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Clonal Propagation Through Nodal Explant Culture of Boerhaavia Diffusa L. a Rare Medicinal Plant

A. Biswas1
Department of Botany, Government B. L. College, Daulatpur,Khulna

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

M. Roy
Department of Botany, Government B. L. College, Daulatpur,Khulna

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

Abstract/ Executive Summary:

Multiple shoots were induced on MS fortified with 2.0 mg/l BAP and 0.2 mg/l NAA within 30 days of culture. Maximum (93%) explants produced multiple shoots with an average 12 shoots per culture after two successive subcultures at 14 days interval in the same medium. Cent per cent rooting was achieved in half strength of MS supplemented with 1.0 mg/l IBA. Following 21 days in vitro rooting period and seven days of ex vitro acclimatization, plantlets were successfully established in natural condition. The survival rate of regenerated plants was 90 per cent.

Keywords: Clonal propagation, Boerhaavia diffusa, Rare medicinal plant, Nodal culture

Location: Rajshahi

Start Date:

End Date:

Program Area: Knowledge Management

Commodity:

Objectives:

The present research study was undertaken to develop an efficient and repeatable in vitro propagation protocol for B. diffusa through nodal explant culture.

Methodology:

Rootstocks of Boerhaavia diffusa L. were collected from Khagrachhari hilly areas and planted in earthen pots in the experimental field of the Institute of Biological Sciences, Rajshahi University. Leaves, internodal and nodal segments (0.5 ‐ 1.0 cm) of young healthy sprouts were used for in vitro culture. Explants were first thoroughly washed under running tap water for 20 min and then treated with liquid detergent (Tween 80) for ten min, followed by dipping in savlon solution (5% v/v) for ten min. The explants were then washed with distilled water. After a 45 second treatment with 70% ethanol the explants were surface sterilized with an aqueous solution of 0.1% HgCl₂for four min and rinsed with sterile distilled water for four ‐ six times. The surface sterilized explants were implanted in 0.6% (w/v) agar solidified MS decant in culture tubes (2.5 × 15 cm) devoid of plant growth regulators (control) or fortified with NAA, IAA and IBA : 0.1, 0.2, 0.3, 0.4 and 0.5 mg/l and BAP and Kn: 1.0, 2.0, 3.0 and 4.0 mg/l either singly or in combination with various concentrations. pH of the medium was adjusted to 5.7 ± 0.1 before adding agar and the medium was autoclaved at 121ºC and 1.1 kg/cm² pressure for 20 min. Initially the explants were cultured individually for 30 days and then subcultured at a regular interval of 14 days. When shoots attained a height of four ‐ six cm these were then subcultured individually for rooting in phytagel solidified half strength of MS with IBA, NAA and IAA (0.1 ‐ 1.0 mg/l + 3% sucrose) and compared to the control (MS without growth regulators). Microcuttings were elongated in the same medium. Culture vessels (shooting and rooting) containing inoculated explants were maintained in the culture room under a regular cycle of 16 hr photoperiod at 3000 lux light intensity of cool white fluorescent light at 25 ± 2ºC. Well‐rooted plantlets were removed from culture tubes and washed thoroughly in running tap water to remove all trace of medium attached to the roots. Those were then transplanted in plastic pots containing 1 : 1 autoclaved garden soil and compost. In order to maintain a high humidity the pots were then covered with transparent polythene bags and acclimatized in the laboratory temperature for two weeks. The pots were irrigated weekly intervals with distilled water. Polythene covers were removed gradually, and the plants were then transferred to earthen pots containing garden soil. After two weeks the plantlets were successfully established in field conditions. Each experiment was repeated thrice using 15 replicates (total 45 cultures per treatment). The data were analyzed statistically. Means were compared using DMRT.

Source of Information: Plant Tissue Cult. & Biotech. 19(1): 53-59, 2009 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In order to induce roots the individual in vitro grown shoot buds were cultured on half‐strength MS fortified with different concentrations of IBA (0,0 0.1 ‐ 1.0 mg/l), NAA (0.1 ‐ 1.0 mg/l) and IAA (0.1 ‐ 1.0 mg/l). In all the concentrations of the plant growth regulators used in the experiments indurooting. Without growth regulators no induction of roots was achieved. A 100% microcuttings rooted in the medium supplemented with 1.0 mg/l IBA. Maximum number (10.48 ± 0.06) and the longest (5.33 ± 0.11) roots were obtained in this concentration within three weeks of inoculation. It indicates that stress condition favours the root induction in B. diffusa. Induction of roots in IBA supplemented medium has also been reported in some other medicinal plants. The plantlets in pots were kept covered by transparent polybags for hardening outside the growth chamber. After seven days the polybags were completely removed and 90 per cent of the transplanted plants survived. Initially the growth performance was slow after transplantation; thereafter it improved gradually and after two weeks new leaves began to emerge from the plants.

Knowledge Management: Journal