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In Vitro Germination and Micropropagation of Geodorum Densiflorum (lam.) Schltr., an Endangered Orchid Species

S. K. Bhadra
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh

M. M. Hossain
Department of Botany, University of Chittagong, Chittagong-4331, Bangladesh

Abstract/ Executive Summary:

Seeds of Geodorum densiflorum (Lam.) Schltr. were aseptically cultured on 0.8% (w/v) agar solidified Murashige and Skoog (MS) and Phytamax (PM) media. Seeds germinated and formed light green globular structures on both the media. These globular structures failed to produce seedlings on PM medium, where it proliferated and developed into irregular-shaped rhizomes with white hairy structures. On the other hand, on MS medium the globular structures directly produced tiny seedlings which underwent elongation in PGRs supplemented MS medium. A composition of 2.0 mg/l NAA and 2.0 mg/l BAP proved best for enhancing elongation. The elongated seedlings were grown on half and full strength MS medium with or without IAA and activated charcoal (AC). For sound development of the root system MS + 3% (w/v) sucrose + 1.0 mg/l IAA + 0.1% (w/v) AC proved best. The irregular shaped rhizomes as developed on PM medium were further used for mass production of seedlings. Rhizome tips produced multiple shoot buds on PGRs (NAA, IAA, IBA, picloram, BAP and zeatin) supplemented media. The highest number of multiple shoot buds per segment was observed on 0.8% (w/v) agar solidified MS + 3% (w/v) sucrose + 2.0 mg/l BAP. The multiple shoot buds underwent elongation on the same media and the maximum rate of elongation was recorded on 0.8% (w/v) agar solidified MS + 3% (w/v) sucrose + 2.0 mg/l NAA + 2.0 mg/l BAP. The elongated seedlings were rooted in the rooting medium and the plantlets after acclimatization were established in pot.

Keywords: In vitro, Micropropagation, Geodorum densiflorum, Orchid

Location: Department of Botany, University of Chittagong, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Orchid

Objectives:

To developing an efficient in vitro cultural technique of germination and micropropagation of G. densiflorum, an endangered orchid species of Bangladesh.

Methodology:

Mature capsules of Geodorum densiflorum were surface sterilized by submerging them in a 0.2% (w/v/) HgCl₂solution for 10 mins with occasional agitation followed by a dip in absolute ethanol for 12 - 15 sec. The sterilized capsules were then washed 5 - 6 times with sterile distilled water. The capsules were then cut with a sterile surgical blade and the seeds were inoculated on to the surface of the medium in the culture vessel. All these operations were done in a laminar airflow cabinet. pH of the medium was adjusted between 5.4 and 5.8 prior to autoclaving at 121⁰C for 20 mins at 1.9 g/cm² pressure. Culture vessels with inoculated seeds were maintained in the culture room with 14/10 h light and dark cycle at 25 ± 2⁰C. After 15 days of germination the tiny seedlings were aseptically taken out of the culture vessels and subcultured at a lower density on the same medium. Further subculturing was done at 20 - 25 days interval. In order to induce rapid growth, the seedlings were transferred to the elongation medium. Different types of 0.8% (w/v) agar solidified media fortified with different concentrations and combinations of PGRs (1.0 mg/l NAA + 0.5 mg/l BAP, 2.0 mg/l NAA + 2.0 mg/l BAP, 2.5 mg/l BAP + 0.1 % (w/v) AC, 1.0 mg/l Pic. + 2.0 mg/l BAP + 0.1% (w/v) AC, 1.0 mg/l IAA + 2.0 mg/l BAP) were used for the purpose. The elongated seedlings at a height of 3 - 4 cm were transferred to three kinds of 0.8% (w/v) agar-solidified rooting media viz. (i) half strength MS + 1.5% (w/v) sucrose, (ii) MS + 3% (w/v) sucrose + 0.5 mg/l IAA and (iii) MS + 3% (w/v) sucrose + 1.0 mg/l IAA + 0.1% (w/v) AC. For micropropagation, tips of seed-derived mini rhizomes were cultured aseptically on different 0.8% (w/v) agar solidified media supplemented with different PGRs. Activated charcoal was also added in some of the media as an additive. Culture vessels were maintained in the culture room under the same conditions as were used for germination. Within 25 - 33 days of culture, new shoot buds emerged from the rhizome tips and those underwent elongation on the same medium. When the multiple shoot buds reached a height of 3 - 4 cm, they were transferred to the rooting medium.

Source of Information: Plant Tissue Cult. 13 (2) : 165-171, 2003 (December)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The irregular shaped rhizomes as developed on PM medium were further used for mass production of seedlings. Rhizome tips produced multiple shoot buds on PGRs (NAA, IAA, IBA, picloram, BAP and zeatin) supplemented media. The highest number of multiple shoot buds per segment was observed on 0.8% (w/v) agar solidified MS + 3% (w/v) sucrose + 2.0 mg/l BAP. The multiple shoot buds underwent elongation on the same media and the maximum rate of elongation was recorded on 0.8% (w/v) agar solidified MS + 3% (w/v) sucrose + 2.0 mg/l NAA + 2.0 mg/l BAP. The elongated seedlings were rooted in the rooting medium and the plantlets after acclimatization were established in pot. Results of the present investigation indicate that, in vitro culture techniques could be formulated for germination and mass scale micropropagation of G. densiflorum. This strategy offers an opportunity for using the techniques in commercial nurseries as well as for ex situ conservation of this endangered orchid species.

Knowledge Management: Journal