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An Efficient Protocol for in Vitro Regeneration of Stevia Rebaudiana

Sheikh Rashel Ahmed
Department of Biotechnology, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh.

Md. Moniruzzaman Shohag Howlader
Department of Biotechnology, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh.

Pijush Sutradhar
Department of Biotechnology, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh.

Sabina Yasmin
Department of Biotechnology, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh.

Abstract/ Executive Summary:

An efficient high frequency plant regeneration protocol through direct organogenesis was developed for Sevia rebaudiana. Shoot tips containing axillary buds were used as an explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.6% (w/v) agar supplemented with various concentrations of benzy-ladenine (BA), kinetin (Kn) and thidiazuron (TDZ). BAP proved to be a better choice than Kn and the maximum number of shoots (3.75) was obtained on 2.0 mgL^-1 BAP concentration. Considering all parameters, combination of BAP and Kn gave comparatively better performance than single BAP or Kn. TDZ was effective for multiple shooting. Though, 1.5 mgL^-1 TDZ gave the best number of shoots (14.5), but 1.0 mgL^-1 TDZ gave best performance in response to all parameters under study. The highest number of shoots was obtained in 60 mlL^-1 coconut water, but 40 mlL^-1 coconut water gave the best result to all parameters. Root induction was tested by using two auxins namely NAA and IBA at different concentrations (1.0, 1.5, 2.0 mgL^-1) on the MS medium. IBA at 1.0 mgL^-1 increased the rooting response (66.67%), number of roots (7.0) and root length (2.9 cm). Higher concentration of IBA and NAA (2.0 mgL^-1) showed poor results of rooting response (33.33%).

Keywords: In vitro regeneration, Stevia, Medicinal plants

Location: Department of Biotechnology and Physics, Central laboratory, Bangladesh Agricultural University, Mymensingh

Start Date: 00-07-2015

End Date: 00-11-2015

Program Area: Crop-Soil-Water Management

Commodity: Medicinal Plants

Objectives:

To evaluate the effect of different concentration of cytokinin hormone (BAP, Kn, Thidiazuron), coconut water on shoot regeneration and the effect of different concentration of auxins (NAA and IBA) on root formation.

Methodology:

The research work was conducted at laboratory of the Department of biotechnology and physics with a common help of central laboratory, Bangladesh Agricultural University, Mymensingh during the period from July to November, 2015. Shoot tips were used as explants for shoot regeneration. All the explants were collected from 4-6 month old yard grown plants from physics department of BAU. Five experiments with different treatments were carried out in this research. Single or combined effect of cytokinin (BAP or Kn), effect of thidiazuron, effect of coconut water concentrations on shoot proliferation and effect of auxin (NAA and IBA) on root induction of Stevia rebaudiana was observed in these experiments. MS medium was used to detect the effect of hormone and coconut water concentration on the shoot and root development of natural sweetener containing herb Stevia rebaudiana. Following procedure was maintained to prepare 1000 ml of MS media with care- a). The appropriate amount of each of stock solutions (macrosalts, microsalts, Fe-EDTA, vitamins, Myo-inositol) b) 500 ml distilled water was added in the flask to dissolve all the ingredients on a heater cum magnetic stirrer. c) 30 gm sucrose was added to this solution and agitated gently to dissolve completely. d) Different concentrations of hormonal supplements were added to the solution either in single or in combinations as required and mixed well. e) The whole mixer was then made up to 1L with adding distilled water. f) The pH of the liquid medium was adjusted to 5.75-5.80 with the aid of 0.1N HCl or 0.1 N NaOH, wherever it necessary. This operation was done using a digital pH meter. g) 0.6% agar was added to the liquid medium to make solid medium. The culture medium was dispensed into culture vial (20 ml/vial) h) Medium was finally sterilized by autoclaving. The explants were cut into small pieces (about 1.5 cm long) and initially washed under running tap water for 6-7 mins. Explants were rinsed with distilled water containing few drops of savlon and tween-20 with continuous shaking for 10-15 seconds and washed thoroughly with distilled water. They were then taken under laminar air flow cabinet to soak them with 70% alcohol for 10 seconds and washed 3-4 times with autoclaved distilled water. Finally, explants were immersed in 0.1% commercial HgCl 2 solution in combination with 2-3 drops tween-20 for 4-5 minutes with continuous shaking followed by adding sterilized distilled water 6-7 times to drain out last particle of HgCl₂. The explants were inoculated on MS medium fortified with cytokinins (BAP, Kinetin and TDZ in single or in combination) for shoot induction and auxins (NAA and IBA) for root induction. The pH of the media was adjusted at 5.75-5.80 before gelling the medium with 0.6% agar. The cap of vial was holded by little finger and was flame sterilized. During inoculation special care was taken so that the explants must touch the medium equally and not dept into the medium. After inoculating the explants onto culture media, cultures were incubated on culture rack at 25 + 2 0 C constant temperature under the white 25 + 2 0 C. The photo period was maintained as 16 hours light and 8 hours dark Data were collected on the effect of different treatments on direct shoot and root regeneration. For shoot induction, data were recorded in terms of days required for shoot initiation, percentage of shoot regeneration, shoot length (cm) and number of shoots per explants. The days required for shoot initiation, percentage of rooted shoots, number of roots formed, average root length (cm) were determined after 3 weeks of culture on the rooting medium. The data for the character under present study were statistically analyzed wherever applicable. The experiment was conducted in growth room and arranged in completely randomized design (CRD). The analysis of variance of different characters was performed and means were compared by the Duncan's multiple range test (DMRT).

Source of Information: Asian J. Med. Biol. Res. 2016, 2 (1), 95-106; ISSN 2411-4472 (Print) 2412-5571 (Online)

Article Link (Online Journal): doi: 10.3329/ajmbr.v2i1.27574

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Present studies were conducted with a view to evaluate the effect of different concentration of cytokinin hormone (BAP, Kn, Thidiazuron), coconut water on shoot regeneration and the effect of different concentration of auxins (NAA and IBA) on root formation. Micropropagation technology of Stevia rebaudiana has been briefly analyzed. Thus, this protocol could be helpful to establish and cultivate stevia as a promising newly introduced non-caloric sweetener and anti-diabetic medicinal plant in Bangladesh.

Knowledge Management: Journal