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

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