopurina (BAP) yang ditambah dengan kitosan berat molekul (MW) rendah, 60 mg/L, selepas 4 minggu dari tempoh kultur. Analisis mikroskop elektron pengimbas (SEM) telah menunjukkan pertumbuhan pucuk primordium baru serta tunas pucuk telah wujud seawal 3 hari dan 5 hari selepas rawatan optimum kitosan. Selanjutnya, asai biokimia telah menunjukkan bahawa kandungan jumlah asid fenolik, kandungan protein dan kandungan gula terhidrolisis adalah lebih tinggi di dalam eksplan yang tumbuh di dalam media optimum berbanding dengan media kawalan. Namun begitu, kandungan klorofil dan flavonoid di dalam eksplan adalah berkurangan dalam media optimum. Tiada perbezaan ketara dapat dilihat di dalam kandungan antioksidan. Semua kultur daripada rawatan optimum telah berjaya ditumbuh semula, diaklimitasi dan tumbuh dengan sihat pada kadar kelangsungan hidup 100%. Kesimpulannya, satu protokol penjanaan semula S. rebaudiana yang ditambahbaik serta berkesan telah berjaya dibangunkan dan berguna untuk pembiakan dengan cepat dan besar-besaran pada masa hadapan.

 

Kata kunci: Kitosan; kultur tisu; mikroperambatan; mikroskop elektron pengimbas (SEM); stevia

 

REFERENCES

Agbodjato, N.A., Noumavo, P.A., Adjanohoun, A., Agbessi, L. & Baba-Moussa, L. 2016. Synergistic effects of plant growth promoting rhizobacteria and chitosan on in vitro seeds germination, greenhouse growth, and nutrient uptake of maize (Zea mays L.). Biotechnology Research International 2016: 1-11.

Ahmed, S.A. & Baig, M.M.V. 2014. Biotic elicitor enhanced production of psoralen in suspension cultures of Psoralea corylifolia L. Saudi Journal of Biological Sciences 21(5): 499-504.

Álvarez-Robles, M.J., López-Orenes, A., Ferrer, M.A. & Calderón, A.A. 2016. Methanol elicits the accumulation of bioactive steviol glycosides and phenolics in Stevia rebaudiana shoot cultures. Industrial Crops and Products 87: 273-279.

Anusuya, S. & Sathiyabama, M. 2016. Effect of chitosan on growth, yield and curcumin content in turmeric under field condition. Biocatalysis and Agricultural Biotechnology 6: 102-106.

Asghari-Zakaria, R., Maleki-Zanjani, B. & Sedghi, E. 2009. Effect of in vitro chitosan application on growth and minituber yield of Solanum tuberosum L. Plant, Soil and Environment 55(6): 252-256.

Bistgani, Z.E., Siadat, S.A., Bakhshandeh, A., Pirbalouti, A.G. & Hashemi, M. 2017a. Interactive effects of drought stress and chitosan application on physiological characteristics and essential oil yield of Thymus daenensis Celak. The Crop Journal 5(5): 407-415.

Bistgani, Z.E., Siadat, S.A., Bakhshandeh, A., Pirbalouti, A.G. & Hashemi, M. 2017b. Morpho-physiological and phytochemical traits of (Thymus daenensis Celak.) in response to deficit irrigation and chitosan application. Acta Physiologiae Plantarum 39(10): 231-244.

Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72(1-2): 248-254.

Büyüktuncel, E., Porgalı, E. & Çolak, C. 2014. Comparison of total phenolic content and total antioxidant activity in local red wines determined by spectrophotometric methods. Food and Nutrition Sciences 5(17): 1660.

Ceunen, S. & Geuns, J.M.C. 2013. Glucose, sucrose, and steviol glycoside accumulation in Stevia rebaudiana grown under diferent photoperiods. Biologia Plantarum 57(2): 390-394.

Chamnanmanoontham, N., Pongprayoon, W., Pichayangkura, R., Roytrakul, S. & Chadchawan, S. 2015. Chitosan enhances rice seedling growth via gene expression network between nucleus and chloroplast. Plant Growth Regulation 75(1): 101-114.

Chawla, S.P., Kanatt, S.R. & Sharma, A.K. 2015. Chitosan. In Polysaccharides: Bioactivity and Biotechnology, edited by Ramawat, K.G. & Mérillon, J.M. Switzerland: Springer. pp. 219-246.

Chiew, M.S., Lai, K.S., Hussein, S. & Abdullah, J.O. 2016. A review on induced mutagenesis of Stevia rebaudiana bertoni. Pertanika Journal of Scholarly Research Reviews 2(3): 77-85.

Chiew, M.S., Lai, K.S., Hussein, S. & Abdullah, J.O. 2019. Acute gamma irradiated Stevia rebaudiana Bertoni enhanced particular types of steviol glycosides. Asia-Pacific Journal of Molecular Biology and Biotechnology 27(1): 56-65.

Das, A., Gantait, S. & Mandal, N. 2011. Micropropagation of an elite medicinal plant: Stevia rebaudiana Bert. International Journal of Agricultural Research 6(1): 40-48.

