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In Vitro Propagation of Stemona Tuberosa Lour. a Rare Medicinal Plant Through High Frequency Shoot Multiplication Using Nodal Explants

Animesh Biswas
Department of Botany, Government B. L. College, Khulna

M. A. Bari
Institute of Biological Sciences, Rajshahi University, Rajshahi

Mohashweta Roy
Institute of Biological Sciences, Rajshahi University, Rajshahi

S. K. Bhadra
Department of Botany, University of Chittagong, Chittagong

Abstract/ Executive Summary:

Nodal segments of Stemona tuberosa Lour. were found to proliferated without any differentiation on to MS supplemented with 3.0 mg/l BAP and 0.5 mg/l NAA under continuous dark condition. After 20 days of inoculation under dark condition the cultures were transferred to a daily cycle of 16/8 hrs light/dark photoperiod and there the proliferated nodal segments underwent direct organogenesis producing huge number of shoot buds (25.87/culture). The shoot buds underwent rapid elongation on a range of BAP (0.1 ‐ 1.5 mg/l) and IBA (0.1 ‐ 1.0 mg/l) supplemented MS. Rooting of elongated shoot buds was successfully achieved (90%) in half strength MS with 1.0 mg/l NAA. The plantlets were finally established in outside environment through a successive phase of acclimatization.

Keywords: Nodal explant, Stemona tuberose, medicinal plant, Direct organogenesis

Location: Chittagong Hill Tracts

Start Date:

End Date:

Program Area: Resource Development and Management

Commodity:

Objectives:

The purpose of the investigation reported here was to develop an efficient in vitro protocol that would maintain and propagate this important species.

Methodology:

Tuberous rootstocks of Stemona tuberosa Lour. were collected from natural habitat of Chittagong Hill Tracts and planted in earthen pots in the experimental field of the Institute of Biological Sciences, Rajshahi University. Nodal, internodal and leaf segments of two months old twigs were used as explants. Those were washed first under running tap water for 20 min. After washing, the explants were dipped into 5% (v/v) Savlon solution with a few drops of Tween 80, shaked gently for 10 min and washed with sterile distilled water following 1 min treatment with 70% ethanol. The explants were then surface sterilized with an aqueous solution of 0.1% HgCl₂ for 6 ‐ 8 min and finally washed in sterile distilled water for five times. Explants were then cut into small segments (0.5 ‐ 0.1 cm) and aseptically cultured on 0.8% (w/v) agar solidified MS with 3% sucrose and fortified with different plant growth regulators (PGRs) viz. BAP (1.0 ‐ 5.0 mg/l), Kn (1.0 ‐ 5.0 mg/l), NAA, IAA and IBA (0.1 ‐ 1.5 mg/l) at different concentrations and combination. pH of the medium was adjusted to 5.7 ± 0.1 before dispensing in culture tubes and autoclaving at 121ºC and 1.1 kg/cm² for 20 min. In order to see the effect of light and dark condition on multiple shoot induction, two sets of experiments were carried out: (i) for 16/8 hrs light dark photoperiod and (ii) for 15 ‐ 20 days continuous dark followed by a 16/18 hrs light/dark photoperiod. All cultures were incubated at 25 ± 2ºC and regular subculturing was done at three weeks interval. For induction of roots the elongated shoots were individually grown on full and half strength MS with different growth regulators (NAA, IAA and IBA) at different concentrations. The experiments were set up in a completely randomized design. Each experiment was repeated thrice using 10 replicates. The data were analyzed statistically following DMRT. These tests were conducted using statistical software package by MSTAT‐C.

Source of Information: Plant Tissue Cult. & Biotech. 21(2): 151-159, 2011 (December)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In order to induce rapid elongation, multiple shoot at a stage of 2 ‐ 4 cm long were individually subcultured on MS supplemented with reduced concentrations of BAP + IBA. Media fortified with 1.5 mg/l BAP + 0.5 mg/l IBA was proved effective for induction of rapid elongation. On elongation media shoot buds did not produce any roots. Thus the induction of strong and stout root systems the elongated shoot buds were individually cultured on full and half‐strength MS with or without IAA, NAA and IBA (0.1‐ 1.5 mg/l). Of the media combinations half strength MS with 0.5 mg/l NAA gave the best response and here 90% of the shoots produced on an average of 4.70 roots per shoot, within two weeks. The effectiveness of NAA at lower concentrations for in vitro rooting has been reported in various medicinal plants. In PGRs free media no root formation occurred. Shoots with well‐developed roots were planted in small pots containing sterile garden soil. Those were acclimatized for another one week in growth chamber. To prevent evaporation and facilitated light transportation, pots were covered by transparent polythene. Eighty per cent of the plantlets survived in out side natural conditions without any abnormal morphological changes

Knowledge Management: Journal