Comparative analyses of stevioside between fresh leaves and in-vitro derived callus tissue from Stevia rebaudiana Bert. using HPLC

Abstract

A protocol was developed to produce large amount of callus in short a period of time from leaf explants of Stevia rebaudiana Bert. The highest amount of white callus was obtained on MS medium supplemented with 2.5 mg/l 2, 4-D and 0.5 mg/l BAP after 3 weeks of inoculating leaf segments. On the other hand, 0.5 mg/l BAP and 1.0 mg/l Kn exhibits poor performance towards callus formation while after using 1.0 mg/l Kn alone did not develop any callus. In this experiment, highest amount of green callus was obtained when MS medium supplemented with 2.5 mg/l NAA and 10% coconut water was used. An improved analytical method HPLC was applied to analyze stevioside extracted from the leaf and callus of Stevia rebaudiana. The stevioside in each sample were analyzed by comparing their retention times with those of the standards. The retention time (RT) of stevioside for leaves were found 14.96 and for callus 13.81 mins. The percentage of stevioside content from leaves and callus was 12.19% and 12.62% respectively Key words: Stevioside; Callus; Natural sweeteners; Growth regulators; Stevia rebaudiana; HPLC Available online at www.banglajol.info Bangladesh J. Sci. Ind. Res. 49(4), [199][200][201][202][203][204] 2014 *Corresponding author: E-mail: trishansu@gmail.com (Mizukami et al., 1982, Nishiyama et al. Ahmed and Dobberstein developed an HPLC method for the determination of eight diterpene glycosides. Satisfactory resolution was achieved on two protein columns in series, after the plant material was extracted with chloroform prior to methanol extraction (Mizukami et al., 1982). Therefore, considering its scope and future need of planting material, the present study was undertaken to find out suitable concentration and combination of cytokinin (BAP) and auxins (NAA, 2, 4-D, Kn) for callus induction and development from leaf explants, to study the production of steviol glycosides in callus culture and to compare the amount of stevioside between induced callus and leaf tissue using HPLC. Materials and methodsThe experiment was conducted at Tissue Culture Section, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka Bangladesh. The experiment was performed in two phases. In the first phase, leaf segments of Stevia rebaudiana, as explants were cultured for callus induction on MS medium (Murashige and Skoog, 1962) supplemented with auxins (2,4-D, NAA) and cytokinin (BAP, Kn) at different concentrations (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/l ) and with 10% coconut water respectively. Regular visual observation was done up to five weeks to record the day of callus initiation and maturation. Then fresh weight of best calluses and fresh leaves were recorded.In the second phase of this study stevioside content was determined in the callus and leaf from which the callus was developed by HPLC method. The HPLC system was a SHIMADJU SPD-M20A chromatograph equipped with a binary pump and UV detection was at 219 am. The analytical column was RP-C18 (250 mm X 4.6 nm). Separation was performed with a water and methano...

Cultivation and uses of stevia (Stevia rebaudiana bertoni): A review

“…No negative clinical reports have appeared in any of these countries where stevia is readily available [41]. Medicinal plants are becoming very popular for the treatment of different diseases all over the world [8].…”

Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves

“…The stevioside content of the leaf sample was found to be 12.19%. 24 Kour 25 developed an HPLC method for determination of stevioside from leafy parts of in vitro and in vivo regenerated plants of S. rebaudiana. Stevioside obtained was 0.0197 mg/mL for in vivo leaf, which was still lower than our study.…”

Role of Plant Growth Regulators on Multiplication of Stevia rebaudiana Inside Manufactured Temporary Immersion Bioreactor and Induction of Callus from Explant

“…Callogenesis originates from wound areas on leaves or plant parts and is an irregular tissue composed of undifferentiated cells [12]. Recent studies have demonstrated that plant growth regulators have an impact on giving different colors to callogenesis, such as light green, dark green, and yellowish white, as well as different textures, such as soft or compact callogenesis [13]. The addition of various concentrations of auxins, cytokinins, and their interaction plays a significant role in callogenesis by enhancing cell activity, division, and the synthesis of essential growth materials [14].…”