Dzung, P.D., Phu, D.V., Du, B.D., Ngoc, L.S., Duy, N.N., Hiet, H.D., Nghia, D.H., Thang, N.T., Le, B.V. & Hien, N.Q. 2017. Effect of foliar application of oligochitosan with different molecular weight on growth promotion and fruit yield enhancement of chili plant. Plant Production Science 20(4): 389-395.

El Hadrami, A., Adam, L.R., El Hadrami, I. & Daayf, F. 2010. Chitosan in plant protection. Marine Drugs 8(4): 968-987.

Future Market Insights. 2014. Stevia Market: Global Industry Analysis and Opportunity Assessment 2014-2020. http://www.futuremarketinsights.com/reports/global-stevia-market.

Gauchan, D.P., Dhakal, A., Sharma, N., Bhandari, S., Maskey, E., Shrestha, N., Gautam, R., Giri, S. & Gurung, S. 2014. Regenerative callus induction and biochemical analysis of Stevia rebaudiana Bertoni. Journal of Advanced Laboratory Research in Biology 5(3): 41-45.

Grobler, S.R. & Perchyonok, V. 2018. Cytotoxicity of low, medium and high molecular weight chitosan’s on balb/c 3t3 mouse fibroblast cells at a 75-85% de-acetylation degree. Material Science and Engineering with Advanced Research 2(2): 27-30.

Hajihashemi, S. & Geuns, J.M.C. 2013. Free radical scavenging activity of steviol glycosides, steviol glucuronide, hydroxytyrosol, metformin, aspirin and leaf extract of Stevia rebaudiana. Free Radicals Antioxidants 3: 34-41.

Hannan, P.A., Khan, J.A., Ullah, I. & Ullah, S. 2016. Synergistic combinatorial antihyperlipidemic study of selected natural antioxidants: Modulatory effects on lipid profile and endogenous antioxidants. Lipids in Health and Disease 15(1): 151-161.

He, Y., Bose, S., Wang, W., Jia, X., Lu, H. & Yin, H. 2018. Pre-harvest treatment of chitosan oligosaccharides improved strawberry fruit quality. International Journal of Molecular Sciences 19(8): 2194-2207.

Jamalian, S., Gholami, M. & Esna-Ashari, M. 2013. Abscisic acid-mediated leaf phenolic compounds, plant growth and yield is strawberry under different salt stress regimes. Theoretical and Experimental Plant Physiology 25(4): 291-299.

Jeong, G.T. & Park, D.H. 2005. Enhancement of growth and secondary metabolite biosynthesis: Effect of elicitors derived from plants and insects. Biotechnology and Bioprocess Engineering 10(1): 73-77.

John, B.I.J.U., Sulaiman, C.T., George, S. & Reddy, V.R.K. 2014. Total phenolics and flavonoids in selected medicinal plants from Kerala. International Journal of Pharmacy and Pharmaceutical Sciences 6(1): 406-408.

Kliebenstein, D.J. 2016. False idolatry of the mythical growth versus immunity tradeoff in molecular systems plant pathology. Physiological and Molecular Plant Pathology 95: 55-59.

Kumar, H., Kaul, K., Bajpai-Gupta, S., Kaul, V.K. & Kumar, S. 2012. A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni). Gene 492(1): 276-284.

Lai, K.S., Yusoff, K. & Maziah, M. 2011. Extracellular matrix as the early structural marker for Centella asiatica embryogenic tissues. Biologia Plantarum 55(3): 549-553.

Lopez-Moya, F., Escudero, N., Zavala-Gonzalez, E., Esteve-Bruna, D., Blázquez, M.A., Alabadi, D. & Lopez-Llorca, L.V. 2017. Induction of auxin biosynthesis and WOX5 repression mediate changes in root development in Arabidopsis exposed to chitosan. Scientific Reports 7(1): 16813.

Mondal, M.M.A., Puteh, A.B., Dafader, N.C., Rafii, M.Y. & Malek, M.A. 2013. Foliar application of chitosan improves growth and yield in maize. Journal of Food, Agriculture and Environment 11: 520-523.

Mondal, M.M.A., Malek, M.A., Puteh, A.B., Ismail, M.R., Ashrafuzzaman, M. & Naher, L. 2012. Effect of foliar application of chitosan on growth and yield in okra. Australian Journal of Crop Science 6(5): 918-921.

Muley, A.B., Shingote, P.R., Patil, A.P., Dalvi, S.G. & Suprasanna, P. 2019. Gamma radiation degradation of chitosan for application in growth promotion and induction of stress tolerance in potato (Solanum tuberosum L.). Carbohydrate Polymers (210): 289-301.

Murashige, T. & Skoog, F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497.

Pornpienpakdee, P., Singhasurasak, R., Chaiyasap, P., Pichyangkura, R., Bunjongrat, R., Chadchawan, P. & Limpanavech, P. 2010. Improving the micropropagation efficiency of hybrid Dendrobium orchids with chitosan. Scientia Horticulturae 124(4): 490-499.

