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Micropropagation of Stevia Rebaudiana Bertoni Through Direct and Indirect Organogenesis

Sweety Majumder*
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh.

Md. Mahbubur Rahman
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh.

Abstract/ Executive Summary:

The present research work was undertaken to develop a protocol for micropropagation of Stevia rebaudiana Bertoni., an important medicinal plant of Bangladesh. Shoot apex, Leaf and Nodal explants were aseptically cultured on agar solidified Murashige and Skoog (MS) medium supplemented with different growth PGRs (Plant growth regulators). Indirect organogenesis was found in leaf segment and direct organogenesis was observed in shoot apex. But in case of nodal segment both direct and indirect organogenesis were found. Maximum amount of green compact callus produced from nodal explants in MS medium supplemented with 1.0mg/l benzylaminopurine (BAP) + 0.5mg/l indole-3-acetic acid (IAA). The induced calli were cultured on MS medium fortified with different PGRs for shoot proliferation. The maximum number of shoot buds (15.30 ± 0.15) formation was observed in MS medium fortified with 1.5mg/l BAP+0.5mg/l IAA. Multiple shoot buds underwent rapid elongation on elongation media and maximum elongation (6.90 cm) took place on MS with 1.0 mg/l BAP + 1.0 mg/l IAA. Elongated shoot buds produced strong and stout roots (4.40 cm) on half strength MS medium fortified with 1.0 mg/l indole-3-beutyric acid (IBA). The well developed plantlets were successfully transferred to hardening and survival rate was 95%.

Keywords: Stevia rebaudiana, Micropropagation, Callus, Organogenesis.

Location: Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh.

Start Date:

End Date:

Program Area: Development of Host and Medicinal Plants

Commodity: Stevia

Objectives:

The present investigation was undertaken with a view to develop reliable and efficient protocol for rapid and mass scale micropropagation of this plant species study for local environment of Bangladesh.

Methodology:

Plant Materials and Surface Sterilization Three months old seedling of S. rebaudiana were collected from a nursery of Bangladesh Council for Scientific and Industrial Research (BCSIR), Chittagong and were established in garden pots of Botany Department, Chittagong University. Shoot Apex, Leaf and nodal segments of garden pots grown plants of S. rebaudiana were collected and thoroughly washed under running tap water for 10 minutes, treated with liquid detergent for 10 minutes, followed by dipping in 5% (v/v) savlon solution for 10 minutes. The materials were then washed 3-4 times with distilled water for complete removal of detergent and taken under running laminar airflow cabinet and transferred to 500ml sterilized conical flask. After rinsing with 70% ethanol for less than 60 Seconds, they were surface sterilized with 0.1% (w/v) HgCl2 for 5 minutes and washed with sterile distilled water 4-5 times. The surface sterilized explants were cut into small pieces (0.5- 1.0cm) with a sterilized surgical blade and then inoculated onto the culture media.

Culture media and conditions for plant regeneration Murashige and Skoog (MS) basal medium supplemented with different concentration and combination of plant growth regulators (PGRs) were used for induction of organogenesis or embryogenesis. In some cases the multiple shoot buds (MSBs) that developed from either nodal explants or from callus elongated on MS supplemented with different PGRs and for rooting, elongated shoots at a height of 2-4 cm were rescued aseptically from the cultured on rooting medium containing half strength and one forth strength of MS basal medium fortified with different concentration and combinations Indole-3-butyric (IBA), indole-3-acetic acid (IAA) and αnaphthaleneacetic acid (NAA). In all cases the media were solidified with 0.8% (w/v) agar and pH was adjusted to 5.8 before autoclaving for 30 minutes at 121⁰C under 1.1kg/cm² pressure. Culture vessels with inoculated explants were maintained under a regular cycle of 14 hours light and 10 hours dark at 25±2⁰ C.

Subculture for multiple shoots Proliferated multiple shoots were rescued very carefully in aseptic conditions and divided into clusters of 2 - 3 shoots using a sterile sharp scalpel. Sub culturing was done on the same or different media for further response at an interval of 15 - 20 days and culture vessels were maintained in the culture room in the same light and temperature conditions.

Rooting of in vitro shoot Experiments of adventitious root formation on the shoots proliferated in vitro were conducted only after having sufficient amount of shoot cultures. Different rooting experiments were carried out with half strength MS medium with or without growth regulators to determine the suitable media composition, optimum growth requirements. After 10-20 days, the proliferated multiple shoots were separated and individual shoots were placed in rooting media. The adventitious roots were produced from the cut ends of micro shoots within 2-3 weeks of culture on suitable medium. Elongated shoots at 2-4 cm height were rescued aseptically from the culture vessels and cultured on freshly prepared rooting medium.

Hardening and Acclimatization of plantlets to soil The well rooted plantlets were transferred to earthen pots containing a mixture of soil and compost(2:1) at relative humidity 90% with light intensity varied 2000-3000 lux and temperature of 28±2⁰ C following successive phases of acclimatization. For the purpose, the month of the culture vessels were kept open for one day in the culture room and they were then kept outside the culture room for 6 hours in the next day. Later on those were kept outside the culture room for 12 hours. Finally, the seedling were taken out of the culture vessels and rinsed with running tap water for complete removal of medium attached to the roots.

Statistical analysis Experiments were set up in a Randomized Block Design (RBD) and each experiment was replicated thrice. Observations were recorded on the percentage of response, number of shoots per explants and number of roots per shoot. A minimum of 10-15 explants were used for each experiment. Means and standard deviations were calculated for each treatment. The data means ± SD of at least three different experiments were represented.

Source of Information: Journal of Innovations in Pharmaceuticals and Biological Sciences Vol 3 (3), 47-56, 2016 e-ISSN: 2349-2759 p-ISSN: 2395- 1095

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, the present investigation reports an efficient and reproducible regeneration protocol via. direct and indirect organogenesis of Stevia rebaudiana Bertoni. The method is flexible, allowing incorporation of different types of explants (nodal segment, shoot apex and leaf segment) with BAP, Kn, NAA, 2,4-D and IAA effective in both callus and multiple shoot buds proliferation. MS medium containing 1.5 mg/l BAP+0.5 mg/l IAA was the best for shoot proliferation. Between the two explants nodal segment gave better response than leaf segments and MS fortified with1.0 mg/l BAP+0.5 mg/l IAA was the best for callus induction. Half strength MS medium fortified with 1.0 mg/l IBA was found to be the best treatment for root formation in S. rebaudiana. The protocol developed for seedlings of S. rebaudiana can be used reliably for propagation in a commercial scale and ex situ conservation of this valuable medicinal plant species.

Knowledge Management: Journal