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Development of an Efficient in Vitro Micropropagation Protocol for Medicinally Important Plant Achyranthes Bidentata Blume

Md. Jakir Hossain
Department of Genetic Engineering and Biotechnology, University of Chittagong, Chittagong-4331, Bangladesh.

Laila Khaleda
Department of Genetic Engineering and Biotechnology, University of Chittagong, Chittagong-4331, Bangladesh.

Mohammad Al-Forkan
Department of Genetic Engineering and Biotechnology, University of Chittagong, Chittagong-4331, Bangladesh.

Abstract/ Executive Summary:

The current situation of medicinal plants and increasing demand of plant derived drugs suggest an immediate need to conserve our medicinal plant resources. The present study attempted to develop in vitro micro propagation protocol for the important medicinal plant Achyranthes bidentata Blume. Shoot tip and nodal segment of healthy plants from natural condition were used as explants for direct organogenesis. Varying concentrations of auxins and cytokinins both alone and in combination were used. Nodal segment explants produced best response in terms of shoot induction percentage and maximum number of shoot than shoot tip. Single cytokinins media was far better than combined effect of cytokinins and auxins. 5.0 mgL^-1 and 4.5 mgL^-1 of BAP (6-Benzyl amino purine) produced the highest number of shoot which was 10.5 and 10.2, respectively from nodal segment explants whereas the same media produced 9.8 and 9.2 shoot from shoot tip. For root induction and root development, IBA (Indole-3-butyric acid) in full strength MS media was better than IBA in ½ MS (Murashige and Skoog) media. Among different concentrations of full and ½ strength MS media tested, 1.5 mgL^-1 of IBA in full strength MS media proved to be the best in terms of root induction (97.78 %) and root number (15.8). Almost 80 % of in vitro grown shoot lets were established and acclimatized in natural soil.

Keywords: In Vitro, Micropropagation, Achyranthes bidentata, Shoot Tip, Nodal segment

Location: Chittagong University campus.

Start Date:

End Date:

Program Area: Development of Host and Medicinal Plants

Commodity: Medicinal Plants

Objectives:

To establish a standard and highly reproducible in vitro propagation and regeneration protocol for the medicinally important herb Achyranthes bidentata through direct organogenesis.

Methodology:

Healthy plants of A. bidentata Blume were collected from Chittagong University campus. Young and green shoots of A. bidentata Blume were harvested and washed with running tap water and rinsed twice with distilled water. Then the explants were surface sterilized with 0.1 % and 0.2 % (w/v) HgCl₂ solutions for different duration (2-7 minutes). After rinsing with sterile distilled water for 4-6 times, shoot tips and nodal segments were cut into smaller segments (1-1.5 cm) and used as the explants. The explants were placed vertically on semi-solid basal MS medium supplemented with 3 % sucrose, 0.8 % (w/v) agar (Hi-Media, Mumbai, India). Different concentrations and combinations of 6-benzyl amino purine (BAP), kinetin (Kin), naphthalene acetic acid (NAA), 2, 4-dichlorophenoxyacetic acid (2,4-D) and indole butyric acid (IBA) for direct regeneration of shoots. For rooting, the in vitro raised shoot lets were sub-cultured on both full strength and 1/2 strength MS medium supplemented with various concentrations and combinations of auxins (IBA). The pH of the medium was adjusted to 5.8 before autoclaving at 121°C for 15 min. The cultures were incubated at (25±2°C) under cool fluorescent light of 16 h/day photo period). When the plant lets attained 4-8 cm heights with few leaves and well developed root system, they were ready for transplantation or acclimatization into soil. Before transfer into soil, the plant lets were taken out from the controlled environment of growth chamber, unplugged and were kept in room temperatures for 2-4 days to bring them in contact with normal temperature for gradual acclimatization. After 2-4 days of hardening the plant lets were taken out from culture vessel and washed the roots under running tap water to remove the agar and media. Then the plant lets were dipped in a beaker with tap water for 30 minutes. Then the plant lets were ready for transfer to natural or garden soil. The soil was prepared in the ratio of 1:1 (Garden soil: compost) and dried. Then this soil was autoclaved to make microorganism free. Finally some sand was mixed with the compost soil and the plant lets were planted into the soil. These plants were observed on 14 th and 28 th day. The data for different parameters were recorded and analyzed using SPSS (Version 16) statistical software by one way ANOVA procedures. Differences among the means were compared following Duncan’s Multiple Range Test (DMRT) at 5 % level of significance.

Source of Information: Journal of Pharmacognosy and Phytochemistry 2013; 2 (4): 6-13, ISSN 2278-4136

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The regeneration protocol developed in this study is superior. This could be exploited for commercial scale mass propagation of A. bidentata by pharmaceutical industries for drug development. Further studies on tissue culture of these species or other species of this family could be benefited from this result. Besides, this protocol can be utilized for cryopreservation study or conservation purpose.

Knowledge Management: Journal