Rahman, M., Mukta, J.A., Sabir, A.A., Gupta, D.R., Mohi-Ud-Din, M., Hasanuzzaman, M., Miah, M.G., Rahman, M. & Islam, M.T. 2018. Chitosan biopolymer promotes yield and stimulates accumulation of antioxidants in strawberry fruit. PLoS One 13(9): e0203769.

Ramírez-Mosqueda, M.A. & Iglesias-Andreu, L.G. 2016. Direct organogenesis of Stevia rebaudiana Bertoni using thin cell layer (TCL) method. Sugar Tech 18(4): 424-428.

Samanta, A., Das, G. & Das, S.K. 2011. Roles of flavonoids in plants. International Journal of Pharmaceutical Sciences and Research 6(1): 12-35.

San José, C., Cernadas, M.J. & Corredoira, E. 2014. Histology of the regeneration of Paulownia tomentosa (Paulowniaceae) by organogenesis. Rev. Biol. Trop. 62(2): 809-818.

Shukla, S., Mehta, A., Bajpai, V.K. & Shukla, S. 2009. In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert. Food Chemical Toxicology 47: 2338-2343.

Singh, S. 2016. Enhancing phytochemical levels, enzymatic and antioxidant activity of spinach leaves by chitosan treatment and an insight into the metabolic pathway using DART-MS technique. Food Chemistry 199: 176-184.

Sivanandhan, G., Dev, G.K., Jeyaraj, M., Rajesh, M., Arjunan, A., Muthuselvam, M., Manickavasagam, M., Selvaraj, N. & Ganapathi, A. 2013. Increased production of withanolide A, withanone, and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell, Tissue and Organ Culture 114(1): 121-129.

Sopalun, K., Thammasiri, K. & Ishikawa, K. 2010. Effects of chitosan as the growth stimulator for Grammatophyllum speciosum in vitro culture. World Academy of Science, Engineering and Technology 4: 11-29.

Sun, Y., Hou, M., Mur, L.A.J., Yang, Y., Zhang, T., Xu, X., Huang, S. & Tong, H. 2019. Nitrogen drives plant growth to the detriment of leaf sugar and steviol glycosides metabolisms in Stevia (Stevia rebaudiana Bertoni). Plant Physiology and Biochemistry 141: 240-249.

Suriyaprabha, R., Karunakaran, G., Yuvakkumar, R., Prabu, P., Rajendran, V. & Kannan, N. 2012. Growth and physiological responses of maize (Zea mays L.) to porous silica nanoparticles in soil. Journal of Nanoparticle Research 14(12): 1294.

Tadhani, M.B., Jadeja, R.P. & Rema, S. 2006. Micropropagation of Stevia rebaudiana using multiple shoot culture. Journal of Cell and Tissue Research 6(1): 545.

Talreja, T. 2011. Biochemical estimation of three primary metabolites from medicinally important plant Moringa oleiferaInternational Journal of Pharmaceutical Sciences Review Research 7: 186-188.

Thiyagarajan, M. & Venkatachalam, P. 2012. Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb. Industrial Crops and Products 37: 111-117.

Totté, N., Charon, L., Rohmer, M., Compernolle, F., Baboeuf, I. & Geuns, J.M. 2000. Biosynthesis of the diterpenoid steviol, an ent-kaurene derivative from Stevia rebaudiana Bertoni, via the methylerythritol phosphate pathway. Tetrahedron Letters 41(33): 6407-6410.

Ummi, N.A.A.R., Ong, C.B., Yu, T.S. & Lau, L.K. 2014. In vitro micropropagation of Stevia rebaudiana Bertoni in Malaysia. Brazilian Archives of Biology and Technology 57(1): 23-28.

Uthairatanakij, A., Teixeira da Silva, J.A. & Obsuwan, K. 2007. Chitosan for improving orchid production and quality. Orchid Science and Biotechnology 1(1): 1-5.

Van Handel, E. 1985. Quenching of carbohydrate reactions by azide. Analytical Biochemistry 148(2): 434-435.

Yoneda, Y., Shimizu, H., Nakashima, H., Miyasaka, J. & Obdoi, K. 2017. Effect of treatment with gibberellin, gebberellin biosynthesis inhibitor, and auxin steviol glycoside content in Stevia rebaudiana Bertoni. Sugar Tech 20(4): 482-491.

Zhang, X., Li, K., Xing, R., Liu, S. & Li, P. 2017. Metabolite profiling of wheat seedlings induced by chitosan: Revelation of the enhanced carbon and nitrogen metabolism. Frontiers in Plant Science 8: 1-13.

Zheng, J., Zhuang, Y., Mao, H.Z. & Jang, I.C. 2019. Overexpression of SrDXS1 and SrKAH enhances steviol glycosides content in transgenic Stevia plants. BMC Plant Biology 19(1): 1-16.

Zou, P., Li, K., Liu, S., Xing, R., Qin, Y., Yu, H., Zhou, M. & Li, P. 2015. Effect of chitooligosaccharides with different degrees of acetylation on wheat seedlings under salt stress. Carbohydrate Polymers 126: 62-69.

 

*Corresponding author; email: lkoksong@hct.ac.ae

 

   

 

previous